Citation
Molecular characterization of citrus tristeza virus genes and their use in plant transformation

Material Information

Title:
Molecular characterization of citrus tristeza virus genes and their use in plant transformation
Creator:
Febres-Rodriguez, Vicente Jose, 1964-
Publication Date:
Language:
English
Physical Description:
vii, 105 leaves : ill., photos ; 29 cm.

Subjects

Subjects / Keywords:
Activating transcription factors ( jstor )
Amino acids ( jstor )
Antiserum ( jstor )
DNA ( jstor )
Open reading frames ( jstor )
Pitting ( jstor )
Plasmids ( jstor )
Polymerase chain reaction ( jstor )
Proteins ( jstor )
RNA ( jstor )
Dissertations, Academic -- Plant Pathology -- UF
Plant Pathology thesis Ph. D
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1995.
Bibliography:
Includes bibliographical references (leaves 95-104)
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Vicente Jose Febres-Rodriguez.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
021943698 ( ALEPH )
33808737 ( OCLC )

Downloads

This item has the following downloads:


Full Text












MOLECULAR CHARACTERIZATION OF CITRUS TRISTEZA VIRUS GENES
AND THEIR USE IN PLANT TRANSFORMATION




















By

VICENTE JOSE FEBRES-RODRIGUEZ

















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1995















ACKNOWLEDGMENTS


I want to express my gratitude to Dr. Chuck Niblett, chairman of my supervisory committee, and Dr. Richard Lee, cochairman, for their support, encouragement and guidance in completing this thesis. I also want to thank Dr. Chuck Powell

and Dr. Jude Grosser, members of my committee, for their assistance and suggestions. Thanks also to Dr. Ken Derrick for his comments and his help in reviewing this manuscript.

I am grateful to Dr. Hanumantha Pappu, Dr. Sita Pappu and Dr. Ed Anderson for their technical support, suggestions and friendship, that certainly helped me complete this work in an easier way.

I also thank Dr. Gloria Moore and members of her lab, especially Alejandra Guti~rrez, for their assistance in the tissue culture portion of this work and for sharing their time, procedures and lab equipment with me. Thanks to Blanca Garagorry for her help in the transformation experiments.

Thanks to Dr. Dave Stark and Monsanto for providing the pMON vectors and Agrobacterium strain used for plant transformation.

I thank Tiffany Niblett for her kindness and patience that allowed this work to be more productive and enjoyable for me and my adviser. And, of course, thanks to Manjunath and ii









David Benscher for all the help and good times spent together in the lab.



Finally, thanks to the faculties, postdocs, technicians, administrative personnel and students in the plant pathology

department that in one way or another collaborated to make this work possible.
















TABLE OF CONTENTS


ACKNOWLEDGMENTS..................... . ... . .. .. .. ...1

ABSTRACT.................................vi

CHAPTER

1. INTRODUCTION.....................1


2. DETECTION OF THE IN VIVO EXPRESSION OF
THE P27, P20 AND P18 PROTEINS...............8

Introduction.....................8
Materials and Methods..............9
Materials...................9
Plant Material..............................10
Cloning of CTV ORFs into pUC118 ......10
Subcloning of CTV ORFs in the
Expression Vector.................16
DNA Sequencing. ........................17
Bacterial Expression of CTV Proteins . 18 Production of Polyclonal. Antisera . . 19
Detection of CTV Proteins in
Infected Citrus Tissue...........20
Results.......................22
Cloning of CTV Genes............22
Expression of Proteins...........23
Detection of p27..............24
Detection of p20..............31
Detection of p18..............31
Discussion.....................33


3. SEQUENCE ANALYSIS OF THE P27 ORF ........40

Introduction....................40
Materials and Methods..............41
Materials.................41
Virus Isolates. ............................42
Cloning and Sequencing...........42
Single Stranded Conformation
Polymorphism (SSCP).............44


iv










Results .:I*I*I**I*IIII*I*14
Sequencing.........................
Single Stranded Conformation
Polymorphism....................54
Discussion.....................63

4. TRANSFORMATION OF SWEET ORANGE WITH P27
AND P20.......................67

Introduction....................67
Materials and Methods..............70
Materials...............................70
Cloning of p27 Into the Transformati~on
Vector. .....*.*......................70
Cloning of p20 Into the Transformati~on
Vector.....................71
Transformation of pVF plasmids
into Agrobacteriun..............73
screening of Transformed Bacteria .... 73
Transformation, Selection and
Regeneration of Citrus Plants.......75
Results.......................80
Cloning of CTV Genes Into the
Transformation Vectors...............80
Transformation and Regeneration of
Sweet Orange....................80
Discussion.........................87

5. SUMMARY AND CONCLUSIONS.............93

LITERATURE CITED.....................95

BIOGRAPHICAL SKETCH...................105





















V















Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

MOLECULAR CHARACTERIZATION OF CITRUS TRISTEZA VIRUS GENES AND THEIR USE IN PLANT TRANSFORMATION By

Vicente Jos6 Febres-Rodriguez

August, 1995

Chairman: C.L. Niblett
Major Department: Plant Pathology

Nucleotide sequence analysis of citrus tristeza closterovirus (CTV) revealed the presence of 12 possible open reading frames (ORFs). The ORF immediately upstream of the coat protein (CP) gene encodes a protein of calculated MW of 27.4 kDa (p27). The deduced amino acid sequence indicated that this gene product is homologous to the CP (41% similarity). Two other genes located toward the 3' end of the CTV genome encode proteins of 18.3 (p18) and 20.5 (p20) kDa, respectively. Deduced amino acid sequence comparisons with protein databases did not reveal any significant relationship that might indicate the possible function of these proteins. The objectives of this research were: 1) to determine if p27, p20 and p18 proteins were expressed in CTV-infected citrus tissue, 2) to identify the sequence variability of the p27 ORF among CTV strains with different biological properties and to vi









determine any possible differences that could correlate with virulence, and 3) to transform citrus plants with p27 and p20 genes in an attempt to confer resistance to CTV. Fusion proteins for p27, p20 and p18 were produced in Escherichia coli and used to raise polyclonal antibodies. Western blot analyses using the antisera indicated that p27 and p20 are expressed in CTV-infected citrus, but not in uninfected plants. Tissue fractionation studies revealed that p27 accumulates in cell wall enriched fractions, whereas p20 accumulates in the soluble protein fraction. The expression of p18 was not detected in CTV-infected citrus tissue. The p27 protein also was detected using tissue blot analysis. RNA sequence homologies of the p27 gene among CTV isolates were between 86 and 99%. The deduced amino acid sequence homologies were 93% or higher. Phylogenetic analysis showed mild and severe, quick decline isolates grouping separately. Agrobacterium-mediated plant transformation experiments were performed using p27 and p20 genes. Approximately 6% of the adventitious shoots obtained on selection media with kanamycin were GUS positive. Between 31 and 37% of the GUS positive shoots were positive using PCR analysis with primers specific to p27 or p20, suggesting that some plants are transformed with each of the genes.







vii
















CHAPTER 1
INTRODUCTION


Citrus tristeza was initially recognized as a decline disease of citrus scions propagated on sour orange (Citrus aurantium L.) rootstock (Lee and Rocha-Pefia, 1992). This disease, caused by citrus tristeza closterovirus (CTV), is one of the major diseases affecting citrus, and an excellent example of an agricultural problem created by man (Bar-Joseph et al., 1989). CTV is now known to cause a variety of symptoms depending on the virus strain and host. Perhaps the most economically important and obvious CTV symptom is the decline of sweet orange [C. sinensis (L.) Osbeck], grapefruit (C. paradisi Macf.) and mandarin (C. reticulata Blanco) grafted on sour orange rootstock (Garnsey et al., 1987; Bar-Joseph and Lee, 1990). Stem pitting in limes [C. aurantifolia (Christm.) Swingle], grapefruit and some sweet oranges is another important reaction and a limiting factor to production in parts of Brazil, South Africa and Australia (Garnsey et al., 1987; Bar-Joseph and Lee, 1990; Lee and Rocha-Pefia, 1992). In addition, some CTV strains can induce seedling yellows when inoculated to sour orange, grapefruit and lemon (C. limon Burm.f) seedlings. The importance of this symptom is not known, except for CTV strain differentiation, because it

1









2

normally is not observed in the field (Garnsey et al., 1987;

Bar-Joseph and Lee, 1990). Some mild strains, on the other hand, do not induce any noticeable symptoms in most of the

cultivated varieties (Garnsey et al., 1987; Bar-Joseph and Lee, 1990). Several strains can be present in the same tree,

as has been determined by cross protection experiments (Powell et al., 1992) and double-stranded RNA analysis (Moreno et al., 1991). This makes the use of biological methods, such as cross-protection, more difficult to control the disease, although they are still possible (Costa and Muller, 1980).

CTV is present in most areas of the world where citrus is grown. In South America it was first described in the 1930s.

Less than two decades later CTV caused the destruction of most of the citrus industries in Argentina, Brazil and Uruguay, killing about 25 million trees planted on the susceptible sour orange rootstock (Muller and Costa, 1992). The spread of the

disease and its impact is increased by the presence of its most ef ficient vector Toxopt era citricida (Kirkaldy) the brown citrus aphid. A well documented case occurred in Venezuela. The disease was reportedly present in the country

in the mid-'50s without having a major impact. In 1976 T. citricida was first found in Venezuela in regions bordering with Colombia and Brazil (Geraud, 1976). Two years later it

was widespread throughout the country. In 1980 the first outbreak of CTV was reported, and since then, at least 6









3

million citrus trees on sour orange rootstock have died from tristeza (Mendt, 1992).

In Florida, approximately 20-25 million trees of sweet orange, grapefruit and mandarin are planted on the susceptible sour orange rootstock (Garnsey, 1991). CTV has been present in Florida since the '50s, and both mild and decline inducing strains have been reported (Brlansky et al., 1986). In addition, 8-10 million trees of grapefruit are also susceptible to stem pitting strains, which have not been detected yet in Florida (Garnsey, 1991). The aphid T. citricida is still not present, although other less efficient vectors such as Aphis gossypii Glover and A. citricola van der Goot are widespread in Florida. However, T. citricida has continued its northward movement from South America. In recent surveys in Central America and the Caribbean the aphid was reported in Nicaragua (Lastra et al., 1991) and Cuba (Yokomi et al., 1994). T. citricida thus represents a serious threat to the citrus industries of Florida and the United States, as well as Mexico, Cuba and other Caribbean countries. This is because of its efficiency to vector CTV (6 to 25 times more efficient than A. gossypii; Yokomi et al., 1994), especially with the decline and stem pitting strains which are not efficiently vectored by the aphid species present in Florida (Yokomi, 1990).

Transmission of CTV by aphids is in a semipersistent manner (Bar-Joseph and Lee, 1990). It also is transmitted by









4

budding and grafting (Bar-Joseph and Lee, 1990). Mechanical transmission is difficult and only possible when concentrated preparations are used and slash inoculated into the stems of young citrus plants (Garnsey et al., 1977).

CTV is a phloem-limited virus. Inclusion bodies are observed inside the phloem, phloem fiber and parenchyma cells adjacent to sieve tubes (Schneider, 1959; Brlansky et al., 1988). They also are detected by immunofluorescence using antibodies to CTV coat protein (CP) (Brlansky et al., 1988), indicating that the inclusion bodies contain virus particles. These virus particles are flexuous, 1900-2000 nm long and 1011 nm in diameter (Bar-Joseph and Lee, 1990).

The genome of CTV is a single-stranded, positive sense RNA 19,296 nucleotides in length (Bar-Joseph and Lee, 1990; Karasev et al., 1995). Sequencing of the entire genome of the Florida, severe, quick decline strain T36 indicates the presence of 12 possible open reading frames (ORFs) (Fig. 1) coding for 17 proteins (Pappu et al., 1994; Karasev et al., 1995). In the 5' to 3' direction the first ORF potentially codes for a polyprotein with an estimated MW of 349 kDa that encodes the putative domains for two papain-like proteases, a methyltransferase and a helicase (Karasev et al., 1995). This polyprotein is speculated to undergo autoproteolytic cleavage to produce three fragments of 54, 55 and 240 kDa, respectively (Karasev et al., 1995). The second ORF encodes a putative RNAdependent RNA polymerase of a calculated MW of 57 kDa that is





















Pol p6 p61 p18 p20





HEL p33 p65 p27 CP p13 p23





Figure 1. Genomic organization of the citrus tristeza virus genome according to Pappu et al. (1994) and Karasev et al. (1995). The ORFs are represented as rectangles and the putative gene products are indicated. (HEL, helicase; Pol, polymerase; CP, coat protein).









6

proposed to be expressed by ribosomal frameshift of the first ORF, resulting in a polyprotein of 401 kDa (Karasev et al., 1995). This polyprotein also would undergo autoproteolytic cleavage producing a 292 kDa fragment with domains for methyltransferase, helicase and RNA polymerase (Karasev et al., 1995). The third ORF potentially encodes a 33 kDa (p33) protein (Karasev et al., 1995) of unknown function, followed by a small ORF encoding a 6.4 kDa protein with a highly hydrophobic domain (Dolja et al., 1994). ORFs 5 and 6 code for proteins of 64.7 (p65) and 61.1 (p61) kDa, respectively (Pappu et al., 1994). Sequence comparisons indicate conservation of several motifs between p65 and the hsp70 group of heat shock proteins (Pappu et al., 1994). Similarly, p61 contains a Cproximal domain that is present in another group of heat shock proteins, the hsp9o group (Pappu et al., 1994). These two proteins have been detected in CTV-infected citrus tissue (S.S. Pappu, personal communication).

The following ORFs, 7 and 8, encode proteins of calculated MW of 27.4 (p27) and 24.9 kDa, respectively (Pappu et al., 1994). ORF 8 has been identified as the CP gene (Sekiya et al., 1991; Pappu et al., 1993b). Based on their deduced amino acid sequence, p27 shows 41% similarity with the CP (Pappu et al., 1994). ORFs 9 to 12 potentially encode proteins with calculated MW of 18.3 (p18), 13.2 (p13), 20.5 (p20) and 23.7 (p23) kDa, respectively (Pappu et al., 1994). The functions of these proteins are not known, and deduced









7

amino acid sequence comparisons with protein databases do not reveal any significant relationships (Pappu et al., 1994). Only p23 seems to contain a motif similar to some RNA-binding

proteins (Dolja et al., 1994). Fig. 1 indicates the genome organization of the 12 potential ORFs described above.

The identification of the functional ORFs of CTV will facilitate understanding the mechanisms of gene expression, replication and pathogenesis of this important virus, and eventually enable the development of better disease control strategies. The expression of viral genes in transgenic

plants, for example, has been a useful strategy to produce plants resistant to several diverse viruses (Powell Abel et al., 1986; Beachy et al., 1990; Pappu et al., 1995).

In this research the expression of the CTV genome was studied to increase the information available on this virus. The specific objectives of this research were as follows:

1) To determine if the p27, p20 and p18 proteins were

expressed in CTV-infected citrus tissue.

2) To identify the sequence variability of the p27 ORF among

CTV strains with different biological properties and determine any possible differences that could correlate

with virulence.

3) To transform citrus plants with the p27 and p20 genes in

an attempt to genetically engineer CTV resistance in

citrus.















CHAPTER 2
DETECTION OF THE IN VIVO EXPRESSION OF THE P27, P20 AND P18 PROTEINS


Introduction

There is significant resemblance between the genomic organization of CTV and two other members of the closterovirus group, beet yellows virus (BYV) and the bipartite lettuce infectious yellows virus (LIYV). CTV ORFs 1 (helicase), 2 (polymerase), 4 (small hydrophobic protein), 5 (p65-hsp70), 6 (p61-hsp90), 7 (p27-CP homologue) and 8 (CP) have homologous ORFs in BYV and LIYV in similar positions (Dolja et al., 1994; Klaassen et al., 1995; Pappu et al., 1994). Interestingly for LIYV, the CP homologue is located downstream of the CP (Dolja et al., 1994; Klaassen et al., 1995). Another CTV ORF, p20, shows similarity in the deduced amino acid sequence only to p21, the 3' terminal ORF in BYV (Pappu et al., 1994). The CTV ORFs 3 (p33), 9 (p18) and 12 (p23) do not have homologues in the two other closteroviruses.

The fact that some of the CTV ORFs have homologues in BYV and LIYV is a strong indication, but does not demonstrate, that those ORFs are functional in vivo. The detection of subgenomic RNAs for 9 of the 12 ORFs, including p27, p20 and p18 (Hilf et al., 1995) also supports that they are expressed



8









9

in vivo. The final evidence, however, is the detection of the protein products in infected tissue.

In order to investigate whether p27, p20 and p18 are expressed in CTV infected plants the genes were separately cloned into Escherichia coli expression vectors. The expressed proteins were used to raise polyclonal antibodies to probe for the proteins in CTV-infected citrus tissue.



Materials and Methods

Materials

Reagents were obtained from Fisher Scientific (Pittsburgh, PA) or Sigma Chemical Company (St. Louis, MO). Enzymes and lambda DNA marker were purchased from Promega Corporation (Madison, WI). Some restriction enzymes, agarose and low melting point (LMP) agarose were from GIBCO BRL (Gaithersburg, MD). Phenol (equilibrated with 0.1 M Tris-HCl, pH>7.8), T4 polynucleotide kinase, dNTPs and Sequenase Version 2.0 DNA sequencing kit were from Amersham (Arlington Heights, IL). The nucleotide [a-35S]-dATP and the Renaissance chemiluminescent detection kit for Western blots were from DuPont NEN Research Products (Boston, MA). Protein standards SDS-6 and SDS-7 were from Sigma Chemical Company. Electrophoresis apparatus for small gels (7 x 8 cm), MiniPROTEAN II dual slab cell, was from Bio-Rad (Richmond, CA). Electrophoretic transfer unit, Mini Trans-Blot, was also from Bio-Rad. The larger electrophoresis unit (14 x 14 cm), VAGE,









10

was from Stratagene (La Jolla, CA). The pETH-3b expression vector was kindly provided by D.R. McCarty. The expression clone for the CP was kindly provided by M.L. Keremane. Plant Material

Mexican lime [C. aurantifolia (Christm.) Swingle] plants infected with CTV T36 were maintained in greenhouses at the University of Florida (Gainesville, FL).

Samples of citrus infected with citrus ringspot virus, citrus variegation virus, citrus leaf rugose virus, psorosis A and concave gum were kindly provided by R.F. Lee. Cloning of CTV ORFs into pUClI8

Extraction of viral RNA templates. Total nucleic acids were extracted from citrus leaves infected with the Florida severe, quick decline strain T36, using the procedure described by Pappu et al. (1993c). Approximately 1 cm2 of tissue was ground in a microcentrifuge tube with a sterile blunt stick (2 x 147 mm) in liquid nitrogen. The samples were thawed in 0.3 ml of extraction buffer [0.1 M Tris-HCl, pH 8.0, 2 mM ethylenediaminetetraacetic acid (EDTA) and 2% sodium dodecyl sulfate (SDS)]. An equal volume (0.3 ml) of a mixture of phenol: chloroform: isoamyl alcohol (25:24:1) were added, followed by vortexing for a few seconds. The samples were incubated at 700C for 5 min and then centrifuged at 14,000 rpm for 5 min at room temperature. The aqueous upper phase was saved.









11

The template was further purified using Sephadex G-50 spin-column chromatography, according to the procedure described in Sambrook et al. (1983). The column (a 1 ml syringe barrel), containing hydrated, sterile Sephadex, was equilibrated by washing three times with 100 Al of sterile TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) and 100 Al of extract applied to it. The column was centrifuged at 1,600g for 4 min at room temperature, and the effluent collected in a microcentrifuge tube. The effluent was used immediately for reverse transcription coupled with polymerase chain reaction (RT-PCR) amplification or quickly frozen in liquid nitrogen and stored at -800C for later use.

PCR amplification. The CTV genes were amplified using RTPCR (Saiki et al., 1988) and specific primers for each gene. Table 1 shows the primers, derived from the T36 sequence (Pappu et al., 1994), used to amplify each of the genes. The RT-PCR reactions were performed successively in a single microcentrifuge tube in a total volume of 100 Al with the following components: 10 mM Tris-HCl pH 9.0 (at 250C), 50 mM KCl, 0.1% Triton X-100, 10 mM dithiothreitol (DTT), 2.5 mM MgCI2, 0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, 0.4 mM dTTP, RNasin (40 u), AMY reverse transcriptase (19 u), Taq polymerase (2.5 u) and primers at a final concentration each of 100 pM. Normally, 30 to 50 Al of the purified template were used in each reaction. It was previously denaturated at 700C for 3 min and quick chilled on ice for a few min. The RT-PCR









12









Table 1. Description of the primers used for
cloning CTV ORFs p27, p20 and p18.



ORF PRIMERa Tmb
(OC)

p27 5' AAGCTTCTAGAACCATGGCAGGTTATACAGTAC 3' 54
5' CTATAAGTACTTACCCAAATC 3' 56
p20 5' AAGCTTCTAGAACCATGCGAGCTTACTTTAGTG 3' 54
5' CTACACGCAAGATGGAGA 3' 58
p18 5' AAGCTTCTAGAACCATGTCAGGCAGCTTGGG 3' 54
5' CTAAGTCACGCTAAACAAAG 3' 56

a Upper sequence is the genome sense primer. Lower
sequence is the genome antisense primer. Bold
letters are HindIII (AAGCTT) and XbaI (TCTAGA) restriction sites. The start codon is underlined.
b Tm= calculated melting temperature.









13

mixtures with the template RNA were incubated in an automatic thermocycler at 420C for 45 min, followed by 40 cycles of incubations at 940C for 1 min, 450C for 1 min and 720C for 1 min, and a final incubation at 720C for 10 min. The same parameters were used for the amplification of all three ORFs.

After the PCR reaction was completed, a 5 Al aliquot was electrophoresed in a 0.8% agarose gel in TBE buffer (9 mM Tris-borate, 2 mM EDTA) to determine the amplification of the DNA fragment of the correct size. Lambda DNA digested with HindIII was used as a molecular weight marker.

Purification of the PCR products. Once the fragments of interest were detected, the rest of the PCR samples (95 Al) were electrophoresed in 0.8% LMP agarose in TBE buffer at 40C and 50 to 75 V for about 1 h. The DNA bands of interest were excised from the gel with a sterile blade under UV light and transferred to a microcentrifuge tube. TE buffer was added to a final volume of 0.6 ml. Samples were then incubated at 650C for 7 min to melt the agarose, and then extracted with phenol, phenol: chloroform, and chloroform followed by ethanol precipitation according to Sambrook at al. (1989). The final DNA pellet was resuspended in 10 il of sterile distilled water.

Initially, the amplified p27, p20 and p18 genes were cloned into the SmaI site of the pUClI8 vector. Restriction sites for HindIII and XbaI were included in the sense primers to facilitate subcloning into the pETH expression vector and









14

the pVF transformation vector once clones with the correct orientation were identified.

Restriction digestion of plasmid DNA. Purification of pUClI8 from E. coli DH5a was done from 3 ml overnight cultures grown at 370C and shaking at 180 rpm in 2 x YT medium (1.6% bacto-tryptone, 1% bacto-yeast extract, 0.5% NaCl, pH 7.0) using a modification of the procedure of Sambrook et al. (1989). The only difference was the use of a half volume of 7.5 M ammonium acetate for the precipitation of bacterial proteins prior to the DNA precipitation. The plasmid DNA was resuspended in 30 Al of sterile distilled water.

For each ligation reaction approximately 0.5 Ag of pUCll8 were digested with SmaI according to the manufacturer's recommendations in a final volume of 30 Al. Digestion was performed for 3 to 4 h, and the DNA was subsequently purified using phenol, phenol: chloroform and chloroform extractions and precipitated with ethanol (Sambrook et al., 1989).

Ligation reaction. For ligation of the PCR products into the SmaI digested pUCII8, the purified PCR products (10 Al) were first treated with 5 units of DNA polymerase I (Klenow fragment) in a final volume of 15 Al containing 90 mM Tris-HCl pH 7.5 (at 370C), 10 mM MgCl2, 50 mM NaCl, 1 mM dNTPs. Incubation was for 10 min at room temperature, 20 min at 370C and 10 min at 700C. The PCR products were then treated with phenol, phenol: chloroform, chloroform and precipitated with









15

ethanol (Sambrook et al. 1989). The dried pellets were resuspended in 16 Al of sterile distilled water.

The total volume of recovered PCR products was mixed with the purified SmaI digested pUClI8 for ligation in 500 mM TrisHC1 pH 8.0, 100 mM MgCl2, 200 mM DTT, 10 mM ATP, 0.05% bovine serum albumin type V (BSA) and T4 polynucleotide kinase (30 u) in a final volume of 28 Al and incubated at 370C for 45 min. After that, 2 Al of T4 DNA ligase (6 u) were added to the reaction and incubated overnight at 160C or, alternatively, incubated at room temperature for 2 h.

Bacterial transformation. After ligation, E. coli DH5a competent cells were prepared and transformed using the calcium chloride procedure (Sambrook et al., 1989).

Transformed bacteria were plated on 2 x YT agar medium containing 100 Ag/ml of ampicillin and 40 Ag/ml of 5-bromo-4chloro-3-idolyl-o-D-galactoside (X-gal) and incubated overnight at 370C. White bacterial colonies were transferred to 2 x YT agar plates containing ampicillin for further analysis.

Detection of recombinant plasmids. The rapid disruption of bacterial colonies method was used to determine the presence of recombinant plasmids containing the gene of interest (Sambrook et al., 1989). Plasmid sizes were compared in 0.8% agarose gel electrophoresis to a standard of pUClI8 without insert; those with insert migrated slower in the gels.









16

Recombinant plasmids were then purified from liquid cultures by the procedure of Sambrook et al. (1989). To confirm the presence of an insert and determine its orientation, approximately 0.5 pg of plasmid DNA were digested with 5 u of HindIII following the manufacturer's instructions. Clones with sense orientation in pUC118 were used for subcloning into the expression vector. Subcloninq of CTV ORFs in the Expression Vector

The plasmid pETH-3b (McCarty et al., 1991) was used for expression of the CTV genes p27, p20 and p18. The inserts were recovered from pUCll8 by digestion with HindIII and EcoRI. Approximately 5 gg of plasmid DNA were digested with HindIII (10 u) and EcoRI (20 u) in a final volume of 30 Al in 6 mM Tris-HCl pH 7.5, 6 mM MgCl2, 50 mM NaCl, 1 mM DTT for 3 to 4 h at 370C. The digestion products were electrophoresed in a 0.8% LMP agarose gel and the inserts purified as described above.

DNA from pETH-3b (0.5 Ag per reaction) was also digested with HindIII (2.5 u) and EcoRI (5 u) and the linearized plasmid similarly purified in LMP agarose. Ligation and bacterial transformation into E. coli DH5a were as described above, omitting only the addition of DNA polymerase I (Klenow fragment) and T4 polynucleotide kinase. Transformed bacteria were grown in 2 x YT agar media containing 100 Ag/ml of ampicillin. Bacterial colonies were screened using the rapid disruption of bacterial colonies method (Sambrook et al.,









17

1989). Colonies, containing the recombinant plasmid, were replicated for later use.

Plasmid DNA was purified (Sambrook et al., 1989) and transformed into E. coli BL21(DE3) (the expression host for pETH plasmids) competent cells prepared by the calcium chloride method (Sambrook et al., 1989). The bacteria were plated on LB agar (1% bacto-tryptone, 0.5% bacto-yeast extract, 1% NaCl, pH 7.0, 1.5% agar) medium supplemented with 100 Ag/ml of ampicillin and 25 Ag/ml of chloramphenicol. DNA SeQuencing

The inserts in the recombinant plasmids were sequenced to confirm the presence of the CTV genes and their correct orientation. Plasmid DNA was purified as before from E. coli DH5a recombinant colonies grown in 2 x YT liquid cultures with antibiotics (Sambrook et al., 1989). Approximately 2 Ag of DNA were used for each labeling reaction. The DNA was first denatured in 20 Al of 1 N NaOH, 25 mM EDTA by incubation at 700C for 5 min and quickly chilled on ice, followed by ethanol precipitation in 0.3 M sodium acetate (Sambrook et al., 1989). The samples were incubated at -800C for 30 min before

centrifugation for 15 min at 14,000 rpm. The DNA was

resuspended in 7 Al of distilled water.

The labeling reaction was performed using the Sequenase Version 2.0 sequencing kit (Amersham) according to the manufacture's instructions. The technique is based on the chain termination method (Sanger et al. 1977). For each









18

reaction 12.5 pCi of [a-35S]-dATP were used. Both strands of DNA were sequenced using primers specific for the pETH expression vector.

Samples were electrophoresed in denaturing 5% polyacrylamide gels (acrylamide/bisacrylamide 19:1, lx TBE, 8 M urea) for 1.5 to 4 h at room temperature and 35 W constant power.

Bacterial Expression of CTV Proteins

Induction. The expression of CTV genes in E. coli BL21(DE3) was induced using isopropyl-O-D-thiogalactoside (IPTG) at a final concentration of 0.4 M, in 25 ml of LB liquid medium supplemented with 100 pg/ml ampicillin and 25 pg/ml chloramphenicol (Sambrook et al., 1989).

To test for expression of recombinant proteins, 1 ml samples were taken before induction and 30 min, 1 h, 2 h and 3 h after induction. All samples were kept on ice in microcentrifuge tubes until the last was collected. Bacterial cells were pelleted by centrifugation at 14,000 rpm for 30 sec at room temperature and resuspended in 50 pl of cracking buffer (300 mM Tris-HCl pH 6.8, 2% SDS, 20% glycerol, 1% v/v 2-mercaptoethanol, 0.025% bromophenol blue). After resuspension, the samples were boiled for 3 min and stored at

-200C for later use.

Detection. Bacterial proteins were separated using 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970). Samples were electrophoresed at 200 V for 45 min in a









19

7 x 8 cm gel at room temperature. Protein standards SDS-6 or

SDS-7 were included. Proteins were stained using Coomassie brilliant blue G-250 for 30 min (Sambrook et al., 1989). Production of Polyclonal Antisera

Protein expression. overnight 3 ml cultures of each

recombinant pETH-3b plasmid containing the CTV p27, p20 or p18 ORFs were used for production of recombinant protein. The medium used was LB with antibiotics as described above and incubation was at 370C with shaking (180 rpm) In the morning, 25 ml of fresh medium were inoculated with 0.5 ml of the overnight cultures and incubated for 2 h. The cultures were then induced with IPTG and incubated for 3 more h. Cells were transferred to a 15 ml tube and centrifuged at 400 for 10 min

and 15,000 g. The bacteria were resuspended in half their original volume in TE buffer and frozen overnight at -200C.

Cells were thawed at 370C, vortexed for a few minutes and centrifuged as before. Both the supernatant and pellet, were collected the first time, and samples were treated with

cracking buffer for analysis in SDS-PAGE to determine the fraction that contains the protein of interest. In those cases in which the protein was insoluble (p27 and p18) the pellet was resuspended in one half volume (6 ml) of TE buf fer and pelleted by centrifugation. This was repeated 2 more times, each time reducing the volume of TE buffer in half. The final volume was 1.5 ml.









20

For p20, most of the protein was soluble. The protein in the supernatant was pelleted with saturated ammonium sulfate (final concentration 33%; Ausubel et al., 1992) and centrifuged 14,500 g for 15 min at 40C. The pellet was resuspended in TE buffer. All three proteins were stored at 200C.

Isolation of recombinant proteins. Proteins of interest were separated from other bacterial components using 12% SDSPAGE in a 15 x 15 cm gel and a single well, 3 mm thick comb. Approximately 5 to 10 mg were separated in each gel. Proteins were detected using ice cold 0.25 M KCl (Hager and Burgess,

1980) which reacted with the bound SDS to form a visible band. The protein band was excised with a sterile blade and stored at 400.

Polyclonal antibodies. The protein (still in the

polyacrylamide gel) was sent to Cocalico Biological, Inc. (Reamstown, PA) for production of polyclonal antibodies in rabbits. Approximately 1.5 to 2 mg of protein were used for

each weekly injection. Antibodies were collected and tested after four weeks of injections. Detection of CTV Proteins in Infected Citrus Tissue

Western and tissue blot analysis. The expression of p27, p20 and p18 in T36 CTV-infected Mexican lime was tested using

Western blot analysis, according to a previously described procedure (Li et al., 1991). Detached midrib segments of approximately 1 cm in length were sliced into small pieces









21

using a blade and combined with 0.5 ml of cracking buffer, boiled for 3 min and stored at -200C. Protein extracts (10 tl) were separated by 15% (p27) or 12% (p20 and p18) SDS-PAGE along with the appropriate molecular weight markers. The proteins were transferred to a nitrocellulose membrane using electrophoretic transfer in 25 mM Tris, 192 mM glycine, 20% v/v methanol at 40C for 1 h at 100 V, 350 mA. Membranes were probed with the different polyclonal antisera using the colorimetric procedure. For p18 a chemiluminescent procedure was also tested using the Renaissance kit. A monoclonal antibody (MCA-13) raised to the T36 CP (Permar et al., 1990) also was used.

Tissue blot analyses also were tested as a rapid method for the detection of p27 and p20 using the procedure of Garnsey et al. (1993). Two incubation conditions (room

temperature and 370C for 1 h) were tested, as well as dilutions of antiserum from 1:500 to 1:20,000.

Cell fractionation. Cell fractions from T36 CTV-infected and uninfected citrus tissue were prepared by a modification of the procedures of Godefroy-Colburn et al. (1986) and Albretch et al. (1988). Approximately 1 g of tissue was ground in liquid nitrogen and thawed in 2 ml of grinding buffer (100 mM Tris-HCl pH 8.1, 10 mM KCl, 0.4 M sucrose, 10% glycerol, 10 mM 2-mercaptoethanol). The mixture was incubated with agitation for 15 min at room temperature and then filtered by centrifugation at 3,000 g for 10 min through a 64-mesh nylon









22

cloth fused to a 3 ml syringe. Both filtrate and solid residue were collected. The liquid filtrate was centrifuged at 1000 g for 10 min; both pellet (fraction P1) and supernatant were collected. The supernatant was further centrifuged at 30,000 g for 20 min, and the pellet (fraction P30) and supernatant

(fraction S30) were collected. The solid residue from the first step was resuspended in 1 ml of grinding buffer and incubated with agitation for 15 min at room temperature. The

extract was filtered again by centrifugation. The filtrate (fraction R1) was collected and the solid residue resuspended in 1 ml of grinding buffer containing 2% Triton X-100 for 15 min with agitation and subsequently filtered again by centrifugation. The filtrate (fraction R2) was collected and

the solid residue was resuspended in 1 ml of 75 mM Tris-HCl pH 6.8, 4.5% SDS, 9 M Urea, 7.2% 2-mercaptoethanol at room

temperature for a final 15 min extraction with agitation, followed by filtration. The filtrate was collected (fraction R3) and the solid residue discarded. All fractions were combined with equal volumes of cracking buffer and boiled for

3 min for analysis by Western blots.



Results

Cloning of CTV Genes

CTV genes for p27, p20 and p18 were amplified by RT-PCR and first cloned into the SinaI restriction site of pUCll8 using blunt-end ligation. A HindIII restriction site was









23

included in the sequence of the sense primer (upstream) to facilitate subcloning in pETH-3b. Identification of the clonal orientation was necessary for subcloning into the pETH vector. Clones with the EcoRI restriction site of the plasmid polylinker located downstream of the gene were used for subcloning. These clones were identified by restriction digestion of the plasmid DNA with HindIII. Clones in the sense orientation were only linearized upon digestion without releasing the inserts, whereas clones in the antisense orientation released the inserts (data not shown).

For subcloning into pETH-3b and to guarantee proper orientation and reading frame of the gene, two noncomplementary restriction enzymes that generated cohesive termini were used: HindIII, located 5' to the start codon and EcoRI, located 3' to the stop codon of each CTV gene. This strategy also avoided self-ligation of the plasmid. Sequencing of the clones (data not shown) confirmed orientation and reading frame for each construct. Expression of Proteins

CTV genes were expressed as fusion proteins, made up of 20 amino acids of the T7 coat protein (Studier et al., 1990; McCarty et al., 1991) and the amino acids of each CTV gene. The fusion part of the proteins increased the MW by 1.9 kDa. The resulting predicted MW of each protein was 29.3 kDa (p27), 22.4 kDa (p20) and 20.2 kDa (p18).









24

Analysis of bacterial proteins by SDS-PAGE showed accumulation of proteins of the expected MW in induced

cultures, but not in non-induced cultures (data not shown). The amount of each protein increased with time. CTV proteins accumulated to approximately 30% of the total bacterial protein.

Detection of P27

The expression of p27 in CTV-infected citrus was tested

using Western blot analysis. The optimal dilution of antiserum, was determined empirically. Best results were obtained at a dilution of 1:500. Because of the amino acid homology between

p27 and the CP the reaction of different antisera to both proteins were compared (Figure 2).

The p27 polyclonal antiserum showed some nonspecific reactivity (as did pre-immune serum) with uninfected citrus tissue (Fig. 2, lane 4). However, a discrete, intense, protein band of approximately 27 kDa was detected in extracts of CTVinfected citrus (Fig. 2, lane 5) but not in those of uninfected citrus (Fig. 2, lane 4). The antiserum also detected E. coil-expressed p27 (Fig. 2, lane 2), which migrates slightly slower than p27 due to its higher MW. While

the p27 polyclonal antibodies reacted with the purified fusion p27 protein (Fig. 2, lane 2), they did not react with a nonfusion, E. coil-expressed CP (Fig. 2, lane 3). Furthermore, no reaction was observed with proteins induced from bacteria containing the pETH plasmid without an insert (Fig. 2, lane









25














1 2 3 4 5 6 7 8 9 10


-27
25










Figure 2. Western blot analysis of CTV-infected and uninfected citrus leaf extracts with p27 polyclonal antiserum (lanes 1-5) and with MCA-13 monoclonal antibody (lanes 6-10). Lanes 1 and 10, pETH-3b without insert; Lanes 2 and 9, E. coli-expressed fusion p27 (lane 9 contains 100 times more protein than lane 2); Lanes 3 and 8, E. coli-expressed CP; Lanes 4 and 7, uninfected citrus extracts; Lanes 5 and 6, CTV T36-infected citrus extracts. Molecular weights in kilodaltons are indicated on the right.









26

1) Finally, no p27 band was detected when preimmune serum was used (data not shown), indicating the specificity of the antibody.

To preclude the possibility that the protein detected in infected tissue was a stress-induced pathogenesis-related

protein, tissue extracts from citrus plants infected with citrus ringspot virus, citrus variegation virus, citrus leaf

rugose virus, psorosis A and concave gum also were analyzed by Western blot, using p27 antiserum. No p27 band was observed in any of the samples (data not shown).

To further demonstrate the specificity of the antibody,

half of the same membrane in Fig. 2 (lanes 6 to 10) was probed with MCA-13. This monoclonal antiserum, specific to the CP, reacted strongly with the E. coli-expressed OP (Fig. 2, lane

8) and with the CP in the extract of CTV-infected citrus tissue (Fig. 2, lane 6), including the lower molecular weight proteolysis products of the CP (Sekiya et al., 1991). No reactions were observed with proteins produced by bacteria containing the pETH plasmid without an insert (Fig. 2, lane 10) or from extracts of uninfected citrus tissue (Fig. 2, lane 7).

Interestingly, MCA-13 gave a weak but consistent reaction with the purified p27 fusion protein, which could be seen only on the membrane (not visible in Fig. 2, lane 9), but not with

p27 from infected tissue. However, to obtain this level of reaction with MCA-13, it was necessary to increase the









27

concentration of p27 protein by 100-fold over the amount used in Fig. 2, lane 2, where it was readily detected by the p27 antiserum.

To determine the cellular localization of p27, cell

fractions from CTV-infected and uninfected citrus tissue were prepared and assayed by Western blots. Similar percentages of total protein for each fraction were analyzed using p27

antiserum (Fig. 3). All cell fractions contained p27. However, most of the p27 accumulated in the cell wall fractions (R1, R2 and R3) (Fig. 3, lanes 6 to 8, respectively). In addition, fraction S30 also contained considerable amounts of p27 (Fig.

3, lanes 5). Fractions Pl and P30 showed much lower levels of p27 (Fig. 3, lanes 3 and 4, respectively) These results indicate that p27 was mostly associated with the cell wall fractions.

The reaction of MCA-13 to the cell fractions was also tested to compare the accumulation of CP and p27. Fig. 4 indicates that most of the CP accumulated in the cell wall fractions R1 and R2 (Fig. 4, lanes 6 and 7), with some also present in the soluble protein fraction S30 (Fig. 4, lane 5).

Fractions P1 and P2 contained less CP (Fig. 4, lanes 3 and 4) The pattern of accumulation of the CP was similar to that observed for p27.

The p27 antiserum also was tested using tissue blot (Fig. 5). The optimal conditions for specific detection were









28













123 456 789
















Figure 3. Western blot analysis of cell fractions prepared from CTV-infected tissue and probed with p27 antiserum. Lane 1, uninfected citrus tissue extract; Lane 2, unfractionated infected citrus extract; Lanes 3 to 8 contained fractions P1, P30, S30, R1, R2 and R3, respectively, of infected citrus tissue as explained in Materials and Methods. Lane 9 is E. coli-expressed p27.









29














1 23 45 67 8














Figure 4. Western blot analysis of cell fractions
prepared from CTV-infected tissue and probed with MCA-13 monoclonal antibody. Lane 1, uninfected citrus tissue extract; Lane 2, unfractionated
infected citrus extract; Lanes 3 to 8 contained fractions P1, P30, S30, R1, R2 and R3, respectively, of infected citrus tissue as explained in Materials and Methods.









30




















A





















B



Figure 5. Tissue blots probed with the p27
polyclonal antiserum. A, CTV-infected citrus
tissue; B, uninfected citrus tissue.









31

incubation at 370C for 1 h, and 1:10,000 dilution. The blots indicate that p27 is present in phloem tissue. Detection of P20

The optimal antiserum concentration for the analysis of p20 was 1:500. Fig. 6 shows the results of the detection of the cell fractions with p20 antiserum. A protein band of the expected size was present in infected, unfractionated tissue (Fig. 6, lane 2) but not in uninfected tissue (Fig. 6, lane 1). Most of the p20 was in the soluble protein fraction (S30) (Fig. 6, lane 5). Cell wall fraction R3 (Fig. 6, lane 8) also

contained some p20. Fractions P1, P30, R1 and R2 contained little or no detectable p20 (Fig 6, lanes 3, 4, 6 and 7, respectively).

The p20 antiserum was not useful for detection of the protein using tissue blots. At high antiserum concentrations (1: 1000) the serum was reactive with uninfected tissue, and at higher dilutions (1: 20,000) positive reactions with CTVinfected tissue were very weak. Detection of P18

Despite several attempts to detect p18 in infected tissue, no band of the expected size was observed in Western

blots. Dilution of the antiserum as low as 1:100 did not produce any favorable results in colorimetric detection (data not shown). The antibody reacted, however, with fusion p2o produced in bacteria, the same protein used as antigen (data not shown). It also reacted with fusion p18 and p27, both of









32
















2 3 4 56 8 9














Figure 6. Western blot analysis of cell fractions prepared from CTV-infected tissue and probed with p20 antiserum. Lane 1, uninfected citrus tissue extract; Lane 2, unfractionated infected citrus extract; Lanes 3 to 8 contained fractions P1, P30, S30, Rl, R2 and R3, respectively, of infected citrus tissue as explained in Materials and Methods. Lane 9 is E. coli-expressed p20.









33

which contained the same 20 N-terminal amino acids. Detection of cell fractions with the antiserum did not reveal any bands of the expected MW. A more sensitive, chemiluminescent

detection system was also used without positive results (data not shown).

Discussion

The levels of bacterial expression obtained with the fusion proteins were similar to those previously reported (Marston, 1986; Studier et al. 1990). With this expression system, it was possible to successfully produce the three CTV proteins and purify them by a relatively simple method. The antisera produced reacted with the antigens and gave low nonspecific reactions when used to probe tissue samples. There was variation in the solubility of the fusion proteins, with p27 and p18 accumulating as insoluble products, and p20 being soluble. This did not affect the levels of expression or the isolation of the proteins.

The presence of the fusion portion seemed to stabilize some of the proteins in E. coi. Attempts to express p27 without this portion rendered very low levels of the protein (data not shown). However, nonfusion CP was expressed in the same bacterial host (K.L. Manjunath, personal communication) The only inconvenience with fusion proteins is that antisera

produced to different fusion proteins cross react with all the antigens, since the fusion part is identical. This might limit the use of the recombinant proteins as positive controls,









34

although it does not have any effect on the detection of the target proteins in vivo (because they lack the fusion portion).

The results using the p27 antiserum showed the presence

of a protein of the expected size (27.4 kDa) in CTV-infected, but not in uninfected tissue. Also, this protein band was not

present in tissue samples infected with other citrus viruses. This indicates that the protein detected is CTV-specific and was expressed during infection, and that its size is in agreement with that predicted from the sequencing data (Pappu et al., 1994).

The protein bands detected with the CP-specific monoclonal antibody MCA-13 are of a lower MW than that

detected with p27 antiserum. Because of this and the fact that the p27 antiserum detected only a single band, and did not react with the nonfusion E. coli-expressed CP, it is concluded that the protein detected with the antiserum is p27, and not the result of a cross-reaction of the p27 antiserum with the CP. This was a definite possibility since both proteins have a high degree of homology in their primary structure.

The monoclonal antibody MCA-13 showed a very low affinity for the E. coli-expressed p27, because its concentration had

to be increased 100-fold compared to the concentration of p27. The region of the CP that is recognized by MCA-13 has been identified (Pappu et al., 1993a) and part of this region is conserved in p27, which may explain its low affinity for p27.









35

The tissue fractionation experiments showed that p27 and the CP are mostly associated with the cell wall and soluble protein fractions. Conversely, in BYV, p24 (the CP homologue) and the CP were shown to accumulate mostly in the cytoplasm or soluble protein fraction (Agranovsky et al., 1994). Comparison of the sequences upstream of the start codons and surrounding the initiation sites for the subgenomic RNAs of BYV p24 and CP revealed a consensus sequence, (Agranovsky et al., 1994), suggesting concerted expression of both genes. This sequence was similar to those found in the CPs of tobamoviruses, brome mosaic virus, cucumber mosaic virus and alfalfa mosaic virus (AlMV; Agranovsky et al., 1994). Comparison of the sequences upstream to the start codons of CTV p27 and CP did not revealed any consensus sequences similar to those of BYV or LIYV (data not shown). This indicates that the expression of these two set of genes is different in CTV and BYV.

Recently, p24 was found to be part of the BYV virions, forming a terminal "tail" at one end of the particles (Agranovsky, 1995). Due to the similarities between the two viruses and the apparent structural conservation between the CP homologues (Pappu et al., 1994), it is possible that p27 may also form part of the CTV virions. This is currently under investigation using the p27 specific antiserum.

The function of p27 in CTV is still not known. Besides its possible structural role, if p27 forms part of the virions, it might also be involved in particle assembly









36

(Agranovsky et al., 1995). This could be through interaction with other viral proteins, including the CP and the heat shock proteins (p65 and p61) or unknown host components. Heat shock proteins work as chaperons helping in proper folding of other proteins (Georgopoulos, 1992).

The p27 might also have a role as a helper component in the transmission of CTV by its aphid vector. This has previously been proposed for BYV p24 (Agranovsky et al., 1995; Boyko et al., 1992). Helper components have been described for potyviruses and caulimoviruses (Mathews, 1991), although they do not form part of the virions. However, in potyviruses the CP is also important for aphid transmission (Atreya et al., 1990, 1991). Interestingly, LIYV also contains a similar duplicate of the CP, with deduced amino acid sequence homologies to BYV and CTV CPs and homologues, however, LIYV is transmitted by the whitefly Bemisia tabaci (Gennadius)

(Klaassen et al., 1995).

Relating to the cellular accumulation of p27, a possible function could be as a movement protein, assisting in the spread of the virus between cells. Movement proteins interact with plasmodesmata causing an increase in their diameter (Wolf et al., 1989; Derrick et al., 1992; Waigmann et al., 1994), thereby enabling the intercellular passage of the large nucleic acid in a naked or coated form, depending on the virus. Putative movement proteins of tobacco mosaic virus (Deom et al., 1987), AIMV (Godefroy-Colburn et al., 1986),









37

cauliflower mosaic virus (Albrecht et al., 1988) and squash leaf curl geminivirus (Pascal et al., 1993) accumulate in the cell wall fractions. The observations that p27 also accumulates in this fraction suggest a possible role in virus movement.

The capacity to bind RNA is another characteristic of movement proteins (Deom et al., 1987; Citovsky et al., 1992; Giesman-Cookmeyer and Lommel, 1993). Due to the apparent structural similarities between p27 and the CP (Pappu et al., 1994), including the conservation of amino acids present in the CPs of other filamentous viruses (Dolja et al., 1991; Boyko et al., 1992), and the finding of p24 as part of the BYV virions, it is likely that p27 might have RNA-binding capacity.

The CP of some RNA viruses also have a role in cell-tocell and long distance movement. In some cases, like cowpea mosaic comovirus (van Lent et al., 1991), the virus moves from cell to cell as virions. In other cases, like AlMV, the CP is required for movement, but virions are not transported (van der Vossen et al., 1994). For brome mosaic bromovirus, the CP is not required for cell-to-cell movement, but is required for long distance movement (Flasinski et al., 1995). It is possible then that p27 acts as a multifunctional protein, forming part of the virus particles and assisting in one or more of the following functions: virus movement, aphid transmission or encapsidation.









38

Using tissue blots, p27 was mostly present in the sieve tubes. This is in agreement with what has been found for the CP (Garnsey et al., 1993) and with the phloem-limited nature of CTV (Bar-Joseph and Lee, 1990). These results also indicate that some of the p27 epitopes are conserved between native and SDS-denatured protein. The antiserum was raised against denatured protein, however, in tissue blots the proteins are not treated with SDS previous to their detection.

Tissue blot is a simple method for assaying the expression of p27, especially when a large number of samples are involved. This technique will be used for the screening of the p27 transformed plants.

The polyclonal antiserum raised using recombinant p20 detected a protein band of the expected size in CTV-infected, but not in uninfected citrus tissue, indicating that the p20 also is CTV specific. Unlike p27, p20 accumulates in the soluble protein fraction. The intensity of the p20 band in Western blots was much lower for p20 than p27. This was not expected since analysis of CTV subgenomic RNAs indicates that p20 transcript is the most abundant (Hilf et al., 1995). One alternative to explain this result is that p20 protein is poorly immunogenic, and so the concentration of specific antibodies in the serum is low. Because the E. coli-expressed p20 contains the fusion portion, it is not possible to conclude anything from the reaction observed with the antiserum to this protein. Another possibility is that p20









39

protein expression is regulated post-transcriptionally and that its RNA plays some role in the virus life cycle.

Regarding the function of p20, it is still difficult to speculate since the protein does not show any similarities to previously described proteins. However, this research demonstrates that the ORF is expressed during CTV infection.

The p18 protein was not detected in CTV-infected tissue samples using Western blot analysis, even when the more

sensitive chemi luminescent method was used. The antiserum, however, reacted with E. coil-expressed p18. Subgenomic RNA analysis indicates that p18 is expressed at a very low level compared to p27, CP or p20 (Hilf et al., 1995). Low protein

expression and/or expression at a specific time during the CTV life cycle may explain the inability to detect p18.















CHAPTER 3
SEQUENCE ANALYSIS OF THE P27 ORF



Introduction

The p27 ORF has been described as a diverged copy of the CP gene (Pappu et al., 1994). Comparison of the deduced amino acid sequences indicates that 19% of the amino acids are identical between p27 and the CP, and 41% of the amino acids are either identical or with similar biochemical properties to those of the CP (Pappu et al., 1994). The first report of the diverged copy of the CP among closteroviruses (and filamentous RNA viruses) was for BYV (Boyko et al., 1992). The BYV p24 ORF also is located 5' to the CP and all four genes of CTV and BYV show various degrees of amino acid sequence similarities, ranging from 38% (CTV p27 and BYV CP) to 50% (CTV p27 and BYV p24) (Boyko et al., 1992; Pappu et al., 1994). Two of the amino acids conserved in the four proteins also are strictly conserved in the CPs of 14 other filamentous positive-stranded RNA viruses (Dolja et al., 1991; Boyko et al., 1992; Pappu et al., 1994). Those two residues are believed to form a salt bridge (Dolja et al., 1991),

suggesting that the CPs and the diverged copies share not only the primary structure, but exhibit similar protein folding (Boyko et al., 1992).

40









41

Detection of sequence variability in viral genes is important since mutations could be linked to particular biological properties. Single stranded conformation polymorphism (SSCP) is a simple and sensitive method used to detect single nucleotide changes in particular sequences

(Orita et al., 1989). This technique is based on migration differences during electrophoresis of short, denatured, DNA fragments in non-denaturing gels, caused by differences in their nucleotide sequences (Orita et al., 1989; Spinardi et al., 1991).

The main purpose of this research was to determine if there was any relationship between RNA or deduced protein sequence and the biological properties of the virus.

Therefore, this gene was cloned and sequenced from CTV strains with distinct geographical origins and different biological activities. In addition, PCR combined with SSCP analysis was

tested for its potential as a rapid method to determine sequence differences among CTV strains.



Materials and Methods

Materials

In addition to the materials mentioned in Chapter 2, the Wizard DNA purification system used was from Promega

Corporation (Madison, WI) and the silver staining kit was from sigma chemical Company (St. Louis, MO). Sequence analysis was

performed using the programs Seqaid, CLUSTALV (Higgins et al.,









42

1992) and Wisconsin Genetic Computer Group Sequencing Analysis Software (GCG) version 7. 0 (Devereux et al. 1984) provided by the Interdisciplinary Center for Biotechnology Research at the University of Florida (Gainesville, FL). Virus Isolates

All CTV strains (except for T30 and T36) were obtained

from the Exotic Citrus Pathogens Collection, maintained by the USDA and IFAS in Beltsville, MD. Strains T30 and T36 were maintained in greenhouses at the University of Florida

(Gainesville, FL) Propagation hosts for the strains were "Madam Vinous" sweet orange [C. sinensis (L.) Osbecki or "Mexican" lime [C. aurantifolia (Christm.) Swingle]. Cloning and Sequencing

Biological properties of the strains used in sequencing

are listed in Table 2. Cloning and sequencing procedures were similar to those described in Materials and Methods, Chapter

2. In some cases (strains B7-1, B185, B227 and B249) PCR products and plasmid DNA were purified using the Wizard DNA Clean-Up System. For B50 and B67, both nucleic acid extracts and dsRNA (Morris and Dodds, 1979) were used for PCR

amplification. When dsRNA was used, denaturation was performed in a solution containing 10 mM dimethylmercury and approximately 100 pM of each primer in a total volume of 10

Al, incubated at room temperature for 8 min and then quick frozen in liquid nitrogen. The mixture was then used in a standard PRC reaction as described in Chapter 2.













Table 2. Characteristics of the CTV strains used for sequencing of the p27 gene.


STRAIN ORIGIN MCAI3a M.L.b DECLINEc Syd SP-Ge SP-MVf COMMENTS

T26 Florida + 0 0 0 0

T30 Florida + 0 0 0 0 Effective f cross protection

T36 Florida + ++ ++ + + 0 Genome sequenced

B7-1 S. Africa + ++ + + ++ 0 Used for cross protection.

B67 China + 0 0 0 0

B128 Colombia + ++ ? 0 ... +

B185 Japan + ... +++ ... +++ ++

B227 India + ++ ... ++ ... ...

B249 Venezuela + ++ . ++ + Vein corking onM.L.

a MCAl3= Reactive with the monoclonal antybody MCA13 (-= no reaction, += positive
reaction).
b M.L.= Symptoms on Mexican lime (+= mild, ++= moderate, +++= severe, ?= unknown). c Decline= Induces decline in sweet orange scions grafted on sour orange rootstock. d SY= Seedling yellows on sour orange seedlings. e SP-G= Stem pitting on grapefruit scions. f SP-MV= Stem pitting on Madame Vinous sweet orange scions.









44

In addition to the p27 genes, the CP from strains B249 was also cloned and sequenced. Sequences for all the other CP genes were kindly provided by H.R. Pappu. Clones for the p27 gene of isolate B128 were kindly provided by S.S. Pappu.

At least two clones of each strain were sequenced in both the sense and antisense directions. Sequences were compared using the programs CLUSTALV and GCG. Deduced amino acid sequences were obtained using Seqaid. Single Stranded Conformation Polymorphism (SSCP)

The same strains employed for sequencing were also used for SSCP analysis. Other strains are described in Table 3. The p27 genes were amplified from tissue extracts or from plasmid DNA and analyzed in non-denaturing polyacrylamide gels. PCR products (2 pl) were denatured in 95% formamide, 20 mM EDTA, 0.05% bromophenol blue by incubation at 1000C for 10 min and electrophoresed in 7 x 8 cm, 8% polyacrylamide gels

(acrylamide: bisacrylamide 30:1, 1 x TBE) for 3 hrs 45 min at 200 V in lx TBE buffer at room temperature. The buffer and gels were pre-chilled at 40C overnight before electrophoresis. DNA was detected using silver staining.



Results

Sequencing

The CTV strains sequenced were selected based on

diversity of biological properties and geographical origins. Table 4 summarizes the overall similarities of nucleotide and













Table 3. Characteristics of the CTV strains used for SSCP analysis. STRAIN ORIGIN MCA-13a M.L.b DECLINEc Syd SP-Ge SP-MVf COMMENTS

B5 Calif. + 0 0 0 ?

B11 Hawaii + + + + 0 0

B28 Florida + ++ ++ ++ ++ ++

B32 Spain + 0 0 0 0

B37 Taiwan + ++ +++ +++ ? +

B148 Florida + ++ ++ ++ 0 0

B192 France + 0 0 0 0 0 Not aphid transmitted

B272 Colombia 0 0 0 0 0

B274 Colombia + 0 0 0 0

a through f, as in Table 2.









Eji









46

deduced amino acid sequences among all the strains studied. At the nucleotide level, homology of the sequences varied from 86.2% to 99.6%. Strains B7-1 (severe from South Africa) and B128 (severe from Colombia) were the most similar. At the protein level, homologies were 93.3% or higher. Strains T30 (mild from Florida) and B67 (mild from China) were identical in deduced amino acid sequence. Strains B128 and B249 (severe

from Venezuela) also were identical in deduced amino acid sequence.

Alignment of the nucleotide sequences (Fig. 7) showed several nucleotide substitutions along the genes. No deletions, insertions or inversions were observed. All

sequences constituted an ORF similar to the one previously reported (Pappu et al., 1994). A total of nine substitutions were conserved in all the severe strains compared to nucleotides conserved in all mild strains. For example,

nucleotides 108 (C -4 T) and 229 (G -+ A) other nine substitutions were present only in stem pitting strains. For example, nucleotide 78 (T--+ C). Most of these substitutions, however were silent.

Comparison of the deduced amino acid sequences (Fig. 8) showed only 4 amino acids exclusive to severe strains (amino

acids 9, 77, 104 and 125) and only one in stem pitting strains (amino acid 102). The C-terminal portion of the protein is the most conserved among the strains sequenced.














Table 4. RNA and deduced amino acid sequence similarities (% of identical bases
or amino acids) between different CTV strains for p27 (upper right is RNA, lower left is amino acid sequences). Values generated using
CLUSTALV.






STRAIN T30 T26 B67 T36 B7-1 B128 B185 B227 B249

T30 98.9 99.2 93.9 92.0 92.1 90.7 88.2 92.1

T26 98.8 98.3 93.8 91.2 91.4 90.2 88.2 91.6

B67 100.0 98.8 94.6 92.5 92.7 91.3 88.8 92.7

T36 96.7 95.4 96.7 92.5 92.7 91.0 87.6 92.4

B7-1 96.7 95.4 96.7 97.5 99.7 98.1 87.3 99.3

B128 97.1 95.8 97.1 97.9 99. 98.3 87.6 99.6

B185 94.6 93.3 94.6 95.4 97.1 97.5 86.2 98.8

B227 95.0 93.8 95.0 95.4 96.7 97.1 94.6 87.7

B249 97.1 95.8 97.1 97.9 99.6 100.0 97.5






.Pb
-13









48

1 I I I I 50
T30 ATGGCAGGTTATACAGTACTTCCTAATACCGATGACAAAGAAATGGATCC
T26 ..----------------------------------------------A---B67
T36 6 ---------------------A ----------B7-1--------------------------------- -------------B128-------------------------------- ------T--------------8185--------------------------- ------ -------------B227 ------------------------- -- ----- ....
B249 ---------------------- ------ -------------51 I I I I 100
T30 GGTGAGTGCCGCTGTACCCGGTAAGTATCCGGATGTCATTGAAAAATTTG
T26 C----------------------B67
T36 T-----....-T--C--------------------
B67--------------------0----------------------------------T128-T--------- --------------------------T-------------B185 -------------------- ---------------------------------B127 ----A--------- -------------------- --------------B22 - .. .. ..... .. .... .-.--.
B249---------------------------------- --------------------B249. .....



101 I I I I 150
T30 TGGCCAACAGGTCCGTAGACGCGTTAATAGAAGGCGTCATAAGTAAGTTG
T26 -----------------------------G--------------------B67
T36-----------------------------------------------------A--T36 ..------- ------.----- --------------------------B7- .. ... ..... ...

B128 --------- T ----- ---------------- T -----------B185 ----------- T .----------------- ------------c
B227 ---.----------------------- T ----- C -----B249 --------- ---- ---- ------------------------------151 I I I I 200
T30 GATACCAATTCAATATACGAAGATTCCACTGAAAAATTTACTGGTGAACA
T26 .........
B67
B6---------------------------------------------------------T36------------------------------------G-----------B7-8 ------- ------------------------ ---------............
B12 .... .... .......
B185 .... .... .... ....
8227 --C--. ----- ------------------------A ----------- -8249 ------P T ---- ------------------------G0------------ GTB2 7 -- .... .... .. . . . . ... .



Fig. 7. Nucleotide sequence alignment of p27 gene from different CTV strains (hyphens, identical nucleotides).
The alignment was constructed using the program
CLUSTALV.









49

201 1 I 1 I 250
T30 CTTGAAATACGTTATGGTTACTATGGATGCTTTCTTATTAGAAAACTACA
T26
B67
T36
T7-1 ---A--G--------------------- CA --------------------B7-1 --- -- ....... K. ....

B128 --- --------------------- ---------------------T
B185 ---A--G --------------------- --------------------B227 4.0 G- -T------------------- ------------------- T
B249 --A---------------------CA!ji---------------------


251 I I I I 300
T30 AGACGAAAACGGAAGATCTGTTGGTTCACTTAGCTATGATCCAAAAGAGG
T26
B67
T36 -A----------------------- ----------------B7-1 ----------------------A
B128 -----------------------------------B185 -.AC-C70-----------------A------B227 -A --------------------- .. -------..--------- G----B249 -A---------------------- -----301 I I I I 350
T30 TTGTACACTACATCCACGAGCACTAAAACCAAGTTCCGCGATAAAGGTTG
T2 6
867
T36 ----------------------B7-1 --A-G--------------.------------------------------8128A ------- T ---------------------------------:----871 ---------------T-------- T ------ --------G -----B128 -------T--------- T -------------------- G ----B84 --A-G --- C-T --------- T-------- T--------------C-B 18 -- .. .. ... .... ......
B227.... .. .. .... .... .... .- -..... .
........... ....:: ....: ::;: ::
B 2 4 ..... ..... iii iiiii . .... ... .... . .



351 I I I I 400
T30 TATTAGTTACGTGCAAGGGGGTTCGCGATATAAGTTAATGGATAAAGTAG
T26 -------------------------------------------------867
TB6------------------------------------------T------------T7-36 -------------- A-----A---- TA------------PT----- C------CB7-1 ..... ....... ....
B128A ... .. ..... .... ....
8718--------------A ------A ----TA---------8185----------------A- ---- A-A----- C------ ------------.B1 5A. ... ... ..... ... ... ...
....: .:.....:::::::::: :::: .. ... :::
B227 ----------T -------------- .. -------------- ... -. .--AB249 ------------------ -A-----..-.. ----- ---- --------Figure 7--continued.









50

401 I I 1 1 450
T30 TTTTTCCTTTCATTATATCGAAATTTACCGACAGGGAGACTCCGAACGCT
T26
B67
B7-1 -------------------------------------------------T36------------T-------------------------------------- -----B71 ---A-------T-----------------------------------B128 -----------T---------------C-------------------- T--B18 -- ..... .. ..... -B2 9.. . ... ... .--B249--------------- T-------- -------------------------- --451 1 I I 1 500
T30 CTACGTAAGTATGCTTGCACTTTCGAGGAGTTACACTTGTGTATGGCTAG
T26
B67
T36----------------B67--------------------------------------------------------T36------------------ --------------------------------------B128 -- - - - - - -- - - - -
B185 -------------------------------------- ----------....7........ ...
B227-- ---------T--T-------------------------B249 ------------------------ -------------- A----------501 I I I I 550
T30 GTTGAGACCCGACTTATACGAAAATAAAAGGACGACTAAAGCCGGGACTC
T26
B67 ---G--T--A ....
T36 ---A--------------------------------- C-GG--T------B7-1 ----------------------------------- ------B128 A--A --------- ------- --------------- C--G--T ------B185 A--------- .------- ---G--------B227 A--A------- -------------- -------- --C ----- T--A ---B249 A- ---------------- -------------- --G--T ------551 1 I I I 600
T30 CACATTTAAAGGGCTATTTATCAGCCGACTTTCTTTCGGGTTCTCTCCCA
T26 --------------------------------------------------B67 ------------------------------------------------T36----------------------C-------- T --------CP--.A-------------1T
B7-1------------ -------T-------- ----------------------B185-----------A- C--- -1B227 ----------- -------- T ---------------------B249Fi e --- At------.

Figure 7--continued.










51


601 I I I I 650
T30 GGGTACTCCGAACATGAACGAGGCATCATTCTTCGAGCGTCTGAGTCTAT
T26 ----------------------------------------------B67
B67---------------------------------------------------------T36 -------------------------- -----------------C0--------T 3 . .. . .......... ...
B7-1 .--------.--G---------------T-------------- C-A ----B127 --------------------------- --------------- --A-----B1859-----------G----------------- T--------------- C--A-----B1 8... I..... .... .. A .. .
... ..... .. .. .... ... . .: .
B249 -- -- ------.. ------ T - - - --A ----651 I I I I 700
T30 GTTAGCTAGACGTCAAGGTTACGAGGAGGCAACCGAGCTTCTTAACCTAC
T 2 6 ..............................
B67
T36 -------------------------------------------------B7-1
B128
B185
B227 -------------------------------- ---------------B249


701 1 723
T30 GTGATTTGGGTAAGTACTTATAG
T26
B67
T36 -C --------------B7-1
B128
B185
B227 ---------------------B249



Figure 7--continued.









52

1 I I I 50
T30 MAGYTVLPNTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKL
T26 ---------------- ----- -------------------- M ------B67
T36 --- --------------B7-1 -------------B128 -- - -K---------------- - - - - -
B185------------K------------------------------------------B227 ----------W------------------- I----- T---------------..... ... . ....
...... -.... ....
B249 -------------51 1 1 1 1 100
T30 DTNSIYEDSTEKFTGEHLKYVMVTMDAFLLENYKTKTEDLLVHLAMIQKR
T26
B67
T36 -------------------------- T -----------------T ----B7-1 --- ----------------------T------------------B128 .-----------------Y --------- T-----------------------B185 H --------------- ----------- T-------- P-------------B227 -------------------Q-------- ----------------------B249 -----------------Y----------- T----------------------101 I I I 150
T30 LYTTSTSTKTKFRDKGCISYVQGGSRYKLMDKVVFPFIISKFTDRETPNA
T26
867
T36 ---I--------------------------L--------------------B7-1 -------------------------L------------------------B 8 -li .... ... ....
8128 -C-I- --... .................- .... --.... .........
B ~ 249 .-. ....
8185 ----------------------L -----L---- M--------------8227 -c-1---------------------L ----- F,---------------------8249 -----------------------Lt---- t-------------------Figure 8. Pairwise alignment of the deduced amino acid sequence of p27 from different CTV strains (hyphens, identical amino acids; periods, similar amino acids). The alignment was constructed using
the program CLUSTALV.










53

151 J J I I 200
T30 LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP
T26 B67 T36 --- .........................-- ....................
B7-1
B128 B185
B 2 2 7 . . .. ... . .... .. .. ... .. . .. . .. . . . . ....
B249





201 I I I 240
T30 GYSEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL
T26 B67 T36 B7-1 B128 B185 -- -------------------------------------B227 B249







Fig. 8--continued.









54

A phylogenetic tree was constructed using the RNA sequences (Fig. 9). Four major groups could be distinguished: l)severe, stem pitting strains B7-1, B128, B249 and B185, 2)

mild strains T30, B67 and T26, 3) severe, quick decline strain T36 and 4) severe, stem pitting strain B227. Mild strains showed some relationship to severe, quick decline and stem pitting strains. Strain B227 (severe from India) was the most distinct and formed a completely separate group. The phylogenetic tree constructed using deduced amino acid sequences (Fig. 10) separated mild from severe strains, each one forming a distinct group.

Comparison of the CP sequences in phylogenetic trees (RNA and amino acid, Figs. 11 and 12, respectively) showed a different grouping of the strains from that of p27.

Sequence alignment of p27 and CP from the different strains is showed in Fig. 13. Approximately 20% of the amino

acids are identical between the two proteins, depending on the strain. Again, the C-terminal regions of the proteins are also the most conserved (Fig. 13).

Single Stranded Conformation Polymorphism

SSCP analysis of the strains used for sequencing (Fig. 14) revealed five groups with similar patterns: 1) severe, stem pitting strains B7-1 and B128, 2) severe, stem pitting strains B249 and B185, 3) mild strains T30, T26 and B67, 4)

quick decline strain T36 and 5) severe, stem pitting strain B227. When all strains were considered (Figs. 14 and 15) they










55









B7-1p27 B128p27
-l-I


_B249p27 8185p27



T30p27



867p27 T26p27 T36p27 B227p27






Figure 9. Phylogenetic tree for p27 based on RNA sequences. Graphic generated using Pileup program in GCG.










56











T30



867



T26 8128



8249 87-1 T36



B185 B227 Figure 10. Phylogenetic tree for p27 based on deduced amino acid sequences. Graphic generated using Pileup program in GCG.









57









T3Ocp T26cp B67cp



T36cp B7-1cp Bl85cp B128cp B227cp B249cp





Figure 11. Phylogenetic tree for CP based on RNA sequences. Graphic generated using Pileup program in GCG.









58









T3Ocp ST26cp 8B67cp T36cp B7-1cp B185cp B1 28cp B227cp 8249cp





Figure 12. Phylogenetic tree for CP based on deduced amino acid sequences. Graphic generated using Pileup program in GCG.











59


CP T30 MDDETKKLKNKNKETKEGDEVVAAESSFGSVNLHIDPTLITMND--VRQL CP T26 MDDETKKLNNKNKETKEGDEVVAAESSFGSVNLHIDPTLITMND--VRQL CP B67 MDDETKKLKNKNKEIKQGDDVVAAESSFGSVNLHIDPTLIT14ND--VRQL CP T36 MDDETKKLKNKNKETKEGDDVVAAESSFSSVNLHIDPTLITMND--VRQL CP B7-1 MDDETKKLKNKNKETKEGDDVVAAESSFGSVNLHIDPTLIAMND--VRQL CP B128 MDDETKKLKNKNKEAKEGDDVVAAESSFGSLNFHIDPTLIAMND--VRQL CP B185 MDDETKKLKNKNKETKEGDDVVAAESSFGSLNLHIDPTLIAMND--VRQL CP B227 MDDETKKLKNKNKETKEGDDVVAAESSFGSMNLHIDPTLIAMND--VRQL CP B249 MDDETKKLKNKNKETKEGDDVVAAESSFGSLNLHIDPTLIAMND--VRQL p27 T30 MAGYTVLPNTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKL p27 T26 MAGYTVLPNTDDKEMNPVSAALPGKYPDVIEKFVANRSVDALMEGVISKL p27 B67 MAGYTVLPNTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKL p27 T36 MAGYTVLPKTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKL p27 B7-1 MAGYTVLPKTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKL p27 B128 MAGYTVLPKTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKL p27 B185 MAGYTVLPKTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKF p27 B227 MAGYTVLPKVDDKEMDPVSAAVPGKYPDIIEKFVTNRSVDALIEGVISKL p27 B249 MAGYTVLPKTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKL




CP T30 STQQNAALNRDLFLALKGKYPNLP ---------- DKDKDF--HIAMMLYR
CP T26 STQQNAALNRDLFIALKGKYPNLP ---------- DKDKDF--HIAMMLYR
CP B67 STQQNAALNRDLFLTLKGKYPNLP ---------- DKDKDF--HIAMMLYR
CP T36 STQQNAALNRDLFLTLKGKHPNLP ---------- DKDKDF--RIAMMLYR
CP B7-1 GTQQNAAVNRDLFLTLKEKYPKLS ---------- DKDKDF--HIAMMLYR
CP B128 STQQNAALNRDLFLTLKGKYPNLS ---------- DKDKDF--HLAMMLYR
CP B185 STQQNAALNRDLFLTLKGKYPNLS ---------- DKDKDF--HLAMMLYR
CP B227 GTQQNAALNRDLFLTLKGKYPNLP ---------- DKDKDF--HIAMMLYR
CP B249 GTQQNAALNRDLFLTLKGKYPNLP ---------- DKDKDF--HIAMMLYR
p27 T30 DTNSIYEDSTEKFTGEHLKYVMVTMDAFLLENYKTKTEDLLVHLAMIQKR p27 T26 DTNSIYEDSTEKFTGEHLKYVMVTMDAFLLENYKTKTEDLLVHLAMIQKR p27 B67 DTNSIYEDSTEKFTGEHLKYVMVTMDAFLLENYKTKTEDLLVHLAMIQKR p27 T36 DTNSIYEDSTEKFTGEHLKYVMVTMDTFLLENYKTKTEDLLVHLTMIQKR p27 B7-1 DTNCIYEDSTEKFTGEYLKYVMVTMDTFLLENYKTKTEDLLVHLAMIQKR p27 B128 DTNSIYEDSTEKFTGEYLKYVMVTMDTFLLENYKTKTEDLLVHLAMIQKR p27 B185 HTNSIYEDSTEKFTGEYLKYVMVTMDTFLLENYKPKTEALLVHLAMIQKR p27 B227 DTNSIYEDSTEKFTGEQLKYVMVTMDTFLLENYKTKTEDLLVHLAMIQNR p27 B249 DTNSIYEDSTEKFTGEYLKYVMVTMDTFLLENYKTKTEDLLVHLAMIQKR







Figure 13. Alignment of CPs and p27 from different
CTV strains. Asterisks, identical amino acids;
periods, similar amino acids. Alignment generated
using CLUSTALV.











60

CP T30 LAVKSSSLQSD-DDTTGITYTREGVEVDLSDKLWTDIVYNSKGIGNRTNA CP T26 LAVKSSSLQSD-DDTTGITYTREGVELDLSDKLWTDIVYNSKGIGNRTNA CP B67 LAVKSSSLQSD-DDTTGITYTREGVEVDLSDKLWTDVVYNSKGIGNRTNA CP T36 LAVKSSSLQSD-DDATGITYTREGVEVDLSDKLWTDVVFNSKGIGNRTNA CP B7-1 LAVKSSSLQSD-DDTTGITYTREGVEVDLSDKLWTDVVFNSKGIGNRRNA CP B128 LAVKSSSLQSD-DDTTGVTYTREGVEVDLSDKLWTDVVFNSKGIGNRTNA CP B185 LAVKSSSLQSD-DDTTGITYTREGVEVDLSDKLWTDVVFNSKGIGNRTNA CP B227 LAVKSSSLQSD-DDTTGITYTREGVEVDLSDKLWTDVVFNSKGIGNRTNA CP B249 LAVKSSSLQSD-DDTTGVTYTREGVEVELSDKLWTDVVFNSKGIGNRTNA p27 T30 LYTTSTSTKTKFRDKGCISYVQGGSRYKLMDKVVFPFIISKFTDRETPNA p27 T26 LYTTSTSTKTKFRDKGCISYVQGGSRYKLMDKVVFPFIISKFTDRETPNA p27 B67 LYTTSTSTKTKFRDKGCISYVQGGSRYKLMDKVVFPFIISKFTDRETPNA p27 T36 LYTISTSTKTKFRDKGCISYVQGGLRYKLLDKVVFPFIISKFTDRETPNA p27 B7-1 LCTISTSTKTKFRDKGCISYVQGGLRYKLLDKVVFPFIISKFTDRETPNA p27 B128 LCTISTSTKTKFRDKGCISYVQGGLRYKLLDKVVFPFIISKFTDRETPNA p27 B185 LCTISTSTKTKFRDKGCISYVQGGLRYKLLDKVVYPFIISKFTDRETPNA p27 B227 LCTISTSTKTKFRDKGCISYVQGGLRYKLFDKVVFPFIISKFTDRETPNA p27 B249 LCTISTSTKTKFRDKGCISYVQGGLRYKLLDKVVFPFIISKFTDRETPNA


CP T30 LRVWGRTNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA-CP T26 LRVWGRTNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA-CP B67 LRVWGRTNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA-CP T36 LRVWGRTNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA-CP B7-1 LRVWGRTNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA-CP B128 LRVWGRSNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA-CP B185 LRVWGRSNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA-CP B227 LRVWGRTNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA-CP B249 LRVWGRTNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA-p27 T30 LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP p27 T26 LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP p27 B67 LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP p27 T36 LRKFACTFEELHLCMARLRPDLYENKRTTRAGTPHLKGYLSADFLSGSLP p27 B7-1 LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP p27 B128 LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP p27 B185 LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP p27 B227 LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLTGSLP p27 B249 LRKYACTFEELHLCHARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP


CP T30 GLTDLECAVYIQAKEQLLKKRGA--DEVVVTNVRQLGKFNTR CP T26 GLTDLECAVYIQAKEQLLKKRGA--DEVVVTNVRQLGKFNTR CP B67 GLTDLECAVYIQAKEQLLKKRGA--DEVVVTNVRQLGKFNTR CP T36 GLTDLECAVYIQAKEQLLKKRGA--DDVVVTNVRQLGKFNTR CP B7-1 GLTDLECAVYIQAKEQLLKKRGA--DEVVVTNVRQLGKFNTR CP B128 GLTDLECAVYIQAKEQLLKKRGA--DEIVVTNVRQLGKFNTR CP B185 GLTDLECAVYVQAKEQLLKKRGA--DEVVVTNVRQLGKFNTR CP B227 GLTDLECAVYIQAKEQLLKKRGA--DEVVVTNVRQLGKFNTR CP B249 GLTDLECAVYLQAKEQLLKKRGA--DEVVVTNVRQLGKFNTR p27 T30 GYSEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL-p27 T26 GYSEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL-p27 B67 GYSEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL-p27 T36 GYSEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL-p27 B7-1 GYSEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL-p27 B128 GYSEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL-p27 B185 GYTEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL-p27 B227 GYSEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL-p27 B249 GYSEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL-Figure 13--continued.









61
































Figure 14. SSCP analysis of the p27 gene from the
same strains used in sequencing. 1, B227; 2, T36; 3, T26; 4, B67; 5, T30; 6, B185; 7, B249; 8, B128 and 9, B7-1.










62


































Figure 15. SSCP analysis of the p27 gene from other CTV strains. 1, T30; 2, B5; 3, B32; 4, B192; 5, B274; 6, B272; 7, T36; 8, B11; 9, B37; 10, B28; 11, B148 and 12, T36.









63

could be separated into nine different groups: 1) mild strains T30, T26, B67, B5, B32, B192 and B274, 2) mild strain B272, 3)

severe, stem pitting B7 and B128, 4) severe, stem pitting strains B249 and B185, 5) severe, stem pitting strain B227, 6) severe, quick decline strain T36, 7) severe, quick decline strain B11, 8) severe, quick decline strain B37, and 9) severe strains B28 (stem pitting) and B148 (quick decline).



Discussion

The p27 genes of the CTV strains sequenced showed 86% or greater homology at the RNA level and 93% or greater at the amino acid level (deduced sequence). These values are similar to those found for the CP (Pappu et al., 1993b). Several of the nucleotide substitutions are consistently conserved only within a particular biological group (mild, severe or severe causing stem pitting). These distinct sequences may have

potential for the development of group-specific molecular probes. Some of the nucleotide substitutions translated as nonsynonymous, although most of them were with functionally conserved amino acids.

The conservation of four amino acid substitutions between the severe strains compared to the mild suggests a possible relationship of p27 with symptom development. However, it is

necessary to note that a limited number of strains were sequenced, and this in no way demonstrates the role of p27, if any, in symptomatology. Further experiments are necessary to









64

establish the relationship between these amino acids and biological activity. For example, transformed plants expressing p27 from severe CTV strains could be used to

determine symptom expression when those plants are infected with mild strains.

Phylogenetic analysis of the amino acid sequences of p27 also indicated a strong relationship between sequence and biological activity, with mild and severe strains forming two

distinct groups. Stem pitting strains B7-1, B128 and B249 also formed a distinct domain within the severe strains. Interestingly, RNA analysis showed a different relationship,

with B227 forming a separate group from all other strains. The decline strain T36 was more similar to the mild strains T26, T30, and B67 than to strains causing stem pitting. This is an indication that the severe strains constitute a polyphyletic

group, and that the similarities in protein sequence (and possible symptom development) represent an example of evolutionary convergence.

Comparison of the phylogenetic trees obtained with CP sequences showed different relationships, indicating distinct evolutionary rates for the two genes. From these results, the amino acid sequence of the p27 gene is more useful in predicting the biological reaction of an strain than CP sequence (RNA or amino acid).

Alignment of p27 and CP deduced amino acid sequences revealed that the C-terminal portion of the two proteins is









65

the most conserved. Located also in this region are the two

consensus amino acids which are conserved in the CPs of filamentous viruses (Pappu et al., 1994). This portion must then contain important active domains for both proteins.

SSCP analysis of the p27 gene of strains T26, T30, T36, B67, B7-1, B128, B185, B227 and B249 produced groupings similar to those obtained in phylogenetic analysis of the RNA sequences. The only exception is the separation of the stem

pitting strains into two groups with different patterns: B7-1 and B128; B249 and B185. Interestingly, B249 is more similar in RNA sequence to B7-1 (99.3%) or B128 (99.6%) than it is to B185 (98.8%). This indicates that certain nucleotide changes have more influence in conformation than others. Sheffield et

al. (1993) found that the position of the base change was more important than the type of nucleotide substitution for the detection of such a change. Strains B227 and T36, the type strain for CTV, showed unique SSCP patterns.

The results for all the strains examined confirmed the sequence analyses indicating that mild strains were very homogeneous in sequence, regardless of the geographical origin. only one strain, B272, a very mild strain from Colombia, showed a different SSCP pattern from all other mild

strains. severe, quick decline strains, on the other hand, were quite variable and separated into four different groups.

Strains T36, B11 and B37 formed distinct SSCP patterns and B148 grouped with the stem pitting strain B28.









66

SSCP can detect single nucleotide changes in short DNA sequences (150 bp) (Orita et al., 1989; Sheffield et al., 1993), but is less efficient when longer fragments are

analyzed. PCR amplification can be followed by restriction digestion to generate short fragments if longer sequences are

amplified (Iwahana et al., 1992). For the CTV p27 gene this technique seems to be useful in differentiating certain strains. Furthermore, some of the patterns were highly conserved within particular biological groups, like the mild

strains. This technique can then be used as a rapid and simple assay for characterization and identification of CTV strains. However, it is necessary to determine the variability in other regions of the genome to expand the possibilities for this characterization. The use of restriction enzymes, together with SSCP can be a useful way of analyzing longer portions of the genome, while reducing the costs in primer synthesis.















CHAPTER 4
TRANSFORMATION OF SWEET ORANGE WITH P27 AND P20 Introduction

Sequence analysis and the identification of ORFs in other plant viruses have permitted the use of some of those genes to transform plants and protect them against viral infections caused by similar strains. The first and most widely used gene has been the CP (Powell Abel et al., 1986; Beachy et al., 1990; Pappu et al., 1995). Transgenic plants expressing CP genes and showing various degrees of resistance have been obtained for members of at least a dozen different virus groups (Hull and Davies, 1992; Pappu et al., 1995), indicating that this is a relatively general phenomenon. Several nonstructural viral genes also have been used to transform plants with mixed results in the induction of resistance. For example, three nonstructural genes of tobacco rattle tobravirus transformed into tobacco plants did not induce resistance to the virus (Angennent et al., 1990). On the other hand, tobacco plants transformed with a 54 kDa putative replicase protein of tobacco mosaic tobamovirus (TMV) were resistant to high concentrations of intact TMV and its RNA (Golemboski et al., 1990).




67









68

The finding of new and varied sources of resistance in

food crops is important. Historically, pathogens frequently overcome the resistance incorporated into those crops, usually obtained by long selection processes. The incorporation of viral genes can also offer a source of resistance when those genes are not naturally present in the crop. In addition, the expression of viral genes in plants, even if those genes are not protective, is an important tool in determining the function of those genes. For example, the 30 kDa protein of

TMV was shown to facilitate the cell-to-cell movement of a mutant strain of TMV (capable of replication but unable to cause systemic symptoms) when expressed in transgenic tobacco plants (Deom et al., 1987).

Most plant transformation experiments, with viral genes

or others, have been accomplished in herbaceous, annual plants and only a few woody species have been successfully

transformed. This is due to the technical difficulties in regenerating those plants, and the length of screening and testing for resistance. Plum (Prurius domestica L.) was recently transformed with the CP gene of plum pox potyvirus

using Agrobacterium (Machado et al., 1994; Scorza et al., 1994). Papaya (Carica papaya L.), a perennial plant, has also

been transformed with the CP gene of papaya ringspot potyvirus (Tennant et al., 1994) Transgenic plants, tested in the field for two years, showed no virus replication or movement when challenged with the same strain of virus used for









69

transformation. However, when the transgenic plants were inoculated with different viral strains they showed only a delay in symptom development (Tennant et al., 1994), a phenomenon observed previously in other CP transgenic plants (Beachy et al., 1990). For annual crops, a delay in symptom development can be sufficient to reduce losses caused by a virus, but for perennial crops this will probably be of little value since they are expected to be in the field for many years.

Several citrus species have reportedly been transformed by different means, including direct uptake of DNA by protoplasts (Schell, 1991), co-cultivation of cell suspensions with Agrobacterium (Hidaka et al., 1990), and infection of epicotyl segments with Agrobacterium (Moore et al., 1989; Kaneyoshi et al., 1994; Pefia et al., 1995). There are two reports of citrus transformation using the CP gene of CTV (Guti~rrez et al., 1992; Schell et al., 1994). The plants obtained in these experiments are being tested currently for resistance to CTV (G.A Moore and J.W. Grosser, personal communication).

To study the feasibility of using some of the non-CP genes of CTV in Agrobacterium-mediated plant transformation, p27 and p20 genes were used to transform "Pineapple" sweet orange plants. Transgenic plantlets were selected after target gene expression was detected in infected citrus tissue.









70

Materials and Methods

Materials

Calf intestinal alkaline phosphatase, Photogene Nucleic Acid Detection System Version 2.0 and biotin-14-dATP were from GIBCO BRL (Gaithersburg, MD). Murashigue and Skoog tissue culture basal salt mixture (MS) and antibiotics were from Sigma Chemical Company (St Louis, MO). Mefoxin was from Merck Sharp & Dohme (West Point, PA). Plasmids pMON10098, pMON755 and Agrobacterium ABI were obtained from Monsanto (St Louis, MO). "Pineapple" sweet orange seeds were from Willits & Newcomb Inc. (Arvin, CA). The 5-bromo-4-chloro-3-indolyl-O-Dglucuronic acid cyclohexylammonium salt (X-gluc) was supplied by Biosynth, Biochemica & Synthetica (Staad, Switzerland) or by Gold Biotechnology, Inc. (St. Louis, MO). Cloning of p27 Into the Transformation Vector

CTV gene p27 was cloned into pMON10098 from the

pUC118/p27 recombinant plasmid (Chapter 2). The gene contains the consensus sequence for optimal context of the AUG start codon (Kozak, 1986; Gallie, 1993). Approximately 0.5 Ag of pMON10098 plasmid DNA and 2 Ag of pUC118/p27 were digested separately with EcoRI (10 u) and XbaI (5 u) in 90 mM Tris-HCl pH 7.5 (at 370C), 10 mM MgC12, 50 mM NaCl at 370C for 4 h. Samples were purified in LMP agarose and ligated as explained in Materials and Methods, Chapter 2, except that the T4 polynucleotide kinase step was omitted. The plasmid was









71

transformed into E. coli DH5a competent cells and plated on 2 x YT agar plates containing 50Ag/ml of spectinomycin.

Plasmid DNA increased and purified from the transformed colonies was screened for presence of the insert by digestion with EcoRI and electrophoresed in a 0.6% agarose gel with lambda/HindIII molecular weight markers. They were also screened by digestion with EcoRI and XbaI and electrophoresed in 0.8 agarose gels.

The gene for 0-glucuronidase (GUS) was cloned into pMON10098/p27 from pMON755. Both plasmids (1 Ag of DNA each) were separately digested with NotI (2.5 u) in 6 mM Tris-HCl pH 7.9 (at 370C), 6 mM MgCl2, 150 mM NaCl, 1 mM DTT and purified in LMP agarose. Linearized pMON10098/p27 was treated with calf intestinal alkaline phosphatase (1.46 u) in 50 mM Tris-HCl pH 8.5, 0.1 mM EDTA according to the manufacturer's instructions and then extracted with phenol, phenol: chloroform, chloroform and precipitated with ethanol (Sambrook et al., 1989). Ligation of the GUS gene into pMON10098/p27 and transformation into E. coli DH5a was as described above.

To confirm presence and orientation of the p27 gene, the insert was sequenced following the protocol explained in Materials and Methods, Chapter 2. This plasmid was designated as pVF1 (Fig. 16).

Cloning of p20 Into the Transformation Vector

The cloning procedure for p20 was similar to that described above, but the GUS gene was cloned in pMON10098 first. The p20













72












x
0
Q. x IM go x hd 40 LU a U 00


p27


bp 720 bp 679 bp.
Pe
Notl Hinall BgIll Petl Petl Nool

Hinall ..........
Left Border Hinell


EcoRl





12,055 bp BarnHI

Notl Xhol Betl Ped
Right Border

Xhol N S PvUl
B*tXl Petl
Claid Clal
PvUl PvUl

















Figure 16. Restriction map of the pVF1 plasmid, containing P27.









73

gene was cloned subsequently from pUCll8/p20 using XbaI and SacI restriction sites. The new plasmid was designated as pVF2 (Fig. 17).

Transformation of pVF plasmids into Agrobacterium

Transformation of Agrobacterium with the pVF plasmids was by using the triple mating procedure. Overnight, 3 ml cultures in LB liquid medium of the following bacteria were used: Agrobacterium ABI (supplemented with 25 yg/ml chloramphenicol, 50 Ag/ml kanamycin), E. coli PRK 2013 (50 Ag/ml kanamycin) and E. coli DH5a with either pVF1 or pVF2 (75 jAg/ml spectinomycin). Samples of 250 jAl from each culture were mixed in a microcentrifuge tube and centrifuged for 30 sec at 14,000 rpm. The pellets were resuspended in 50 Al of 10 mM MgSO4 and placed on single LB agar plates free of antibiotics and without spreading the bacteria. The plates were incubated at 280C for 24 h. Bacteria were collected from single plates and resuspended in 1 ml of 10 mM MgSO4. Samples of 1, 5 and 10 Al were plated on LB agar containing 25 Ag/ml chloramphenicol, 50 Ag/ml kanamycin and 75 pg/ml spectinomycin and incubated for 3 to 4 days at 280C. Surviving bacterial cultures were then kept on YEP (1% yeast extract, 1% peptone, 0.5% NaCl pH 7.0) agar plates containing the three antibiotics. Screening of Transformed Bacteria

Agrobacterium colonies obtained in the transformation event were screened using PCR to determine the presence of the pVF plasmids. Samples from colonies were resuspended in 50 Al












74













E EE XI weOXUQ0 e


e35S p20 E
A~
.524 bp 546 bp 679 p









Notl

Petl Xhol















continin p20.i









75

of 20 mM Tris-HCl pH 8.5, 2 mM EDTA, 1% Triton X-100, boiled 75for 15 min and centrifuged for a few seconds. PCR reactions contained the normal constituents plus 1 jil of the bacterial extracts and the appropriate primers for p27, p20 or the GUS gene.

Transformation. Selection and Regeneration of Citrus Plants

Seed germination. The procedure for seed germination was modified from Moore et al. (1992, 1993). Intact seeds were

surface sterilized (10 min in 70% ethanol, 20 min in 20% Clorox and 2 drops of Tween-20, rinsed 3 times with sterile

distilled water) and germinated individually in 150 x 25 mm capped tubes containing 10 ml of half-strength MS basal salt

medium, 2.5% sucrose, 0.005% myo-inositol, 0.8% agar pH 5.7 and maintained at 270C with 16 h fluorescent light. Seedlings were used 3 to 6 months after sowing.

Bacterial culture. Individual colonies of Agrobacteriun

with either pVFl or pVF2 were increased in 50 ml flasks containing 5 ml YEP medium with the three antibiotics and incubated overnight at 280C and 200 rpm to post log phase. The bacteria were collected by centrifugation at 3,500 g for 15 min at 40C and resuspended in 1.5 ml of YEP without antibiotics. The solution was kept on ice until its use for transformation.

Plant transformation. Nodal, internodal and root segments from "Pineapple" sweet orange seedlings were used as explants for transformation (Moore et al., 1992, 1993). The internodal









76

and root pieces (1 cm) were inserted on MS medium (MS basal salt, 5% sucrose, 0.01% myo-inositol, 0.8% agar pH 7.5) with the apical end protruding. Nodal segments were placed lengthways on the MS medium. The explants were inoculated with one drop of Agrobacterium using a syringe and co-cultivated for 3 days at 270C and 16 h of fluorescent light (Fig. 18, A and B).

Selection. After 3 days, the explants were transferred to petri plates (20 x 100) with MS medium, without growth regulators, and supplemented with 100 Ag/ml of kanamycin and 200 Ag/ml of mefoxin. After 4 to 6 weeks, shoots began to emerge from the segments. Shoots were harvested and explants transferred to fresh selection medium every 4 weeks for up to three months, when they were discarded (Fig. 18, C).

Analysis of shoots. Harvested shoots were assayed histochemically for GUS activity (Stomp., 1992). Segments excised from the basal end of the shoots were incubated, using microtiter plates, in 25 Al of 0.1 M NaPO4 buffer, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.25 mg/ml X-gluc at 370C overnight. Transformed tobacco leaf segments, containing the GUS gene (kindly provided by G. Moore), were included as positive controls. The tissue samples were fixed and destained with 50 Al of 95% ethanol: acetic acid (3:1) at room

temperature for approximately 1 h before examination under the microscope.

PCR analysis. Some of the GUS positive (GUS+) tissue samples were further analyzed by PCR using p27 or p20 specific









Figure 18. Schematic representation of the transformation protocol for sweet orange plants. A), seedlings (3 to 6 months old), germinated in culture tubes, were pruned to eliminate leaves. Segments of approximately 1 cm were cut out from roots and stems (separating nodal and internodal portions). B), explants were placed in culture plates containing MS medium with the apical end protruding (internodes and roots) or length-ways on the medium (nodes). Explants were then inoculated with one drop of Agrobacterium. After three days of incubation the explants were transferred to selection medium (MS with antibiotics). C), approximately four to six weeks after the transformation shoots started to regenerate. The shoots were excised from the explants and assayed for GUS activity.







78 F7









79

primers (Chapter 2). Fixed materials were washed 4 times (30 min each) with 200 il of sterile distilled water and DNA extracted using the procedure of Rogers and Bendich (1988). The total, unquantified DNA extracted from each sample was used in a 100 pl PCR reaction containing 10 mM Tris-HCl pH 9.0, 50 mM KCI, 0.1% Triton X-100, 2.5 mM MgCl2, 10 mM DTT, 0.1 mM dNTPs, 2.5 u Taq polymerase and 40 pM of each primer. The mixtures were given 25 cycles of incubation at 940C for 1 min, 500C for 1 min, 720C for 1 min and a final incubation cycle at 940C for 1 min, 500C for 1 min and 720C for 10 min. Approximately 30 pl of the PCR product were analyzed by 0.8% agarose gel electrophoresis.

Southern analysis of the PCR products (1 pl) was

conducted using the procedure described in Sambrook et al. (1989). The membranes were probed using the Photogene system. Biotinylated p27 probes were produced using pVFI clones as templates in PCR reactions (Rashtchian and Mackey, 1992; Mertz et al., 1994).

Regeneration of shoots. GUS positive shoots were transferred to sterile baby food jars containing 40 ml of sterile soil (Metro-mix 200) and irrigated with 15 ml of sterile two-thirds strength MS basal salt medium. A solution of 100 pg/ml kanamycin, 200 pg/ml mefoxin and RooTone (Green Light Co., San Antonio, Texas) was applied to the base of the shoots before planting into soil. Plants were maintained at 270C with 16 h fluorescent light.









80

Results

Cloninq of CTV Genes Into the Transformation Vectors

Several approaches were used to determine the presence and copy number of the CTV genes in the pVF plasmids. Restriction digestion of pVFl and pVF2 with EcoRI and XbaI released inserts of the expected sizes indicating the presence of the gene. Comparison of the plasmid sizes after linearization with EcoRI indicated the presence of only one copy in each plasmid. Finally, sequence analysis also

confirmed the presence and orientation of the CTV genes in the pVF plasmids (data not shown).

PCR with p27, p20 (Fig. 19) or GUS specific primers indicated the presence of the pVF plasmids in Agrobacterium. This assay was employed routinely to determine the presence of the insert of interest in the cultures to be used in transformation experiments.

Transformation and Regeneration of Sweet Orange

Three different types of explants were used for transformation. Nodes and internodes were used with both pVFl and pVF2, and roots were only used with pVFI. This may be the first report of the use of root segments for transformation and regeneration of sweet orange. They were included for two reasons, to increase the number of explants obtainable per seedling and to determine their regenerative capacity. Figure 20 shows adventitious shoots on internodal segments and GUS+ shoots on soil for rooting.









81
































Figure 19. PCR of Agrobacterium ABI cultures to confirm the presence of pVF plasmids. Lane 1, Agrobacterium/pVF1 bacterial extract amplified using p27 primers; Lane 2, Agrobacterium/pVF2
bacterial extract amplified using p20 primers; Lane 3, purified pVF1 plasmid DNA amplified with p27 primers; Lane 4, purified pVF2 plasmid DNA amplified with p20 primers; Lane 5, Lambda DNA digested with HindIIIl used as a MW marker.









82






















A





















B




Figure 20. Adventitious shoot formation on internodal segments (A) and GUS+ shoots in soil for
rooting (B).









83

The results in Table 5 indicate that more adventitious shoots were produced from root segments compared to nodes or internodes. Nodes produced the lowest average number of adventitious shoots. There was no difference in shoot production when explants were inoculated with pVFl or pVF2.

However, comparison of the efficiencies of transformation (as percentage of GUS+ shoots obtained from the total) showed that internodes were more efficiently transformed with pVFl (Table 6) than nodes or roots. This indicates that proportionately, internodes generated twice as many GUS+ (Fig. 21) shoots than

the root segments. Interestingly, transformation with pVF2 was more successful on nodes than on internodes (Table 6). Again,

taking regeneration and transformation efficiency together, internode segments produced three times as many GUS+ shoots than nodes.

Transformation efficiency was quite variable between

experiments, ranging from 0 to 15%. The overall efficiency was lower than desirable. Taking the results from all the transformation experiments using pVFl, from 2,481 shoots obtained, 148 (5.97%) were GUS+. When pVF2 was used from 1181 shoots obtained, 71 (6.0%) were GUS+.

Survival of the GUS+ shoots in soil (Table 6) ranged from 25 to 45%, seven months after transfer, although none of them have rooted at the time this is written. The results from the shoots obtained from root segments are not included as they have been in soil only two months.









84







Table 5. Effects of explant type and plasmid
on adventitious shoot formation in
"Pineapple" sweet orange.



No. OF SHOOTS
EXPLANT PLASMID SEGMENTS PER SEGMENT

Nodes pVF1 350 0.30 0.11

pVF2 378 0.34 0.15

Total 728 0.32 0.14


Internodes pVF1 910 1.14 0.50

pVF2 434 1.00 0.38

Total 1344 1.10 0.47


Roots pVF1 42 1.64 0.45









85






















A




















B



Figure 21. GUS assay on adventitious shoots of "Pineapple" sweet orange regenerated from Agrobacterium-inoculated internodal segments. A),
GUS- segment. B), GUS+ segment.














Table 6. Transformation efficiencies and survival of shoots of "Pineapple" sweet
orange using pVFl (p27) and pVF2 (p20).



No. OF No. OF % GUS+ No.

PLASMIDa EXPLANT SHOOTS GUS+ SHOOTS SHOOTS SURVIVING SURVIVINGb

pVF1 Nodes 148 1 0.7 0 0.0

Internodes 1037 71 6.8 18 25.4

Roots 208 5 2.4 _C __C


pVF2 Nodes 215 19 8.8 7 37.8

Internodes 886 49 5.3 22 44.9



a pVFl=p27, pVF2=p20.
b Survival in soil seven months after transfer. c Adventitious shoots derived from root segments have been in soil less than seven months.






00









87

To confirm presence of the CTV genes in the GUS+ plants, samples were analyzed using PCR (Fig. 22, Table 7). For those GUS+ shoots transformed with pVF1, 31% were also PCR+ For pVF2, 37% of the GUS+ shoots were also PCR+. Transformed, GUS- segments were used as negative controls to rule out the possibility of obtaining PCR products from contaminating Agrobacterium. Hybridization of the PCR products obtained from the plants with p27 or p20 probes demonstrated they were CTV specific sequences (Fig. 23).



Discussion

The number of shoots produced by the internodes is comparable to what was obtained previously for Carrizo citrange [C. sinensis (L.) Osb. x Poncirus trifoliata (L.) Raf.] under similar experimental conditions (Moore et al., 1992). There are no published values for root and node segments. Comparing the three different types of explants used to transform with pVFI, root segments produced more adventitious shoots than internodes, and internodes more than nodes. However, a higher percentage of GUS+ shoots were obtained from internodes than from roots, making internode segments more efficient. For pVF2 internode segments also produced more GUS+ shoots than nodes.

Kanamycin was used in the media for selection of transformed shoots, however, only an average of 6% (considering all experiments together) were GUS+. "Escapes"









88







1234 5 6 70 01 2 31 51












A





















B


Figure 22. PCR amplification of DNA extracts from "Pineapple" sweet orange GUS+ segments transformed
with p27 (A) or p20 (B).- A), Lane 1, Lambda DNA digested with HindIII used as a MW marker; Lanes 2
and 7, GUS- segments; Lanes 3 to 6 and 8 to 15, GUS+ segments; Lane 16, pVFl. B) Lane 1, Lambda DNA digested with HindIII used as a MW marker; Lane 2, GUS- segment; Lanes 3 to 10, GUS+ segments; Lane
11, pVF2.









89





Table 7. Number and percentage of "Pineapple"
sweet orange shoots that tested
positive for GUS and by PCR.








PLASMID GUS+ PCR+ % PCR+
pVF1 79 25 31.2
pVF2 8 3 37.5









90
















1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

















Figure 23. Southern analysis of PCR products from GUS+ segments of "Pineapple" sweet orange hybridized with a p27 specific probe. Lanes 1 and
12, GUS- segment; Lanes 2 to 11 and 13, 14, GUS+ segments transformed with p27 gene; Lane 15, Lambda
DNA digested with HindIII; Lane 16, amplified p27 from pVFl.









91

have been reported previously (Moore et al., 1992; Peia et al., 1995) and constitute a serious problem, increasing the work necessary in screening the shoots. This ineffective Kanamycin selection may be explained by one or several possibilities: 1) protection of non-transformed cells by the surrounding transformed cells, 2) contamination with Agrobacterium or 3) by endogenous levels of NPT II activity (Jordan and McHughen, 1988; Dandekar et al., 1988; Moore et al., 1992; Pefia et al., 1995).

Transformation efficiencies, expressed as percentage of GUS+ shoots from the total adventitious shoots produced, were very variable between experiments and in general low. Values ranged from 0 to 15%, with an average of 6%. These results are comparable to those obtained by Moore et al. (1992, 1993), but lower than the 55% obtained with Carrizo citrange by Pefia et al. (1995) or the 55% to 87% reported by Kaneyoshi et al. (1994) with Poncirus trifoliata Raf. One of the differences in the transformation protocol used here and the ones mentioned above is that the segments are inoculated by submersion for several minutes to a few days in a solution containing the Agrobacterium. Another difference is that in those cases the explants were obtained from relatively young seedlings (3 to 5 weeks old) instead of 3 to 6 months old as reported here. Finally, the procedure of Kaneyoshi et al. (1994) includes acetosyringone in the inoculation media to increase the efficiency of bacterial infection.









92

Rooting of the transformed shoots has been difficult, with lower survival rates than reported by other authors (Moore et al., 1992; Kaneyoshi et al. 1994; Pefia et al., 1995). This may be due to a detrimental effect of the antibiotics on the explants or possibly to the expression of

the CTV gene products in the shoots. CTV infection reduces root and bud formation of shoots in vitro (Duran-Vila, 1989).

It is possible that the constitutive expression p27 or p20 causes the reduction of root formation in the explants.

PCR analysis of the same segments used in the histochemical GUS assay confirmed the presence of p27 and p20 in the putatively transformed shoots. Only 31% (p27) or 37% (p20) of the GUS+ segments were also PCR+. This may be due to

true false positives, or to deficiencies in the DNA extraction procedure because of the small amount of tissue used.

Hybridization experiments confirmed the specificity of the bands obtained in the PCR analysis, demonstrating that they are p27 and p20 genes.















CHAPTER 5
SUMMARY AND CONCLUSIONS



1) The expression of CTV ORFs p27 and p20 was detected in

vivo in CTV-infected plants using specific polyclonal

antibodies raised against the recombinant proteins.



2) Cell fractionation analysis indicated that p27

accumulates in the cell wall fraction, whereas p20

accumulates in the soluble protein fraction.



3) Tissue blots indicated that p27 is present in the phloem.

This is in agreement with CTV being a phloem limited

virus.



4) The function of p27 is still unknown, but based on

sequence homology and cell fraction studies, p27 could be involved in any or all of three functions: virus

assembly, virus movement and aphid transmission.



5) Based on sequence conservation, the active site of p27

appears to be located in the C-terminal portion of the

protein.



93




Full Text
27
concentration of p27 protein by 100-fold over the amount used
in Fig. 2, lane 2, where it was readily detected by the p27
antiserum.
To determine the cellular localization of p27, cell
fractions from CTV-infected and uninfected citrus tissue were
prepared and assayed by Western blots. Similar percentages of
total protein for each fraction were analyzed using p27
antiserum (Fig. 3). All cell fractions contained p27. However,
most of the p27 accumulated in the cell wall fractions (Rl, R2
and R3) (Fig. 3, lanes 6 to 8, respectively). In addition,
fraction S30 also contained considerable amounts of p27 (Fig.
3, lanes 5). Fractions PI and P30 showed much lower levels of
p27 (Fig. 3, lanes 3 and 4, respectively). These results
indicate that p27 was mostly associated with the cell wall
fractions.
The reaction of MCA-13 to the cell fractions was also
tested to compare the accumulation of CP and p27. Fig. 4
indicates that most of the CP accumulated in the cell wall
fractions Rl and R2 (Fig. 4, lanes 6 and 7), with some also
present in the soluble protein fraction S30 (Fig. 4, lane 5).
Fractions PI and P2 contained less CP (Fig. 4, lanes 3 and 4).
The pattern of accumulation of the CP was similar to that
observed for p27.
The p27 antiserum also was tested using tissue blot (Fig.
5). The optimal conditions for specific detection were


Table 3. Characteristics of the CTV strains used for SSCP analysis
STRAIN
ORIGIN
MCA-13a
M.L.b
DECLINE0
SYd
SPGe
SP-MVf
COMMENTS
B5
Calif.
-
+
0
0
0
7
Bll
Hawaii
+
+
+
+
0
0
B28
Florida
+
++
++
++
++
++
B32
Spain
-
+
0
0
0
0
B37
Taiwan
+
++
+++
+++
7
+
B14 8
Florida
+
++
++
++
0
0
B192
France
+
0
0
0
0
0
Not aphid transmitted
B272
Colombia
-
0
0
0
0
0
B274
Colombia
-
+
0
0
0
0
a through f, as in Table 2


LITERATURE CITED
Agranovsky, A.A., Koening, R. Maiss, E. Boyko, V.P., Casper,
R. and Atabekov, J.G. 1994. Expression of the beet
yellows closterovirus capsid protein and p24, a capsid
protein homologue, in vitro and in vivo. J. Gen. Virol.
75: 1431-1439.
Agranovsky, A.A., Lesemann, D.E., Maiss, E. Hull, R. and
Atabekov, J.G. 1995. "Rattlesnake" structure of a
filamentous plant RNA virus built of two capsid proteins.
Proc. Natl. Acad. Sci. USA 92: 2470-2473.
Albrecht, H., Geldreich, A., Menissier de Murcia, J.,
Kirchherr, D., Mesnard, J.M. and Lebeurier, G. 1988.
Cauliflower mosaic virus gene I product detected in a
cell-wall-enriched fraction. Virology 163:503-508.
Angenent, G.C., van den Ouweland, J.M.W. and Bol, J.F. 1990.
Susceptibility to virus infection of transgenic tobacco
plants expressing structural and nonstructural genes of
tobacco rattle virus. Virology 175:271-274.
Atreya, P.L., Atreya, C.D. and Pirone, T.P. 1991. Amino acid
substitutions in the coat protein results in loss of
insect transmissibility of a plant virus. Proc. Natl.
Acad. Sci. USA 88: 7887-7891.
Atreya, C.D., Raccah, B. and Pirone, T.P. 1990. A point
mutation in the coat protein abolishes aphid
transmissibility of a potyvirus. Virology 178:161-165.
Ausubel, F.M., Brent, R. Kingston, R.E., Moore, D.D.,
Seidman, J.G., Smith, J.A. and Struhl, K. 1992. Short
Protocols in Molecular Biology, Second Edition. John
Wiley & Sons. New York, NY.
Bar-Joseph, M. and Lee, R.F. 1990. Citrus tristeza virus.
Description of Plant Viruses No. 353. Commonwealth
Mycological Institute/Association of Applied Biologist.
Kew Surrey, England. 7 p.
Bar-Joseph, M. Marcus, R. and Lee, R.F. 1989. The continuous
challenge of citrus tristeza virus control. Ann. Rev.
Phytopathol. 27:291-316.
95


Table 4
RNA and deduced amino acid sequence similarities (% of identical bases
or amino acids) between different CTV strains for p27 (upper right is
RNA, lower left is amino acid sequences). Values generated using
CLUSTALV.
STRAIN
T30
T26
B67
T36
B7-1
B128
B185
B227
B249
T30
98.9
99.2
93.9
92.0
92.1
90.7
88.2
92.1
T26
98.8
98.3
93.8
91.2
91.4
90.2
88.2
91.6
B67
100.0
98.8
94.6
92.5
92.7
91.3
88.8
92.7
T36
96.7
95.4
96.7
92.5
92.7
91.0
87.6
92.4
B7-1
96.7
95.4
96.7
97.5
99.7
98.1
87.3
99.3
B128
97.1
95.8
97.1
97.9
99.6
98.3
87.6
99.6
B185
94.6
93.3
94.6
95.4
97.1
97.5
86.2
98.8
B227
95.0
93.8
95.0
95.4
96.7
97.1
94.6
87.7
B249
97.1
95.8
97.1
97.9
99.6
100.0
97.5
97.1


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Charles L.Niblett, Chair
Professor of Plant
Pathology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Richard F. Lee ,'-C5chair
Professor of Plant
Pathology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
(~¡LCL %&U)44JL/
Jude W. Grosser
Professor of Horticultural
Science


34
although it does not have any effect on the detection of the
target proteins in vivo (because they lack the fusion
portion).
The results using the p27 antiserum showed the presence
of a protein of the expected size (27.4 kDa) in CTV-infected,
but not in uninfected tissue. Also, this protein band was not
present in tissue samples infected with other citrus viruses.
This indicates that the protein detected is CTV-specific and
was expressed during infection, and that its size is in
agreement with that predicted from the sequencing data (Pappu
et al., 1994).
The protein bands detected with the CP-specific
monoclonal antibody MCA-13 are of a lower MW than that
detected with p27 antiserum. Because of this and the fact that
the p27 antiserum detected only a single band, and did not
react with the nonfusion E. coli-expressed CP, it is concluded
that the protein detected with the antiserum is p27, and not
the result of a cross-reaction of the p27 antiserum with the
CP. This was a definite possibility since both proteins have
a high degree of homology in their primary structure.
The monoclonal antibody MCA-13 showed a very low affinity
for the E. coli-expressed p27, because its concentration had
to be increased 100-fold compared to the concentration of p27.
The region of the CP that is recognized by MCA-13 has been
identified (Pappu et al., 1993a) and part of this region is
conserved in p27, which may explain its low affinity for p27.


T30cp
T26cp
B67cp
T36cp
B71cp
B185cp
B128cp
B227cp
B249cp
Figure 12. Phylogenetic tree for CP based on
deduced amino acid sequences. Graphic generated
using Pileup program in GCG.


102
Powell Abel, P., Nelson, R.S., De, B., Hoffmann, N., Rogers,
S.G., Fraley, R.T. and Beachy, R.N. 1986. Delay of
disease development in transgenic plants that express the
tobacco mosaic virus coat protein gene. Science 232:738-
743 .
Rashtchian, A. and Mackey, J. 1992. Efficient synthesis of
biotinylated DNA probes using polymerase chain reaction.
Focus 14:64-65.
Rogers, S.O. and Bendich, A.J. 1988. Extraction of DNA from
plant tissues. Plant Molecular Biology Manual A6:l-10.
Kluwer Academic Publisher, Dordretch, Belgium.
Saiki, R.K., Gelfand, D.H., Stoffe, S., Sharf, S.J., Higuchi,
R. Horn, G.T. and Mullis, K.B. 1988. Primer-directed
enzymatic amplification of DNA with a thermostable DNA
polymerase. Science 239:487-491.
Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular
Cloning: A Laboratory Manual. Second Edition. Cold Spring
Harbor Press, Cold Spring Harbor, NY.
Sanger, F., Niklen, S. and Coulson, A.R. 1977. DNA sequencing
with chain-terminating inhibitors. Proc. Natl. Acad. Sci.
USA 74:5463-5467.
Schell, J. 1991. Genetic transformation of citrus protoplasts
by PEG-mediated direct DNA uptake and regeneration of
putative transgenic plants. MS. Thesis. University of
Florida, Gainesville, FL. 127 p.
Schell, J.L., Pappu, H.R., Pappu, S.S., Derrick, K.S., Lee,
R.F., Niblett, C.L. and Grosser, J.W. 1994.
Transformation of 'Nova' tngelo with the coat protein
gene of citrus tristeza closterovirus. Phytopathology
84:1076.
Schneider, H. 1959. The anatomy of tristeza-virus-infected
citrus. Pages 73-84, in: Wallace, J.M. (ed.) Citrus Virus
Diseases. University of California, Berkeley, CA.
Scorza, R. Ravelonandro, M. Callahan, A.M., Cordts, J.M. ,
Fuchs, M., Dunez, J. and Gonsalves, D. 1994. Transgenic
plums (Prunus domestica L.) express the plum pox virus
coat protein. Plant Cell Reports 14: 18-22.
Sekiya, M.E., Lawrence, S.D. McCaffery, M. and Cline, K. 1991.
Molecular cloning and nucleotide sequencing of the coat
protein gene of citrus tristeza virus. J. Gen. Virol.
72:1013-1020.


70
Materials and Methods
Materials
Calf intestinal alkaline phosphatase, Photogene Nucleic
Acid Detection System Version 2.0 and biotin-14-dATP were
from GIBCO BRL (Gaithersburg, MD) Murashigue and Skoog tissue
culture basal salt mixture (MS) and antibiotics were from
Sigma Chemical Company (St Louis, MO). Mefoxin was from Merck
Sharp & Dohme (West Point, PA). Plasmids pMON10098, pMON755
and Agrobacterium ABI were obtained from Monsanto (St Louis,
MO) "Pineapple" sweet orange seeds were from Willits &
Newcomb Inc. (Arvin, CA) The 5-bromo-4-chloro-3-indolyl-j8-D-
glucuronic acid cyclohexylammonium salt (X-gluc) was supplied
by Biosynth, Biochemica & Synthetica (Staad, Switzerland) or
by Gold Biotechnology, Inc. (St. Louis, MO).
Cloning of p27 Into the Transformation Vector
CTV gene p27 was cloned into pMON10098 from the
pUC118/p27 recombinant plasmid (Chapter 2). The gene contains
the consensus sequence for optimal context of the AUG start
codon (Kozak, 1986; Gallie, 1993). Approximately 0.5 /xg of
pMON10098 plasmid DNA and 2 ¡iq of pUC118/p27 were digested
separately with EcoRI (10 u) and Xbal (5 u) in 90 mM Tris-HCl
pH 7.5 (at 37C) 10 mM MgCl2, 50 mM NaCl at 37C for 4 h.
Samples were purified in LMP agarose and ligated as explained
in Materials and Methods, Chapter 2, except that the T4
polynucleotide kinase step was omitted. The plasmid was


51
T30
T26
B67
T36
B7-1
B128
B185
B227
B249
601 lili 650
GGGTACTCCGAACATGAACGAGGCATCATTCTTCGAGCGTCTGAGTCTAT
1
TG
A-T--G
TG
T §
1 cA
T !A
_____| CA
651 lili 700
T30 GTTAGCTAGACGTCAAGGTTACGAGGAGGCAACCGAGCTTCTTAACCTAC
T2 6 1
B67
T36 G
B7-1
B12 8
B185
B227 G i
B249
T30
T26
B67
T36
B7-1
B128
B185
B227
B249
701 I 723
GTGATTTGGGTAAGTACTTATAG
-C
Figure 7continued


20
For p20, most of the protein was soluble. The protein in
the supernatant was pelleted with saturated ammonium sulfate
(final concentration 33%; Ausubel et al., 1992) and
centrifuged 14,500 g for 15 min at 4C. The pellet was
resuspended in TE buffer. All three proteins were stored at -
20C.
Isolation of recombinant proteins. Proteins of interest
were separated from other bacterial components using 12% SDS-
PAGE in a 15 x 15 cm gel and a single well, 3 mm thick comb.
Approximately 5 to 10 mg were separated in each gel. Proteins
were detected using ice cold 0.25 M KC1 (Hager and Burgess,
1980) which reacted with the bound SDS to form a visible band.
The protein band was excised with a sterile blade and stored
at 4 0 C.
Polyclonal antibodies. The protein (still in the
polyacrylamide gel) was sent to Cocalico Biological, Inc.
(Reamstown, PA) for production of polyclonal antibodies in
rabbits. Approximately 1.5 to 2 mg of protein were used for
each weekly injection. Antibodies were collected and tested
after four weeks of injections.
Detection of CTV Proteins in Infected Citrus Tissue
Western and tissue blot analysis. The expression of p27,
p20 and pl8 in T36 CTV-infected Mexican lime was tested using
Western blot analysis, according to a previously described
procedure (Li et al., 1991). Detached midrib segments of
approximately 1 cm in length were sliced into small pieces


determine any possible differences that could correlate with
virulence, and 3) to transform citrus plants with p27 and p20
genes in an attempt to confer resistance to CTV. Fusion
proteins for p27, p2 0 and pl8 were produced in Escherichia
coli and used to raise polyclonal antibodies. Western blot
analyses using the antisera indicated that p27 and p20 are
expressed in CTV-infected citrus, but not in uninfected
plants. Tissue fractionation studies revealed that p27
accumulates in cell wall enriched fractions, whereas p20
accumulates in the soluble protein fraction. The expression of
pl8 was not detected in CTV-infected citrus tissue. The p27
protein also was detected using tissue blot analysis. RNA
seguence homologies of the p27 gene among CTV isolates were
between 86 and 99%. The deduced amino acid seguence homologies
were 93% or higher. Phylogenetic analysis showed mild and
severe, quick decline isolates grouping separately.
Agrobacterium-mediated plant transformation experiments were
performed using p27 and p20 genes. Approximately 6% of the
adventitious shoots obtained on selection media with kanamycin
were GUS positive. Between 31 and 37% of the GUS positive
shoots were positive using PCR analysis with primers specific
to p27 or p20, suggesting that some plants are transformed
with each of the genes.
vii


99
Karasev, A.V., Boyko, V.P., Gowda, S., Nikolaeva, O.V., Hilf,
M.E., Koonin, E.V., Niblett, C.L., Cline, K. Gumpf,
D.J., Lee, R.F., Garnsey, S.M., Lewandowski, D.J. and
Dawson, W.O. 1995. Complete sequence of the citrus
tristeza virus RNA genome. Virology 208:511-520:
Klaassen, V.A., Boeshore, M., Koonin, E.V., Tian, T. and Falk,
B.W. 1995. Genome structure and phylogenetic analysis of
lettuce infectious yellows virus, a whitefly-transmitted,
bipartite closterovirus. Virology 208: 99-110.
Kozak, M. 1986. Point mutations define a sequence flanking the
AUG initiator codon that modulates translation by
eukaryotic ribosomes. Cell 44: 283-292.
Laemmli, U.K. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680-
685.
Lastra, R., Meneses, R., Still, P.E. and Niblett, C.L. 1991.
The CTV situation in Central America. Pages 146-149 in
Brlansky, R.H., Lee, R.F. and Timmer, L.W. (eds.) Proc.
11th Conf. of Int. Organ. Citrus Virol. IOCV. Riverside,
CA.
Lee, R.F. and Rocha-Pea, M.A. 1992. Citrus tristeza virus.
Pages 226-249, in: Kumar, J. Chaube, H.S., Singh, U.S.,
Mukhopadhyay, A.N. (eds.). Plant Diseases of
International Importance. Vol III. Diseases of Fruit
Crops. Prentice Hall. Englewood Cliffs, NJ.
Li, R.H., Zettler, F.W., Elliot, M.S., Petersen, M.A., Still,
P.A., Baker, C.A. and Mink, G.I. 1991. A strain of peanut
mottle virus seedborne in bambarra groundnut. Plant Dis.
75:130-133.
Machado, A.D., Katinger, H. and Machado, M.L.D. 1994. Coat
protein-mediated protection against plum pox virus in
herbaceous model plants and transformation of apricot and
plum. Euphytica 77: 129-134.
Marston, F.A.O. 1986. The purification of eukaryotic
polypeptides synthesized in Escherichia coli. Biochem. J.
240:1-12.
Mathews, R.E.F. 1991. Plant Virology. Third edition. Academic
Press, Inc. San Diego, CA. 835 pp.
McCarty, D.R., Hattori, T., Carson, C.B., Vasil, V., Lazar, M.
and Vasil I.K. The viviparous-1 developmental gene of
maize encodes a novel transcriptional activator. Cell
66:895-905.


9
in vivo. The final evidence, however, is the detection of the
protein products in infected tissue.
In order to investigate whether p27, p20 and pl8 are
expressed in CTV infected plants the genes were separately
cloned into Escherichia coli expression vectors. The expressed
proteins were used to raise polyclonal antibodies to probe for
the proteins in CTV-infected citrus tissue.
Materials and Methods
Materials
Reagents were obtained from Fisher Scientific
(Pittsburgh, PA) or Sigma Chemical Company (St. Louis, MO).
Enzymes and lambda DNA marker were purchased from Promega
Corporation (Madison, WI). Some restriction enzymes, agarose
and low melting point (LMP) agarose were from GIBCO BRL
(Gaithersburg, MD). Phenol (equilibrated with 0.1 M Tris-HCl,
pH>7.8), T4 polynucleotide kinase, dNTPs and Sequenase Version
2.0 DNA sequencing kit were from Amersham (Arlington Heights,
IL) The nucleotide [a-35S]-dATP and the Renaissance
chemiluminescent detection kit for Western blots were from
DuPont NEN Research Products (Boston, MA). Protein standards
SDS-6 and SDS-7 were from Sigma Chemical Company.
Electrophoresis apparatus for small gels (7x8 cm), Mini-
PROTEAN II dual slab cell, was from Bio-Rad (Richmond, CA).
Electrophoretic transfer unit, Mini Trans-Blot, was also from
Bio-Rad. The larger electrophoresis unit (14 x 14 cm), VAGE,


49
201 lili 250
T 3 0 CTTGAAATACGTTATGGTTACTATGGATGCTTTCTTATTAGAAAACTACA
T26
B67
T3 6 CA
B7-1 AG CA 1
B128 A1 CA 1
B185 AG CA ¡h
B227 AC GT ~A 1
B249 AG Cl T-
251 lili 300
T3 0 AGACGAAAACGGAAGATCTGTTGGTTCACTTAGCTATGATCCAAAAGAGG
T26
B67
T36 -A
B7-1 -A
B128 -A
B185 -AC-C
B227 -A
B249 -A
A
1 -
i
i 1
i TC-G 1GC
A _______
T30
T26
B67
T36
B7-1
B128
B185
B227
B249
301 lili 350
TTGTACACTACATCCACGAGCACTAAAACCAAGTTCCGCGATAAAGGTTG
A i i
i-ii-i 1 i
A-l 1- T
I-i1-1 T T
G T T aTT
A-G C-T T i---
G
I
G
I
G
351 lili 400
T 3 0 TATTAGTTACGTGCAAGGGGGTTCGCGATATAAGTTAATGGATAAAGTAG
T2 6 C
B67
T36 A TA GT C
B7-1 A A TA C T G-
312 8 A A TA 1 T G-
3185 A A TA C T G-
B227 T TA 111 A-
32 4 9 A A TA C T- G-
Figure 7continued


50
T30
T26
B67
T3 6
B7-1
B128
B185
B227
B249
401 lili 450
TTTTTCCTTTCATTATATCGAAATTTACCGACAGGGAGACTCCGAACGCT
i
1
|
|
-i~!
c-
A
GA-
T
T
T
T
451 lili 500
T3 0 CTACGTAAGTATGCTTGCACTTTCGAGGAGTTACACTTGTGTATGGCTAG
T26
B67
T3 6 1
B7-1 A
B128
B185 A
B227 111
B249 _______ A
T30
T26
B67
T36
B7-1
B12 8
B185
B227
B249
501 lili 550
GTTGAGACCCGACTTATACGAAAATAAAAGGACGACTAAAGCCGGGACTC
AA 1-
i1 CI
A-A CI
A-A 1-
AA C-
CGTA
C-GGT
-G CGT
-G 11T
1 i! 1A
-I 1i1
T30
T2 6
B67
T36
B7-1
B128
B185
B227
B249
551 lili 600
CACATTTAAAGGGCTATTTATCAGCCGACTTTCTTTCGGGTTCTCTCCCA
T
c T
cc T CA T
ATC- T
1! t
Ai1 1
-G CG TT A T
AI T
Figure 7continued


36
(Agranovsky et al., 1995). This could be through interaction
with other viral proteins, including the CP and the heat shock
proteins (p65 and p61) or unknown host components. Heat shock
proteins work as chaperons helping in proper folding of other
proteins (Georgopoulos, 1992).
The p27 might also have a role as a helper component in
the transmission of CTV by its aphid vector. This has
previously been proposed for BYV p24 (Agranovsky et al., 1995;
Boyko et al., 1992). Helper components have been described for
potyviruses and caulimoviruses (Mathews, 1991), although they
do not form part of the virions. However, in potyviruses the
CP is also important for aphid transmission (Atreya et al.,
1990, 1991). Interestingly, LIYV also contains a similar
duplicate of the CP, with deduced amino acid sequence
homologies to BYV and CTV CPs and homologues, however, LIYV is
transmitted by the whitefly Bemisia tabaci (Gennadius)
(Klaassen et al., 1995).
Relating to the cellular accumulation of p27, a possible
function could be as a movement protein, assisting in the
spread of the virus between cells. Movement proteins interact
with plasmodesmata causing an increase in their diameter (Wolf
et al., 1989; Derrick et al., 1992; Waigmann et al., 1994),
thereby enabling the intercellular passage of the large
nucleic acid in a naked or coated form, depending on the
virus. Putative movement proteins of tobacco mosaic virus
(Deom et al., 1987), A1MV (Godefroy-Colburn et al., 1986),


6
proposed to be expressed by ribosomal frameshift of the first
ORF, resulting in a polyprotein of 401 kDa (Karasev et al.,
1995). This polyprotein also would undergo autoproteolytic
cleavage producing a 292 kDa fragment with domains for
methyltransferase, helicase and RNA polymerase (Karasev et
al., 1995). The third ORF potentially encodes a 33 kDa (p33)
protein (Karasev et al., 1995) of unknown function, followed
by a small ORF encoding a 6.4 kDa protein with a highly
hydrophobic domain (Dolja et al., 1994). ORFs 5 and 6 code for
proteins of 64.7 (p65) and 61.1 (p61) kDa, respectively (Pappu
et al., 1994). Sequence comparisons indicate conservation of
several motifs between p65 and the hsp70 group of heat shock
proteins (Pappu et al., 1994). Similarly, p61 contains a C-
proximal domain that is present in another group of heat shock
proteins, the hsp90 group (Pappu et al., 1994). These two
proteins have been detected in CTV-infected citrus tissue
(S.S. Pappu, personal communication).
The following ORFs, 7 and 8, encode proteins of
calculated MW of 27.4 (p27) and 24.9 kDa, respectively (Pappu
et al., 1994). ORF 8 has been identified as the CP gene
(Sekiya et al., 1991; Pappu et al., 1993b). Based on their
deduced amino acid sequence, p27 shows 41% similarity with the
CP (Pappu et al., 1994). ORFs 9 to 12 potentially encode
proteins with calculated MW of 18.3 (pl8), 13.2 (pl3), 20.5
(p20) and 23.7 (p23) kDa, respectively (Pappu et al., 1994).
The functions of these proteins are not known, and deduced


94
6) The function of p2 0 is also unknown. Its in vivo
expression and accumulation did not give any indications
about its possible role in the CTV life cycle.
7) Sequence and phylogenetic analysis of the p27 gene from
different CTV strains indicated a relationship between
deduced amino acid sequence and biological activity of
the strains.
8) SSCP analysis permitted the differentiation of CTV
strains. Most mild strains could to be identified using
this method.
9) The genes encoding p27 and p20 were transformed into
"Pineapple" sweet orange using Agrobacterium.
Transformation was confirmed using GUS and PCR assays.
10) Internodal segments from seedling epicotyls were the most
efficient in producing GUS+ adventitious shoots.


10
was from Stratagene (La Jolla, CA). The pETH-3b expression
vector was kindly provided by D.R. McCarty. The expression
clone for the CP was kindly provided by M.L. Keremane.
Plant Material
Mexican lime [C. aurantifolia (Christm.) Swingle] plants
infected with CTV T36 were maintained in greenhouses at the
University of Florida (Gainesville, FL).
Samples of citrus infected with citrus ringspot virus,
citrus variegation virus, citrus leaf rugose virus, psorosis
A and concave gum were kindly provided by R.F. Lee.
Cloning of CTV ORFs into PUC118
Extraction of viral RNA templates. Total nucleic acids
were extracted from citrus leaves infected with the Florida
severe, quick decline strain T36, using the procedure
described by Pappu et al. (1993c). Approximately 1 cm2 of
tissue was ground in a microcentrifuge tube with a sterile
blunt stick (2 x 147 mm) in liquid nitrogen. The samples were
thawed in 0.3 ml of extraction buffer [0.1 M Tris-HCl, pH 8.0,
2 mM ethylenediaminetetraacetic acid (EDTA) and 2% sodium
dodecyl sulfate (SDS)]. An equal volume (0.3 ml) of a mixture
of phenol: chloroform: isoamyl alcohol (25:24:1) were added,
followed by vortexing for a few seconds. The samples were
incubated at 70C for 5 min and then centrifuged at 14,000
rpm for 5 min at room temperature. The aqueous upper phase was
saved.


48
1 I I I I 50
T3 0 ATGGCAGGTTATACAGTACTTCCTAATACCGATGACAAAGAAATGGATCC
T2 6 1
B67
T3 6 1 1
B7-1 1 1
B128 11 1
B185 11 T
B2 2 7 CAGTT C
B2 4 9 GA T
51 I I I I 100
T30 GGTGAGTGCCGCTGTACCCGGTAAGTATCCGGATGTCATTGAAAAATTTG
T2 6 1
B67
T3 6 1 11| 1-
B7-1 1 1 1
B128 1 1 1
B185 C 1
B2 27 A 1 1I1
B2 4 9 -1 1
T30
T26
B67
T36
B7-1
B128
B185
B227
B249
101 I I I I 150
TGGCCAACAGGTCCGTAGACGCGTTAATAGAAGGCGTCATAAGTAAGTTG
1
-
-1 I
-1 T
-T 1
-1 1
-I I
T
T
T
T
-i-
-i 1
-!
-T
-I c
-1 c
_T
T30
T26
B67
T36
B7-1
B128
B185
B227
B249
151 I I I I 200
GATACCAATTCAATATACGAAGATTCCACTGAAAAATTTACTGGTGAACA
Q
_!
c t 1
CT G
1 i
-G
-1 GT-
-1 11-
-I C GT-
-i G
-i GT-
Fig. 7. Nucleotide sequence alignment of p27 gene from
different CTV strains (hyphens, identical nucleotides).
The alignment was constructed using the program
CLUSTALV.


CHAPTER 1
INTRODUCTION
Citrus tristeza was initially recognized as a decline
disease of citrus scions propagated on sour orange (Citrus
aurantium L. ) rootstock (Lee and Rocha-Pea, 1992). This
disease, caused by citrus tristeza closterovirus (CTV), is one
of the major diseases affecting citrus, and an excellent
example of an agricultural problem created by man (Bar-Joseph
et al., 1989) CTV is now known to cause a variety of symptoms
depending on the virus strain and host. Perhaps the most
economically important and obvious CTV symptom is the decline
of sweet orange [C. sinensis (L.) Osbeck], grapefruit (C.
paradisi Macf.) and mandarin (C. reticulata Blanco) grafted on
sour orange rootstock (Garnsey et al., 1987; Bar-Joseph and
Lee, 1990). Stem pitting in limes [C. aurantifolia (Christm.)
Swingle], grapefruit and some sweet oranges is another
important reaction and a limiting factor to production in
parts of Brazil, South Africa and Australia (Garnsey et al.,
1987; Bar-Joseph and Lee, 1990; Lee and Rocha-Pea, 1992). In
addition, some CTV strains can induce seedling yellows when
inoculated to sour orange, grapefruit and lemon (C. limn
Burm.f) seedlings. The importance of this symptom is not
known, except for CTV strain differentiation, because it
1


74
Figure 17. Restriction map of the pVF2
containing p20.
plasmid,


Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MOLECULAR CHARACTERIZATION OF CITRUS TRISTEZA VIRUS GENES
AND THEIR USE IN PLANT TRANSFORMATION
By
Vicente Jos Febres-Rodriguez
August, 1995
Chairman: C.L. Niblett
Major Department: Plant Pathology
Nucleotide sequence analysis of citrus tristeza
closterovirus (CTV) revealed the presence of 12 possible open
reading frames (ORFs). The ORF immediately upstream of the
coat protein (CP) gene encodes a protein of calculated MW of
27.4 kDa (p27). The deduced amino acid sequence indicated that
this gene product is homologous to the CP (41% similarity).
Two other genes located toward the 3' end of the CTV genome
encode proteins of 18.3 (pl8) and 20.5 (p20) kDa,
respectively. Deduced amino acid sequence comparisons with
protein databases did not reveal any significant relationship
that might indicate the possible function of these proteins.
The objectives of this research were: 1) to determine if p27,
p20 and pl8 proteins were expressed in CTV-infected citrus
tissue, 2) to identify the sequence variability of the p27 ORF
among CTV strains with different biological properties and to
vi


79
primers (Chapter 2). Fixed materials were washed 4 times (30
min each) with 200 /I of sterile distilled water and DNA
extracted using the procedure of Rogers and Bendich (1988).
The total, unquantified DNA extracted from each sample was
used in a 100 il PCR reaction containing 10 mM Tris-HCl pH
9.0, 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl2, 10 mM DTT,
0.1 mM dNTPs, 2.5 u Taq polymerase and 40 pM of each primer.
The mixtures were given 25 cycles of incubation at 94C for 1
min, 50C for 1 min, 72C for 1 min and a final incubation
cycle at 94C for 1 min, 50C for 1 min and 72C for 10 min.
Approximately 30 /til of the PCR product were analyzed by 0.8%
agarose gel electrophoresis.
Southern analysis of the PCR products (1 /xl) was
conducted using the procedure described in Sambrook et al.
(1989). The membranes were probed using the Photogene system.
Biotinylated p27 probes were produced using pVFl clones as
templates in PCR reactions (Rashtchian and Mackey, 1992; Mertz
et al., 1994).
Regeneration of shoots. GUS positive shoots were transferred
to sterile baby food jars containing 40 ml of sterile soil
(Metro-mix 200) and irrigated with 15 ml of sterile two-thirds
strength MS basal salt medium. A solution of 100 ^g/ml
kanamycin, 200 jug/ml mefoxin and RooTone (Green Light Co., San
Antonio, Texas) was applied to the base of the shoots before
planting into soil. Plants were maintained at 27C with 16 h
fluorescent light.


66
SSCP can detect single nucleotide changes in short DNA
sequences (150 bp) (Orita et al., 1989; Sheffield et al.,
1993), but is less efficient when longer fragments are
analyzed. PCR amplification can be followed by restriction
digestion to generate short fragments if longer sequences are
amplified (Iwahana et al., 1992). For the CTV p27 gene this
technique seems to be useful in differentiating certain
strains. Furthermore, some of the patterns were highly
conserved within particular biological groups, like the mild
strains. This technique can then be used as a rapid and simple
assay for characterization and identification of CTV strains.
However, it is necessary to determine the variability in other
regions of the genome to expand the possibilities for this
characterization. The use of restriction enzymes, together
with SSCP can be a useful way of analyzing longer portions of
the genome, while reducing the costs in primer synthesis.


12
Table 1.
Description of the primers used
cloning CTV ORFs p27, p20 and pl8.
for
ORF
PRIMER3
Tmb
(C)
p2 7
5' AAGCTTCTAGAACCATGGCAGGTTATACAGTAC 3'
54
5' CTATAAGTACTTACCCAAATC 3'
56
p2 0
5' AAGCTTCTAGAACCATGCGAGCTTACTTTAGTG 3'
54
5' CTACACGCAAGATGGAGA 3'
58
pl8
5' AAGCTTCTAGAACCATGTCAGGCAGCTTGGG 3'
54
5' CTAAGTCACGCTAAACAAAG 3'
56
a Upper sequence is the genome sense primer. Lower
sequence is the genome antisense primer. Bold
letters are HindIII (AAGCTT) and Xbal (TCTAGA)
restriction sites. The start codon is underlined,
b Tm= calculated melting temperature.


ACKNOWLEDGMENTS
I want to express my gratitude to Dr. Chuck Niblett,
chairman of my supervisory committee, and Dr. Richard Lee,
cochairman, for their support, encouragement and guidance in
completing this thesis. I also want to thank Dr. Chuck Powell
and Dr. Jude Grosser, members of my committee, for their
assistance and suggestions. Thanks also to Dr. Ken Derrick for
his comments and his help in reviewing this manuscript.
I am grateful to Dr. Hanumantha Pappu, Dr. Sita Pappu and
Dr. Ed Anderson for their technical support, suggestions and
friendship, that certainly helped me complete this work in an
easier way.
I also thank Dr. Gloria Moore and members of her lab,
especially Alejandra Gutirrez, for their assistance in the
tissue culture portion of this work and for sharing their
time, procedures and lab equipment with me. Thanks to Blanca
Garagorry for her help in the transformation experiments.
Thanks to Dr. Dave Stark and Monsanto for providing the
pMON vectors and Agrobacterium strain used for plant
transformation.
I thank Tiffany Niblett for her kindness and patience
that allowed this work to be more productive and enjoyable for
me and my adviser. And, of course, thanks to Manjunath and
ii


15
ethanol (Sambrook et al. 1989). The dried pellets were
resuspended in 16 il of sterile distilled water.
The total volume of recovered PCR products was mixed with
the purified Smal digested pUC118 for ligation in 500 mM Tris-
HC1 pH 8.0, 100 mM MgCl2, 200 mM DTT, 10 mM ATP, 0.05% bovine
serum albumin type V (BSA) and T4 polynucleotide kinase (30 u)
in a final volume of 28 /I and incubated at 37C for 45 min.
After that, 2 ¡j.1 of T4 DNA ligase (6 u) were added to the
reaction and incubated overnight at 16C or, alternatively,
incubated at room temperature for 2 h.
Bacterial transformation. After ligation, E. coli DH5a
competent cells were prepared and transformed using the
calcium chloride procedure (Sambrook et al., 1989).
Transformed bacteria were plated on 2 x YT agar medium
containing 100 /xg/ml of ampicillin and 40 /g/ml of 5-bromo-4-
chloro-3-idolyl-/3-D-galactoside (X-gal) and incubated
overnight at 37C. White bacterial colonies were transferred
to 2 x YT agar plates containing ampicillin for further
analysis.
Detection of recombinant plasmids. The rapid disruption
of bacterial colonies method was used to determine the
presence of recombinant plasmids containing the gene of
interest (Sambrook et al., 1989). Plasmid sizes were compared
in 0.8% agarose gel electrophoresis to a standard of pUC118
without insert; those with insert migrated slower in the gels.


61
Figure 14. SSCP analysis of the p27 gene from the
same strains used in sequencing. 1, B227; 2, T36;
3, T26; 4, B67; 5, T30; 6, B185; 7, B249; 8, B128
and 9, B7-1.


80
Results
Cloning of CTV Genes Into the Transformation Vectors
Several approaches were used to determine the presence
and copy number of the CTV genes in the pVF plasmids.
Restriction digestion of pVFl and pVF2 with EcoRI and Xbal
released inserts of the expected sizes indicating the presence
of the gene. Comparison of the plasmid sizes after
linearization with EcoRI indicated the presence of only one
copy in each plasmid. Finally, sequence analysis also
confirmed the presence and orientation of the CTV genes in the
pVF plasmids (data not shown).
PCR with p27, p2 0 (Fig. 19) or GUS specific primers
indicated the presence of the pVF plasmids in Agrobacterium.
This assay was employed routinely to determine the presence of
the insert of interest in the cultures to be used in
transformation experiments.
Transformation and Regeneration of Sweet Orange
Three different types of explants were used for
transformation. Nodes and internodes were used with both pVFl
and pVF2, and roots were only used with pVFl. This may be the
first report of the use of root segments for transformation
and regeneration of sweet orange. They were included for two
reasons, to increase the number of explants obtainable per
seedling and to determine their regenerative capacity. Figure
20 shows adventitious shoots on internodal segments and GUS+
shoots on soil for rooting.


17
1989) Colonies, containing the recombinant plasmid, were
replicated for later use.
Plasmid DNA was purified (Sambrook et al., 1989) and
transformed into E. coli BL21(DE3) (the expression host for
pETH plasmids) competent cells prepared by the calcium
chloride method (Sambrook et al., 1989). The bacteria were
plated on LB agar (1% bacto-tryptone, 0.5% bacto-yeast
extract, 1% NaCl, pH 7.0, 1.5% agar) medium supplemented with
100 /xg/ml of ampicillin and 25 tg/ml of chloramphenicol.
DNA Sequencing
The inserts in the recombinant plasmids were sequenced to
confirm the presence of the CTV genes and their correct
orientation. Plasmid DNA was purified as before from E. coli
DH5a recombinant colonies grown in 2 x YT liquid cultures with
antibiotics (Sambrook et al., 1989). Approximately 2 /xg of DNA
were used for each labeling reaction. The DNA was first
denatured in 20 /*1 of 1 N NaOH, 25 mM EDTA by incubation at
70C for 5 min and quickly chilled on ice, followed by ethanol
precipitation in 0.3 M sodium acetate (Sambrook et al., 1989) .
The samples were incubated at -80C for 30 min before
centrifugation for 15 min at 14,000 rpm. The DNA was
resuspended in 7 /xl of distilled water.
The labeling reaction was performed using the Sequenase
Version 2.0 sequencing kit (Amersham) according to the
manufacture's instructions. The technique is based on the
chain termination method (Sanger et al. 1977) For each


MOLECULAR CHARACTERIZATION OF CITRUS TRISTEZA VIRUS GENES
AND THEIR USE IN PLANT TRANSFORMATION
By
VICENTE JOSE FEBRES-RODRIGUEZ
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1995


7
amino acid sequence comparisons with protein databases do not
reveal any significant relationships (Pappu et al., 1994).
Only p23 seems to contain a motif similar to some RNA-binding
proteins (Dolja et al., 1994). Fig. 1 indicates the genome
organization of the 12 potential ORFs described above.
The identification of the functional ORFs of CTV will
facilitate understanding the mechanisms of gene expression,
replication and pathogenesis of this important virus, and
eventually enable the development of better disease control
strategies. The expression of viral genes in transgenic
plants, for example, has been a useful strategy to produce
plants resistant to several diverse viruses (Powell Abel et
al., 1986; Beachy et al., 1990; Pappu et al., 1995).
In this research the expression of the CTV genome was
studied to increase the information available on this virus.
The specific objectives of this research were as follows:
1) To determine if the p27, p20 and pl8 proteins were
expressed in CTV-infected citrus tissue.
2) To identify the sequence variability of the p27 ORF among
CTV strains with different biological properties and
determine any possible differences that could correlate
with virulence.
3) To transform citrus plants with the p27 and p20 genes in
an attempt to genetically engineer CTV resistance in
citrus.


CHAPTER 2
DETECTION OF THE IN VIVO EXPRESSION OF THE P27, P20 AND P18
PROTEINS
Introduction
There is significant resemblance between the genomic
organization of CTV and two other members of the closterovirus
group, beet yellows virus (BYV) and the bipartite lettuce
infectious yellows virus (LIYV). CTV ORFs 1 (helicase), 2
(polymerase), 4 (small hydrophobic protein), 5 (p65-hsp70), 6
(p61-hsp90), 7 (p27 CP homologue) and 8 (CP) have homologous
ORFs in BYV and LIYV in similar positions (Dolja et al., 1994;
Klaassen et al., 1995; Pappu et al., 1994). Interestingly for
LIYV, the CP homologue is located downstream of the CP (Dolja
et al., 1994; Klaassen et al., 1995). Another CTV ORF, p20,
shows similarity in the deduced amino acid sequence only to
p21, the 3' terminal ORF in BYV (Pappu et al., 1994). The CTV
ORFs 3 (p33), 9 (pl8) and 12 (p23) do not have homologues in
the two other closteroviruses.
The fact that some of the CTV ORFs have homologues in BYV
and LIYV is a strong indication, but does not demonstrate,
that those ORFs are functional in vivo. The detection of
subgenomic RNAs for 9 of the 12 ORFs, including p27, p20 and
pl8 (Hilf et al., 1995) also supports that they are expressed
8


71
transformed into E. coli DH5a competent cells and plated on 2
x YT agar plates containing 50/g/ml of spectinomycin.
Plasmid DNA increased and purified from the transformed
colonies was screened for presence of the insert by digestion
with EcoRI and electrophoresed in a 0.6% agarose gel with
lambda/Hindlll molecular weight markers. They were also
screened by digestion with EcoRI and Xbal and electrophoresed
in 0.8 agarose gels.
The gene for (3-glucuronidase (GUS) was cloned into
pMON10098/p27 from pMON755. Both plasmids (1 /ug of DNA each)
were separately digested with Notl (2.5 u) in 6 mM Tris-HCl pH
7.9 (at 37C) 6 mM MgCl2, 150 mM NaCl, 1 mM DTT and purified
in LMP agarose. Linearized pMON10098/p27 was treated with calf
intestinal alkaline phosphatase (1.46 u) in 50 mM Tris-HCl pH
8.5, 0.1 mM EDTA according to the manufacturer's instructions
and then extracted with phenol, phenol: chloroform, chloroform
and precipitated with ethanol (Sambrook et al., 1989).
Ligation of the GUS gene into pMON10098/p27 and transformation
into E. coli DH5a was as described above.
To confirm presence and orientation of the p27 gene, the
insert was seguenced following the protocol explained in
Materials and Methods, Chapter 2. This plasmid was designated
as pVFl (Fig. 16).
Cloning of p20 Into the Transformation Vector
The cloning procedure for p20 was similar to that described
above, but the GUS gene was cloned in pMON10098 first. The p20


42
1992) and Wisconsin Genetic Computer Group Sequencing Analysis
Software (GCG) version 7.0 (Devereux et al., 1984) provided by
the Interdisciplinary Center for Biotechnology Research at the
University of Florida (Gainesville, FL).
Virus Isolates
All CTV strains (except for T30 and T36) were obtained
from the Exotic Citrus Pathogens Collection, maintained by the
USDA and IFAS in Beltsville, MD. Strains T30 and T36 were
maintained in greenhouses at the University of Florida
(Gainesville, FL) Propagation hosts for the strains were
"Madam Vinous" sweet orange [C. sinensis (L.) Osbeck] or
"Mexican" lime [C. aurantifolia (Christm.) Swingle].
Cloning and Sequencing
Biological properties of the strains used in sequencing
are listed in Table 2. Cloning and sequencing procedures were
similar to those described in Materials and Methods, Chapter
2. In some cases (strains B7-1, B185, B227 and B249) PCR
products and plasmid DNA were purified using the Wizard DNA
Clean-Up System. For B50 and B67, both nucleic acid extracts
and dsRNA (Morris and Dodds, 1979) were used for PCR
amplification. When dsRNA was used, denaturation was performed
in a solution containing 10 mM dimethylmercury and
approximately 100 pM of each primer in a total volume of 10
Hi, incubated at room temperature for 8 min and then quick
frozen in liquid nitrogen. The mixture was then used in a
standard PRC reaction as described in Chapter 2.


68
The finding of new and varied sources of resistance in
food crops is important. Historically, pathogens frequently
overcome the resistance incorporated into those crops, usually
obtained by long selection processes. The incorporation of
viral genes can also offer a source of resistance when those
genes are not naturally present in the crop. In addition, the
expression of viral genes in plants, even if those genes are
not protective, is an important tool in determining the
function of those genes. For example, the 30 kDa protein of
TMV was shown to facilitate the cell-to-cell movement of a
mutant strain of TMV (capable of replication but unable to
cause systemic symptoms) when expressed in transgenic tobacco
plants (Deom et al., 1987).
Most plant transformation experiments, with viral genes
or others, have been accomplished in herbaceous, annual plants
and only a few woody species have been successfully
transformed. This is due to the technical difficulties in
regenerating those plants, and the length of screening and
testing for resistance. Plum (Prunus domestica L.) was
recently transformed with the CP gene of plum pox potyvirus
using Agrobacterium (Machado et al., 1994; Scorza et al.,
1994). Papaya (Carica papaya L.), a perennial plant, has also
been transformed with the CP gene of papaya ringspot potyvirus
(Tennant et al., 1994). Transgenic plants, tested in the field
for two years, showed no virus replication or movement when
challenged with the same strain of virus used for


26
1) Finally, no p27 band was detected when preimmune serum was
used (data not shown), indicating the specificity of the
antibody.
To preclude the possibility that the protein detected in
infected tissue was a stress-induced pathogenesis-related
protein, tissue extracts from citrus plants infected with
citrus ringspot virus, citrus variegation virus, citrus leaf
rugose virus, psorosis A and concave gum also were analyzed by
Western blot, using p27 antiserum. No p27 band was observed in
any of the samples (data not shown).
To further demonstrate the specificity of the antibody,
half of the same membrane in Fig. 2 (lanes 6 to 10) was probed
with MCA-13. This monoclonal antiserum, specific to the CP,
reacted strongly with the E. coli-expressed CP (Fig. 2, lane
8) and with the CP in the extract of CTV-infected citrus
tissue (Fig. 2, lane 6), including the lower molecular weight
proteolysis products of the CP (Sekiya et al., 1991). No
reactions were observed with proteins produced by bacteria
containing the pETH plasmid without an insert (Fig. 2, lane
10) or from extracts of uninfected citrus tissue (Fig. 2, lane
7) .
Interestingly, MCA-13 gave a weak but consistent reaction
with the purified p27 fusion protein, which could be seen only
on the membrane (not visible in Fig. 2, lane 9), but not with
p27 from infected tissue. However, to obtain this level of
reaction with MCA-13, it was necessary to increase the


David Benscher for all the help and good times spent together
in the lab.
Finally, thanks to the faculties, postdocs, technicians,
administrative personnel and students in the plant pathology
department that in one way or another collaborated to make
this work possible.
iii


16
Recombinant plasmids were then purified from liquid
cultures by the procedure of Sambrook et al. (1989). To
confirm the presence of an insert and determine its
orientation, approximately 0.5 xg of plasmid DNA were digested
with 5 u of Hindlll following the manufacturer's instructions.
Clones with sense orientation in pUC118 were used for
subcloning into the expression vector.
Subcloning of CTV ORFs in the Expression Vector
The plasmid pETH-3b (McCarty et al., 1991) was used for
expression of the CTV genes p27, p20 and pl8. The inserts were
recovered from pUC118 by digestion with Hindlll and EcoRI.
Approximately 5 /xg of plasmid DNA were digested with Hindlll
(10 u) and EcoRI (20 u) in a final volume of 30 ¡xl in 6 mM
Tris-HCl pH 7.5, 6 mM MgCl2, 50 mM NaCl, 1 mM DTT for 3 to 4
h at 37C. The digestion products were electrophoresed in a
0.8% LMP agarose gel and the inserts purified as described
above.
DNA from pETH-3b (0.5 fxq per reaction) was also digested
with Hindlll (2.5 u) and EcoRI (5 u) and the linearized
plasmid similarly purified in LMP agarose. Ligation and
bacterial transformation into E. coli DH5a were as described
above, omitting only the addition of DNA polymerase I (Klenow
fragment) and T4 polynucleotide kinase. Transformed bacteria
were grown in 2 x YT agar media containing 100 /xg/ml of
ampicillin. Bacterial colonies were screened using the rapid
disruption of bacterial colonies method (Sambrook et al.,


82
Figure 20. Adventitious shoot formation on
internodal segments (A) and GUS+ shoots in soil for
rooting (B).


28
123 456 789
Figure 3. Western blot analysis of cell fractions
prepared from CTV-infected tissue and probed with
p27 antiserum. Lane 1, uninfected citrus tissue
extract; Lane 2, unfractionated infected citrus
extract; Lanes 3 to 8 contained fractions PI, P30,
S30, Rl, R2 and R3, respectively, of infected
citrus tissue as explained in Materials and
Methods. Lane 9 is E. coli-expressed p27.


85
Figure 21. GUS assay on adventitious shoots of
"Pineapple" sweet orange regenerated from
Agrobacterium-inoculated internodal segments. A),
GUS- segment. B), GUS+ segment.


21
using a blade and combined with 0.5 ml of cracking buffer,
boiled for 3 min and stored at -20C. Protein extracts (10 fxl)
were separated by 15% (p27) or 12% (p20 and pl8) SDS-PAGE
along with the appropriate molecular weight markers. The
proteins were transferred to a nitrocellulose membrane using
electrophoretic transfer in 25 mM Tris, 192 mM glycine, 20%
v/v methanol at 4C for 1 h at 100 V, 350 mA. Membranes were
probed with the different polyclonal antisera using the
colorimetric procedure. For pl8 a chemiluminescent procedure
was also tested using the Renaissance kit. A monoclonal
antibody (MCA-13) raised to the T36 CP (Permar et al., 1990)
also was used.
Tissue blot analyses also were tested as a rapid method
for the detection of p27 and p20 using the procedure of
Garnsey et al. (1993). Two incubation conditions (room
temperature and 37 C for 1 h) were tested, as well as
dilutions of antiserum from 1:500 to 1:20,000.
Cell fractionation. Cell fractions from T36 CTV-infected
and uninfected citrus tissue were prepared by a modification
of the procedures of Godefroy-Colburn et al. (1986) and
Albretch et al. (1988). Approximately 1 g of tissue was ground
in liguid nitrogen and thawed in 2 ml of grinding buffer (100
mM Tris-HCl pH 8.1, 10 mM KC1, 0.4 M sucrose, 10% glycerol, 10
mM 2-mercaptoethanol). The mixture was incubated with
agitation for 15 min at room temperature and then filtered by
centrifugation at 3,000 g for 10 min through a 64-mesh nylon


98
Godefroy-Colburn, T., Gagey, M.T., Berna, A., Stussi-Garaud,
C. 1986. A non-structural protein of alfalfa mosaic virus
in the walls of infected tobacco cells. J.Gen. Virol.
67:2233-2239.
Golemboski, D.B., Lomonossoff, G.P. and Zaitlin, M. 1990.
Plants transformed with a tobacco mosaic virus non-
structural gene sequence are resistant to the virus.
Proc. Natl. Acad. Sci. U.S.A. 87:6311-6315.
Gutirrez-E, A., Moore, G.A., Jacono, C., McCaffery, M. and
Cline, K. 1992. Production of transgenic citrus plants
expressing the citrus tristeza virus coat protein gene.
Phytopathology 82: 1148.
Hager, D.A. and Burgess, R.R. 1980. Elution of proteins from
sodium dodecyl sulfate-polyacrylamide gels, removal of
sodium dodecyl sulfate, and renaturation of enzymatic
activity: results with sigma subunit of Escherichia coli
RNA polymerase, and other enzymes. Anal. Biochem. 109:76.
Hidaka, T. Omura, M. Ugaki, M. Tomiyawa, M. Kato, A.,
Oshima, M. and Motoyoshi, F. 1990. Agrobacterium-mediated
transformation and regeneration of Citrus spp. from
suspension cells. Japan J. Breed. 40:199-207.
Higgins, D.G., Bleasby, A.G. and Fuchs, R. 1992. Clustal V:
Improved software for multiple sequence alignment. Comp.
Appl. Biosci. 8:189-191.
Hilf, M.E., Karasev, A.V., Pappu, H.R., Gumpf, D.J., Niblett,
C.L. and Garnsey, S.M. 1995. Characterization of citrus
tristeza virus subgenomic RNAs in infected tissue.
Virology, 208:576- 582.
Hull, R. and Davies, J.W. 1992. Approaches to nonconventional
control of plant virus diseases. Critical Reviews in
Plant Sciences 11:17-33.
Iwahana, H., Yoshimoto, K. and Itakura, M. 1992. Detection of
point mutations by SSCP of PCR-amplif ied DNA after
endonuclease digestion. Biotechniques 12:64-66.
Jorda, M.C. and McHughen, A. 1988. Transformed callus does not
necessarily regenerate transformed shoots. Plant Cell
Rep. 7: 285-287.
Kaneyoshi H.J., Kobayashi, S., Nakamura, Y., Shigemoto, N. and
Doi, Y. 1994. A simple and efficient gene transfer system
of trifoliate orange (Poncirus trifoliata Raf.) Plant
Cell Reports 13:541-545.


30
Figure 5. Tissue blots probed with the p27
polyclonal antiserum. A, CTV-infected citrus
tissue; B, uninfected citrus tissue.


39
protein expression is regulated post-transcriptionally and
that its RNA plays some role in the virus life cycle.
Regarding the function of p20, it is still difficult to
speculate since the protein does not show any similarities to
previously described proteins. However, this research
demonstrates that the ORF is expressed during CTV infection.
The pl8 protein was not detected in CTV-infected tissue
samples using Western blot analysis, even when the more
sensitive chemiluminescent method was used. The antiserum,
however, reacted with E. coli-expressed pl8. Subgenomic RNA
analysis indicates that pl8 is expressed at a very low level
compared to p27, CP or p20 (Hilf et al., 1995). Low protein
expression and/or expression at a specific time during the CTV
life cycle may explain the inability to detect pl8.


63
could be separated into nine different groups: 1) mild strains
T30, T26, B67, B5, B32, B192 and B274, 2) mild strain B272, 3)
severe, stem pitting B7 and B128, 4) severe, stem pitting
strains B249 and B185, 5) severe, stem pitting strain B227, 6)
severe, quick decline strain T36, 7) severe, quick decline
strain Bll, 8) severe, quick decline strain B37, and 9) severe
strains B28 (stem pitting) and B148 (quick decline) .
Discussion
The p27 genes of the CTV strains sequenced showed 86% or
greater homology at the RNA level and 93% or greater at the
amino acid level (deduced sequence). These values are similar
to those found for the CP (Pappu et al., 1993b). Several of
the nucleotide substitutions are consistently conserved only
within a particular biological group (mild, severe or severe
causing stem pitting). These distinct sequences may have
potential for the development of group-specific molecular
probes. Some of the nucleotide substitutions translated as
nonsynonymous, although most of them were with functionally
conserved amino acids.
The conservation of four amino acid substitutions between
the severe strains compared to the mild suggests a possible
relationship of p27 with symptom development. However, it is
necessary to note that a limited number of strains were
sequenced, and this in no way demonstrates the role of p27, if
any, in symptomatology. Further experiments are necessary to


22
cloth fused to a 3 ml syringe. Both filtrate and solid residue
were collected. The liquid filtrate was centrifuged at 1000 g
for 10 min; both pellet (fraction PI) and supernatant were
collected. The supernatant was further centrifuged at 30,000
g for 20 min, and the pellet (fraction P30) and supernatant
(fraction S30) were collected. The solid residue from the
first step was resuspended in 1 ml of grinding buffer and
incubated with agitation for 15 min at room temperature. The
extract was filtered again by centrifugation. The filtrate
(fraction Rl) was collected and the solid residue resuspended
in 1 ml of grinding buffer containing 2% Triton X-100 for 15
min with agitation and subsequently filtered again by
centrifugation. The filtrate (fraction R2) was collected and
the solid residue was resuspended in 1 ml of 75 mM Tris-HCl pH
6.8, 4.5% SDS, 9 M Urea, 7.2% 2-mercaptoethanol at room
temperature for a final 15 min extraction with agitation,
followed by filtration. The filtrate was collected (fraction
R3) and the solid residue discarded. All fractions were
combined with equal volumes of cracking buffer and boiled for
3 min for analysis by Western blots.
Results
Cloning of CTV Genes
CTV genes for p27, p20 and pl8 were amplified by RT-PCR
and first cloned into the Smal restriction site of pUC118
using blunt-end ligation. A Hindlll restriction site was


BIOGRAPHICAL SKETCH
Vicente Jos Febres-Rodriguez was born in Caracas,
Venezuela, in 1964. He obtained a bachelor's degree in biology
in 1981 from the Simn Bolivar University in Caracas. After
his graduation he worked at the Venezuelan Institute for
Scientific Research (IVIC) until he moved to Costa Rica in
1988 to continue studies at the Tropical Agronomic Center for
Research and Teaching (CATIE) in Turrialba. He obtained his
master's in plant breeding in 1990 and continued working at
CATIE as a consultant. In 1991 he started his studies for a
Ph.D. in plant pathology at the University of Florida, from
which he graduated in 1995.
105


4
budding and grafting (Bar-Joseph and Lee, 1990). Mechanical
transmission is difficult and only possible when concentrated
preparations are used and slash inoculated into the stems of
young citrus plants (Garnsey et al., 1977).
CTV is a phloem-limited virus. Inclusion bodies are
observed inside the phloem, phloem fiber and parenchyma cells
adjacent to sieve tubes (Schneider, 1959; Brlansky et al.,
1988) They also are detected by immunofluorescence using
antibodies to CTV coat protein (CP) (Brlansky et al., 1988),
indicating that the inclusion bodies contain virus particles.
These virus particles are flexuous, 1900-2000 nm long and 10-
11 nm in diameter (Bar-Joseph and Lee, 1990).
The genome of CTV is a single-stranded, positive sense
RNA 19,296 nucleotides in length (Bar-Joseph and Lee, 1990;
Karasev et al., 1995). Seguencing of the entire genome of the
Florida, severe, guick decline strain T36 indicates the
presence of 12 possible open reading frames (ORFs) (Fig. 1)
coding for 17 proteins (Pappu et al., 1994; Karasev et al.,
1995). In the 5' to 3' direction the first ORF potentially
codes for a polyprotein with an estimated MW of 349 kDa that
encodes the putative domains for two papain-like proteases, a
methyltransferase and a helicase (Karasev et al., 1995). This
polyprotein is speculated to undergo autoproteolytic cleavage
to produce three fragments of 54, 55 and 24 0 kDa, respectively
(Karasev et al., 1995). The second ORF encodes a putative RNA-
dependent RNA polymerase of a calculated MW of 57 kDa that is


65
the most conserved. Located also in this region are the two
consensus amino acids which are conserved in the CPs of
filamentous viruses (Pappu et al., 1994). This portion must
then contain important active domains for both proteins.
SSCP analysis of the p27 gene of strains T26, T30, T36,
B67, B7-1, B128, B185, B227 and B249 produced groupings
similar to those obtained in phylogenetic analysis of the RNA
sequences. The only exception is the separation of the stem
pitting strains into two groups with different patterns: B7-1
and B128; B249 and B185. Interestingly, B249 is more similar
in RNA sequence to B7-1 (99.3%) or B128 (99.6%) than it is to
B185 (98.8%). This indicates that certain nucleotide changes
have more influence in conformation than others. Sheffield et
al. (1993) found that the position of the base change was more
important than the type of nucleotide substitution for the
detection of such a change. Strains B227 and T36, the type
strain for CTV, showed unique SSCP patterns.
The results for all the strains examined confirmed the
sequence analyses indicating that mild strains were very
homogeneous in sequence, regardless of the geographical
origin. Only one strain, B272, a very mild strain from
Colombia, showed a different SSCP pattern from all other mild
strains. Severe, quick decline strains, on the other hand,
were quite variable and separated into four different groups.
Strains T36, Bll and B37 formed distinct SSCP patterns and
B148 grouped with the stem pitting strain B28.


44
In addition to the p27 genes, the CP from strains B249
was also cloned and sequenced. Sequences for all the other CP
genes were kindly provided by H.R. Pappu. Clones for the p27
gene of isolate B128 were kindly provided by S.S. Pappu.
At least two clones of each strain were sequenced in both
the sense and antisense directions. Sequences were compared
using the programs CLUSTALV and GCG. Deduced amino acid
sequences were obtained using Seqaid.
Single Stranded Conformation Polymorphism (SSCP)
The same strains employed for sequencing were also used
for SSCP analysis. Other strains are described in Table 3. The
p27 genes were amplified from tissue extracts or from plasmid
DNA and analyzed in non-denaturing polyacrylamide gels. PCR
products (2 ¡J. 1) were denatured in 95% formamide, 20 mM EDTA,
0.05% bromophenol blue by incubation at 100C for 10 min and
electrophoresed in 7 x 8 cm, 8% polyacrylamide gels
(acrylamide: bisacrylamide 30:1, lx TBE) for 3 hrs 45 min at
200 V in lx TBE buffer at room temperature. The buffer and
gels were pre-chilled at 4C overnight before electrophoresis.
DNA was detected using silver staining.
Results
Sequencing
The CTV strains sequenced were selected based on
diversity of biological properties and geographical origins.
Table 4 summarizes the overall similarities of nucleotide and


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
(3 ^
Charles A. Powell
Associate Professor of
Plant Pathology
This dissertation was submitted to the Graduate Faculty
of the College of Agriculture and to the Graduate School and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.
August, 1995
Dean, College of
Agriculture
Dean, Graduate School


88
1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
M
y
Figure 22. PCR amplification of DNA extracts from
"Pineapple" sweet orange GUS+ segments transformed
with p27 (A) or p20 (B) A) Lane 1, Lambda DNA
digested with Hindlll used as a MW marker; Lanes 2
and 7, GUS- segments; Lanes 3 to 6 and 8 to 15,
GUS+ segments; Lane 16, pVFl. B) Lane 1, Lambda
DNA digested with Hindlll used as a MW marker; Lane
2, GUS- segment; Lanes 3 to 10, GUS+ segments; Lane
11, pVF2.


11
The template was further purified using Sephadex G-50
spin-column chromatography, according to the procedure
described in Sambrook et al. (1983). The column (a 1 ml
syringe barrel), containing hydrated, sterile Sephadex, was
equilibrated by washing three times with 100 fil of sterile TE
buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) and 100 /I of
extract applied to it. The column was centrifuged at l,600g
for 4 min at room temperature, and the effluent collected in
a microcentrifuge tube. The effluent was used immediately for
reverse transcription coupled with polymerase chain reaction
(RT-PCR) amplification or quickly frozen in liquid nitrogen
and stored at -80C for later use.
PCR amplification. The CTV genes were amplified using RT-
PCR (Saiki et al., 1988) and specific primers for each gene.
Table 1 shows the primers, derived from the T3 6 sequence
(Pappu et al., 1994), used to amplify each of the genes. The
RT-PCR reactions were performed successively in a single
microcentrifuge tube in a total volume of 100 n1 with the
following components: 10 mM Tris-HCl pH 9.0 (at 25C), 50 mM
KC1, 0.1% Triton X-100, 10 mM dithiothreitol (DTT) 2.5 mM
MgCl2, 0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, 0.4 mM dTTP,
RNasin (40 u), AMV reverse transcriptase (19 u), Taq
polymerase (2.5 u) and primers at a final concentration each
of 100 pM. Normally, 30 to 50 /x 1 of the purified template were
used in each reaction. It was previously denaturated at 70C
for 3 min and guick chilled on ice for a few min. The RT-PCR


Figure 18. Schematic representation of the
transformation protocol for sweet orange plants.
A) seedlings (3 to 6 months old), germinated in
culture tubes, were pruned to eliminate leaves.
Segments of approximately 1 cm were cut out from
roots and stems (separating nodal and internodal
portions). B) explants were placed in culture
plates containing MS medium with the apical end
protruding (internodes and roots) or length-ways on
the medium (nodes). Explants were then inoculated
with one drop of Agrobacterium. After three days of
incubation the explants were transferred to
selection medium (MS with antibiotics). C) ,
approximately four to six weeks after the
transformation shoots started to regenerate. The
shoots were excised from the explants and assayed
for GUS activity.


29
1 2 3 4 5 6 7 8
Figure 4. Western blot analysis of cell fractions
prepared from CTV-infected tissue and probed with
MCA-13 monoclonal antibody. Lane 1, uninfected
citrus tissue extract; Lane 2, unfractionated
infected citrus extract; Lanes 3 to 8 contained
fractions PI, P30, S30, Rl, R2 and R3,
respectively, of infected citrus tissue as
explained in Materials and Methods.


14
the pVF transformation vector once clones with the correct
orientation were identified.
Restriction digestion of plasmid DNA. Purification of
pUCl 18 from E. coli DH5a was done from 3 ml overnight cultures
grown at 37C and shaking at 180 rpm in 2 x YT medium (1.6%
bacto-tryptone, 1% bacto-yeast extract, 0.5% NaCl, pH 7.0)
using a modification of the procedure of Sambrook et al.
(1989) The only difference was the use of a half volume of
7.5 M ammonium acetate for the precipitation of bacterial
proteins prior to the DNA precipitation. The plasmid DNA was
resuspended in 30 /xl of sterile distilled water.
For each ligation reaction approximately 0.5 ig of pUC118
were digested with Smal according to the manufacturer's
recommendations in a final volume of 30 /l. Digestion was
performed for 3 to 4 h, and the DNA was subsequently purified
using phenol, phenol: chloroform and chloroform extractions
and precipitated with ethanol (Sambrook et al., 1989).
Ligation reaction. For ligation of the PCR products into
the Smal digested pUC118, the purified PCR products (10 /x 1)
were first treated with 5 units of DNA polymerase I (Klenow
fragment) in a final volume of 15 il containing 90 mM Tris-HCl
pH 7.5 (at 37 C) 10 mM MgCl2, 50 mM NaCl, 1 mM dNTPs.
Incubation was for 10 min at room temperature, 20 min at 37C
and 10 min at 70C. The PCR products were then treated with
phenol, phenol: chloroform, chloroform and precipitated with


100
Mendt, R. 1992. History of CTV in Venezuela. Pages 137-141,
in: Lastra, R., Lee, R., Rocha-Pea, M., Niblett, C.L.,
Ochoa, F., Garnsey, S.M. and Yokomi, R.K. (eds.).
Workshop on Citrus Tristeza Virus and Toxoptera citricida
in Central America: Development of Management Strategies
and Use of Biotechnology for Control. Maracay, Venezuela.
Mertz, L.M., Westfall, B. and Rashtchian, A. 1994. PCR
nonradioactive labeling system for synthesis of
biotinylated DNA probes. Focus 16: 49-51.
Moore, G.A., DeWald, M.G. and Cline, K. 1989. Agrobacterium-
mediated transformation of Citrus. Journal of Cellular
Biochemistry, Supplement 3D, p255.
Moore, G.A., Jacomo, C.C., Neidigh, S.D., Lawrence, S.D. and
Kline, K. 1992. Agrobacterium-mediated transformation of
Citrus stems segments and regeneration of transformed
plants. Plant Cell Reports 11:238-242.
Moore, G.A., Jacomo, C.C., Neidigh, S.D., Lawrence, S.D. and
Kline, K. 1993. Transformation in Citrus. Pages 194-208,
in: Bajaj, Y.P.S. (ed.). Biotechnology in Agriculture and
Forestry, Vol. 23. Plant Protoplasts and Genetic
Engineering IV. Springen-Verlag, Berlin, Germany.
Moreno, P., Guerri, J., Ballester-Olmos, J.F. and Martinez,
M.E. 1991. Segregation of citrus tristeza virus strains
evidenced by double stranded RNA (dsRNA) analysis. Pages
20-24, in: Brlansky, R.H. Lee, R.F. and Timmer, L.W.
(eds) Proc. 11th Conf. Int. Organ.Citrus Virol. IOCV.
Riverside, CA.
Morris, T.J. and Dodds, J.A. 1979. Isolation and analysis of
double stranded RNA from virus infected plant and fungal
tissue. Phytopathology 69:854-858.
Muller, G.W. and Costa, A.S. 1992. History of citrus tristeza
virus (CTV) in Brazil. Pages 126-131, in: Lastra, R. ,
Lee, R. Rocha-Pea, M. Niblett, C.L., Ochoa, F. ,
Garnsey, S.M. and Yokomi, R.K. (eds.). Workshop on Citrus
Tristeza Virus and Toxoptera citricida in Central
America: Development of Management Strategies and Use of
Biotechnology for Control. Maracay, Venezuela.
Orita, M., Suzuki, Y., Sekiya, T. and Hayashi, K. 1989. Rapid
and sensitive detection of point mutations and DNA
polymorphism using the polymerase chain reaction.
Genomics 5: 874-879.


55
Figure 9. Phylogenetic tree for p27 based on RNA
sequences. Graphic generated using Pileup program
in GCG


19
7 x 8 cm gel at room temperature. Protein standards SDS-6 or
SDS-7 were included. Proteins were stained using Coomassie
brilliant blue G-250 for 30 min (Sambrook et al., 1989).
Production of Polyclonal Antisera
Protein expression. Overnight 3 ml cultures of each
recombinant pETH-3b plasmid containing the CTV p27, p20 or pl8
ORFs were used for production of recombinant protein. The
medium used was LB with antibiotics as described above and
incubation was at 37C with shaking (180 rpm) In the morning,
25 ml of fresh medium were inoculated with 0.5 ml of the
overnight cultures and incubated for 2 h. The cultures were
then induced with IPTG and incubated for 3 more h. Cells were
transferred to a 15 ml tube and centrifuged at 4C for 10 min
and 15,000 g. The bacteria were resuspended in half their
original volume in TE buffer and frozen overnight at -20C.
Cells were thawed at 37C, vortexed for a few minutes and
centrifuged as before. Both the supernatant and pellet, were
collected the first time, and samples were treated with
cracking buffer for analysis in SDS-PAGE to determine the
fraction that contains the protein of interest. In those cases
in which the protein was insoluble (p27 and pl8) the pellet
was resuspended in one half volume (6 ml) of TE buffer and
pelleted by centrifugation. This was repeated 2 more times,
each time reducing the volume of TE buffer in half. The final
volume was 1.5 ml.


Results 44
Sequencing 44
Single Stranded Conformation
Polymorphism 54
Discussion 63
4. TRANSFORMATION OF SWEET ORANGE WITH P27
AND P20 67
Introduction 67
Materials and Methods 70
Materials 70
Cloning of p27 Into the Transformation
Vector 70
Cloning of p20 Into the Transformation
Vector 71
Transformation of pVF plasmids
into Agrobacterium 73
Screening of Transformed Bacteria .... 73
Transformation, Selection and
Regeneration of Citrus Plants 75
Results 80
Cloning of CTV Genes Into the
Transformation Vectors 80
Transformation and Regeneration of
Sweet Orange 8 0
Discussion 87
5. SUMMARY AND CONCLUSIONS 93
LITERATURE CITED 95
BIOGRAPHICAL SKETCH 105
V


33
which contained the same 20 N-terminal amino acids. Detection
of cell fractions with the antiserum did not reveal any bands
of the expected MW. A more sensitive, chemiluminescent
detection system was also used without positive results (data
not shown).
Discussion
The levels of bacterial expression obtained with the
fusion proteins were similar to those previously reported
(Marston, 1986; Studier et al., 1990). With this expression
system, it was possible to successfully produce the three CTV
proteins and purify them by a relatively simple method. The
antisera produced reacted with the antigens and gave low non
specific reactions when used to probe tissue samples. There
was variation in the solubility of the fusion proteins, with
p27 and pl8 accumulating as insoluble products, and p20 being
soluble. This did not affect the levels of expression or the
isolation of the proteins.
The presence of the fusion portion seemed to stabilize
some of the proteins in E. coli. Attempts to express p27
without this portion rendered very low levels of the protein
(data not shown). However, nonfusion CP was expressed in the
same bacterial host (K.L. Manjunath, personal communication).
The only inconvenience with fusion proteins is that antisera
produced to different fusion proteins cross react with all the
antigens, since the fusion part is identical. This might limit
the use of the recombinant proteins as positive controls,


TABLE OF CONTENTS
ACKNOWLEDGMENTS
ABSTRACT vi
CHAPTER
1. INTRODUCTION 1
2. DETECTION OF THE IN VIVO EXPRESSION OF
THE P27, P20 AND P18 PROTEINS 8
Introduction 8
Materials and Methods 9
Materials 9
Plant Material 10
Cloning of CTV ORFs into pUC118 10
Subcloning of CTV ORFs in the
Expression Vector 16
DNA Sequencing 17
Bacterial Expression of CTV Proteins ... 18
Production of Polyclonal Antisera .... 19
Detection of CTV Proteins in
Infected Citrus Tissue 20
Results 22
Cloning of CTV Genes 22
Expression of Proteins 23
Detection of p27 24
Detection of p2 0 31
Detection of pl8 31
Discussion 33
3. SEQUENCE ANALYSIS OF THE P27 ORF 40
Introduction 40
Materials and Methods 41
Materials 41
Virus Isolates 42
Cloning and Sequencing 42
Single Stranded Conformation
Polymorphism (SSCP) 44
iv


75
of 20 mM Tris-HCl pH 8.5, 2 mM EDTA, 1% Triton X-100, boiled
75for 15 min and centrifuged for a few seconds. PCR reactions
contained the normal constituents plus 1 /I of the bacterial
extracts and the appropriate primers for p27, p20 or the GUS
gene.
Transformation. Selection and Regeneration of Citrus Plants
Seed germination. The procedure for seed germination was
modified from Moore et al. (1992, 1993). Intact seeds were
surface sterilized (10 min in 70% ethanol, 20 min in 20%
Clorox and 2 drops of Tween-20, rinsed 3 times with sterile
distilled water) and germinated individually in 150 x 25 mm
capped tubes containing 10 ml of half-strength MS basal salt
medium, 2.5% sucrose, 0.005% myo-inositol, 0.8% agar pH 5.7
and maintained at 27C with 16 h fluorescent light. Seedlings
were used 3 to 6 months after sowing.
Bacterial culture. Individual colonies of Agrobacterium
with either pVFl or pVF2 were increased in 50 ml flasks
containing 5 ml YEP medium with the three antibiotics and
incubated overnight at 28C and 200 rpm to post log phase. The
bacteria were collected by centrifugation at 3,500 g for 15
min at 4C and resuspended in 1.5 ml of YEP without
antibiotics. The solution was kept on ice until its use for
transformation.
Plant transformation. Nodal, internodal and root segments
from "Pineapple" sweet orange seedlings were used as explants
for transformation (Moore et al., 1992, 1993). The internodal


64
establish the relationship between these amino acids and
biological activity. For example, transformed plants
expressing p27 from severe CTV strains could be used to
determine symptom expression when those plants are infected
with mild strains.
Phylogenetic analysis of the amino acid sequences of p27
also indicated a strong relationship between sequence and
biological activity, with mild and severe strains forming two
distinct groups. Stem pitting strains B7-1, B128 and B249 also
formed a distinct domain within the severe strains.
Interestingly, RNA analysis showed a different relationship,
with B227 forming a separate group from all other strains. The
decline strain T36 was more similar to the mild strains T26,
T30, and B67 than to strains causing stem pitting. This is an
indication that the severe strains constitute a polyphyletic
group, and that the similarities in protein sequence (and
possible symptom development) represent an example of
evolutionary convergence.
Comparison of the phylogenetic trees obtained with CP
sequences showed different relationships, indicating distinct
evolutionary rates for the two genes. From these results, the
amino acid sequence of the p27 gene is more useful in
predicting the biological reaction of an strain than CP
sequence (RNA or amino acid).
Alignment of p27 and CP deduced amino acid sequences
revealed that the C-terminal portion of the two proteins is


53
T30
T26
B67
T36
B7-1
B128
B185
B227
B249
151 lili 200
LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP
T
T30
T26
B67
T36
B7-1
B12 8
B185
B227
B249
201 I I I 240
GYSEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL
Fig. 8continued


23
included in the sequence of the sense primer (upstream) to
facilitate subcloning in pETH-3b. Identification of the clonal
orientation was necessary for subcloning into the pETH vector.
Clones with the EcoRI restriction site of the plasmid
polylinker located downstream of the gene were used for
subcloning. These clones were identified by restriction
digestion of the plasmid DNA with Hindlll. Clones in the sense
orientation were only linearized upon digestion without
releasing the inserts, whereas clones in the antisense
orientation released the inserts (data not shown).
For subcloning into pETH-3b and to guarantee proper
orientation and reading frame of the gene, two non
complementary restriction enzymes that generated cohesive
termini were used: Hindlll, located 5' to the start codon and
EcoRI, located 3' to the stop codon of each CTV gene. This
strategy also avoided self-ligation of the plasmid. Sequencing
of the clones (data not shown) confirmed orientation and
reading frame for each construct.
Expression of Proteins
CTV genes were expressed as fusion proteins, made up of
20 amino acids of the T7 coat protein (Studier et al., 1990;
McCarty et al., 1991) and the amino acids of each CTV gene.
The fusion part of the proteins increased the MW by 1.9 kDa.
The resulting predicted MW of each protein was 29.3 kDa (p27) ,
22.4 kDa (p20) and 20.2 kDa (pl8).


101
Pappu, H.R., Karasev, A.V., Anderson, E.J., Pappu, S.S., Hilf,
M.E. Febres, V.J., Eckloff, R.M.G., McCaffery, M., Boyko,
V. Gowda, S., Dolja, V.V., Koonin, E.V., Gumpf, D.J.,
Cline, K.C., Garnsey, S.M. Dawson, W.O., Lee, R.F. and
Niblett, C.L. 1994. Nucleotide sequence and organization
of eight 3' open reading frames of the citrus tristeza
closterovirus genome. Virology 199:35-46.
Pappu, H.R., Niblett, C.L., Lee, R.F. 1995. Application of
recombinant DNA technology to plant protection: molecular
approaches to engineering virus resistance in crop
plants. World Journal of Microbiology and Biotechnology.
In press.
Pappu, H.R., Pappu, S.S., Manjunath, K.L., Lee, R.F., and
Niblett, C.L. 1993a. Molecular characterization of a
structural epitope that is largely conserved among severe
isolates of a plant virus. Proc. Natl. Acad. Sci. USA
90:3641-3644.
Pappu, H.R., Pappu, S.S., Niblett, C.L., Lee, R.F. and
Civerolo, E. 1993b. Comparative sequence analysis of the
coat proteins of biologically distinct citrus tristeza
virus isolates. Virus Genes 7:255-264.
Pappu, S.S., Brand, R., Pappu, H.R., Rybicki, E., Gough, M.J.,
Frenkel, M.J. and Niblett, C.L. 1993c. A polymerase chain
reaction method adapted for selective cloning of 3' non-
translated regions of potyviruses: Application to dasheen
mosaic virus. J. Virological Methods 41:9-20.
Pascal, E., Goodlove, P.E., Wu, C. and Lazarowitz, S.G. 1993.
Transgenic tobacco plants expressing the geminivirus BL1
protein exhibits symptoms of viral disease. Plant Cell
5:795-807.
Pea, L. Cervera, M. Jurez, J. Ortega, C. Pina, J.A. ,
Durn-Vila, N. and Navarro, L. 1995. High efficiency
Agrobacterium-mediated transformation and regeneration
of citrus. Plant Science 104:183-191.
Permar, T., Garnsey, S.M., Gumpf, D.J. and Lee, R.F. 1990. A
monoclonal antibody that discriminates strains of citrus
tristeza virus. Phytopathology 80:224-228.
Powell, C.A., Pelosi, R.R. and Cohen, M. 1992. Superinfection
of orange trees containing mild isolates of citrus
tristeza virus with severe isolates of citrus tristeza
virus. Plant Disease 76:141-144.


35
The tissue fractionation experiments showed that p27 and
the CP are mostly associated with the cell wall and soluble
protein fractions. Conversely, in BYV, p24 (the CP homologue)
and the CP were shown to accumulate mostly in the cytoplasm or
soluble protein fraction (Agranovsky et al., 1994) Comparison
of the sequences upstream of the start codons and surrounding
the initiation sites for the subgenomic RNAs of BYV p24 and CP
revealed a consensus sequence, (Agranovsky et al., 1994),
suggesting concerted expression of both genes. This sequence
was similar to those found in the CPs of tobamoviruses, brome
mosaic virus, cucumber mosaic virus and alfalfa mosaic virus
(A1MV; Agranovsky et al., 1994). Comparison of the sequences
upstream to the start codons of CTV p27 and CP did not
revealed any consensus sequences similar to those of BYV or
LIYV (data not shown). This indicates that the expression of
these two set of genes is different in CTV and BYV.
Recently, p24 was found to be part of the BYV virions,
forming a terminal "tail" at one end of the particles
(Agranovsky, 1995). Due to the similarities between the two
viruses and the apparent structural conservation between the
CP homologues (Pappu et al., 1994), it is possible that p27
may also form part of the CTV virions. This is currently under
investigation using the p27 specific antiserum.
The function of p27 in CTV is still not known. Besides
its possible structural role, if p27 forms part of the
virions, it might also be involved in particle assembly


54
A phylogenetic tree was constructed using the RNA
sequences (Fig. 9) Four major groups could be distinguished:
l)severe; stem pitting strains B7-1, B128, B249 and B185, 2)
mild strains T30, B67 and T26, 3) severe, quick decline strain
T36 and 4) severe, stem pitting strain B227. Mild strains
showed some relationship to severe, quick decline and stem
pitting strains. Strain B227 (severe from India) was the most
distinct and formed a completely separate group. The
phylogenetic tree constructed using deduced amino acid
sequences (Fig. 10) separated mild from severe strains, each
one forming a distinct group.
Comparison of the CP sequences in phylogenetic trees (RNA
and amino acid, Figs. 11 and 12, respectively) showed a
different grouping of the strains from that of p27.
Sequence alignment of p27 and CP from the different
strains is showed in Fig. 13. Approximately 20% of the amino
acids are identical between the two proteins, depending on the
strain. Again, the C-terminal regions of the proteins are also
the most conserved (Fig. 13) .
Single Stranded Conformation Polymorphism
SSCP analysis of the strains used for sequencing (Fig.
14) revealed five groups with similar patterns: 1) severe,
stem pitting strains B7-1 and B128, 2) severe, stem pitting
strains B249 and B185, 3) mild strains T30, T26 and B67, 4)
quick decline strain T36 and 5) severe, stem pitting strain
B227. When all strains were considered (Figs. 14 and 15) they


84
Table 5.
Effects of explant type and plasmid
on adventitious shoot formation in
'Pineapple" sweet orange.
EXPLANT
PLASMID
No. OF
SEGMENTS
SHOOTS
PER SEGMENT
Nodes
pVFl
350
0.30 0.11
pVF2
378
0.34 0.15
Total
728
0.32 0.14
Internodes
pVFl
910
1.14 0.50
pVF2
434
1.00 0.38
Total
1344
1.10 0.47
Roots
pVFl
42
1.64 0.45


Pol p6 p61 pl8 p20
HEL P3 3 p6 5
p2 7 CP pi 3 p2 3
Figure 1. Genomic organization of the citrus tristeza virus genome according to
Pappu et al. (1994) and Karasev et al. (1995). The ORFs are represented as
rectangles and the putative gene products are indicated. (HEL, helicase; Pol,
polymerase; CP, coat protein).
m


73
gene was cloned subsequently from pUC118/p20 using Xbal and
SacI restriction sites. The new plasmid was designated as pVF2
(Fig. 17).
Transformation of pVF plasmids into Aarobacterium
Transformation of Agrobacterium with the pVF plasmids was
by using the triple mating procedure. Overnight, 3 ml cultures
in LB liquid medium of the following bacteria were used:
Agrobacterium ABI (supplemented with 25 /g/ml chloramphenicol,
50 jug/ml kanamycin) E. coli PRK 2013 (50 pig/ml kanamycin) and
E. coli DH5a with either pVFl or pVF2 (75 ig/ml
spectinomycin) Samples of 250 ¡xl from each culture were mixed
in a microcentrifuge tube and centrifuged for 30 sec at 14,000
rpm. The pellets were resuspended in 50 ¡xl of 10 mM MgS04 and
placed on single LB agar plates free of antibiotics and
without spreading the bacteria. The plates were incubated at
28C for 24 h. Bacteria were collected from single plates and
resuspended in 1 ml of 10 mM MgS04. Samples of 1, 5 and 10 ¡xl
were plated on LB agar containing 25 ^g/ml chloramphenicol, 50
¡Xg/ml kanamycin and 75 ig/ml spectinomycin and incubated for
3 to 4 days at 28C. Surviving bacterial cultures were then
kept on YEP (1% yeast extract, 1% peptone, 0.5% NaCl pH 7.0)
agar plates containing the three antibiotics.
Screening of Transformed Bacteria
Agrobacterium colonies obtained in the transformation
event were screened using PCR to determine the presence of the
pVF plasmids. Samples from colonies were resuspended in 50 il


60
CP T30
CP T26
CP B67
CP T36
CP B7-1
CP B128
CP B185
CP B227
CP B249
p27 T30
p27 T26
p27 B67
p27 T36
p27 B7-1
p27 B128
p27 B185
p27 B227
p27 B249
LAVKSSSLQSD-DDTTGITYTREGVEVDLSDKLWTDIVYNSKGIGNRTNA
LAVKSSSLQSD-DDTTGITYTREGVELDLSDKLWTDIVYNSKGIGNRTNA
LAVKSSSLQSD-DDTTGITYTREGVEVDLSDKLWTDWYNSKGIGNRTNA
LAVKSSSLQSD-DDATGITYTREGVEVDLSDKLWTDWFNSKGIGNRTNA
LAVKSSSLQSD-DDTTGITYTREGVEVDLSDKLWTDWFNSKGIGNRRNA
LAVKSSSLQSD-DDTTGVTYTREGVEVDLSDKLWTDWFNSKGIGNRTNA
LAVKSSSLQSD-DDTTGITYTREGVEVDLSDKLWTDWFNSKGIGNRTNA
LAVKSSSLQSD-DDTTGITYTREGVEVDLSDKLWTDWFNSKGIGNRTNA
LAVKSSSLQSD-DDTTGVTYTREGVEVELSDKLWTDWFNSKGIGNRTNA
LYTTSTSTKTKFRDKGCISYVQGGSRYKLMDKWFPFIISKFTDRETPNA
LYTTSTSTKTKFRDKGCISYVQGGSRYKLMDKWFPFIISKFTDRETPNA
LYTTSTSTKTKFRDKGCISYVQGGSRYKLMDKWFPFIISKFTDRETPNA
LYTISTSTKTKFRDKGCISYVQGGLRYKLLDKWFPFIISKFTDRETPNA
LCTISTSTKTKFRDKGCISYVQGGLRYKLLDKWFPFIISKFTDRETPNA
LCTISTSTKTKFRDKGCISYVQGGLRYKLLDKWFPFIISKFTDRETPNA
LCTISTSTKTKFRDKGCISYVQGGLRYKLLDKWYPFIISKFTDRETPNA
LCTISTSTKTKFRDKGCISYVQGGLRYKLFDKWFPFIISKFTDRETPNA
LCTISTSTKTKFRDKGCISYVQGGLRYKLLDKWFPFIISKFTDRETPNA
* *.* .. ..* * **. . **
CP T30
CP T26
CP B67
CP T36
CP B7-1
CP B128
CP B185
CP B227
CP B249
p27 T30
p27 T26
p27 B67
p27 T36
p27 B7-1
p27 B128
p27 B185
p27 B227
p27 B249
LRVWGRTNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA
LRVWGRTNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA
LRVWGRTNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA
LRVWGRTNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA
LRVWGRTNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA
LRVWGRSNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA
LRVWGRSNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA
LRVWGRTNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCADFLTGA
LRVWGRTNDALYLAFCRQNRNLSYGGRPLDAGIPAGYHYLCAD FLTGA
LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP
LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP
LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP
LRKFACTFEELHLCMARLRPDLYENKRTTRAGTPHLKGYLSADFLSGSLP
LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP
LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP
LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP
LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLTGSLP
LRKYACTFEELHLCMARLRPDLYENKRTTKAGTPHLKGYLSADFLSGSLP
* ** * ******#*#
CP T30
CP T26
CP B67
CP T36
CP B7-1
CP B128
CP B185
CP B227
CP B249
p27 T30
p27 T26
p27 B67
p27 T36
p27 B7-1
p27 B128
p27 B185
p27 B227
p27 B249
GLTDLECAVYIQAKEQLLKKRGADEVWTNVRQLGKFNTR
GLTDLECAVYIQAKEQLLKKRGADEVWTNVRQLGKFNTR
GLTDLECAVYIQAKEQLLKKRGADEVWTNVRQLGKFNTR
GLTDLECAVYIQAKEQLLKKRGADDVWTNVRQLGKFNTR
GLTDLECAVYIQAKEQLLKKRGADEVWTNVRQLGKFNTR
GLTDLECAVYIQAKEQLLKKRGADEIWTNVRQLGKFNTR
GLTDLECAVYVQAKEQLLKKRGADEVWTNVRQLGKFNTR
GLTDLECAVYIQAKEQLLKKRGADEVWTNVRQLGKFNTR
GLTDLECAVYLQAKEQLLKKRGADEVWTNVRQLGKFNTR
GY SEHERG11LRASESMLARRQGYEEATELLNLRDLGKYL
GYSEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL
GYSEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL
GY SEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL
GYSEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL
GY SEHERG11LRASESMLARRQGYEEATELLNLRDLGKYL
GYTEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL
GYSEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL
GYSEHERGIILRASESMLARRQGYEEATELLNLRDLGKYL
* ***

Figure
13continued.


52
T30
12 6
B67
T36
B7-1
B128
B185
B227
B249
1 I I I I 50
MAGYTVLPNTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKL
N i 1
K
K
K
K 1
KV 1 1
K
51 I I I I 100
T30 DTNSIYEDSTEKFTGEHLKYVMVTMDAFLLENYKTKTEDLLVHLAMIQKR
T26
B67
T3 6 1 1
B7-1 C Y 1
B128 Y 1
B185 H 1 1 S ft
B227 §j 1 N-
B249 1 1
T30
126
B67
T36
B7-1
B128
B185
B227
B249
101 I I I I 150
LYTTSTSTKTKFRDKGCISYVQGGSRYKLMDKWFPFIISKFTDRETPNA
L 1
-C-X L L
-C-X b L
-C-X 1 I
-C-I L Xi
Figure 8. Pairwise alignment of the deduced amino
acid sequence of p27 from different CTV strains
(hyphens, identical amino acids; periods, similar
amino acids). The alignment was constructed using
the program CLUSTALV.


CHAPTER 5
SUMMARY AND CONCLUSIONS
1) The expression of CTV ORFs p27 and p20 was detected in
vivo in CTV-infected plants using specific polyclonal
antibodies raised against the recombinant proteins.
2) Cell fractionation analysis indicated that p27
accumulates in the cell wall fraction, whereas p20
accumulates in the soluble protein fraction.
3) Tissue blots indicated that p27 is present in the phloem.
This is in agreement with CTV being a phloem limited
virus.
4) The function of p27 is still unknown, but based on
seguence homology and cell fraction studies, p27 could be
involved in any or all of three functions: virus
assembly, virus movement and aphid transmission.
5) Based on seguence conservation, the active site of p27
appears to be located in the C-terminal portion of the
protein.
93


Table 2.
Characteristics of
the CTV
strains used
for sequencing of
the p27
gene.
STRAIN
ORIGIN
MCA13a
M.L.b
DECLINE0
SYd
SP-Ge
SP-MVf
COMMENTS
T26
Florida
-
+
0
0
0
0
T30
Florida
-
+
0
0
0
0
Effective for cross protection
T36
Florida
+
++
++
+
+
0
Genome sequenced
B7-1
S. Africa
+
++
+
+
++
0
Used for cross protection.
B67
China
-
+
0
0
0
0
B12 8
Colombia
+
++
?
0
+++
+
B185
Japan
+
+++
+++
+++
+++
++
B227
India
+
++
+++
++
+++
+++
B249
Venezuela
+
++
+++
+++
++
+
Vein corking on M.L.
a MCA13= Reactive with the monoclonal antybody MCA13 (-= no reaction, += positive
reaction).
b M.L.= Symptoms on Mexican lime (+= mild, ++= moderate, +++= severe, ?= unknown).
c Decline= Induces decline in sweet orange scions grafted on sour orange rootstock,
d SY= Seedling yellows on sour orange seedlings,
e SP-G= Stem pitting on grapefruit scions.
f SP-MV= Stem pitting on Madame Vinous sweet orange scions.


103
Sheffield, V.C., Beck, J.S., Kwitek, A.E., Sandstrom, D.W. and
Stone, E.M. 1993. The sensitivity of single-stranded
conformation polymorphism analysis for the detection of
singles base substitutions. Genomics 16:325-332.
Spinardi, L., Mazars, R. and Theillet, C. 1991. Protocols for
an improved detection of point mutations by SSCP. Nucleic
Acids Research 19:4009.
Stomp, A. M. 1992. Histochemical localization of /3-
glucuronidase. Pages 103-124, in: Gallagher, S.R. (ed.)
GUS Protocols: Using the GUS Gene as a Reporter of Gene
Expression. Academic Press, Inc. San Diego, CA.
Studier, F.W., Rosenberg, A.H., Dunn, J.J. and Dubendorff,
J.W. 1990. Use of T7 RNA polymerase to direct expression
of cloned genes. Pages 60-89, in Goeddel, D.V. (ed.)
Methods in Enzymology, Vol. 185, Gene Expression
Technology. Academic Press Inc, San Diego, CA.
Tennant, P.F., Gonsalves, C., Ling, K.S., Fitch, M. ,
Manshardt, R. Slightom, J.L. and Gonsalves, D. 1994.
Differential protection against papaya ringspot virus
isolates in coat protein gene transgenic papaya and
classically cross-protected papaya. Phytopathology
84:1359-1366.
van der Vossen, E.A.G., Neeleman, L. and Bol, J.F. 1994. Early
and late functions of alfalfa mosaic virus coat protein
can be mutated separately. Virology 202:891-903.
van Lent, J., Storms, M., van der Meer, F., Wellink, J. and
Goldbach, R. 1991. Tubular structures in movement of
cowpea mosaic virus are also formed in infected cowpea
protoplasts. J. Gen. Virol. 72: 2625-2623.
Waigmann, E. Lucas, W.J., Citovsky, V. and Zambrynski, P.
1994. Direct functional assay for tobacco mosaic virus
cell-to-cell movement protein and identification of a
domain involved in increasing plasmodesmatal
permeability. Proc. Natl. Acad. Sci. USA 91:1433-1437.
Wolf, S., Deom, C.M., Beachy, R.N., Lucas, W.J. 1989. Movement
protein of tobacco mosaic virus modifies plasmodesmata
size exclusion limit. Science: 337-339.


92
Rooting of the transformed shoots has been difficult,
with lower survival rates than reported by other authors
(Moore et al., 1992; Kaneyoshi et al. 1994; Pea et al.,
1995) This may be due to a detrimental effect of the
antibiotics on the explants or possibly to the expression of
the CTV gene products in the shoots. CTV infection reduces
root and bud formation of shoots in vitro (Duran-Vila, 1989).
It is possible that the constitutive expression p27 or p20
causes the reduction of root formation in the explants.
PCR analysis of the same segments used in the
histochemical GUS assay confirmed the presence of p27 and p20
in the putatively transformed shoots. Only 31% (p27) or 37%
(p20) of the GUS+ segments were also PCR+. This may be due to
true false positives, or to deficiencies in the DNA extraction
procedure because of the small amount of tissue used.
Hybridization experiments confirmed the specificity of the
bands obtained in the PCR analysis, demonstrating that they
are p27 and p20 genes.


3
million citrus trees on sour orange rootstock have died from
tristeza (Mendt, 1992).
In Florida, approximately 20-25 million trees of sweet
orange, grapefruit and mandarin are planted on the susceptible
sour orange rootstock (Garnsey, 1991). CTV has been present in
Florida since the '50s, and both mild and decline inducing
strains have been reported (Brlansky et al., 1986). In
addition, 8-10 million trees of grapefruit are also
susceptible to stem pitting strains, which have not been
detected yet in Florida (Garnsey, 1991). The aphid T.
citricida is still not present, although other less efficient
vectors such as Aphis gossypii Glover and A. citricola van der
Goot are widespread in Florida. However, T. citricida has
continued its northward movement from South America. In recent
surveys in Central America and the Caribbean the aphid was
reported in Nicaragua (Lastra et al., 1991) and Cuba (Yokomi
et al., 1994). T. citricida thus represents a serious threat
to the citrus industries of Florida and the United States, as
well as Mexico, Cuba and other Caribbean countries. This is
because of its efficiency to vector CTV (6 to 25 times more
efficient than A. gossypii; Yokomi et al., 1994), especially
with the decline and stem pitting strains which are not
efficiently vectored by the aphid species present in Florida
(Yokomi, 1990).
Transmission of CTV by aphids is in a semipersistent
manner (Bar-Joseph and Lee, 1990). It also is transmitted by


24
Analysis of bacterial proteins by SDS-PAGE showed
accumulation of proteins of the expected MW in induced
cultures, but not in non-induced cultures (data not shown).
The amount of each protein increased with time. CTV proteins
accumulated to approximately 30% of the total bacterial
protein.
Detection of p27
The expression of p27 in CTV-infected citrus was tested
using Western blot analysis. The optimal dilution of antiserum
was determined empirically. Best results were obtained at a
dilution of 1:500. Because of the amino acid homology between
p27 and the CP the reaction of different antisera to both
proteins were compared (Figure 2).
The p27 polyclonal antiserum showed some nonspecific
reactivity (as did pre-immune serum) with uninfected citrus
tissue (Fig. 2, lane 4). However, a discrete, intense, protein
band of approximately 27 kDa was detected in extracts of CTV-
infected citrus (Fig. 2, lane 5), but not in those of
uninfected citrus (Fig. 2, lane 4). The antiserum also
detected E. coli-expressed p27 (Fig. 2, lane 2), which
migrates slightly slower than p27 due to its higher MW. While
the p27 polyclonal antibodies reacted with the purified fusion
p27 protein (Fig. 2, lane 2), they did not react with a non
fusion, E. coli-expressed CP (Fig. 2, lane 3). Furthermore, no
reaction was observed with proteins induced from bacteria
containing the pETH plasmid without an insert (Fig. 2, lane


18
reaction 12.5 ¡xC i of [a-35S]-dATP were used. Both strands of
DNA were sequenced using primers specific for the pETH
expression vector.
Samples were electrophoresed in denaturing 5%
polyacrylamide gels (acrylamide/bisacrylamide 19:1, lx TBE, 8
M urea) for 1.5 to 4 h at room temperature and 35 W constant
power.
Bacterial Expression of CTV Proteins
Induction. The expression of CTV genes in E. coli
BL21 (DE3) was induced using isopropyl-/3-D-thiogalactoside
(IPTG) at a final concentration of 0.4 M, in 25 ml of LB
liquid medium supplemented with 100 /g/ml ampicillin and 25
ixg/xal chloramphenicol (Sambrook et al., 1989).
To test for expression of recombinant proteins, 1 ml
samples were taken before induction and 30 min, 1 h, 2 h and
3 h after induction. All samples were kept on ice in
microcentrifuge tubes until the last was collected. Bacterial
cells were pelleted by centrifugation at 14,000 rpm for 30 sec
at room temperature and resuspended in 50 ti of cracking
buffer (300 mM Tris-HCl pH 6.8, 2% SDS, 20% glycerol, 1% v/v
2-mercaptoethanol, 0.025% bromophenol blue). After
resuspension, the samples were boiled for 3 min and stored at
-20C for later use.
Detection. Bacterial proteins were separated using 12%
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli,
1970). Samples were electrophoresed at 200 V for 45 min in a


91
have been reported previously (Moore et al., 1992; Pea et
al., 1995) and constitute a serious problem, increasing the
work necessary in screening the shoots. This ineffective
Kanamycin selection may be explained by one or several
possibilities: 1) protection of non-transformed cells by the
surrounding transformed cells, 2) contamination with
Agrobacterium or 3) by endogenous levels of NPT II activity
(Jordan and McHughen, 1988; Dandekar et al., 1988; Moore et
al., 1992; Pea et al., 1995).
Transformation efficiencies, expressed as percentage of
GUS+ shoots from the total adventitious shoots produced, were
very variable between experiments and in general low. Values
ranged from 0 to 15%, with an average of 6%. These results are
comparable to those obtained by Moore et al. (1992, 1993), but
lower than the 55% obtained with Carrizo citrange by Pea et
al. (1995) or the 55% to 87% reported by Kaneyoshi et al.
(1994) with Poncirus trifoliata Raf. One of the differences in
the transformation protocol used here and the ones mentioned
above is that the segments are inoculated by submersion for
several minutes to a few days in a solution containing the
Agrobacterium. Another difference is that in those cases the
explants were obtained from relatively young seedlings (3 to
5 weeks old) instead of 3 to 6 months old as reported here.
Finally, the procedure of Kaneyoshi et al. (1994) includes
acetosyringone in the inoculation media to increase the
efficiency of bacterial infection.


38
Using tissue blots, p27 was mostly present in the sieve
tubes. This is in agreement with what has been found for the
CP (Garnsey et al., 1993) and with the phloem-limited nature
of CTV (Bar-Joseph and Lee, 1990). These results also indicate
that some of the p2 7 epitopes are conserved between native and
SDS-denatured protein. The antiserum was raised against
denatured protein, however, in tissue blots the proteins are
not treated with SDS previous to their detection.
Tissue blot is a simple method for assaying the
expression of p27, especially when a large number of samples
are involved. This technique will be used for the screening of
the p27 transformed plants.
The polyclonal antiserum raised using recombinant p20
detected a protein band of the expected size in CTV-infected,
but not in uninfected citrus tissue, indicating that the p20
also is CTV specific. Unlike p27, p20 accumulates in the
soluble protein fraction. The intensity of the p20 band in
Western blots was much lower for p20 than p27. This was not
expected since analysis of CTV subgenomic RNAs indicates that
p20 transcript is the most abundant (Hilf et al., 1995). One
alternative to explain this result is that p20 protein is
poorly immunogenic, and so the concentration of specific
antibodies in the serum is low. Because the E. coli-expressed
p2 0 contains the fusion portion, it is not possible to
conclude anything from the reaction observed with the
antiserum to this protein. Another possibility is that p20


59
CP T30
CP T26
CP B67
CP T36
CP B7-1
CP B128
CP B185
CP B227
CP B249
p27 T30
p27 T26
p27 B67
p27 T36
p27 B7-1
p27 B128
p27 B185
p27 B227
p27 B249
MDDETKKLKNKNKETKEGDEWAAESSFGSVNLHIDPTLITMNDVRQL
MDDETKKLNNKNKETKEGDEWAAESSFGSVNLHIDPTLITMNDVRQL
MDDETKKLKNKNKEIKQGDDWAAESSFGSVNLHIDPTLITMNDVRQL
MDDETKKLKNKNKETKEGDDWAAESSFSSVNLHIDPTLITMNDVRQL
MDDETKKLKNKNKETKEGDDWAAESSFGSVNLHIDPTLIAMNDVRQL
MDDETKKLKNKNKEAKEGDDWAAESSFGSLNFHIDPTLIAMNDVRQL
MDDETKKLKNKNKETKEGDDWAAESSFGSLNLHIDPTLIAMNDVRQL
MDDETKKLKNKNKETKEGDDWAAESSFGSMNLHIDPTLIAMNDVRQL
MDDETKKLKNKNKETKEGDDWAAESSFGSLNLHIDPTLIAMNDVRQL
MAGYTVLPNTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKL
MAGYTVLPNTDDKEMNPVSAALPGKYPDVIEKFVANRSVDALMEGVISKL
MAGYTVLPNTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKL
MAGYTVLPKTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKL
MAGYTVLPKTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKL
MAGYTVLPKTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKL
MAGYTVLPKTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKF
MAGYTVLPKVDDKEMDPVSAAVPGKYPDIIEKFVTNRSVDALIEGVISKL
MAGYTVLPKTDDKEMDPVSAAVPGKYPDVIEKFVANRSVDALIEGVISKL
* .** ... .
CP T30 STQQNAALNRDLFLALKGKYPNLP DKDKDFHIAMMLYR
CP T26 STQQNAALNRDLFIALKGKYPNLP DKDKDFHIAMMLYR
CP B67 STQQNAALNRDLFLTLKGKYPNLP DKDKDFHIAMMLYR
CP T36 STQQNAALNRDLFLTLKGKHPNLP DKDKDFRIAMMLYR
CP B7-1 GTQQNAAVNRDLFLTLKEKYPKLS DKDKDFHIAMMLYR
CP B128 STQQNAALNRDLFLTLKGKYPNLS DKDKDFHLAMMLYR
CP B185 STQQNAALNRDLFLTLKGKYPNLS DKDKDFHLAMMLYR
CP B227 GTQQNAALNRDLFLTLKGKYPNLP DKDKDFHIAMMLYR
CP B249 GTQQNAALNRDLFLTLKGKYPNLP DKDKDFHIAMMLYR
p27 T30 DTNSIYEDSTEKFTGEHLKYVMVTMDAFLLENYKTKTEDLLVHLAMIQKR
p27 T26 DTNSIYEDSTEKFTGEHLKYVMVTMDAFLLENYKTKTEDLLVHLAMIQKR
p27 B67 DTNSIYEDSTEKFTGEHLKYVMVTMDAFLLENYKTKTEDLLVHLAMIQKR
p27 T36 DTNSIYEDSTEKFTGEHLKYVMVTMDTFLLENYKTKTEDLLVHLTMIQKR
p27 B7-1 DTNCIYEDSTEKFTGEYLKYVMVTMDTFLLENYKTKTEDLLVHLAMIQKR
p27 B128 DTNSIYEDSTEKFTGEYLKYVMVTMDTFLLENYKTKTEDLLVHLAMIQKR
p27 B185 HTNSIYEDSTEKFTGEYLKYVMVTMDTFLLENYKPKTEALLVHLAMIQKR
p27 B227 DTNSIYEDSTEKFTGEQLKYVMVTMDTFLLENYKTKTEDLLVHLAMIQNR
p27 B249 DTNSIYEDSTEKFTGEYLKYVMVTMDTFLLENYKTKTEDLLVHLAMIQKR
* *.*
Figure 13. Alignment of CPs and p27 from different
CTV strains. Asterisks, identical amino acids;
periods, similar amino acids. Alignment generated
using CLUSTALV.


104
Yokomi, R.K. and Damsteegt, V.D. 1990. Comparison of citrus
tristeza virus transmission efficiency between Toxoptera
citricidus and Aphis gossypii. Page 319, in: Peters,
D.C., Webster, J.A., Chlouber C.S. (eds.). Proceedings
Aphid-Plant Interactions: Populations to Molecules.
Stillwater, OK.
Yokomi, R.K., Lastra, R., Stoetzel, M.B., Damsteegt, V.D.,
Lee, R.F., Garnsey, S.M., Gottwald, T.R., Rocha-Pea,
M.A. and Niblett, C.L. 1994. Establishment of the brown
citrus aphid (Homoptera: Aphididae) in Central America
and the Caribbean Basin and transmission of citrus
tristeza virus. J. Econ. Entomol. 87:1078-1085


31
incubation at 37C for 1 h, and 1:10,000 dilution. The blots
indicate that p27 is present in phloem tissue.
Detection of p20
The optimal antiserum concentration for the analysis of
p20 was 1:500. Fig. 6 shows the results of the detection of
the cell fractions with p20 antiserum. A protein band of the
expected size was present in infected, unfractionated tissue
(Fig. 6, lane 2) but not in uninfected tissue (Fig. 6, lane
1) Most of the p20 was in the soluble protein fraction (S30)
(Fig. 6, lane 5). Cell wall fraction R3 (Fig. 6, lane 8) also
contained some p20. Fractions PI, P30, R1 and R2 contained
little or no detectable p20 (Fig 6, lanes 3, 4, 6 and 7,
respectively).
The p20 antiserum was not useful for detection of the
protein using tissue blots. At high antiserum concentrations
(1: 1000) the serum was reactive with uninfected tissue, and
at higher dilutions (1: 20,000) positive reactions with CTV-
infected tissue were very weak.
Detection of p!8
Despite several attempts to detect pl8 in infected
tissue, no band of the expected size was observed in Western
blots. Dilution of the antiserum as low as 1:100 did not
produce any favorable results in colorimetric detection (data
not shown). The antibody reacted, however, with fusion p20
produced in bacteria, the same protein used as antigen (data
not shown). It also reacted with fusion pl8 and p27, both of


Figure 16. Restriction map of the pVFl plasmid,
containing p27.


97
Dolja, V.V., Karasev, A.V. and Koonin, E.V. 1994. Molecular
biology and evolution of closteroviruses: Sophisticated
build-up of large RNA genomes. Annu. Rev. Phytopathol.
32: 261-285.
Duran-Vila, N., Cambra, M. Medina, V. Ortega, C. and
Navarro, L. 1989. Growth and morphogenesis of citrus
tissue cultures infected with citrus tristeza virus and
citrus infectious variegation virus. Phytopathology
79:820-826.
Flasinski, S., Dzianott, A., Pratt, S. and Bujarski, J.J.
1995. Mutational analysis of the coat protein gene of
brome mosaic virus: Effects on replication and movement
in barley and Chenopodium hybridum. Molecular Plant-
Microbe Interactions 8:23-31.
Gallei, D.R. 1993. Posttranscriptional regulation of gene
expression in plants. Annu. Rev. Plant Physiol. Plant
Mol. Biol. 44: 77-105.
Garnsey, S.M. 1991. Citrus tristeza virus. In: Lastra R. Lee,
R., Rocha-Pea, M. and Niblett, C.L. (eds.) Workshop on
citrus tristeza virus/Toxoptera citricida in Central
America and the Caribbean Basin. Turrialba, Costa Rica.
Garnsey, S.M., Gumpf, D.J., Roistacher, C.N., Civerolo, E.L.,
Lee, R.F., Yokomi, R.K. and Bar-Joseph, M. 1987. Toward
a standardized evaluation of the biological properties of
citrus tristeza virus. Phytophylactica 29:151-157.
Garnsey, S.M., Permar, T.A., Cambra, M. and Henderson, C.T.
1993. Direct tissue blot immunoassay (DTBIA) for
detection of citrus tristeza virus. Pages 39-50, in
Moreno, P., da Graga, J.V. and Timmer, L.W. (eds.) Proc.
12th Conf. of Int. Organ. Citrus Virol. IOCV. Riverside,
CA.
Georgopoulos, C. 1992. The emergence of the chaperone
machines. Trends in Biochemical Sciences 17:295-299.
Geraud, F. 1976. El fido negro de los ctricos Toxoptera
citricida Kirkaldy en Venezuela. I Encuentro Venezolano
de Entomologa. U.C.V. Facultad de Agronoma. Instituto
de Zoologa Agrcola. Maracay, Venezuela.
Giesman-Cookmeyer, D. and Lommel, S.A. 1993. Alanine scanning
mutagenesis of a plant virus movement protein identifies
three functional domains. Plant Cell 5:973-982.


46
deduced amino acid sequences among all the strains studied. At
the nucleotide level, homology of the sequences varied from
86.2% to 99.6%. Strains B7-1 (severe from South Africa) and
B128 (severe from Colombia) were the most similar. At the
protein level, homologies were 93.3% or higher. Strains T30
(mild from Florida) and B67 (mild from China) were identical
in deduced amino acid sequence. Strains B128 and B249 (severe
from Venezuela) also were identical in deduced amino acid
sequence.
Alignment of the nucleotide sequences (Fig. 7) showed
several nucleotide substitutions along the genes. No
deletions, insertions or inversions were observed. All
sequences constituted an ORF similar to the one previously
reported (Pappu et al., 1994). A total of nine substitutions
were conserved in all the severe strains compared to
nucleotides conserved in all mild strains. For example,
nucleotides 108 (C T) and 229 (G A) Other nine
substitutions were present only in stem pitting strains. For
example, nucleotide 78 (T C) Most of these substitutions,
however were silent.
Comparison of the deduced amino acid sequences (Fig. 8)
showed only 4 amino acids exclusive to severe strains (amino
acids 9, 77, 104 and 125),and only one in stem pitting strains
(amino acid 102) The C-terminal portion of the protein is the
most conserved among the strains sequenced.


41
Detection of sequence variability in viral genes is
important since mutations could be linked to particular
biological properties. Single stranded conformation
polymorphism (SSCP) is a simple and sensitive method used to
detect single nucleotide changes in particular sequences
(Orita et al., 1989). This technique is based on migration
differences during electrophoresis of short, denatured, DNA
fragments in non-denaturing gels, caused by differences in
their nucleotide sequences (Orita et al., 1989; Spinardi et
al., 1991).
The main purpose of this research was to determine if
there was any relationship between RNA or deduced protein
sequence and the biological properties of the virus.
Therefore, this gene was cloned and sequenced from CTV strains
with distinct geographical origins and different biological
activities. In addition, PCR combined with SSCP analysis was
tested for its potential as a rapid method to determine
sequence differences among CTV strains.
Materials and Methods
Materials
In addition to the materials mentioned in Chapter 2, the
Wizard DNA purification system used was from Promega
Corporation (Madison, WI) and the silver staining kit was from
Sigma Chemical Company (St. Louis, MO). Sequence analysis was
performed using the programs Seqaid, CLUSTALV (Higgins et al.,


CHAPTER 4
TRANSFORMATION OF SWEET ORANGE WITH P27 AND P20
Introduction
Sequence analysis and the identification of ORFs in other
plant viruses have permitted the use of some of those genes to
transform plants and protect them against viral infections
caused by similar strains. The first and most widely used gene
has been the CP (Powell Abel et al., 1986; Beachy et al.,
1990; Pappu et al., 1995). Transgenic plants expressing CP
genes and showing various degrees of resistance have been
obtained for members of at least a dozen different virus
groups (Hull and Davies, 1992; Pappu et al., 1995), indicating
that this is a relatively general phenomenon. Several non-
structural viral genes also have been used to transform plants
with mixed results in the induction of resistance. For
example, three nonstructural genes of tobacco rattle
tobravirus transformed into tobacco plants did not induce
resistance to the virus (Angennent et al., 1990) On the other
hand, tobacco plants transformed with a 54 kDa putative
replicase protein of tobacco mosaic tobamovirus (TMV) were
resistant to high concentrations of intact TMV and its RNA
(Golemboski et al., 1990).
67


96
Beachy, R.N., Loesch-Fries, S. and Turner, N.E. 1990. Coat
protein mediated resistance against virus infection. Ann.
Rev. Phytopathol. 28:451-474.
Boyko, V.P., Karasev, A.V., Agranovsky, A.A., Koonin, E.V. and
Dolja, V.V. 1992. Coat protein gene duplication in a
filamentous RNA virus of plants. Proc. Natl. Acad. Sci.
USA 89:9156-9160.
Brlansky, R.H., Lee, R.F. and Garnsey, S.M. 1988. In situ
immunofluorescence for the detection of citrus tristeza
virus inclusion bodies. Plant Disease 72: 1039-1041.
Brlansky, R.H., Pelosi, R.R., Garnsey, G.M., Youtsey, C.O.,
Lee, R.F., Yokomi, R.K. and Sonoda, R.M. 1986. Tristeza
quick decline epidemic in South Florida. Proc. Fla. State
Hort. Soc. 99:66-69.
Citovsky, V. Wong, M.L., Shaw, A.L., Prasad, B.V. and
Zambryski, P. 1992. Visualization and characterization of
tobacco mosaic virus movement protein binding to single
stranded nucleic acids. Plant Cell 4:397-411.
Costa, A.S. and Muller, G.W. 1980. Tristeza control by cross
protection: a US-Brazil cooperation success. Plant
Disease 64:538-541.
Dandekar, A.M., Martin, L.A. and McGranahan, G.H. 1988.
Genetic transformation and foreign gene expression in
walnut tissue. J. Am. Soc. Hort. Sci. 113: 945-949.
Deom, C.M. Oliver, M.J. and Beachy, R.N. 1987. The 30-
kilodalton gene product of tobacco mosaic virus
potentiates virus movement. Science 237: 389-393.
Derrick, P.M., Barker, H. and Oparka, K.J. 1992. Increase in
plasmodesmatal permeability during cell-to-cell spread of
tobacco rattle virus from individually inoculated cells.
Plant Cell 4:1405-1412.
Deveraux, J., Haerbeli, P. and Smithies, 0. 1984. A
comprehensive set of sequence analysis programs for the
VAX. Nucleic Acids Research 12:387-395.
Dolja, V.V., Boyko, V.P., Agranovsky, A.A. and Koonin, E.V.
1991. Phylogeny of capsid proteins of rod-shaped and
filamentous RNA plant viruses: Two families with distinct
patterns of sequence and probably structure conservation.
Virology 184:79-86.


2
normally is not observed in the field (Garnsey et al., 1987;
Bar-Joseph and Lee, 1990) Some mild strains, on the other
hand, do not induce any noticeable symptoms in most of the
cultivated varieties (Garnsey et al., 1987; Bar-Joseph and
Lee, 1990). Several strains can be present in the same tree,
as has been determined by cross protection experiments (Powell
et al., 1992) and double-stranded RNA analysis (Moreno et al.,
1991) This makes the use of biological methods, such as
cross-protection, more difficult to control the disease,
although they are still possible (Costa and Muller, 1980).
CTV is present in most areas of the world where citrus is
grown. In South America it was first described in the 1930s.
Less than two decades later CTV caused the destruction of most
of the citrus industries in Argentina, Brazil and Uruguay,
killing about 25 million trees planted on the susceptible sour
orange rootstock (Muller and Costa, 1992). The spread of the
disease and its impact is increased by the presence of its
most efficient vector Toxoptera citricida (Kirkaldy), the
brown citrus aphid. A well documented case occurred in
Venezuela. The disease was reportedly present in the country
in the mid-'50s without having a major impact. In 1976 T.
citricida was first found in Venezuela in regions bordering
with Colombia and Brazil (Geraud, 1976). Two years later it
was widespread throughout the country. In 1980 the first
outbreak of CTV was reported, and since then, at least 6


Table 6. Transformation efficiencies and survival of shoots of "Pineapple" sweet
orange using pVFl (p27) and pVF2 (p20).
PLASMID3
EXPLANT
No. OF
SHOOTS
No. OF
GUS+ SHOOTS
% GUS+
SHOOTS
No.
SURVIVING
%
SURVIVING1
pVFl
Nodes
148
1
0.7
0
0.0
Internodes
1037
71
6.8
18
25.4
Roots
208
5
2.4
c
c
pVF2
Nodes
215
19
8.8
7
37.8
Internodes
886
49
5.3
22
44.9
a pVFl=p27, pVF2=p20.
b Survival in soil seven months after transfer.
c Adventitious shoots derived from root segments have been in soil less than seven months.
00
CT<


90
12 3 4
5 6 7 8 9
10 11 12 13 14 15 16
Figure 23. Southern analysis of PCR products from
GUS+ segments of "Pineapple" sweet orange
hybridized with a p27 specific probe. Lanes 1 and
12, GUS- segment; Lanes 2 to 11 and 13, 14, GUS+
segments transformed with p27 gene; Lane 15, Lambda
DNA digested with Hindlll; Lane 16, amplified p27
from pVFl.


83
The results in Table 5 indicate that more adventitious
shoots were produced from root segments compared to nodes or
internodes. Nodes produced the lowest average number of
adventitious shoots. There was no difference in shoot
production when explants were inoculated with pVFl or pVF2.
However, comparison of the efficiencies of transformation (as
percentage of GUS+ shoots obtained from the total) showed that
internodes were more efficiently transformed with pVFl (Table
6) than nodes or roots. This indicates that proportionately,
internodes generated twice as many GUS+ (Fig. 21) shoots than
the root segments. Interestingly, transformation with pVF2 was
more successful on nodes than on internodes (Table 6). Again,
taking regeneration and transformation efficiency together,
internode segments produced three times as many GUS+ shoots
than nodes.
Transformation efficiency was quite variable between
experiments, ranging from 0 to 15%. The overall efficiency was
lower than desirable. Taking the results from all the
transformation experiments using pVFl, from 2,481 shoots
obtained, 148 (5.97%) were GUS+. When pVF2 was used from 1181
shoots obtained, 71 (6.0%) were GUS+.
Survival of the GUS+ shoots in soil (Table 6) ranged from
25 to 45%, seven months after transfer, although none of them
have rooted at the time this is written. The results from the
shoots obtained from root segments are not included as they
have been in soil only two months.


37
cauliflower mosaic virus (Albrecht et al., 1988) and squash
leaf curl geminivirus (Pascal et al., 1993) accumulate in the
cell wall fractions. The observations that p27 also
accumulates in this fraction suggest a possible role in virus
movement.
The capacity to bind RNA is another characteristic of
movement proteins (Deom et al., 1987; Citovsky et al., 1992;
Giesman-Cookmeyer and Lommel, 1993). Due to the apparent
structural similarities between p27 and the CP (Pappu et al.,
1994), including the conservation of amino acids present in
the CPs of other filamentous viruses (Dolja et al., 1991;
Boyko et al., 1992), and the finding of p24 as part of the BYV
virions, it is likely that p27 might have RNA-binding
capacity.
The CP of some RNA viruses also have a role in cell-to-
cell and long distance movement. In some cases, like cowpea
mosaic comovirus (van Lent et al., 1991), the virus moves from
cell to cell as virions. In other cases, like A1MV, the CP is
required for movement, but virions are not transported (van
der Vossen et al., 1994). For brome mosaic bromovirus, the CP
is not required for cell-to-cell movement, but is required for
long distance movement (Flasinski et al., 1995). It is
possible then that p27 acts as a multifunctional protein,
forming part of the virus particles and assisting in one or
more of the following functions: virus movement, aphid
transmission or encapsidation.


78
/
c


32
Figure 6. Western blot analysis of cell fractions
prepared from CTV-infected tissue and probed with
p20 antiserum. Lane 1, uninfected citrus tissue
extract; Lane 2, unfractionated infected citrus
extract; Lanes 3 to 8 contained fractions PI, P30,
S30, Rl, R2 and R3, respectively, of infected
citrus tissue as explained in Materials and
Methods. Lane 9 is . coli-expressed p20.


87
To confirm presence of the CTV genes in the GUS+ plants,
samples were analyzed using PCR (Fig. 22, Table 7). For those
GUS+ shoots transformed with pVFl, 31% were also PCR+ For
pVF2, 37% of the GUS+ shoots were also PCR+. Transformed,
GUS' segments were used as negative controls to rule out the
possibility of obtaining PCR products from contaminating
Agrobacterium. Hybridization of the PCR products obtained from
the plants with p27 or p20 probes demonstrated they were CTV
specific seguences (Fig. 23).
Discussion
The number of shoots produced by the internodes is
comparable to what was obtained previously for Carrizo
citrange [C. sinensis (L.) Osb. x Poncirus trifoliata (L.)
Raf.] under similar experimental conditions (Moore et al.,
1992) There are no published values for root and node
segments. Comparing the three different types of explants used
to transform with pVFl, root segments produced more
adventitious shoots than internodes, and internodes more than
nodes. However, a higher percentage of GUS+ shoots were
obtained from internodes than from roots, making internode
segments more efficient. For pVF2 internode segments also
produced more GUS+ shoots than nodes.
Kanamycin was used in the media for selection of
transformed shoots, however, only an average of 6%
(considering all experiments together) were GUS+. "Escapes"


81
Figure 19. PCR of Agrobacterium ABI cultures to
confirm the presence of pVF plasmids. Lane 1,
Agrobacterium/pVFl bacterial extract amplified
using p27 primers; Lane 2, Agrobacterium/pVF2
bacterial extract amplified using p20 primers; Lane
3, purified pVFl plasmid DNA amplified with p27
primers; Lane 4, purified pVF2 plasmid DNA
amplified with p2 0 primers; Lane 5, Lambda DNA
digested with Hindlll used as a MW marker.


CHAPTER 3
SEQUENCE ANALYSIS OF THE P27 ORF
Introduction
The p27 ORF has been described as a diverged copy of the
CP gene (Pappu et al., 1994). Comparison of the deduced amino
acid seguences indicates that 19% of the amino acids are
identical between p27 and the CP, and 41% of the amino acids
are either identical or with similar biochemical properties to
those of the CP (Pappu et al., 1994). The first report of the
diverged copy of the CP among closteroviruses (and filamentous
RNA viruses) was for BYV (Boyko et al., 1992). The BYV p24
ORF also is located 5' to the CP and all four genes of CTV
and BYV show various degrees of amino acid sequence
similarities, ranging from 38% (CTV p27 and BYV CP) to 50%
(CTV p27 and BYV p24) (Boyko et al., 1992; Pappu et al.,
1994). Two of the amino acids conserved in the four proteins
also are strictly conserved in the CPs of 14 other filamentous
positive-stranded RNA viruses (Dolja et al., 1991; Boyko et
al., 1992; Pappu et al., 1994). Those two residues are
believed to form a salt bridge (Dolja et al., 1991),
suggesting that the CPs and the diverged copies share not only
the primary structure, but exhibit similar protein folding
(Boyko et al., 1992).
40


89
Table 7. Number and percentage of "Pineapple"
sweet orange shoots that tested
positive for GUS and by PCR.
PLASMID
GUS+
PCR+
% PCR+
pVFl
79
25
31.2
pVF2
8
3
37.5


T30cp
T26cp
B67cp
T36cp
B71cp
B185cp
B128cp
B227cp
B249cp
Figure 11. Phylogenetic tree for CP based on RNA
sequences. Graphic generated using Pileup program
in GCG.


69
transformation. However, when the transgenic plants were
inoculated with different viral strains they showed only a
delay in symptom development (Tennant et al., 1994), a
phenomenon observed previously in other CP transgenic plants
(Beachy et al., 1990). For annual crops, a delay in symptom
development can be sufficient to reduce losses caused by a
virus, but for perennial crops this will probably be of little
value since they are expected to be in the field for many
years.
Several citrus species have reportedly been transformed
by different means, including direct uptake of DNA by
protoplasts (Schell, 1991), co-cultivation of cell suspensions
with Agrobacterium (Hidaka et al., 1990), and infection of
epicotyl segments with Agrobacterium (Moore et al., 1989;
Kaneyoshi et al., 1994; Pea et al., 1995). There are two
reports of citrus transformation using the CP gene of CTV
(Gutirrez et al., 1992; Schell et al., 1994). The plants
obtained in these experiments are being tested currently for
resistance to CTV (G.A Moore and J.W. Grosser, personal
communication).
To study the feasibility of using some of the non-CP
genes of CTV in Agrobacterium-mediated plant transformation,
p27 and p20 genes were used to transform "Pineapple" sweet
orange plants. Transgenic plantlets were selected after target
gene expression was detected in infected citrus tissue.


T30
B67
T26
Figure 10. Phylogenetic tree for p27 based on
deduced amino acid sequences. Graphic generated
using Pileup program in GCG.



PAGE 1

02/(&8/$5 &+$5$&7(5,=$7,21 2) &,7586 75,67(=$ 9,586 *(1(6 $1' 7+(,5 86( ,1 3/$17 75$16)250$7,21 %\ 9,&(17( -26( )(%5(652'5,*8(= $ ',66(57$7,21 35(6(17(' 72 7+( *5$'8$7( 6&+22/ 2) 7+( 81,9(56,7< 2) )/25,'$ ,1 3$57,$/ )8/),//0(17 2) 7+( 5(48,5(0(176 )25 7+( '(*5(( 2) '2&725 2) 3+,/2623+< 81,9(56,7< 2) )/25,'$

PAGE 2

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

PAGE 3

'DYLG %HQVFKHU IRU DOO WKH KHOS DQG JRRG WLPHV VSHQW WRJHWKHU LQ WKH ODE )LQDOO\ WKDQNV WR WKH IDFXOWLHV SRVWGRFV WHFKQLFLDQV DGPLQLVWUDWLYH SHUVRQQHO DQG VWXGHQWV LQ WKH SODQW SDWKRORJ\ GHSDUWPHQW WKDW LQ RQH ZD\ RU DQRWKHU FROODERUDWHG WR PDNH WKLV ZRUN SRVVLEOH LLL

PAGE 4

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f LY

PAGE 5

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

PAGE 6

$EVWUDFW RI 'LVVHUWDWLRQ 3UHVHQWHG WR WKH *UDGXDWH 6FKRRO RI WKH 8QLYHUVLW\ RI )ORULGD LQ 3DUWLDO )XOILOOPHQW RI WKH 5HTXLUHPHQWV IRU WKH 'HJUHH RI 'RFWRU RI 3KLORVRSK\ 02/(&8/$5 &+$5$&7(5,=$7,21 2) &,7586 75,67(=$ 9,586 *(1(6 $1' 7+(,5 86( ,1 3/$17 75$16)250$7,21 %\ 9LFHQWH -RV )HEUHV5RGULJXH] $XJXVW &KDLUPDQ &/ 1LEOHWW 0DMRU 'HSDUWPHQW 3ODQW 3DWKRORJ\ 1XFOHRWLGH VHTXHQFH DQDO\VLV RI FLWUXV WULVWH]D FORVWHURYLUXV &79f UHYHDOHG WKH SUHVHQFH RI SRVVLEOH RSHQ UHDGLQJ IUDPHV 25)Vf 7KH 25) LPPHGLDWHO\ XSVWUHDP RI WKH FRDW SURWHLQ &3f JHQH HQFRGHV D SURWHLQ RI FDOFXODWHG 0: RI N'D Sf 7KH GHGXFHG DPLQR DFLG VHTXHQFH LQGLFDWHG WKDW WKLV JHQH SURGXFW LV KRPRORJRXV WR WKH &3 b VLPLODULW\f 7ZR RWKHU JHQHV ORFDWHG WRZDUG WKH n HQG RI WKH &79 JHQRPH HQFRGH SURWHLQV RI SOf DQG Sf N'D UHVSHFWLYHO\ 'HGXFHG DPLQR DFLG VHTXHQFH FRPSDULVRQV ZLWK SURWHLQ GDWDEDVHV GLG QRW UHYHDO DQ\ VLJQLILFDQW UHODWLRQVKLS WKDW PLJKW LQGLFDWH WKH SRVVLEOH IXQFWLRQ RI WKHVH SURWHLQV 7KH REMHFWLYHV RI WKLV UHVHDUFK ZHUH f WR GHWHUPLQH LI S S DQG SO SURWHLQV ZHUH H[SUHVVHG LQ &79LQIHFWHG FLWUXV WLVVXH f WR LGHQWLI\ WKH VHTXHQFH YDULDELOLW\ RI WKH S 25) DPRQJ &79 VWUDLQV ZLWK GLIIHUHQW ELRORJLFDO SURSHUWLHV DQG WR YL

PAGE 7

GHWHUPLQH DQ\ SRVVLEOH GLIIHUHQFHV WKDW FRXOG FRUUHODWH ZLWK YLUXOHQFH DQG f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b 7KH GHGXFHG DPLQR DFLG VHJXHQFH KRPRORJLHV ZHUH b RU KLJKHU 3K\ORJHQHWLF DQDO\VLV VKRZHG PLOG DQG VHYHUH TXLFN GHFOLQH LVRODWHV JURXSLQJ VHSDUDWHO\ $JUREDFWHULXPPHGLDWHG SODQW WUDQVIRUPDWLRQ H[SHULPHQWV ZHUH SHUIRUPHG XVLQJ S DQG S JHQHV $SSUR[LPDWHO\ b RI WKH DGYHQWLWLRXV VKRRWV REWDLQHG RQ VHOHFWLRQ PHGLD ZLWK NDQDP\FLQ ZHUH *86 SRVLWLYH %HWZHHQ DQG b RI WKH *86 SRVLWLYH VKRRWV ZHUH SRVLWLYH XVLQJ 3&5 DQDO\VLV ZLWK SULPHUV VSHFLILF WR S RU S VXJJHVWLQJ WKDW VRPH SODQWV DUH WUDQVIRUPHG ZLWK HDFK RI WKH JHQHV YLL

PAGE 8

&+$37(5 ,1752'8&7,21 &LWUXV WULVWH]D ZDV LQLWLDOO\ UHFRJQL]HG DV D GHFOLQH GLVHDVH RI FLWUXV VFLRQV SURSDJDWHG RQ VRXU RUDQJH &LWUXV DXUDQWLXP / f URRWVWRFN /HH DQG 5RFKD3HD f 7KLV GLVHDVH FDXVHG E\ FLWUXV WULVWH]D FORVWHURYLUXV &79f LV RQH RI WKH PDMRU GLVHDVHV DIIHFWLQJ FLWUXV DQG DQ H[FHOOHQW H[DPSOH RI DQ DJULFXOWXUDO SUREOHP FUHDWHG E\ PDQ %DU-RVHSK HW DO f &79 LV QRZ NQRZQ WR FDXVH D YDULHW\ RI V\PSWRPV GHSHQGLQJ RQ WKH YLUXV VWUDLQ DQG KRVW 3HUKDSV WKH PRVW HFRQRPLFDOO\ LPSRUWDQW DQG REYLRXV &79 V\PSWRP LV WKH GHFOLQH RI VZHHW RUDQJH >& VLQHQVLV /f 2VEHFN@ JUDSHIUXLW & SDUDGLVL 0DFIf DQG PDQGDULQ & UHWLFXODWD %ODQFRf JUDIWHG RQ VRXU RUDQJH URRWVWRFN *DUQVH\ HW DO %DU-RVHSK DQG /HH f 6WHP SLWWLQJ LQ OLPHV >& DXUDQWLIROLD &KULVWPf 6ZLQJOH@ JUDSHIUXLW DQG VRPH VZHHW RUDQJHV LV DQRWKHU LPSRUWDQW UHDFWLRQ DQG D OLPLWLQJ IDFWRU WR SURGXFWLRQ LQ SDUWV RI %UD]LO 6RXWK $IULFD DQG $XVWUDOLD *DUQVH\ HW DO %DU-RVHSK DQG /HH /HH DQG 5RFKD3HD f ,Q DGGLWLRQ VRPH &79 VWUDLQV FDQ LQGXFH VHHGOLQJ \HOORZV ZKHQ LQRFXODWHG WR VRXU RUDQJH JUDSHIUXLW DQG OHPRQ & OLPQ %XUPIf VHHGOLQJV 7KH LPSRUWDQFH RI WKLV V\PSWRP LV QRW NQRZQ H[FHSW IRU &79 VWUDLQ GLIIHUHQWLDWLRQ EHFDXVH LW

PAGE 9

QRUPDOO\ LV QRW REVHUYHG LQ WKH ILHOG *DUQVH\ HW DO %DU-RVHSK DQG /HH f 6RPH PLOG VWUDLQV RQ WKH RWKHU KDQG GR QRW LQGXFH DQ\ QRWLFHDEOH V\PSWRPV LQ PRVW RI WKH FXOWLYDWHG YDULHWLHV *DUQVH\ HW DO %DU-RVHSK DQG /HH f 6HYHUDO VWUDLQV FDQ EH SUHVHQW LQ WKH VDPH WUHH DV KDV EHHQ GHWHUPLQHG E\ FURVV SURWHFWLRQ H[SHULPHQWV 3RZHOO HW DO f DQG GRXEOHVWUDQGHG 51$ DQDO\VLV 0RUHQR HW DO f 7KLV PDNHV WKH XVH RI ELRORJLFDO PHWKRGV VXFK DV FURVVSURWHFWLRQ PRUH GLIILFXOW WR FRQWURO WKH GLVHDVH DOWKRXJK WKH\ DUH VWLOO SRVVLEOH &RVWD DQG 0XOOHU f &79 LV SUHVHQW LQ PRVW DUHDV RI WKH ZRUOG ZKHUH FLWUXV LV JURZQ ,Q 6RXWK $PHULFD LW ZDV ILUVW GHVFULEHG LQ WKH V /HVV WKDQ WZR GHFDGHV ODWHU &79 FDXVHG WKH GHVWUXFWLRQ RI PRVW RI WKH FLWUXV LQGXVWULHV LQ $UJHQWLQD %UD]LO DQG 8UXJXD\ NLOOLQJ DERXW PLOOLRQ WUHHV SODQWHG RQ WKH VXVFHSWLEOH VRXU RUDQJH URRWVWRFN 0XOOHU DQG &RVWD f 7KH VSUHDG RI WKH GLVHDVH DQG LWV LPSDFW LV LQFUHDVHG E\ WKH SUHVHQFH RI LWV PRVW HIILFLHQW YHFWRU 7R[RSWHUD FLWULFLGD .LUNDOG\f WKH EURZQ FLWUXV DSKLG $ ZHOO GRFXPHQWHG FDVH RFFXUUHG LQ 9HQH]XHOD 7KH GLVHDVH ZDV UHSRUWHGO\ SUHVHQW LQ WKH FRXQWU\ LQ WKH PLGnV ZLWKRXW KDYLQJ D PDMRU LPSDFW ,Q 7 FLWULFLGD ZDV ILUVW IRXQG LQ 9HQH]XHOD LQ UHJLRQV ERUGHULQJ ZLWK &RORPELD DQG %UD]LO *HUDXG f 7ZR \HDUV ODWHU LW ZDV ZLGHVSUHDG WKURXJKRXW WKH FRXQWU\ ,Q WKH ILUVW RXWEUHDN RI &79 ZDV UHSRUWHG DQG VLQFH WKHQ DW OHDVW

PAGE 10

PLOOLRQ FLWUXV WUHHV RQ VRXU RUDQJH URRWVWRFN KDYH GLHG IURP WULVWH]D 0HQGW f ,Q )ORULGD DSSUR[LPDWHO\ PLOOLRQ WUHHV RI VZHHW RUDQJH JUDSHIUXLW DQG PDQGDULQ DUH SODQWHG RQ WKH VXVFHSWLEOH VRXU RUDQJH URRWVWRFN *DUQVH\ f &79 KDV EHHQ SUHVHQW LQ )ORULGD VLQFH WKH nV DQG ERWK PLOG DQG GHFOLQH LQGXFLQJ VWUDLQV KDYH EHHQ UHSRUWHG %UODQVN\ HW DO f ,Q DGGLWLRQ PLOOLRQ WUHHV RI JUDSHIUXLW DUH DOVR VXVFHSWLEOH WR VWHP SLWWLQJ VWUDLQV ZKLFK KDYH QRW EHHQ GHWHFWHG \HW LQ )ORULGD *DUQVH\ f 7KH DSKLG 7 FLWULFLGD LV VWLOO QRW SUHVHQW DOWKRXJK RWKHU OHVV HIILFLHQW YHFWRUV VXFK DV $SKLV JRVV\SLL *ORYHU DQG $ FLWULFROD YDQ GHU *RRW DUH ZLGHVSUHDG LQ )ORULGD +RZHYHU 7 FLWULFLGD KDV FRQWLQXHG LWV QRUWKZDUG PRYHPHQW IURP 6RXWK $PHULFD ,Q UHFHQW VXUYH\V LQ &HQWUDO $PHULFD DQG WKH &DULEEHDQ WKH DSKLG ZDV UHSRUWHG LQ 1LFDUDJXD /DVWUD HW DO f DQG &XED
PAGE 11

EXGGLQJ DQG JUDIWLQJ %DU-RVHSK DQG /HH f 0HFKDQLFDO WUDQVPLVVLRQ LV GLIILFXOW DQG RQO\ SRVVLEOH ZKHQ FRQFHQWUDWHG SUHSDUDWLRQV DUH XVHG DQG VODVK LQRFXODWHG LQWR WKH VWHPV RI \RXQJ FLWUXV SODQWV *DUQVH\ HW DO f &79 LV D SKORHPOLPLWHG YLUXV ,QFOXVLRQ ERGLHV DUH REVHUYHG LQVLGH WKH SKORHP SKORHP ILEHU DQG SDUHQFK\PD FHOOV DGMDFHQW WR VLHYH WXEHV 6FKQHLGHU %UODQVN\ HW DO f 7KH\ DOVR DUH GHWHFWHG E\ LPPXQRIOXRUHVFHQFH XVLQJ DQWLERGLHV WR &79 FRDW SURWHLQ &3f %UODQVN\ HW DO f LQGLFDWLQJ WKDW WKH LQFOXVLRQ ERGLHV FRQWDLQ YLUXV SDUWLFOHV 7KHVH YLUXV SDUWLFOHV DUH IOH[XRXV QP ORQJ DQG QP LQ GLDPHWHU %DU-RVHSK DQG /HH f 7KH JHQRPH RI &79 LV D VLQJOHVWUDQGHG SRVLWLYH VHQVH 51$ QXFOHRWLGHV LQ OHQJWK %DU-RVHSK DQG /HH .DUDVHY HW DO f 6HJXHQFLQJ RI WKH HQWLUH JHQRPH RI WKH )ORULGD VHYHUH JXLFN GHFOLQH VWUDLQ 7 LQGLFDWHV WKH SUHVHQFH RI SRVVLEOH RSHQ UHDGLQJ IUDPHV 25)Vf )LJ f FRGLQJ IRU SURWHLQV 3DSSX HW DO .DUDVHY HW DO f ,Q WKH n WR n GLUHFWLRQ WKH ILUVW 25) SRWHQWLDOO\ FRGHV IRU D SRO\SURWHLQ ZLWK DQ HVWLPDWHG 0: RI N'D WKDW HQFRGHV WKH SXWDWLYH GRPDLQV IRU WZR SDSDLQOLNH SURWHDVHV D PHWK\OWUDQVIHUDVH DQG D KHOLFDVH .DUDVHY HW DO f 7KLV SRO\SURWHLQ LV VSHFXODWHG WR XQGHUJR DXWRSURWHRO\WLF FOHDYDJH WR SURGXFH WKUHH IUDJPHQWV RI DQG N'D UHVSHFWLYHO\ .DUDVHY HW DO f 7KH VHFRQG 25) HQFRGHV D SXWDWLYH 51$ GHSHQGHQW 51$ SRO\PHUDVH RI D FDOFXODWHG 0: RI N'D WKDW LV

PAGE 12

3RO S S SO S +(/ 3 S S &3 SL S )LJXUH *HQRPLF RUJDQL]DWLRQ RI WKH FLWUXV WULVWH]D YLUXV JHQRPH DFFRUGLQJ WR 3DSSX HW DO f DQG .DUDVHY HW DO f 7KH 25)V DUH UHSUHVHQWHG DV UHFWDQJOHV DQG WKH SXWDWLYH JHQH SURGXFWV DUH LQGLFDWHG +(/ KHOLFDVH 3RO SRO\PHUDVH &3 FRDW SURWHLQf P

PAGE 13

SURSRVHG WR EH H[SUHVVHG E\ ULERVRPDO IUDPHVKLIW RI WKH ILUVW 25) UHVXOWLQJ LQ D SRO\SURWHLQ RI N'D .DUDVHY HW DO f 7KLV SRO\SURWHLQ DOVR ZRXOG XQGHUJR DXWRSURWHRO\WLF FOHDYDJH SURGXFLQJ D N'D IUDJPHQW ZLWK GRPDLQV IRU PHWK\OWUDQVIHUDVH KHOLFDVH DQG 51$ SRO\PHUDVH .DUDVHY HW DO f 7KH WKLUG 25) SRWHQWLDOO\ HQFRGHV D N'D Sf SURWHLQ .DUDVHY HW DO f RI XQNQRZQ IXQFWLRQ IROORZHG E\ D VPDOO 25) HQFRGLQJ D N'D SURWHLQ ZLWK D KLJKO\ K\GURSKRELF GRPDLQ 'ROMD HW DO f 25)V DQG FRGH IRU SURWHLQV RI Sf DQG Sf N'D UHVSHFWLYHO\ 3DSSX HW DO f 6HTXHQFH FRPSDULVRQV LQGLFDWH FRQVHUYDWLRQ RI VHYHUDO PRWLIV EHWZHHQ S DQG WKH KVS JURXS RI KHDW VKRFN SURWHLQV 3DSSX HW DO f 6LPLODUO\ S FRQWDLQV D & SUR[LPDO GRPDLQ WKDW LV SUHVHQW LQ DQRWKHU JURXS RI KHDW VKRFN SURWHLQV WKH KVS JURXS 3DSSX HW DO f 7KHVH WZR SURWHLQV KDYH EHHQ GHWHFWHG LQ &79LQIHFWHG FLWUXV WLVVXH 66 3DSSX SHUVRQDO FRPPXQLFDWLRQf 7KH IROORZLQJ 25)V DQG HQFRGH SURWHLQV RI FDOFXODWHG 0: RI Sf DQG N'D UHVSHFWLYHO\ 3DSSX HW DO f 25) KDV EHHQ LGHQWLILHG DV WKH &3 JHQH 6HNL\D HW DO 3DSSX HW DO Ef %DVHG RQ WKHLU GHGXFHG DPLQR DFLG VHTXHQFH S VKRZV b VLPLODULW\ ZLWK WKH &3 3DSSX HW DO f 25)V WR SRWHQWLDOO\ HQFRGH SURWHLQV ZLWK FDOFXODWHG 0: RI SOf SOf Sf DQG Sf N'D UHVSHFWLYHO\ 3DSSX HW DO f 7KH IXQFWLRQV RI WKHVH SURWHLQV DUH QRW NQRZQ DQG GHGXFHG

PAGE 14

DPLQR DFLG VHTXHQFH FRPSDULVRQV ZLWK SURWHLQ GDWDEDVHV GR QRW UHYHDO DQ\ VLJQLILFDQW UHODWLRQVKLSV 3DSSX HW DO f 2QO\ S VHHPV WR FRQWDLQ D PRWLI VLPLODU WR VRPH 51$ELQGLQJ SURWHLQV 'ROMD HW DO f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f ,Q WKLV UHVHDUFK WKH H[SUHVVLRQ RI WKH &79 JHQRPH ZDV VWXGLHG WR LQFUHDVH WKH LQIRUPDWLRQ DYDLODEOH RQ WKLV YLUXV 7KH VSHFLILF REMHFWLYHV RI WKLV UHVHDUFK ZHUH DV IROORZV f 7R GHWHUPLQH LI WKH S S DQG SO SURWHLQV ZHUH H[SUHVVHG LQ &79LQIHFWHG FLWUXV WLVVXH f 7R LGHQWLI\ WKH VHTXHQFH YDULDELOLW\ RI WKH S 25) DPRQJ &79 VWUDLQV ZLWK GLIIHUHQW ELRORJLFDO SURSHUWLHV DQG GHWHUPLQH DQ\ SRVVLEOH GLIIHUHQFHV WKDW FRXOG FRUUHODWH ZLWK YLUXOHQFH f 7R WUDQVIRUP FLWUXV SODQWV ZLWK WKH S DQG S JHQHV LQ DQ DWWHPSW WR JHQHWLFDOO\ HQJLQHHU &79 UHVLVWDQFH LQ FLWUXV

PAGE 15

&+$37(5 '(7(&7,21 2) 7+( ,1 9,92 (;35(66,21 2) 7+( 3 3 $1' 3 3527(,16 ,QWURGXFWLRQ 7KHUH LV VLJQLILFDQW UHVHPEODQFH EHWZHHQ WKH JHQRPLF RUJDQL]DWLRQ RI &79 DQG WZR RWKHU PHPEHUV RI WKH FORVWHURYLUXV JURXS EHHW \HOORZV YLUXV %<9f DQG WKH ELSDUWLWH OHWWXFH LQIHFWLRXV \HOORZV YLUXV /,<9f &79 25)V KHOLFDVHf SRO\PHUDVHf VPDOO K\GURSKRELF SURWHLQf SKVSf SKVSf S f§&3 KRPRORJXHf DQG &3f KDYH KRPRORJRXV 25)V LQ %<9 DQG /,<9 LQ VLPLODU SRVLWLRQV 'ROMD HW DO .ODDVVHQ HW DO 3DSSX HW DO f ,QWHUHVWLQJO\ IRU /,<9 WKH &3 KRPRORJXH LV ORFDWHG GRZQVWUHDP RI WKH &3 'ROMD HW DO .ODDVVHQ HW DO f $QRWKHU &79 25) S VKRZV VLPLODULW\ LQ WKH GHGXFHG DPLQR DFLG VHTXHQFH RQO\ WR S WKH n WHUPLQDO 25) LQ %<9 3DSSX HW DO f 7KH &79 25)V Sf SOf DQG Sf GR QRW KDYH KRPRORJXHV LQ WKH WZR RWKHU FORVWHURYLUXVHV 7KH IDFW WKDW VRPH RI WKH &79 25)V KDYH KRPRORJXHV LQ %<9 DQG /,<9 LV D VWURQJ LQGLFDWLRQ EXW GRHV QRW GHPRQVWUDWH WKDW WKRVH 25)V DUH IXQFWLRQDO LQ YLYR 7KH GHWHFWLRQ RI VXEJHQRPLF 51$V IRU RI WKH 25)V LQFOXGLQJ S S DQG SO +LOI HW DO f DOVR VXSSRUWV WKDW WKH\ DUH H[SUHVVHG

PAGE 16

LQ YLYR 7KH ILQDO HYLGHQFH KRZHYHU LV WKH GHWHFWLRQ RI WKH SURWHLQ SURGXFWV LQ LQIHFWHG WLVVXH ,Q RUGHU WR LQYHVWLJDWH ZKHWKHU S S DQG SO DUH H[SUHVVHG LQ &79 LQIHFWHG SODQWV WKH JHQHV ZHUH VHSDUDWHO\ FORQHG LQWR (VFKHULFKLD FROL H[SUHVVLRQ YHFWRUV 7KH H[SUHVVHG SURWHLQV ZHUH XVHG WR UDLVH SRO\FORQDO DQWLERGLHV WR SUREH IRU WKH SURWHLQV LQ &79LQIHFWHG FLWUXV WLVVXH 0DWHULDOV DQG 0HWKRGV 0DWHULDOV 5HDJHQWV ZHUH REWDLQHG IURP )LVKHU 6FLHQWLILF 3LWWVEXUJK 3$f RU 6LJPD &KHPLFDO &RPSDQ\ 6W /RXLV 02f (Q]\PHV DQG ODPEGD '1$ PDUNHU ZHUH SXUFKDVHG IURP 3URPHJD &RUSRUDWLRQ 0DGLVRQ :,f 6RPH UHVWULFWLRQ HQ]\PHV DJDURVH DQG ORZ PHOWLQJ SRLQW /03f DJDURVH ZHUH IURP *,%&2 %5/ *DLWKHUVEXUJ 0'f 3KHQRO HTXLOLEUDWHG ZLWK 0 7ULV+&O S+!f 7 SRO\QXFOHRWLGH NLQDVH G173V DQG 6HTXHQDVH 9HUVLRQ '1$ VHTXHQFLQJ NLW ZHUH IURP $PHUVKDP $UOLQJWRQ +HLJKWV ,/f 7KH QXFOHRWLGH >D6@G$73 DQG WKH 5HQDLVVDQFH FKHPLOXPLQHVFHQW GHWHFWLRQ NLW IRU :HVWHUQ EORWV ZHUH IURP 'X3RQW 1(1 5HVHDUFK 3URGXFWV %RVWRQ 0$f 3URWHLQ VWDQGDUGV 6'6 DQG 6'6 ZHUH IURP 6LJPD &KHPLFDO &RPSDQ\ (OHFWURSKRUHVLV DSSDUDWXV IRU VPDOO JHOV [ FPf 0LQL 3527($1 ,, GXDO VODE FHOO ZDV IURP %LR5DG 5LFKPRQG &$f (OHFWURSKRUHWLF WUDQVIHU XQLW 0LQL 7UDQV%ORW ZDV DOVR IURP %LR5DG 7KH ODUJHU HOHFWURSKRUHVLV XQLW [ FPf 9$*(

PAGE 17

ZDV IURP 6WUDWDJHQH /D -ROOD &$f 7KH S(7+E H[SUHVVLRQ YHFWRU ZDV NLQGO\ SURYLGHG E\ '5 0F&DUW\ 7KH H[SUHVVLRQ FORQH IRU WKH &3 ZDV NLQGO\ SURYLGHG E\ 0/ .HUHPDQH 3ODQW 0DWHULDO 0H[LFDQ OLPH >& DXUDQWLIROLD &KULVWPf 6ZLQJOH@ SODQWV LQIHFWHG ZLWK &79 7 ZHUH PDLQWDLQHG LQ JUHHQKRXVHV DW WKH 8QLYHUVLW\ RI )ORULGD *DLQHVYLOOH )/f 6DPSOHV RI FLWUXV LQIHFWHG ZLWK FLWUXV ULQJVSRW YLUXV FLWUXV YDULHJDWLRQ YLUXV FLWUXV OHDI UXJRVH YLUXV SVRURVLV $ DQG FRQFDYH JXP ZHUH NLQGO\ SURYLGHG E\ 5) /HH &ORQLQJ RI &79 25)V LQWR 38& ([WUDFWLRQ RI YLUDO 51$ WHPSODWHV 7RWDO QXFOHLF DFLGV ZHUH H[WUDFWHG IURP FLWUXV OHDYHV LQIHFWHG ZLWK WKH )ORULGD VHYHUH TXLFN GHFOLQH VWUDLQ 7 XVLQJ WKH SURFHGXUH GHVFULEHG E\ 3DSSX HW DO Ff $SSUR[LPDWHO\ FP RI WLVVXH ZDV JURXQG LQ D PLFURFHQWULIXJH WXEH ZLWK D VWHULOH EOXQW VWLFN [ PPf LQ OLTXLG QLWURJHQ 7KH VDPSOHV ZHUH WKDZHG LQ PO RI H[WUDFWLRQ EXIIHU > 0 7ULV+&O S+ P0 HWK\OHQHGLDPLQHWHWUDDFHWLF DFLG ('7$f DQG b VRGLXP GRGHF\O VXOIDWH 6'6f@ $Q HTXDO YROXPH POf RI D PL[WXUH RI SKHQRO FKORURIRUP LVRDP\O DOFRKRO f ZHUH DGGHG IROORZHG E\ YRUWH[LQJ IRU D IHZ VHFRQGV 7KH VDPSOHV ZHUH LQFXEDWHG DW r& IRU PLQ DQG WKHQ FHQWULIXJHG DW USP IRU PLQ DW URRP WHPSHUDWXUH 7KH DTXHRXV XSSHU SKDVH ZDV VDYHG

PAGE 18

7KH WHPSODWH ZDV IXUWKHU SXULILHG XVLQJ 6HSKDGH[ VSLQFROXPQ FKURPDWRJUDSK\ DFFRUGLQJ WR WKH SURFHGXUH GHVFULEHG LQ 6DPEURRN HW DO f 7KH FROXPQ D PO V\ULQJH EDUUHOf FRQWDLQLQJ K\GUDWHG VWHULOH 6HSKDGH[ ZDV HTXLOLEUDWHG E\ ZDVKLQJ WKUHH WLPHV ZLWK ILO RI VWHULOH 7( EXIIHU P0 7ULV+&O S+ P0 ('7$f DQG , RI H[WUDFW DSSOLHG WR LW 7KH FROXPQ ZDV FHQWULIXJHG DW OJ IRU PLQ DW URRP WHPSHUDWXUH DQG WKH HIIOXHQW FROOHFWHG LQ D PLFURFHQWULIXJH WXEH 7KH HIIOXHQW ZDV XVHG LPPHGLDWHO\ IRU UHYHUVH WUDQVFULSWLRQ FRXSOHG ZLWK SRO\PHUDVH FKDLQ UHDFWLRQ 573&5f DPSOLILFDWLRQ RU TXLFNO\ IUR]HQ LQ OLTXLG QLWURJHQ DQG VWRUHG DW r& IRU ODWHU XVH 3&5 DPSOLILFDWLRQ 7KH &79 JHQHV ZHUH DPSOLILHG XVLQJ 57 3&5 6DLNL HW DO f DQG VSHFLILF SULPHUV IRU HDFK JHQH 7DEOH VKRZV WKH SULPHUV GHULYHG IURP WKH 7 VHTXHQFH 3DSSX HW DO f XVHG WR DPSOLI\ HDFK RI WKH JHQHV 7KH 573&5 UHDFWLRQV ZHUH SHUIRUPHG VXFFHVVLYHO\ LQ D VLQJOH PLFURFHQWULIXJH WXEH LQ D WRWDO YROXPH RI Q ZLWK WKH IROORZLQJ FRPSRQHQWV P0 7ULV+&O S+ DW r&f P0 .& b 7ULWRQ ; P0 GLWKLRWKUHLWRO '77f P0 0J&O P0 G$73 P0 G&73 P0 G*73 P0 G773 51DVLQ Xf $09 UHYHUVH WUDQVFULSWDVH Xf 7DT SRO\PHUDVH Xf DQG SULPHUV DW D ILQDO FRQFHQWUDWLRQ HDFK RI S0 1RUPDOO\ WR [ RI WKH SXULILHG WHPSODWH ZHUH XVHG LQ HDFK UHDFWLRQ ,W ZDV SUHYLRXVO\ GHQDWXUDWHG DW r& IRU PLQ DQG JXLFN FKLOOHG RQ LFH IRU D IHZ PLQ 7KH 573&5

PAGE 19

7DEOH 'HVFULSWLRQ RI WKH SULPHUV XVHG FORQLQJ &79 25)V S S DQG SO IRU 25) 35,0(5 7PE r&f S n $$*&77&7$*$$&&$7**&$**77$7$&$*7$& n n &7$7$$*7$&77$&&&$$$7& n S n $$*&77&7$*$$&&$7*&*$*&77$&777$*7* n n &7$&$&*&$$*$7**$*$ n SO n $$*&77&7$*$$&&$7*7&$**&$*&77*** n n &7$$*7&$&*&7$$$&$$$* n D 8SSHU VHTXHQFH LV WKH JHQRPH VHQVH SULPHU /RZHU VHTXHQFH LV WKH JHQRPH DQWLVHQVH SULPHU %ROG OHWWHUV DUH +LQG,,, $$*&77f DQG ;EDO 7&7$*$f UHVWULFWLRQ VLWHV 7KH VWDUW FRGRQ LV XQGHUOLQHG E 7P FDOFXODWHG PHOWLQJ WHPSHUDWXUH

PAGE 20

PL[WXUHV ZLWK WKH WHPSODWH 51$ ZHUH LQFXEDWHG LQ DQ DXWRPDWLF WKHUPRF\FOHU DW r& IRU PLQ IROORZHG E\ F\FOHV RI LQFXEDWLRQV DW r& IRU PLQ r& IRU PLQ DQG r& IRU PLQ DQG D ILQDO LQFXEDWLRQ DW r& IRU PLQ 7KH VDPH SDUDPHWHUV ZHUH XVHG IRU WKH DPSOLILFDWLRQ RI DOO WKUHH 25)V $IWHU WKH 3&5 UHDFWLRQ ZDV FRPSOHWHG D M8O DOLTXRW ZDV HOHFWURSKRUHVHG LQ D b DJDURVH JHO LQ 7%( EXIIHU P0 7ULVERUDWH P0 ('7$f WR GHWHUPLQH WKH DPSOLILFDWLRQ RI WKH '1$ IUDJPHQW RI WKH FRUUHFW VL]H /DPEGD '1$ GLJHVWHG ZLWK +LQGOOO ZDV XVHG DV D PROHFXODU ZHLJKW PDUNHU 3XULILFDWLRQ RI WKH 3&5 SURGXFWV 2QFH WKH IUDJPHQWV RI LQWHUHVW ZHUH GHWHFWHG WKH UHVW RI WKH 3&5 VDPSOHV Of ZHUH HOHFWURSKRUHVHG LQ b /03 DJDURVH LQ 7%( EXIIHU DW r& DQG WR 9 IRU DERXW K 7KH '1$ EDQGV RI LQWHUHVW ZHUH H[FLVHG IURP WKH JHO ZLWK D VWHULOH EODGH XQGHU 89 OLJKW DQG WUDQVIHUUHG WR D PLFURFHQWULIXJH WXEH 7( EXIIHU ZDV DGGHG WR D ILQDO YROXPH RI PO 6DPSOHV ZHUH WKHQ LQFXEDWHG DW r& IRU PLQ WR PHOW WKH DJDURVH DQG WKHQ H[WUDFWHG ZLWK SKHQRO SKHQRO FKORURIRUP DQG FKORURIRUP IROORZHG E\ HWKDQRO SUHFLSLWDWLRQ DFFRUGLQJ WR 6DPEURRN DW DO f 7KH ILQDO '1$ SHOOHW ZDV UHVXVSHQGHG LQ cMO RI VWHULOH GLVWLOOHG ZDWHU ,QLWLDOO\ WKH DPSOLILHG S S DQG SO JHQHV ZHUH FORQHG LQWR WKH 6PDO VLWH RI WKH S8& YHFWRU 5HVWULFWLRQ VLWHV IRU +LQGOOO DQG ;EDO ZHUH LQFOXGHG LQ WKH VHQVH SULPHUV WR IDFLOLWDWH VXEFORQLQJ LQWR WKH S(7+ H[SUHVVLRQ YHFWRU DQG

PAGE 21

WKH S9) WUDQVIRUPDWLRQ YHFWRU RQFH FORQHV ZLWK WKH FRUUHFW RULHQWDWLRQ ZHUH LGHQWLILHG 5HVWULFWLRQ GLJHVWLRQ RI SODVPLG '1$ 3XULILFDWLRQ RI S8&O IURP ( FROL '+D ZDV GRQH IURP PO RYHUQLJKW FXOWXUHV JURZQ DW r& DQG VKDNLQJ DW USP LQ [ <7 PHGLXP b EDFWRWU\SWRQH b EDFWR\HDVW H[WUDFW b 1D&O S+ f XVLQJ D PRGLILFDWLRQ RI WKH SURFHGXUH RI 6DPEURRN HW DO f 7KH RQO\ GLIIHUHQFH ZDV WKH XVH RI D KDOI YROXPH RI 0 DPPRQLXP DFHWDWH IRU WKH SUHFLSLWDWLRQ RI EDFWHULDO SURWHLQV SULRU WR WKH '1$ SUHFLSLWDWLRQ 7KH SODVPLG '1$ ZDV UHVXVSHQGHG LQ [O RI VWHULOH GLVWLOOHG ZDWHU )RU HDFK OLJDWLRQ UHDFWLRQ DSSUR[LPDWHO\ LJ RI S8& ZHUH GLJHVWHG ZLWK 6PDO DFFRUGLQJ WR WKH PDQXIDFWXUHUnV UHFRPPHQGDWLRQV LQ D ILQDO YROXPH RI O 'LJHVWLRQ ZDV SHUIRUPHG IRU WR K DQG WKH '1$ ZDV VXEVHTXHQWO\ SXULILHG XVLQJ SKHQRO SKHQRO FKORURIRUP DQG FKORURIRUP H[WUDFWLRQV DQG SUHFLSLWDWHG ZLWK HWKDQRO 6DPEURRN HW DO f /LJDWLRQ UHDFWLRQ )RU OLJDWLRQ RI WKH 3&5 SURGXFWV LQWR WKH 6PDO GLJHVWHG S8& WKH SXULILHG 3&5 SURGXFWV [ f ZHUH ILUVW WUHDWHG ZLWK XQLWV RI '1$ SRO\PHUDVH .OHQRZ IUDJPHQWf LQ D ILQDO YROXPH RI LO FRQWDLQLQJ P0 7ULV+&O S+ DW r&f P0 0J&O P0 1D&O P0 G173V ,QFXEDWLRQ ZDV IRU PLQ DW URRP WHPSHUDWXUH PLQ DW r& DQG PLQ DW r& 7KH 3&5 SURGXFWV ZHUH WKHQ WUHDWHG ZLWK SKHQRO SKHQRO FKORURIRUP FKORURIRUP DQG SUHFLSLWDWHG ZLWK

PAGE 22

HWKDQRO 6DPEURRN HW DO f 7KH GULHG SHOOHWV ZHUH UHVXVSHQGHG LQ LO RI VWHULOH GLVWLOOHG ZDWHU 7KH WRWDO YROXPH RI UHFRYHUHG 3&5 SURGXFWV ZDV PL[HG ZLWK WKH SXULILHG 6PDO GLJHVWHG S8& IRU OLJDWLRQ LQ P0 7ULV +& S+ P0 0J&O P0 '77 P0 $73 b ERYLQH VHUXP DOEXPLQ W\SH 9 %6$f DQG 7 SRO\QXFOHRWLGH NLQDVH Xf LQ D ILQDO YROXPH RI , DQG LQFXEDWHG DW r& IRU PLQ $IWHU WKDW cM RI 7 '1$ OLJDVH Xf ZHUH DGGHG WR WKH UHDFWLRQ DQG LQFXEDWHG RYHUQLJKW DW r& RU DOWHUQDWLYHO\ LQFXEDWHG DW URRP WHPSHUDWXUH IRU K %DFWHULDO WUDQVIRUPDWLRQ $IWHU OLJDWLRQ ( FROL '+D FRPSHWHQW FHOOV ZHUH SUHSDUHG DQG WUDQVIRUPHG XVLQJ WKH FDOFLXP FKORULGH SURFHGXUH 6DPEURRN HW DO f 7UDQVIRUPHG EDFWHULD ZHUH SODWHG RQ [ <7 DJDU PHGLXP FRQWDLQLQJ [JPO RI DPSLFLOOLQ DQG JPO RI EURPR FKORURLGRO\O'JDODFWRVLGH ;JDOf DQG LQFXEDWHG RYHUQLJKW DW r& :KLWH EDFWHULDO FRORQLHV ZHUH WUDQVIHUUHG WR [ <7 DJDU SODWHV FRQWDLQLQJ DPSLFLOOLQ IRU IXUWKHU DQDO\VLV 'HWHFWLRQ RI UHFRPELQDQW SODVPLGV 7KH UDSLG GLVUXSWLRQ RI EDFWHULDO FRORQLHV PHWKRG ZDV XVHG WR GHWHUPLQH WKH SUHVHQFH RI UHFRPELQDQW SODVPLGV FRQWDLQLQJ WKH JHQH RI LQWHUHVW 6DPEURRN HW DO f 3ODVPLG VL]HV ZHUH FRPSDUHG LQ b DJDURVH JHO HOHFWURSKRUHVLV WR D VWDQGDUG RI S8& ZLWKRXW LQVHUW WKRVH ZLWK LQVHUW PLJUDWHG VORZHU LQ WKH JHOV

PAGE 23

5HFRPELQDQW SODVPLGV ZHUH WKHQ SXULILHG IURP OLTXLG FXOWXUHV E\ WKH SURFHGXUH RI 6DPEURRN HW DO f 7R FRQILUP WKH SUHVHQFH RI DQ LQVHUW DQG GHWHUPLQH LWV RULHQWDWLRQ DSSUR[LPDWHO\ [J RI SODVPLG '1$ ZHUH GLJHVWHG ZLWK X RI +LQGOOO IROORZLQJ WKH PDQXIDFWXUHUnV LQVWUXFWLRQV &ORQHV ZLWK VHQVH RULHQWDWLRQ LQ S8& ZHUH XVHG IRU VXEFORQLQJ LQWR WKH H[SUHVVLRQ YHFWRU 6XEFORQLQJ RI &79 25)V LQ WKH ([SUHVVLRQ 9HFWRU 7KH SODVPLG S(7+E 0F&DUW\ HW DO f ZDV XVHG IRU H[SUHVVLRQ RI WKH &79 JHQHV S S DQG SO 7KH LQVHUWV ZHUH UHFRYHUHG IURP S8& E\ GLJHVWLRQ ZLWK +LQGOOO DQG (FR5, $SSUR[LPDWHO\ [J RI SODVPLG '1$ ZHUH GLJHVWHG ZLWK +LQGOOO Xf DQG (FR5, Xf LQ D ILQDO YROXPH RI c[O LQ P0 7ULV+&O S+ P0 0J&O P0 1D&O P0 '77 IRU WR K DW r& 7KH GLJHVWLRQ SURGXFWV ZHUH HOHFWURSKRUHVHG LQ D b /03 DJDURVH JHO DQG WKH LQVHUWV SXULILHG DV GHVFULEHG DERYH '1$ IURP S(7+E I[T SHU UHDFWLRQf ZDV DOVR GLJHVWHG ZLWK +LQGOOO Xf DQG (FR5, Xf DQG WKH OLQHDUL]HG SODVPLG VLPLODUO\ SXULILHG LQ /03 DJDURVH /LJDWLRQ DQG EDFWHULDO WUDQVIRUPDWLRQ LQWR ( FROL '+D ZHUH DV GHVFULEHG DERYH RPLWWLQJ RQO\ WKH DGGLWLRQ RI '1$ SRO\PHUDVH .OHQRZ IUDJPHQWf DQG 7 SRO\QXFOHRWLGH NLQDVH 7UDQVIRUPHG EDFWHULD ZHUH JURZQ LQ [ <7 DJDU PHGLD FRQWDLQLQJ [JPO RI DPSLFLOOLQ %DFWHULDO FRORQLHV ZHUH VFUHHQHG XVLQJ WKH UDSLG GLVUXSWLRQ RI EDFWHULDO FRORQLHV PHWKRG 6DPEURRN HW DO

PAGE 24

f &RORQLHV FRQWDLQLQJ WKH UHFRPELQDQW SODVPLG ZHUH UHSOLFDWHG IRU ODWHU XVH 3ODVPLG '1$ ZDV SXULILHG 6DPEURRN HW DO f DQG WUDQVIRUPHG LQWR ( FROL %/'(f WKH H[SUHVVLRQ KRVW IRU S(7+ SODVPLGVf FRPSHWHQW FHOOV SUHSDUHG E\ WKH FDOFLXP FKORULGH PHWKRG 6DPEURRN HW DO f 7KH EDFWHULD ZHUH SODWHG RQ /% DJDU b EDFWRWU\SWRQH b EDFWR\HDVW H[WUDFW b 1D&O S+ b DJDUf PHGLXP VXSSOHPHQWHG ZLWK [JPO RI DPSLFLOOLQ DQG WJPO RI FKORUDPSKHQLFRO '1$ 6HTXHQFLQJ 7KH LQVHUWV LQ WKH UHFRPELQDQW SODVPLGV ZHUH VHTXHQFHG WR FRQILUP WKH SUHVHQFH RI WKH &79 JHQHV DQG WKHLU FRUUHFW RULHQWDWLRQ 3ODVPLG '1$ ZDV SXULILHG DV EHIRUH IURP ( FROL '+D UHFRPELQDQW FRORQLHV JURZQ LQ [ <7 OLTXLG FXOWXUHV ZLWK DQWLELRWLFV 6DPEURRN HW DO f $SSUR[LPDWHO\ [J RI '1$ ZHUH XVHG IRU HDFK ODEHOLQJ UHDFWLRQ 7KH '1$ ZDV ILUVW GHQDWXUHG LQ r RI 1 1D2+ P0 ('7$ E\ LQFXEDWLRQ DW r& IRU PLQ DQG TXLFNO\ FKLOOHG RQ LFH IROORZHG E\ HWKDQRO SUHFLSLWDWLRQ LQ 0 VRGLXP DFHWDWH 6DPEURRN HW DO f 7KH VDPSOHV ZHUH LQFXEDWHG DW r& IRU PLQ EHIRUH FHQWULIXJDWLRQ IRU PLQ DW USP 7KH '1$ ZDV UHVXVSHQGHG LQ [O RI GLVWLOOHG ZDWHU 7KH ODEHOLQJ UHDFWLRQ ZDV SHUIRUPHG XVLQJ WKH 6HTXHQDVH 9HUVLRQ VHTXHQFLQJ NLW $PHUVKDPf DFFRUGLQJ WR WKH PDQXIDFWXUHnV LQVWUXFWLRQV 7KH WHFKQLTXH LV EDVHG RQ WKH FKDLQ WHUPLQDWLRQ PHWKRG 6DQJHU HW DO f )RU HDFK

PAGE 25

UHDFWLRQ c[& L RI >D6@G$73 ZHUH XVHG %RWK VWUDQGV RI '1$ ZHUH VHTXHQFHG XVLQJ SULPHUV VSHFLILF IRU WKH S(7+ H[SUHVVLRQ YHFWRU 6DPSOHV ZHUH HOHFWURSKRUHVHG LQ GHQDWXULQJ b SRO\DFU\ODPLGH JHOV DFU\ODPLGHELVDFU\ODPLGH O[ 7%( 0 XUHDf IRU WR K DW URRP WHPSHUDWXUH DQG : FRQVWDQW SRZHU %DFWHULDO ([SUHVVLRQ RI &79 3URWHLQV ,QGXFWLRQ 7KH H[SUHVVLRQ RI &79 JHQHV LQ ( FROL %/ '(f ZDV LQGXFHG XVLQJ LVRSURS\O'WKLRJDODFWRVLGH ,37*f DW D ILQDO FRQFHQWUDWLRQ RI 0 LQ PO RI /% OLTXLG PHGLXP VXSSOHPHQWHG ZLWK JPO DPSLFLOOLQ DQG L[J[DO FKORUDPSKHQLFRO 6DPEURRN HW DO f 7R WHVW IRU H[SUHVVLRQ RI UHFRPELQDQW SURWHLQV PO VDPSOHV ZHUH WDNHQ EHIRUH LQGXFWLRQ DQG PLQ K K DQG K DIWHU LQGXFWLRQ $OO VDPSOHV ZHUH NHSW RQ LFH LQ PLFURFHQWULIXJH WXEHV XQWLO WKH ODVW ZDV FROOHFWHG %DFWHULDO FHOOV ZHUH SHOOHWHG E\ FHQWULIXJDWLRQ DW USP IRU VHF DW URRP WHPSHUDWXUH DQG UHVXVSHQGHG LQ WL RI FUDFNLQJ EXIIHU P0 7ULV+&O S+ b 6'6 b JO\FHURO b YY PHUFDSWRHWKDQRO b EURPRSKHQRO EOXHf $IWHU UHVXVSHQVLRQ WKH VDPSOHV ZHUH ERLOHG IRU PLQ DQG VWRUHG DW r& IRU ODWHU XVH 'HWHFWLRQ %DFWHULDO SURWHLQV ZHUH VHSDUDWHG XVLQJ b 6'6SRO\DFU\ODPLGH JHO HOHFWURSKRUHVLV 6'63$*(f /DHPPOL f 6DPSOHV ZHUH HOHFWURSKRUHVHG DW 9 IRU PLQ LQ D

PAGE 26

[ FP JHO DW URRP WHPSHUDWXUH 3URWHLQ VWDQGDUGV 6'6 RU 6'6 ZHUH LQFOXGHG 3URWHLQV ZHUH VWDLQHG XVLQJ &RRPDVVLH EULOOLDQW EOXH IRU PLQ 6DPEURRN HW DO f 3URGXFWLRQ RI 3RO\FORQDO $QWLVHUD 3URWHLQ H[SUHVVLRQ 2YHUQLJKW PO FXOWXUHV RI HDFK UHFRPELQDQW S(7+E SODVPLG FRQWDLQLQJ WKH &79 S S RU SO 25)V ZHUH XVHG IRU SURGXFWLRQ RI UHFRPELQDQW SURWHLQ 7KH PHGLXP XVHG ZDV /% ZLWK DQWLELRWLFV DV GHVFULEHG DERYH DQG LQFXEDWLRQ ZDV DW r& ZLWK VKDNLQJ USPf ,Q WKH PRUQLQJ PO RI IUHVK PHGLXP ZHUH LQRFXODWHG ZLWK PO RI WKH RYHUQLJKW FXOWXUHV DQG LQFXEDWHG IRU K 7KH FXOWXUHV ZHUH WKHQ LQGXFHG ZLWK ,37* DQG LQFXEDWHG IRU PRUH K &HOOV ZHUH WUDQVIHUUHG WR D PO WXEH DQG FHQWULIXJHG DW r& IRU PLQ DQG J 7KH EDFWHULD ZHUH UHVXVSHQGHG LQ KDOI WKHLU RULJLQDO YROXPH LQ 7( EXIIHU DQG IUR]HQ RYHUQLJKW DW r& &HOOV ZHUH WKDZHG DW r& YRUWH[HG IRU D IHZ PLQXWHV DQG FHQWULIXJHG DV EHIRUH %RWK WKH VXSHUQDWDQW DQG SHOOHW ZHUH FROOHFWHG WKH ILUVW WLPH DQG VDPSOHV ZHUH WUHDWHG ZLWK FUDFNLQJ EXIIHU IRU DQDO\VLV LQ 6'63$*( WR GHWHUPLQH WKH IUDFWLRQ WKDW FRQWDLQV WKH SURWHLQ RI LQWHUHVW ,Q WKRVH FDVHV LQ ZKLFK WKH SURWHLQ ZDV LQVROXEOH S DQG SOf WKH SHOOHW ZDV UHVXVSHQGHG LQ RQH KDOI YROXPH POf RI 7( EXIIHU DQG SHOOHWHG E\ FHQWULIXJDWLRQ 7KLV ZDV UHSHDWHG PRUH WLPHV HDFK WLPH UHGXFLQJ WKH YROXPH RI 7( EXIIHU LQ KDOI 7KH ILQDO YROXPH ZDV PO

PAGE 27

)RU S PRVW RI WKH SURWHLQ ZDV VROXEOH 7KH SURWHLQ LQ WKH VXSHUQDWDQW ZDV SHOOHWHG ZLWK VDWXUDWHG DPPRQLXP VXOIDWH ILQDO FRQFHQWUDWLRQ b $XVXEHO HW DO f DQG FHQWULIXJHG J IRU PLQ DW r& 7KH SHOOHW ZDV UHVXVSHQGHG LQ 7( EXIIHU $OO WKUHH SURWHLQV ZHUH VWRUHG DW r& ,VRODWLRQ RI UHFRPELQDQW SURWHLQV 3URWHLQV RI LQWHUHVW ZHUH VHSDUDWHG IURP RWKHU EDFWHULDO FRPSRQHQWV XVLQJ b 6'6 3$*( LQ D [ FP JHO DQG D VLQJOH ZHOO PP WKLFN FRPE $SSUR[LPDWHO\ WR PJ ZHUH VHSDUDWHG LQ HDFK JHO 3URWHLQV ZHUH GHWHFWHG XVLQJ LFH FROG 0 .& +DJHU DQG %XUJHVV f ZKLFK UHDFWHG ZLWK WKH ERXQG 6'6 WR IRUP D YLVLEOH EDQG 7KH SURWHLQ EDQG ZDV H[FLVHG ZLWK D VWHULOH EODGH DQG VWRUHG DW & 3RO\FORQDO DQWLERGLHV 7KH SURWHLQ VWLOO LQ WKH SRO\DFU\ODPLGH JHOf ZDV VHQW WR &RFDOLFR %LRORJLFDO ,QF 5HDPVWRZQ 3$f IRU SURGXFWLRQ RI SRO\FORQDO DQWLERGLHV LQ UDEELWV $SSUR[LPDWHO\ WR PJ RI SURWHLQ ZHUH XVHG IRU HDFK ZHHNO\ LQMHFWLRQ $QWLERGLHV ZHUH FROOHFWHG DQG WHVWHG DIWHU IRXU ZHHNV RI LQMHFWLRQV 'HWHFWLRQ RI &79 3URWHLQV LQ ,QIHFWHG &LWUXV 7LVVXH :HVWHUQ DQG WLVVXH EORW DQDO\VLV 7KH H[SUHVVLRQ RI S S DQG SO LQ 7 &79LQIHFWHG 0H[LFDQ OLPH ZDV WHVWHG XVLQJ :HVWHUQ EORW DQDO\VLV DFFRUGLQJ WR D SUHYLRXVO\ GHVFULEHG SURFHGXUH /L HW DO f 'HWDFKHG PLGULE VHJPHQWV RI DSSUR[LPDWHO\ FP LQ OHQJWK ZHUH VOLFHG LQWR VPDOO SLHFHV

PAGE 28

XVLQJ D EODGH DQG FRPELQHG ZLWK PO RI FUDFNLQJ EXIIHU ERLOHG IRU PLQ DQG VWRUHG DW r& 3URWHLQ H[WUDFWV I[Of ZHUH VHSDUDWHG E\ b Sf RU b S DQG SOf 6'63$*( DORQJ ZLWK WKH DSSURSULDWH PROHFXODU ZHLJKW PDUNHUV 7KH SURWHLQV ZHUH WUDQVIHUUHG WR D QLWURFHOOXORVH PHPEUDQH XVLQJ HOHFWURSKRUHWLF WUDQVIHU LQ P0 7ULV P0 JO\FLQH b YY PHWKDQRO DW r& IRU K DW 9 P$ 0HPEUDQHV ZHUH SUREHG ZLWK WKH GLIIHUHQW SRO\FORQDO DQWLVHUD XVLQJ WKH FRORULPHWULF SURFHGXUH )RU SO D FKHPLOXPLQHVFHQW SURFHGXUH ZDV DOVR WHVWHG XVLQJ WKH 5HQDLVVDQFH NLW $ PRQRFORQDO DQWLERG\ 0&$f UDLVHG WR WKH 7 &3 3HUPDU HW DO f DOVR ZDV XVHG 7LVVXH EORW DQDO\VHV DOVR ZHUH WHVWHG DV D UDSLG PHWKRG IRU WKH GHWHFWLRQ RI S DQG S XVLQJ WKH SURFHGXUH RI *DUQVH\ HW DO f 7ZR LQFXEDWLRQ FRQGLWLRQV URRP WHPSHUDWXUH DQG r& IRU Kf ZHUH WHVWHG DV ZHOO DV GLOXWLRQV RI DQWLVHUXP IURP WR &HOO IUDFWLRQDWLRQ &HOO IUDFWLRQV IURP 7 &79LQIHFWHG DQG XQLQIHFWHG FLWUXV WLVVXH ZHUH SUHSDUHG E\ D PRGLILFDWLRQ RI WKH SURFHGXUHV RI *RGHIUR\&ROEXUQ HW DO f DQG $OEUHWFK HW DO f $SSUR[LPDWHO\ J RI WLVVXH ZDV JURXQG LQ OLJXLG QLWURJHQ DQG WKDZHG LQ PO RI JULQGLQJ EXIIHU P0 7ULV+&O S+ P0 .& 0 VXFURVH b JO\FHURO P0 PHUFDSWRHWKDQROf 7KH PL[WXUH ZDV LQFXEDWHG ZLWK DJLWDWLRQ IRU PLQ DW URRP WHPSHUDWXUH DQG WKHQ ILOWHUHG E\ FHQWULIXJDWLRQ DW J IRU PLQ WKURXJK D PHVK Q\ORQ

PAGE 29

FORWK IXVHG WR D PO V\ULQJH %RWK ILOWUDWH DQG VROLG UHVLGXH ZHUH FROOHFWHG 7KH OLTXLG ILOWUDWH ZDV FHQWULIXJHG DW J IRU PLQ ERWK SHOOHW IUDFWLRQ 3,f DQG VXSHUQDWDQW ZHUH FROOHFWHG 7KH VXSHUQDWDQW ZDV IXUWKHU FHQWULIXJHG DW J IRU PLQ DQG WKH SHOOHW IUDFWLRQ 3f DQG VXSHUQDWDQW IUDFWLRQ 6f ZHUH FROOHFWHG 7KH VROLG UHVLGXH IURP WKH ILUVW VWHS ZDV UHVXVSHQGHG LQ PO RI JULQGLQJ EXIIHU DQG LQFXEDWHG ZLWK DJLWDWLRQ IRU PLQ DW URRP WHPSHUDWXUH 7KH H[WUDFW ZDV ILOWHUHG DJDLQ E\ FHQWULIXJDWLRQ 7KH ILOWUDWH IUDFWLRQ 5Of ZDV FROOHFWHG DQG WKH VROLG UHVLGXH UHVXVSHQGHG LQ PO RI JULQGLQJ EXIIHU FRQWDLQLQJ b 7ULWRQ ; IRU PLQ ZLWK DJLWDWLRQ DQG VXEVHTXHQWO\ ILOWHUHG DJDLQ E\ FHQWULIXJDWLRQ 7KH ILOWUDWH IUDFWLRQ 5f ZDV FROOHFWHG DQG WKH VROLG UHVLGXH ZDV UHVXVSHQGHG LQ PO RI P0 7ULV+&O S+ b 6'6 0 8UHD b PHUFDSWRHWKDQRO DW URRP WHPSHUDWXUH IRU D ILQDO PLQ H[WUDFWLRQ ZLWK DJLWDWLRQ IROORZHG E\ ILOWUDWLRQ 7KH ILOWUDWH ZDV FROOHFWHG IUDFWLRQ 5f DQG WKH VROLG UHVLGXH GLVFDUGHG $OO IUDFWLRQV ZHUH FRPELQHG ZLWK HTXDO YROXPHV RI FUDFNLQJ EXIIHU DQG ERLOHG IRU PLQ IRU DQDO\VLV E\ :HVWHUQ EORWV 5HVXOWV &ORQLQJ RI &79 *HQHV &79 JHQHV IRU S S DQG SO ZHUH DPSOLILHG E\ 573&5 DQG ILUVW FORQHG LQWR WKH 6PDO UHVWULFWLRQ VLWH RI S8& XVLQJ EOXQWHQG OLJDWLRQ $ +LQGOOO UHVWULFWLRQ VLWH ZDV

PAGE 30

LQFOXGHG LQ WKH VHTXHQFH RI WKH VHQVH SULPHU XSVWUHDPf WR IDFLOLWDWH VXEFORQLQJ LQ S(7+E ,GHQWLILFDWLRQ RI WKH FORQDO RULHQWDWLRQ ZDV QHFHVVDU\ IRU VXEFORQLQJ LQWR WKH S(7+ YHFWRU &ORQHV ZLWK WKH (FR5, UHVWULFWLRQ VLWH RI WKH SODVPLG SRO\OLQNHU ORFDWHG GRZQVWUHDP RI WKH JHQH ZHUH XVHG IRU VXEFORQLQJ 7KHVH FORQHV ZHUH LGHQWLILHG E\ UHVWULFWLRQ GLJHVWLRQ RI WKH SODVPLG '1$ ZLWK +LQGOOO &ORQHV LQ WKH VHQVH RULHQWDWLRQ ZHUH RQO\ OLQHDUL]HG XSRQ GLJHVWLRQ ZLWKRXW UHOHDVLQJ WKH LQVHUWV ZKHUHDV FORQHV LQ WKH DQWLVHQVH RULHQWDWLRQ UHOHDVHG WKH LQVHUWV GDWD QRW VKRZQf )RU VXEFORQLQJ LQWR S(7+E DQG WR JXDUDQWHH SURSHU RULHQWDWLRQ DQG UHDGLQJ IUDPH RI WKH JHQH WZR QRQn FRPSOHPHQWDU\ UHVWULFWLRQ HQ]\PHV WKDW JHQHUDWHG FRKHVLYH WHUPLQL ZHUH XVHG +LQGOOO ORFDWHG n WR WKH VWDUW FRGRQ DQG (FR5, ORFDWHG n WR WKH VWRS FRGRQ RI HDFK &79 JHQH 7KLV VWUDWHJ\ DOVR DYRLGHG VHOIOLJDWLRQ RI WKH SODVPLG 6HTXHQFLQJ RI WKH FORQHV GDWD QRW VKRZQf FRQILUPHG RULHQWDWLRQ DQG UHDGLQJ IUDPH IRU HDFK FRQVWUXFW ([SUHVVLRQ RI 3URWHLQV &79 JHQHV ZHUH H[SUHVVHG DV IXVLRQ SURWHLQV PDGH XS RI DPLQR DFLGV RI WKH 7 FRDW SURWHLQ 6WXGLHU HW DO 0F&DUW\ HW DO f DQG WKH DPLQR DFLGV RI HDFK &79 JHQH 7KH IXVLRQ SDUW RI WKH SURWHLQV LQFUHDVHG WKH 0: E\ N'D 7KH UHVXOWLQJ SUHGLFWHG 0: RI HDFK SURWHLQ ZDV N'D Sf N'D Sf DQG N'D SOf

PAGE 31

$QDO\VLV RI EDFWHULDO SURWHLQV E\ 6'63$*( VKRZHG DFFXPXODWLRQ RI SURWHLQV RI WKH H[SHFWHG 0: LQ LQGXFHG FXOWXUHV EXW QRW LQ QRQLQGXFHG FXOWXUHV GDWD QRW VKRZQf 7KH DPRXQW RI HDFK SURWHLQ LQFUHDVHG ZLWK WLPH &79 SURWHLQV DFFXPXODWHG WR DSSUR[LPDWHO\ b RI WKH WRWDO EDFWHULDO SURWHLQ 'HWHFWLRQ RI S 7KH H[SUHVVLRQ RI S LQ &79LQIHFWHG FLWUXV ZDV WHVWHG XVLQJ :HVWHUQ EORW DQDO\VLV 7KH RSWLPDO GLOXWLRQ RI DQWLVHUXP ZDV GHWHUPLQHG HPSLULFDOO\ %HVW UHVXOWV ZHUH REWDLQHG DW D GLOXWLRQ RI %HFDXVH RI WKH DPLQR DFLG KRPRORJ\ EHWZHHQ S DQG WKH &3 WKH UHDFWLRQ RI GLIIHUHQW DQWLVHUD WR ERWK SURWHLQV ZHUH FRPSDUHG )LJXUH f 7KH S SRO\FORQDO DQWLVHUXP VKRZHG VRPH QRQVSHFLILF UHDFWLYLW\ DV GLG SUHLPPXQH VHUXPf ZLWK XQLQIHFWHG FLWUXV WLVVXH )LJ ODQH f +RZHYHU D GLVFUHWH LQWHQVH SURWHLQ EDQG RI DSSUR[LPDWHO\ N'D ZDV GHWHFWHG LQ H[WUDFWV RI &79 LQIHFWHG FLWUXV )LJ ODQH f EXW QRW LQ WKRVH RI XQLQIHFWHG FLWUXV )LJ ODQH f 7KH DQWLVHUXP DOVR GHWHFWHG ( FROLH[SUHVVHG S )LJ ODQH f ZKLFK PLJUDWHV VOLJKWO\ VORZHU WKDQ S GXH WR LWV KLJKHU 0: :KLOH WKH S SRO\FORQDO DQWLERGLHV UHDFWHG ZLWK WKH SXULILHG IXVLRQ S SURWHLQ )LJ ODQH f WKH\ GLG QRW UHDFW ZLWK D QRQn IXVLRQ ( FROLH[SUHVVHG &3 )LJ ODQH f )XUWKHUPRUH QR UHDFWLRQ ZDV REVHUYHG ZLWK SURWHLQV LQGXFHG IURP EDFWHULD FRQWDLQLQJ WKH S(7+ SODVPLG ZLWKRXW DQ LQVHUW )LJ ODQH

PAGE 32

)LJXUH :HVWHUQ EORW DQDO\VLV RI &79LQIHFWHG DQG XQLQIHFWHG FLWUXV OHDI H[WUDFWV ZLWK S SRO\FORQDO DQWLVHUXP ODQHV f DQG ZLWK 0&$ PRQRFORQDO DQWLERG\ ODQHV f /DQHV DQG S(7+E ZLWKRXW LQVHUW /DQHV DQG ( FROLH[SUHVVHG IXVLRQ S ODQH FRQWDLQV WLPHV PRUH SURWHLQ WKDQ ODQH f /DQHV DQG ( FROLH[SUHVVHG &3 /DQHV DQG XQLQIHFWHG FLWUXV H[WUDFWV /DQHV DQG &79 7LQIHFWHG FLWUXV H[WUDFWV 0ROHFXODU ZHLJKWV LQ NLORGDOWRQV DUH LQGLFDWHG RQ WKH ULJKW

PAGE 33

f )LQDOO\ QR S EDQG ZDV GHWHFWHG ZKHQ SUHLPPXQH VHUXP ZDV XVHG GDWD QRW VKRZQf LQGLFDWLQJ WKH VSHFLILFLW\ RI WKH DQWLERG\ 7R SUHFOXGH WKH SRVVLELOLW\ WKDW WKH SURWHLQ GHWHFWHG LQ LQIHFWHG WLVVXH ZDV D VWUHVVLQGXFHG SDWKRJHQHVLVUHODWHG SURWHLQ WLVVXH H[WUDFWV IURP FLWUXV SODQWV LQIHFWHG ZLWK FLWUXV ULQJVSRW YLUXV FLWUXV YDULHJDWLRQ YLUXV FLWUXV OHDI UXJRVH YLUXV SVRURVLV $ DQG FRQFDYH JXP DOVR ZHUH DQDO\]HG E\ :HVWHUQ EORW XVLQJ S DQWLVHUXP 1R S EDQG ZDV REVHUYHG LQ DQ\ RI WKH VDPSOHV GDWD QRW VKRZQf 7R IXUWKHU GHPRQVWUDWH WKH VSHFLILFLW\ RI WKH DQWLERG\ KDOI RI WKH VDPH PHPEUDQH LQ )LJ ODQHV WR f ZDV SUREHG ZLWK 0&$ 7KLV PRQRFORQDO DQWLVHUXP VSHFLILF WR WKH &3 UHDFWHG VWURQJO\ ZLWK WKH ( FROLH[SUHVVHG &3 )LJ ODQH f DQG ZLWK WKH &3 LQ WKH H[WUDFW RI &79LQIHFWHG FLWUXV WLVVXH )LJ ODQH f LQFOXGLQJ WKH ORZHU PROHFXODU ZHLJKW SURWHRO\VLV SURGXFWV RI WKH &3 6HNL\D HW DO f 1R UHDFWLRQV ZHUH REVHUYHG ZLWK SURWHLQV SURGXFHG E\ EDFWHULD FRQWDLQLQJ WKH S(7+ SODVPLG ZLWKRXW DQ LQVHUW )LJ ODQH f RU IURP H[WUDFWV RI XQLQIHFWHG FLWUXV WLVVXH )LJ ODQH f ,QWHUHVWLQJO\ 0&$ JDYH D ZHDN EXW FRQVLVWHQW UHDFWLRQ ZLWK WKH SXULILHG S IXVLRQ SURWHLQ ZKLFK FRXOG EH VHHQ RQO\ RQ WKH PHPEUDQH QRW YLVLEOH LQ )LJ ODQH f EXW QRW ZLWK S IURP LQIHFWHG WLVVXH +RZHYHU WR REWDLQ WKLV OHYHO RI UHDFWLRQ ZLWK 0&$ LW ZDV QHFHVVDU\ WR LQFUHDVH WKH

PAGE 34

FRQFHQWUDWLRQ RI S SURWHLQ E\ IROG RYHU WKH DPRXQW XVHG LQ )LJ ODQH ZKHUH LW ZDV UHDGLO\ GHWHFWHG E\ WKH S DQWLVHUXP 7R GHWHUPLQH WKH FHOOXODU ORFDOL]DWLRQ RI S FHOO IUDFWLRQV IURP &79LQIHFWHG DQG XQLQIHFWHG FLWUXV WLVVXH ZHUH SUHSDUHG DQG DVVD\HG E\ :HVWHUQ EORWV 6LPLODU SHUFHQWDJHV RI WRWDO SURWHLQ IRU HDFK IUDFWLRQ ZHUH DQDO\]HG XVLQJ S DQWLVHUXP )LJ f $OO FHOO IUDFWLRQV FRQWDLQHG S +RZHYHU PRVW RI WKH S DFFXPXODWHG LQ WKH FHOO ZDOO IUDFWLRQV 5O 5 DQG 5f )LJ ODQHV WR UHVSHFWLYHO\f ,Q DGGLWLRQ IUDFWLRQ 6 DOVR FRQWDLQHG FRQVLGHUDEOH DPRXQWV RI S )LJ ODQHV f )UDFWLRQV 3, DQG 3 VKRZHG PXFK ORZHU OHYHOV RI S )LJ ODQHV DQG UHVSHFWLYHO\f 7KHVH UHVXOWV LQGLFDWH WKDW S ZDV PRVWO\ DVVRFLDWHG ZLWK WKH FHOO ZDOO IUDFWLRQV 7KH UHDFWLRQ RI 0&$ WR WKH FHOO IUDFWLRQV ZDV DOVR WHVWHG WR FRPSDUH WKH DFFXPXODWLRQ RI &3 DQG S )LJ LQGLFDWHV WKDW PRVW RI WKH &3 DFFXPXODWHG LQ WKH FHOO ZDOO IUDFWLRQV 5O DQG 5 )LJ ODQHV DQG f ZLWK VRPH DOVR SUHVHQW LQ WKH VROXEOH SURWHLQ IUDFWLRQ 6 )LJ ODQH f )UDFWLRQV 3, DQG 3 FRQWDLQHG OHVV &3 )LJ ODQHV DQG f 7KH SDWWHUQ RI DFFXPXODWLRQ RI WKH &3 ZDV VLPLODU WR WKDW REVHUYHG IRU S 7KH S DQWLVHUXP DOVR ZDV WHVWHG XVLQJ WLVVXH EORW )LJ f 7KH RSWLPDO FRQGLWLRQV IRU VSHFLILF GHWHFWLRQ ZHUH

PAGE 35

)LJXUH :HVWHUQ EORW DQDO\VLV RI FHOO IUDFWLRQV SUHSDUHG IURP &79LQIHFWHG WLVVXH DQG SUREHG ZLWK S DQWLVHUXP /DQH XQLQIHFWHG FLWUXV WLVVXH H[WUDFW /DQH XQIUDFWLRQDWHG LQIHFWHG FLWUXV H[WUDFW /DQHV WR FRQWDLQHG IUDFWLRQV 3, 3 6 5O 5 DQG 5 UHVSHFWLYHO\ RI LQIHFWHG FLWUXV WLVVXH DV H[SODLQHG LQ 0DWHULDOV DQG 0HWKRGV /DQH LV ( FROLH[SUHVVHG S

PAGE 36

)LJXUH :HVWHUQ EORW DQDO\VLV RI FHOO IUDFWLRQV SUHSDUHG IURP &79LQIHFWHG WLVVXH DQG SUREHG ZLWK 0&$ PRQRFORQDO DQWLERG\ /DQH XQLQIHFWHG FLWUXV WLVVXH H[WUDFW /DQH XQIUDFWLRQDWHG LQIHFWHG FLWUXV H[WUDFW /DQHV WR FRQWDLQHG IUDFWLRQV 3, 3 6 5O 5 DQG 5 UHVSHFWLYHO\ RI LQIHFWHG FLWUXV WLVVXH DV H[SODLQHG LQ 0DWHULDOV DQG 0HWKRGV

PAGE 37

)LJXUH 7LVVXH EORWV SUREHG ZLWK WKH S SRO\FORQDO DQWLVHUXP $ &79LQIHFWHG FLWUXV WLVVXH % XQLQIHFWHG FLWUXV WLVVXH

PAGE 38

LQFXEDWLRQ DW r& IRU K DQG GLOXWLRQ 7KH EORWV LQGLFDWH WKDW S LV SUHVHQW LQ SKORHP WLVVXH 'HWHFWLRQ RI S 7KH RSWLPDO DQWLVHUXP FRQFHQWUDWLRQ IRU WKH DQDO\VLV RI S ZDV )LJ VKRZV WKH UHVXOWV RI WKH GHWHFWLRQ RI WKH FHOO IUDFWLRQV ZLWK S DQWLVHUXP $ SURWHLQ EDQG RI WKH H[SHFWHG VL]H ZDV SUHVHQW LQ LQIHFWHG XQIUDFWLRQDWHG WLVVXH )LJ ODQH f EXW QRW LQ XQLQIHFWHG WLVVXH )LJ ODQH f 0RVW RI WKH S ZDV LQ WKH VROXEOH SURWHLQ IUDFWLRQ 6f )LJ ODQH f &HOO ZDOO IUDFWLRQ 5 )LJ ODQH f DOVR FRQWDLQHG VRPH S )UDFWLRQV 3, 3 5 DQG 5 FRQWDLQHG OLWWOH RU QR GHWHFWDEOH S )LJ ODQHV DQG UHVSHFWLYHO\f 7KH S DQWLVHUXP ZDV QRW XVHIXO IRU GHWHFWLRQ RI WKH SURWHLQ XVLQJ WLVVXH EORWV $W KLJK DQWLVHUXP FRQFHQWUDWLRQV f WKH VHUXP ZDV UHDFWLYH ZLWK XQLQIHFWHG WLVVXH DQG DW KLJKHU GLOXWLRQV f SRVLWLYH UHDFWLRQV ZLWK &79 LQIHFWHG WLVVXH ZHUH YHU\ ZHDN 'HWHFWLRQ RI S 'HVSLWH VHYHUDO DWWHPSWV WR GHWHFW SO LQ LQIHFWHG WLVVXH QR EDQG RI WKH H[SHFWHG VL]H ZDV REVHUYHG LQ :HVWHUQ EORWV 'LOXWLRQ RI WKH DQWLVHUXP DV ORZ DV GLG QRW SURGXFH DQ\ IDYRUDEOH UHVXOWV LQ FRORULPHWULF GHWHFWLRQ GDWD QRW VKRZQf 7KH DQWLERG\ UHDFWHG KRZHYHU ZLWK IXVLRQ S SURGXFHG LQ EDFWHULD WKH VDPH SURWHLQ XVHG DV DQWLJHQ GDWD QRW VKRZQf ,W DOVR UHDFWHG ZLWK IXVLRQ SO DQG S ERWK RI

PAGE 39

)LJXUH :HVWHUQ EORW DQDO\VLV RI FHOO IUDFWLRQV SUHSDUHG IURP &79LQIHFWHG WLVVXH DQG SUREHG ZLWK S DQWLVHUXP /DQH XQLQIHFWHG FLWUXV WLVVXH H[WUDFW /DQH XQIUDFWLRQDWHG LQIHFWHG FLWUXV H[WUDFW /DQHV WR FRQWDLQHG IUDFWLRQV 3, 3 6 5O 5 DQG 5 UHVSHFWLYHO\ RI LQIHFWHG FLWUXV WLVVXH DV H[SODLQHG LQ 0DWHULDOV DQG 0HWKRGV /DQH LV  FROLH[SUHVVHG S

PAGE 40

ZKLFK FRQWDLQHG WKH VDPH 1WHUPLQDO DPLQR DFLGV 'HWHFWLRQ RI FHOO IUDFWLRQV ZLWK WKH DQWLVHUXP GLG QRW UHYHDO DQ\ EDQGV RI WKH H[SHFWHG 0: $ PRUH VHQVLWLYH FKHPLOXPLQHVFHQW GHWHFWLRQ V\VWHP ZDV DOVR XVHG ZLWKRXW SRVLWLYH UHVXOWV GDWD QRW VKRZQf 'LVFXVVLRQ 7KH OHYHOV RI EDFWHULDO H[SUHVVLRQ REWDLQHG ZLWK WKH IXVLRQ SURWHLQV ZHUH VLPLODU WR WKRVH SUHYLRXVO\ UHSRUWHG 0DUVWRQ 6WXGLHU HW DO f :LWK WKLV H[SUHVVLRQ V\VWHP LW ZDV SRVVLEOH WR VXFFHVVIXOO\ SURGXFH WKH WKUHH &79 SURWHLQV DQG SXULI\ WKHP E\ D UHODWLYHO\ VLPSOH PHWKRG 7KH DQWLVHUD SURGXFHG UHDFWHG ZLWK WKH DQWLJHQV DQG JDYH ORZ QRQn VSHFLILF UHDFWLRQV ZKHQ XVHG WR SUREH WLVVXH VDPSOHV 7KHUH ZDV YDULDWLRQ LQ WKH VROXELOLW\ RI WKH IXVLRQ SURWHLQV ZLWK S DQG SO DFFXPXODWLQJ DV LQVROXEOH SURGXFWV DQG S EHLQJ VROXEOH 7KLV GLG QRW DIIHFW WKH OHYHOV RI H[SUHVVLRQ RU WKH LVRODWLRQ RI WKH SURWHLQV 7KH SUHVHQFH RI WKH IXVLRQ SRUWLRQ VHHPHG WR VWDELOL]H VRPH RI WKH SURWHLQV LQ ( FROL $WWHPSWV WR H[SUHVV S ZLWKRXW WKLV SRUWLRQ UHQGHUHG YHU\ ORZ OHYHOV RI WKH SURWHLQ GDWD QRW VKRZQf +RZHYHU QRQIXVLRQ &3 ZDV H[SUHVVHG LQ WKH VDPH EDFWHULDO KRVW ./ 0DQMXQDWK SHUVRQDO FRPPXQLFDWLRQf 7KH RQO\ LQFRQYHQLHQFH ZLWK IXVLRQ SURWHLQV LV WKDW DQWLVHUD SURGXFHG WR GLIIHUHQW IXVLRQ SURWHLQV FURVV UHDFW ZLWK DOO WKH DQWLJHQV VLQFH WKH IXVLRQ SDUW LV LGHQWLFDO 7KLV PLJKW OLPLW WKH XVH RI WKH UHFRPELQDQW SURWHLQV DV SRVLWLYH FRQWUROV

PAGE 41

DOWKRXJK LW GRHV QRW KDYH DQ\ HIIHFW RQ WKH GHWHFWLRQ RI WKH WDUJHW SURWHLQV LQ YLYR EHFDXVH WKH\ ODFN WKH IXVLRQ SRUWLRQf 7KH UHVXOWV XVLQJ WKH S DQWLVHUXP VKRZHG WKH SUHVHQFH RI D SURWHLQ RI WKH H[SHFWHG VL]H N'Df LQ &79LQIHFWHG EXW QRW LQ XQLQIHFWHG WLVVXH $OVR WKLV SURWHLQ EDQG ZDV QRW SUHVHQW LQ WLVVXH VDPSOHV LQIHFWHG ZLWK RWKHU FLWUXV YLUXVHV 7KLV LQGLFDWHV WKDW WKH SURWHLQ GHWHFWHG LV &79VSHFLILF DQG ZDV H[SUHVVHG GXULQJ LQIHFWLRQ DQG WKDW LWV VL]H LV LQ DJUHHPHQW ZLWK WKDW SUHGLFWHG IURP WKH VHTXHQFLQJ GDWD 3DSSX HW DO f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f DQG SDUW RI WKLV UHJLRQ LV FRQVHUYHG LQ S ZKLFK PD\ H[SODLQ LWV ORZ DIILQLW\ IRU S

PAGE 42

7KH WLVVXH IUDFWLRQDWLRQ H[SHULPHQWV VKRZHG WKDW S DQG WKH &3 DUH PRVWO\ DVVRFLDWHG ZLWK WKH FHOO ZDOO DQG VROXEOH SURWHLQ IUDFWLRQV &RQYHUVHO\ LQ %<9 S WKH &3 KRPRORJXHf DQG WKH &3 ZHUH VKRZQ WR DFFXPXODWH PRVWO\ LQ WKH F\WRSODVP RU VROXEOH SURWHLQ IUDFWLRQ $JUDQRYVN\ HW DO f &RPSDULVRQ RI WKH VHTXHQFHV XSVWUHDP RI WKH VWDUW FRGRQV DQG VXUURXQGLQJ WKH LQLWLDWLRQ VLWHV IRU WKH VXEJHQRPLF 51$V RI %<9 S DQG &3 UHYHDOHG D FRQVHQVXV VHTXHQFH $JUDQRYVN\ HW DO f VXJJHVWLQJ FRQFHUWHG H[SUHVVLRQ RI ERWK JHQHV 7KLV VHTXHQFH ZDV VLPLODU WR WKRVH IRXQG LQ WKH &3V RI WREDPRYLUXVHV EURPH PRVDLF YLUXV FXFXPEHU PRVDLF YLUXV DQG DOIDOID PRVDLF YLUXV $09 $JUDQRYVN\ HW DO f &RPSDULVRQ RI WKH VHTXHQFHV XSVWUHDP WR WKH VWDUW FRGRQV RI &79 S DQG &3 GLG QRW UHYHDOHG DQ\ FRQVHQVXV VHTXHQFHV VLPLODU WR WKRVH RI %<9 RU /,<9 GDWD QRW VKRZQf 7KLV LQGLFDWHV WKDW WKH H[SUHVVLRQ RI WKHVH WZR VHW RI JHQHV LV GLIIHUHQW LQ &79 DQG %<9 5HFHQWO\ S ZDV IRXQG WR EH SDUW RI WKH %<9 YLULRQV IRUPLQJ D WHUPLQDO WDLO DW RQH HQG RI WKH SDUWLFOHV $JUDQRYVN\ f 'XH WR WKH VLPLODULWLHV EHWZHHQ WKH WZR YLUXVHV DQG WKH DSSDUHQW VWUXFWXUDO FRQVHUYDWLRQ EHWZHHQ WKH &3 KRPRORJXHV 3DSSX HW DO f LW LV SRVVLEOH WKDW S PD\ DOVR IRUP SDUW RI WKH &79 YLULRQV 7KLV LV FXUUHQWO\ XQGHU LQYHVWLJDWLRQ XVLQJ WKH S VSHFLILF DQWLVHUXP 7KH IXQFWLRQ RI S LQ &79 LV VWLOO QRW NQRZQ %HVLGHV LWV SRVVLEOH VWUXFWXUDO UROH LI S IRUPV SDUW RI WKH YLULRQV LW PLJKW DOVR EH LQYROYHG LQ SDUWLFOH DVVHPEO\

PAGE 43

$JUDQRYVN\ HW DO f 7KLV FRXOG EH WKURXJK LQWHUDFWLRQ ZLWK RWKHU YLUDO SURWHLQV LQFOXGLQJ WKH &3 DQG WKH KHDW VKRFN SURWHLQV S DQG Sf RU XQNQRZQ KRVW FRPSRQHQWV +HDW VKRFN SURWHLQV ZRUN DV FKDSHURQV KHOSLQJ LQ SURSHU IROGLQJ RI RWKHU SURWHLQV *HRUJRSRXORV f 7KH S PLJKW DOVR KDYH D UROH DV D KHOSHU FRPSRQHQW LQ WKH WUDQVPLVVLRQ RI &79 E\ LWV DSKLG YHFWRU 7KLV KDV SUHYLRXVO\ EHHQ SURSRVHG IRU %<9 S $JUDQRYVN\ HW DO %R\NR HW DO f +HOSHU FRPSRQHQWV KDYH EHHQ GHVFULEHG IRU SRW\YLUXVHV DQG FDXOLPRYLUXVHV 0DWKHZV f DOWKRXJK WKH\ GR QRW IRUP SDUW RI WKH YLULRQV +RZHYHU LQ SRW\YLUXVHV WKH &3 LV DOVR LPSRUWDQW IRU DSKLG WUDQVPLVVLRQ $WUH\D HW DO f ,QWHUHVWLQJO\ /,<9 DOVR FRQWDLQV D VLPLODU GXSOLFDWH RI WKH &3 ZLWK GHGXFHG DPLQR DFLG VHTXHQFH KRPRORJLHV WR %<9 DQG &79 &3V DQG KRPRORJXHV KRZHYHU /,<9 LV WUDQVPLWWHG E\ WKH ZKLWHIO\ %HPLVLD WDEDFL *HQQDGLXVf .ODDVVHQ HW DO f 5HODWLQJ WR WKH FHOOXODU DFFXPXODWLRQ RI S D SRVVLEOH IXQFWLRQ FRXOG EH DV D PRYHPHQW SURWHLQ DVVLVWLQJ LQ WKH VSUHDG RI WKH YLUXV EHWZHHQ FHOOV 0RYHPHQW SURWHLQV LQWHUDFW ZLWK SODVPRGHVPDWD FDXVLQJ DQ LQFUHDVH LQ WKHLU GLDPHWHU :ROI HW DO 'HUULFN HW DO :DLJPDQQ HW DO f WKHUHE\ HQDEOLQJ WKH LQWHUFHOOXODU SDVVDJH RI WKH ODUJH QXFOHLF DFLG LQ D QDNHG RU FRDWHG IRUP GHSHQGLQJ RQ WKH YLUXV 3XWDWLYH PRYHPHQW SURWHLQV RI WREDFFR PRVDLF YLUXV 'HRP HW DO f $09 *RGHIUR\&ROEXUQ HW DO f

PAGE 44

FDXOLIORZHU PRVDLF YLUXV $OEUHFKW HW DO f DQG VTXDVK OHDI FXUO JHPLQLYLUXV 3DVFDO HW DO f DFFXPXODWH LQ WKH FHOO ZDOO IUDFWLRQV 7KH REVHUYDWLRQV WKDW S DOVR DFFXPXODWHV LQ WKLV IUDFWLRQ VXJJHVW D SRVVLEOH UROH LQ YLUXV PRYHPHQW 7KH FDSDFLW\ WR ELQG 51$ LV DQRWKHU FKDUDFWHULVWLF RI PRYHPHQW SURWHLQV 'HRP HW DO &LWRYVN\ HW DO *LHVPDQ&RRNPH\HU DQG /RPPHO f 'XH WR WKH DSSDUHQW VWUXFWXUDO VLPLODULWLHV EHWZHHQ S DQG WKH &3 3DSSX HW DO f LQFOXGLQJ WKH FRQVHUYDWLRQ RI DPLQR DFLGV SUHVHQW LQ WKH &3V RI RWKHU ILODPHQWRXV YLUXVHV 'ROMD HW DO %R\NR HW DO f DQG WKH ILQGLQJ RI S DV SDUW RI WKH %<9 YLULRQV LW LV OLNHO\ WKDW S PLJKW KDYH 51$ELQGLQJ FDSDFLW\ 7KH &3 RI VRPH 51$ YLUXVHV DOVR KDYH D UROH LQ FHOOWR FHOO DQG ORQJ GLVWDQFH PRYHPHQW ,Q VRPH FDVHV OLNH FRZSHD PRVDLF FRPRYLUXV YDQ /HQW HW DO f WKH YLUXV PRYHV IURP FHOO WR FHOO DV YLULRQV ,Q RWKHU FDVHV OLNH $09 WKH &3 LV UHTXLUHG IRU PRYHPHQW EXW YLULRQV DUH QRW WUDQVSRUWHG YDQ GHU 9RVVHQ HW DO f )RU EURPH PRVDLF EURPRYLUXV WKH &3 LV QRW UHTXLUHG IRU FHOOWRFHOO PRYHPHQW EXW LV UHTXLUHG IRU ORQJ GLVWDQFH PRYHPHQW )ODVLQVNL HW DO f ,W LV SRVVLEOH WKHQ WKDW S DFWV DV D PXOWLIXQFWLRQDO SURWHLQ IRUPLQJ SDUW RI WKH YLUXV SDUWLFOHV DQG DVVLVWLQJ LQ RQH RU PRUH RI WKH IROORZLQJ IXQFWLRQV YLUXV PRYHPHQW DSKLG WUDQVPLVVLRQ RU HQFDSVLGDWLRQ

PAGE 45

8VLQJ WLVVXH EORWV S ZDV PRVWO\ SUHVHQW LQ WKH VLHYH WXEHV 7KLV LV LQ DJUHHPHQW ZLWK ZKDW KDV EHHQ IRXQG IRU WKH &3 *DUQVH\ HW DO f DQG ZLWK WKH SKORHPOLPLWHG QDWXUH RI &79 %DU-RVHSK DQG /HH f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f 2QH DOWHUQDWLYH WR H[SODLQ WKLV UHVXOW LV WKDW S SURWHLQ LV SRRUO\ LPPXQRJHQLF DQG VR WKH FRQFHQWUDWLRQ RI VSHFLILF DQWLERGLHV LQ WKH VHUXP LV ORZ %HFDXVH WKH ( FROLH[SUHVVHG S FRQWDLQV WKH IXVLRQ SRUWLRQ LW LV QRW SRVVLEOH WR FRQFOXGH DQ\WKLQJ IURP WKH UHDFWLRQ REVHUYHG ZLWK WKH DQWLVHUXP WR WKLV SURWHLQ $QRWKHU SRVVLELOLW\ LV WKDW S

PAGE 46

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f /RZ SURWHLQ H[SUHVVLRQ DQGRU H[SUHVVLRQ DW D VSHFLILF WLPH GXULQJ WKH &79 OLIH F\FOH PD\ H[SODLQ WKH LQDELOLW\ WR GHWHFW SO

PAGE 47

&+$37(5 6(48(1&( $1$/<6,6 2) 7+( 3 25) ,QWURGXFWLRQ 7KH S 25) KDV EHHQ GHVFULEHG DV D GLYHUJHG FRS\ RI WKH &3 JHQH 3DSSX HW DO f &RPSDULVRQ RI WKH GHGXFHG DPLQR DFLG VHJXHQFHV LQGLFDWHV WKDW b RI WKH DPLQR DFLGV DUH LGHQWLFDO EHWZHHQ S DQG WKH &3 DQG b RI WKH DPLQR DFLGV DUH HLWKHU LGHQWLFDO RU ZLWK VLPLODU ELRFKHPLFDO SURSHUWLHV WR WKRVH RI WKH &3 3DSSX HW DO f 7KH ILUVW UHSRUW RI WKH GLYHUJHG FRS\ RI WKH &3 DPRQJ FORVWHURYLUXVHV DQG ILODPHQWRXV 51$ YLUXVHVf ZDV IRU %<9 %R\NR HW DO f 7KH %<9 S 25) DOVR LV ORFDWHG n WR WKH &3 DQG DOO IRXU JHQHV RI &79 DQG %<9 VKRZ YDULRXV GHJUHHV RI DPLQR DFLG VHTXHQFH VLPLODULWLHV UDQJLQJ IURP b &79 S DQG %<9 &3f WR b &79 S DQG %<9 Sf %R\NR HW DO 3DSSX HW DO f 7ZR RI WKH DPLQR DFLGV FRQVHUYHG LQ WKH IRXU SURWHLQV DOVR DUH VWULFWO\ FRQVHUYHG LQ WKH &3V RI RWKHU ILODPHQWRXV SRVLWLYHVWUDQGHG 51$ YLUXVHV 'ROMD HW DO %R\NR HW DO 3DSSX HW DO f 7KRVH WZR UHVLGXHV DUH EHOLHYHG WR IRUP D VDOW EULGJH 'ROMD HW DO f VXJJHVWLQJ WKDW WKH &3V DQG WKH GLYHUJHG FRSLHV VKDUH QRW RQO\ WKH SULPDU\ VWUXFWXUH EXW H[KLELW VLPLODU SURWHLQ IROGLQJ %R\NR HW DO f

PAGE 48

'HWHFWLRQ RI VHTXHQFH YDULDELOLW\ LQ YLUDO JHQHV LV LPSRUWDQW VLQFH PXWDWLRQV FRXOG EH OLQNHG WR SDUWLFXODU ELRORJLFDO SURSHUWLHV 6LQJOH VWUDQGHG FRQIRUPDWLRQ SRO\PRUSKLVP 66&3f LV D VLPSOH DQG VHQVLWLYH PHWKRG XVHG WR GHWHFW VLQJOH QXFOHRWLGH FKDQJHV LQ SDUWLFXODU VHTXHQFHV 2ULWD HW DO f 7KLV WHFKQLTXH LV EDVHG RQ PLJUDWLRQ GLIIHUHQFHV GXULQJ HOHFWURSKRUHVLV RI VKRUW GHQDWXUHG '1$ IUDJPHQWV LQ QRQGHQDWXULQJ JHOV FDXVHG E\ GLIIHUHQFHV LQ WKHLU QXFOHRWLGH VHTXHQFHV 2ULWD HW DO 6SLQDUGL HW DO f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f DQG WKH VLOYHU VWDLQLQJ NLW ZDV IURP 6LJPD &KHPLFDO &RPSDQ\ 6W /RXLV 02f 6HTXHQFH DQDO\VLV ZDV SHUIRUPHG XVLQJ WKH SURJUDPV 6HTDLG &/867$/9 +LJJLQV HW DO

PAGE 49

f DQG :LVFRQVLQ *HQHWLF &RPSXWHU *URXS 6HTXHQFLQJ $QDO\VLV 6RIWZDUH *&*f YHUVLRQ 'HYHUHX[ HW DO f SURYLGHG E\ WKH ,QWHUGLVFLSOLQDU\ &HQWHU IRU %LRWHFKQRORJ\ 5HVHDUFK DW WKH 8QLYHUVLW\ RI )ORULGD *DLQHVYLOOH )/f 9LUXV ,VRODWHV $OO &79 VWUDLQV H[FHSW IRU 7 DQG 7f ZHUH REWDLQHG IURP WKH ([RWLF &LWUXV 3DWKRJHQV &ROOHFWLRQ PDLQWDLQHG E\ WKH 86'$ DQG ,)$6 LQ %HOWVYLOOH 0' 6WUDLQV 7 DQG 7 ZHUH PDLQWDLQHG LQ JUHHQKRXVHV DW WKH 8QLYHUVLW\ RI )ORULGD *DLQHVYLOOH )/f 3URSDJDWLRQ KRVWV IRU WKH VWUDLQV ZHUH 0DGDP 9LQRXV VZHHW RUDQJH >& VLQHQVLV /f 2VEHFN@ RU 0H[LFDQ OLPH >& DXUDQWLIROLD &KULVWPf 6ZLQJOH@ &ORQLQJ DQG 6HTXHQFLQJ %LRORJLFDO SURSHUWLHV RI WKH VWUDLQV XVHG LQ VHTXHQFLQJ DUH OLVWHG LQ 7DEOH &ORQLQJ DQG VHTXHQFLQJ SURFHGXUHV ZHUH VLPLODU WR WKRVH GHVFULEHG LQ 0DWHULDOV DQG 0HWKRGV &KDSWHU ,Q VRPH FDVHV VWUDLQV % % % DQG %f 3&5 SURGXFWV DQG SODVPLG '1$ ZHUH SXULILHG XVLQJ WKH :L]DUG '1$ &OHDQ8S 6\VWHP )RU % DQG % ERWK QXFOHLF DFLG H[WUDFWV DQG GV51$ 0RUULV DQG 'RGGV f ZHUH XVHG IRU 3&5 DPSOLILFDWLRQ :KHQ GV51$ ZDV XVHG GHQDWXUDWLRQ ZDV SHUIRUPHG LQ D VROXWLRQ FRQWDLQLQJ P0 GLPHWK\OPHUFXU\ DQG DSSUR[LPDWHO\ S0 RI HDFK SULPHU LQ D WRWDO YROXPH RI +L LQFXEDWHG DW URRP WHPSHUDWXUH IRU PLQ DQG WKHQ TXLFN IUR]HQ LQ OLTXLG QLWURJHQ 7KH PL[WXUH ZDV WKHQ XVHG LQ D VWDQGDUG 35& UHDFWLRQ DV GHVFULEHG LQ &KDSWHU

PAGE 50

7DEOH &KDUDFWHULVWLFV RI WKH &79 VWUDLQV XVHG IRU VHTXHQFLQJ RI WKH S JHQH 675$,1 25,*,1 0&$D 0/E '(&/,1( 6
PAGE 51

,Q DGGLWLRQ WR WKH S JHQHV WKH &3 IURP VWUDLQV % ZDV DOVR FORQHG DQG VHTXHQFHG 6HTXHQFHV IRU DOO WKH RWKHU &3 JHQHV ZHUH NLQGO\ SURYLGHG E\ +5 3DSSX &ORQHV IRU WKH S JHQH RI LVRODWH % ZHUH NLQGO\ SURYLGHG E\ 66 3DSSX $W OHDVW WZR FORQHV RI HDFK VWUDLQ ZHUH VHTXHQFHG LQ ERWK WKH VHQVH DQG DQWLVHQVH GLUHFWLRQV 6HTXHQFHV ZHUH FRPSDUHG XVLQJ WKH SURJUDPV &/867$/9 DQG *&* 'HGXFHG DPLQR DFLG VHTXHQFHV ZHUH REWDLQHG XVLQJ 6HTDLG 6LQJOH 6WUDQGHG &RQIRUPDWLRQ 3RO\PRUSKLVP 66&3f 7KH VDPH VWUDLQV HPSOR\HG IRU VHTXHQFLQJ ZHUH DOVR XVHG IRU 66&3 DQDO\VLV 2WKHU VWUDLQV DUH GHVFULEHG LQ 7DEOH 7KH S JHQHV ZHUH DPSOLILHG IURP WLVVXH H[WUDFWV RU IURP SODVPLG '1$ DQG DQDO\]HG LQ QRQGHQDWXULQJ SRO\DFU\ODPLGH JHOV 3&5 SURGXFWV cf ZHUH GHQDWXUHG LQ b IRUPDPLGH P0 ('7$ b EURPRSKHQRO EOXH E\ LQFXEDWLRQ DW r& IRU PLQ DQG HOHFWURSKRUHVHG LQ [ FP b SRO\DFU\ODPLGH JHOV DFU\ODPLGH ELVDFU\ODPLGH O[ 7%(f IRU KUV PLQ DW 9 LQ O[ 7%( EXIIHU DW URRP WHPSHUDWXUH 7KH EXIIHU DQG JHOV ZHUH SUHFKLOOHG DW r& RYHUQLJKW EHIRUH HOHFWURSKRUHVLV '1$ ZDV GHWHFWHG XVLQJ VLOYHU VWDLQLQJ 5HVXOWV 6HTXHQFLQJ 7KH &79 VWUDLQV VHTXHQFHG ZHUH VHOHFWHG EDVHG RQ GLYHUVLW\ RI ELRORJLFDO SURSHUWLHV DQG JHRJUDSKLFDO RULJLQV 7DEOH VXPPDUL]HV WKH RYHUDOO VLPLODULWLHV RI QXFOHRWLGH DQG

PAGE 52

7DEOH &KDUDFWHULVWLFV RI WKH &79 VWUDLQV XVHG IRU 66&3 DQDO\VLV 675$,1 25,*,1 0&$D 0/E '(&/,1( 6
PAGE 53

GHGXFHG DPLQR DFLG VHTXHQFHV DPRQJ DOO WKH VWUDLQV VWXGLHG $W WKH QXFOHRWLGH OHYHO KRPRORJ\ RI WKH VHTXHQFHV YDULHG IURP b WR b 6WUDLQV % VHYHUH IURP 6RXWK $IULFDf DQG % VHYHUH IURP &RORPELDf ZHUH WKH PRVW VLPLODU $W WKH SURWHLQ OHYHO KRPRORJLHV ZHUH b RU KLJKHU 6WUDLQV 7 PLOG IURP )ORULGDf DQG % PLOG IURP &KLQDf ZHUH LGHQWLFDO LQ GHGXFHG DPLQR DFLG VHTXHQFH 6WUDLQV % DQG % VHYHUH IURP 9HQH]XHODf DOVR ZHUH LGHQWLFDO LQ GHGXFHG DPLQR DFLG VHTXHQFH $OLJQPHQW RI WKH QXFOHRWLGH VHTXHQFHV )LJ f VKRZHG VHYHUDO QXFOHRWLGH VXEVWLWXWLRQV DORQJ WKH JHQHV 1R GHOHWLRQV LQVHUWLRQV RU LQYHUVLRQV ZHUH REVHUYHG $OO VHTXHQFHV FRQVWLWXWHG DQ 25) VLPLODU WR WKH RQH SUHYLRXVO\ UHSRUWHG 3DSSX HW DO f $ WRWDO RI QLQH VXEVWLWXWLRQV ZHUH FRQVHUYHG LQ DOO WKH VHYHUH VWUDLQV FRPSDUHG WR QXFOHRWLGHV FRQVHUYHG LQ DOO PLOG VWUDLQV )RU H[DPSOH QXFOHRWLGHV & f§ 7f DQG f§ $f 2WKHU QLQH VXEVWLWXWLRQV ZHUH SUHVHQW RQO\ LQ VWHP SLWWLQJ VWUDLQV )RU H[DPSOH QXFOHRWLGH 7 f§ &f 0RVW RI WKHVH VXEVWLWXWLRQV KRZHYHU ZHUH VLOHQW &RPSDULVRQ RI WKH GHGXFHG DPLQR DFLG VHTXHQFHV )LJ f VKRZHG RQO\ DPLQR DFLGV H[FOXVLYH WR VHYHUH VWUDLQV DPLQR DFLGV DQG fDQG RQO\ RQH LQ VWHP SLWWLQJ VWUDLQV DPLQR DFLG f 7KH &WHUPLQDO SRUWLRQ RI WKH SURWHLQ LV WKH PRVW FRQVHUYHG DPRQJ WKH VWUDLQV VHTXHQFHG

PAGE 54

7DEOH 51$ DQG GHGXFHG DPLQR DFLG VHTXHQFH VLPLODULWLHV b RI LGHQWLFDO EDVHV RU DPLQR DFLGVf EHWZHHQ GLIIHUHQW &79 VWUDLQV IRU S XSSHU ULJKW LV 51$ ORZHU OHIW LV DPLQR DFLG VHTXHQFHVf 9DOXHV JHQHUDWHG XVLQJ &/867$/9 675$,1 7 7 % 7 % % % % % 7 7 % 7 % % % % %

PAGE 55

, , 7 $7**&$**77$7$&$*7$&77&&7$$7$&&*$7*$&$$$*$$$7**$7&& 7 % 7 % f§ % f§ % f§ 7 % &f§$*77 & % f§*f§$ 7 , , 7 **7*$*7*&&*&7*7$&&&**7$$*7$7&&**$7*7&$77*$$$$$777* 7 % 7 f§f§_ f§ % f§ % % & % $ f§,f§ f§f§ % f§f§ f§ 7 7 % 7 % % % % % , , 7**&&$$&$**7&&*7$*$&*&*77$$7$*$$**&*7&$7$$*7$$*77*  ,f§ 7f§ 7 f§ ,f§ 7 7 7 7 L L f§ 7 F F B7 7 7 % 7 % % % % % , , *$7$&&$$77&$$7$7$&*$$*$77&&$&7*$$$$$777$&7**7*$$&$ 4 ‘B F W f§&f§7 L *7 & *7 L *f§ L *7 )LJ 1XFOHRWLGH VHTXHQFH DOLJQPHQW RI S JHQH IURP GLIIHUHQW &79 VWUDLQV K\SKHQV LGHQWLFDO QXFOHRWLGHVf 7KH DOLJQPHQW ZDV FRQVWUXFWHG XVLQJ WKH SURJUDP &/867$/9

PAGE 56

OLOL 7 &77*$$$7$&*77$7**77$&7$7**$7*&777&77$77$*$$$$&7$&$ 7 % 7 &$ % $f§* &$ f % $f§ &$ f % $f§* &$ cK % $& *f§7 a$ % $f§* &O 7 OLOL 7 $*$&*$$$$&**$$*$7&7*77**77&$&77$*&7$7*$7&&$$$$*$** 7 % 7 $ % $ % $ % $&& % $ % $ $ L fL Lf§ 7&* f§*f§& f§$ BBBBBBB 7 7 % 7 % % % % % OLOL 77*7$&$&7$&$7&&$&*$*&$&7$$$$&&$$*77&&*&*$7$$$**77* f§$ L L f§LLf§LL L f§$O   7 f§,Lf§ 7 7 7 7 Df§7f§7 f§$* &7 7 Lf§ * OLOL 7 7$77$*77$&*7*&$$*****77&*&*$7$7$$*77$$7**$7$$$*7$* 7 & % 7 $ 7$ *7 & % $ $ 7$ & 7 $ $ 7$ 7 $ $ 7$ & 7 % 7 7$ $ f§$ $ 7$ & 7 )LJXUH f§FRQWLQXHG

PAGE 57

7 7 % 7 % % % % % OLOL 77777&&777&$77$7$7&*$$$777$&&*$&$***$*$&7&&*$$&*&7 L f§ f§_ f§_ La F $ f§*f§$ 7 7 7 7 OLOL 7 &7$&*7$$*7$7*&77*&$&777&*$**$*77$&$&77*7*7$7**&7$* 7 % 7 % $ % ƒ % $ % f§f§ % BBBBBBB $ 7 7 % 7 % % % % % OLOL *77*$*$&&&*$&77$7$&*$$$$7$$$$**$&*$&7$$$*&&***$&7& $f§$ Lf§ &,n $$ &,n $$ $f§$ & &f§*f§7f§$ &**f§7 &f§*f§7 f§f§7 f§f§ Lf§ f§$ ,f§ f§Lf§ 7 7 % 7 % % % % % OLOL &$&$777$$$***&7$777$7&$*&&*$&777&777&***77&7&7&&&$ 7 F 7 FF 7 &f§$ 7 $f§7f§& 7 f§f§ W $f§Lf§ &f§* 7f§7 $ 7 $f§f§,f§ 7 )LJXUH f§FRQWLQXHG

PAGE 58

7 7 % 7 % % % % % OLOL ***7$&7&&*$$&$7*$$&*$**&$7&$77&77&*$*&*7&7*$*7&7$7 7f§* $7* f§ 7f§* 7 i Ff§$ 7 f§$ BBBBBf§_ &f§$ OLOL 7 *77$*&7$*$&*7&$$**77$&*$**$**&$$&&*$*&77&77$$&&7$& 7 % 7 % % % % L % 7 7 % 7 % % % % % *7*$777***7$$*7$&77$7$* & )LJXUH f§FRQWLQXHG

PAGE 59

7 % 7 % % % % % , , 0$*<79/317''.(0'396$$93*.<3'9,(.)9$1569'$/,(*9,6./ 1 L . . .9 , , 7 '716,<('67(.)7*(+/.<90970'$)//(1<.7.7('//9+/$0,4.5 7 % 7 % & < % < % + 6 IW % iM 1 % 7 % 7 % % % % % , , /<776767.7.)5'.*&,6<94**65<./0'.:)3),,6.)7'5(731$ / &; / / &; E / &; &, / ;L )LJXUH 3DLUZLVH DOLJQPHQW RI WKH GHGXFHG DPLQR DFLG VHTXHQFH RI S IURP GLIIHUHQW &79 VWUDLQV K\SKHQV LGHQWLFDO DPLQR DFLGV SHULRGV VLPLODU DPLQR DFLGVf 7KH DOLJQPHQW ZDV FRQVWUXFWHG XVLQJ WKH SURJUDP &/867$/9

PAGE 60

7 7 % 7 % % % % % OLOL /5.<$&7)((/+/&0$5/53'/<(1.577.$*73+/.*
PAGE 61

$ SK\ORJHQHWLF WUHH ZDV FRQVWUXFWHG XVLQJ WKH 51$ VHTXHQFHV )LJ f )RXU PDMRU JURXSV FRXOG EH GLVWLQJXLVKHG OfVHYHUH VWHP SLWWLQJ VWUDLQV % % % DQG % f PLOG VWUDLQV 7 % DQG 7 f VHYHUH TXLFN GHFOLQH VWUDLQ 7 DQG f VHYHUH VWHP SLWWLQJ VWUDLQ % 0LOG VWUDLQV VKRZHG VRPH UHODWLRQVKLS WR VHYHUH TXLFN GHFOLQH DQG VWHP SLWWLQJ VWUDLQV 6WUDLQ % VHYHUH IURP ,QGLDf ZDV WKH PRVW GLVWLQFW DQG IRUPHG D FRPSOHWHO\ VHSDUDWH JURXS 7KH SK\ORJHQHWLF WUHH FRQVWUXFWHG XVLQJ GHGXFHG DPLQR DFLG VHTXHQFHV )LJ f VHSDUDWHG PLOG IURP VHYHUH VWUDLQV HDFK RQH IRUPLQJ D GLVWLQFW JURXS &RPSDULVRQ RI WKH &3 VHTXHQFHV LQ SK\ORJHQHWLF WUHHV 51$ DQG DPLQR DFLG )LJV DQG UHVSHFWLYHO\f VKRZHG D GLIIHUHQW JURXSLQJ RI WKH VWUDLQV IURP WKDW RI S 6HTXHQFH DOLJQPHQW RI S DQG &3 IURP WKH GLIIHUHQW VWUDLQV LV VKRZHG LQ )LJ $SSUR[LPDWHO\ b RI WKH DPLQR DFLGV DUH LGHQWLFDO EHWZHHQ WKH WZR SURWHLQV GHSHQGLQJ RQ WKH VWUDLQ $JDLQ WKH &WHUPLQDO UHJLRQV RI WKH SURWHLQV DUH DOVR WKH PRVW FRQVHUYHG )LJ f 6LQJOH 6WUDQGHG &RQIRUPDWLRQ 3RO\PRUSKLVP 66&3 DQDO\VLV RI WKH VWUDLQV XVHG IRU VHTXHQFLQJ )LJ f UHYHDOHG ILYH JURXSV ZLWK VLPLODU SDWWHUQV f VHYHUH VWHP SLWWLQJ VWUDLQV % DQG % f VHYHUH VWHP SLWWLQJ VWUDLQV % DQG % f PLOG VWUDLQV 7 7 DQG % f TXLFN GHFOLQH VWUDLQ 7 DQG f VHYHUH VWHP SLWWLQJ VWUDLQ % :KHQ DOO VWUDLQV ZHUH FRQVLGHUHG )LJV DQG f WKH\

PAGE 62

)LJXUH 3K\ORJHQHWLF WUHH IRU S EDVHG RQ 51$ VHTXHQFHV *UDSKLF JHQHUDWHG XVLQJ 3LOHXS SURJUDP LQ *&*f

PAGE 63

7 % 7 )LJXUH 3K\ORJHQHWLF WUHH IRU S EDVHG RQ GHGXFHG DPLQR DFLG VHTXHQFHV *UDSKLF JHQHUDWHG XVLQJ 3LOHXS SURJUDP LQ *&*

PAGE 64

7FS 7FS %FS 7FS %f§FS %FS %FS %FS %FS )LJXUH 3K\ORJHQHWLF WUHH IRU &3 EDVHG RQ 51$ VHTXHQFHV *UDSKLF JHQHUDWHG XVLQJ 3LOHXS SURJUDP LQ *&*

PAGE 65

f§ 7FS 7FS %FS 7FS %f§FS %FS %FS %FS %FS )LJXUH 3K\ORJHQHWLF WUHH IRU &3 EDVHG RQ GHGXFHG DPLQR DFLG VHTXHQFHV *UDSKLF JHQHUDWHG XVLQJ 3LOHXS SURJUDP LQ *&*

PAGE 66

&3 7 &3 7 &3 % &3 7 &3 % &3 % &3 % &3 % &3 % S 7 S 7 S % S 7 S % S % S % S % S % 0''(7../.1.1.(7.(*'(:$$(66)*691/+,'37/,701'f§954/ 0''(7../11.1.(7.(*'(:$$(66)*691/+,'37/,701'f§954/ 0''(7../.1.1.(,.4*'':$$(66)*691/+,'37/,701'f§954/ 0''(7../.1.1.(7.(*'':$$(66)6691/+,'37/,701'f§954/ 0''(7../.1.1.(7.(*'':$$(66)*691/+,'37/,$01'f§954/ 0''(7../.1.1.($.(*'':$$(66)*6/1)+,'37/,$01'f§954/ 0''(7../.1.1.(7.(*'':$$(66)*6/1/+,'37/,$01'f§954/ 0''(7../.1.1.(7.(*'':$$(66)*601/+,'37/,$01'f§954/ 0''(7../.1.1.(7.(*'':$$(66)*6/1/+,'37/,$01'f§954/ 0$*<79/317''.(0'396$$93*.<3'9,(.)9$1569'$/,(*9,6./ 0$*<79/317''.(01396$$/3*.<3'9,(.)9$1569'$/0(*9,6./ 0$*<79/317''.(0'396$$93*.<3'9,(.)9$1569'$/,(*9,6./ 0$*<79/3.7''.(0'396$$93*.<3'9,(.)9$1569'$/,(*9,6./ 0$*<79/3.7''.(0'396$$93*.<3'9,(.)9$1569'$/,(*9,6./ 0$*<79/3.7''.(0'396$$93*.<3'9,(.)9$1569'$/,(*9,6./ 0$*<79/3.7''.(0'396$$93*.<3'9,(.)9$1569'$/,(*9,6.) 0$*<79/3.9''.(0'396$$93*.<3',,(.)971569'$/,(*9,6./ 0$*<79/3.7''.(0'396$$93*.<3'9,(.)9$1569'$/,(*9,6./ r r rr &3 7 67441$$/15'/)/$/.*.<31/3 '.'.')f§+,$00/<5 &3 7 67441$$/15'/),$/.*.<31/3 '.'.')f§+,$00/<5 &3 % 67441$$/15'/)/7/.*.<31/3 '.'.')f§+,$00/<5 &3 7 67441$$/15'/)/7/.*.+31/3 '.'.')f§5,$00/<5 &3 % *7441$$915'/)/7/.(.<3./6 '.'.')f§+,$00/<5 &3 % 67441$$/15'/)/7/.*.<31/6 '.'.')f§+/$00/<5 &3 % 67441$$/15'/)/7/.*.<31/6 '.'.')f§+/$00/<5 &3 % *7441$$/15'/)/7/.*.<31/3 '.'.')f§+,$00/<5 &3 % *7441$$/15'/)/7/.*.<31/3 '.'.')f§+,$00/<5 S 7 '716,<('67(.)7*(+/.<90970'$)//(1<.7.7('//9+/$0,4.5 S 7 '716,<('67(.)7*(+/.<90970'$)//(1<.7.7('//9+/$0,4.5 S % '716,<('67(.)7*(+/.<90970'$)//(1<.7.7('//9+/$0,4.5 S 7 '716,<('67(.)7*(+/.<90970'7)//(1<.7.7('//9+/70,4.5 S % '71&,<('67(.)7*(
PAGE 67

&3 7 &3 7 &3 % &3 7 &3 % &3 % &3 % &3 % &3 % S 7 S 7 S % S 7 S % S % S % S % S % /$9.666/46'''77*,7<75(*9(9'/6'./:7',9<16.*,*1571$ /$9.666/46'''77*,7<75(*9(/'/6'./:7',9<16.*,*1571$ /$9.666/46'''77*,7<75(*9(9'/6'./:7':<16.*,*1571$ /$9.666/46'''$7*,7<75(*9(9'/6'./:7':)16.*,*1571$ /$9.666/46'''77*,7<75(*9(9'/6'./:7':)16.*,*1551$ /$9.666/46'''77*97<75(*9(9'/6'./:7':)16.*,*1571$ /$9.666/46'''77*,7<75(*9(9'/6'./:7':)16.*,*1571$ /$9.666/46'''77*,7<75(*9(9'/6'./:7':)16.*,*1571$ /$9.666/46'''77*97<75(*9(9(/6'./:7':)16.*,*1571$ /<776767.7.)5'.*&,6<94**65<./0'.:)3),,6.)7'5(731$ /<776767.7.)5'.*&,6<94**65<./0'.:)3),,6.)7'5(731$ /<776767.7.)5'.*&,6<94**65<./0'.:)3),,6.)7'5(731$ /<7,6767.7.)5'.*&,6<94**/5<.//'.:)3),,6.)7'5(731$ /&7,6767.7.)5'.*&,6<94**/5<.//'.:)3),,6.)7'5(731$ /&7,6767.7.)5'.*&,6<94**/5<.//'.:)3),,6.)7'5(731$ /&7,6767.7.)5'.*&,6<94**/5<.//'.:<3),,6.)7'5(731$ /&7,6767.7.)5'.*&,6<94**/5<./)'.:)3),,6.)7'5(731$ /&7,6767.7.)5'.*&,6<94**/5<.//'.:)3),,6.)7'5(731$ r rr r r r r rr rr &3 7 &3 7 &3 % &3 7 &3 % &3 % &3 % &3 % &3 % S 7 S 7 S % S 7 S % S % S % S % S % /59:*571'$/
PAGE 68

)LJXUH 66&3 DQDO\VLV RI WKH S JHQH IURP WKH VDPH VWUDLQV XVHG LQ VHTXHQFLQJ % 7 7 % 7 % % % DQG %

PAGE 69

)LJXUH 66&3 DQDO\VLV RI WKH S JHQH IURP RWKHU &79 VWUDLQV 7 % % % % % 7 %OO % % % DQG 7

PAGE 70

FRXOG EH VHSDUDWHG LQWR QLQH GLIIHUHQW JURXSV f PLOG VWUDLQV 7 7 % % % % DQG % f PLOG VWUDLQ % f VHYHUH VWHP SLWWLQJ % DQG % f VHYHUH VWHP SLWWLQJ VWUDLQV % DQG % f VHYHUH VWHP SLWWLQJ VWUDLQ % f VHYHUH TXLFN GHFOLQH VWUDLQ 7 f VHYHUH TXLFN GHFOLQH VWUDLQ %OO f VHYHUH TXLFN GHFOLQH VWUDLQ % DQG f VHYHUH VWUDLQV % VWHP SLWWLQJf DQG % TXLFN GHFOLQHf 'LVFXVVLRQ 7KH S JHQHV RI WKH &79 VWUDLQV VHTXHQFHG VKRZHG b RU JUHDWHU KRPRORJ\ DW WKH 51$ OHYHO DQG b RU JUHDWHU DW WKH DPLQR DFLG OHYHO GHGXFHG VHTXHQFHf 7KHVH YDOXHV DUH VLPLODU WR WKRVH IRXQG IRU WKH &3 3DSSX HW DO Ef 6HYHUDO RI WKH QXFOHRWLGH VXEVWLWXWLRQV DUH FRQVLVWHQWO\ FRQVHUYHG RQO\ ZLWKLQ D SDUWLFXODU ELRORJLFDO JURXS PLOG VHYHUH RU VHYHUH FDXVLQJ VWHP SLWWLQJf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

PAGE 71

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f UHSUHVHQW DQ H[DPSOH RI HYROXWLRQDU\ FRQYHUJHQFH &RPSDULVRQ RI WKH SK\ORJHQHWLF WUHHV REWDLQHG ZLWK &3 VHTXHQFHV VKRZHG GLIIHUHQW UHODWLRQVKLSV LQGLFDWLQJ GLVWLQFW HYROXWLRQDU\ UDWHV IRU WKH WZR JHQHV )URP WKHVH UHVXOWV WKH DPLQR DFLG VHTXHQFH RI WKH S JHQH LV PRUH XVHIXO LQ SUHGLFWLQJ WKH ELRORJLFDO UHDFWLRQ RI DQ VWUDLQ WKDQ &3 VHTXHQFH 51$ RU DPLQR DFLGf $OLJQPHQW RI S DQG &3 GHGXFHG DPLQR DFLG VHTXHQFHV UHYHDOHG WKDW WKH &WHUPLQDO SRUWLRQ RI WKH WZR SURWHLQV LV

PAGE 72

WKH PRVW FRQVHUYHG /RFDWHG DOVR LQ WKLV UHJLRQ DUH WKH WZR FRQVHQVXV DPLQR DFLGV ZKLFK DUH FRQVHUYHG LQ WKH &3V RI ILODPHQWRXV YLUXVHV 3DSSX HW DO f 7KLV SRUWLRQ PXVW WKHQ FRQWDLQ LPSRUWDQW DFWLYH GRPDLQV IRU ERWK SURWHLQV 66&3 DQDO\VLV RI WKH S JHQH RI VWUDLQV 7 7 7 % % % % % DQG % SURGXFHG JURXSLQJV VLPLODU WR WKRVH REWDLQHG LQ SK\ORJHQHWLF DQDO\VLV RI WKH 51$ VHTXHQFHV 7KH RQO\ H[FHSWLRQ LV WKH VHSDUDWLRQ RI WKH VWHP SLWWLQJ VWUDLQV LQWR WZR JURXSV ZLWK GLIIHUHQW SDWWHUQV % DQG % % DQG % ,QWHUHVWLQJO\ % LV PRUH VLPLODU LQ 51$ VHTXHQFH WR % bf RU % bf WKDQ LW LV WR % bf 7KLV LQGLFDWHV WKDW FHUWDLQ QXFOHRWLGH FKDQJHV KDYH PRUH LQIOXHQFH LQ FRQIRUPDWLRQ WKDQ RWKHUV 6KHIILHOG HW DO f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

PAGE 73

66&3 FDQ GHWHFW VLQJOH QXFOHRWLGH FKDQJHV LQ VKRUW '1$ VHTXHQFHV ESf 2ULWD HW DO 6KHIILHOG HW DO f EXW LV OHVV HIILFLHQW ZKHQ ORQJHU IUDJPHQWV DUH DQDO\]HG 3&5 DPSOLILFDWLRQ FDQ EH IROORZHG E\ UHVWULFWLRQ GLJHVWLRQ WR JHQHUDWH VKRUW IUDJPHQWV LI ORQJHU VHTXHQFHV DUH DPSOLILHG ,ZDKDQD HW DO f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

PAGE 74

&+$37(5 75$16)250$7,21 2) 6:((7 25$1*( :,7+ 3 $1' 3 ,QWURGXFWLRQ 6HTXHQFH DQDO\VLV DQG WKH LGHQWLILFDWLRQ RI 25)V LQ RWKHU SODQW YLUXVHV KDYH SHUPLWWHG WKH XVH RI VRPH RI WKRVH JHQHV WR WUDQVIRUP SODQWV DQG SURWHFW WKHP DJDLQVW YLUDO LQIHFWLRQV FDXVHG E\ VLPLODU VWUDLQV 7KH ILUVW DQG PRVW ZLGHO\ XVHG JHQH KDV EHHQ WKH &3 3RZHOO $EHO HW DO %HDFK\ HW DO 3DSSX HW DO f 7UDQVJHQLF SODQWV H[SUHVVLQJ &3 JHQHV DQG VKRZLQJ YDULRXV GHJUHHV RI UHVLVWDQFH KDYH EHHQ REWDLQHG IRU PHPEHUV RI DW OHDVW D GR]HQ GLIIHUHQW YLUXV JURXSV +XOO DQG 'DYLHV 3DSSX HW DO f LQGLFDWLQJ WKDW WKLV LV D UHODWLYHO\ JHQHUDO SKHQRPHQRQ 6HYHUDO QRQ VWUXFWXUDO YLUDO JHQHV DOVR KDYH EHHQ XVHG WR WUDQVIRUP SODQWV ZLWK PL[HG UHVXOWV LQ WKH LQGXFWLRQ RI UHVLVWDQFH )RU H[DPSOH WKUHH QRQVWUXFWXUDO JHQHV RI WREDFFR UDWWOH WREUDYLUXV WUDQVIRUPHG LQWR WREDFFR SODQWV GLG QRW LQGXFH UHVLVWDQFH WR WKH YLUXV $QJHQQHQW HW DO f 2Q WKH RWKHU KDQG WREDFFR SODQWV WUDQVIRUPHG ZLWK D N'D SXWDWLYH UHSOLFDVH SURWHLQ RI WREDFFR PRVDLF WREDPRYLUXV 709f ZHUH UHVLVWDQW WR KLJK FRQFHQWUDWLRQV RI LQWDFW 709 DQG LWV 51$ *ROHPERVNL HW DO f

PAGE 75

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f ZKHQ H[SUHVVHG LQ WUDQVJHQLF WREDFFR SODQWV 'HRP HW DO f 0RVW SODQW WUDQVIRUPDWLRQ H[SHULPHQWV ZLWK YLUDO JHQHV RU RWKHUV KDYH EHHQ DFFRPSOLVKHG LQ KHUEDFHRXV DQQXDO SODQWV DQG RQO\ D IHZ ZRRG\ VSHFLHV KDYH EHHQ VXFFHVVIXOO\ WUDQVIRUPHG 7KLV LV GXH WR WKH WHFKQLFDO GLIILFXOWLHV LQ UHJHQHUDWLQJ WKRVH SODQWV DQG WKH OHQJWK RI VFUHHQLQJ DQG WHVWLQJ IRU UHVLVWDQFH 3OXP 3UXQXV GRPHVWLFD /f ZDV UHFHQWO\ WUDQVIRUPHG ZLWK WKH &3 JHQH RI SOXP SR[ SRW\YLUXV XVLQJ $JUREDFWHULXP 0DFKDGR HW DO 6FRU]D HW DO f 3DSD\D &DULFD SDSD\D /f D SHUHQQLDO SODQW KDV DOVR EHHQ WUDQVIRUPHG ZLWK WKH &3 JHQH RI SDSD\D ULQJVSRW SRW\YLUXV 7HQQDQW HW DO f 7UDQVJHQLF SODQWV WHVWHG LQ WKH ILHOG IRU WZR \HDUV VKRZHG QR YLUXV UHSOLFDWLRQ RU PRYHPHQW ZKHQ FKDOOHQJHG ZLWK WKH VDPH VWUDLQ RI YLUXV XVHG IRU

PAGE 76

WUDQVIRUPDWLRQ +RZHYHU ZKHQ WKH WUDQVJHQLF SODQWV ZHUH LQRFXODWHG ZLWK GLIIHUHQW YLUDO VWUDLQV WKH\ VKRZHG RQO\ D GHOD\ LQ V\PSWRP GHYHORSPHQW 7HQQDQW HW DO f D SKHQRPHQRQ REVHUYHG SUHYLRXVO\ LQ RWKHU &3 WUDQVJHQLF SODQWV %HDFK\ HW DO f )RU DQQXDO FURSV D GHOD\ LQ V\PSWRP GHYHORSPHQW FDQ EH VXIILFLHQW WR UHGXFH ORVVHV FDXVHG E\ D YLUXV EXW IRU SHUHQQLDO FURSV WKLV ZLOO SUREDEO\ EH RI OLWWOH YDOXH VLQFH WKH\ DUH H[SHFWHG WR EH LQ WKH ILHOG IRU PDQ\ \HDUV 6HYHUDO FLWUXV VSHFLHV KDYH UHSRUWHGO\ EHHQ WUDQVIRUPHG E\ GLIIHUHQW PHDQV LQFOXGLQJ GLUHFW XSWDNH RI '1$ E\ SURWRSODVWV 6FKHOO f FRFXOWLYDWLRQ RI FHOO VXVSHQVLRQV ZLWK $JUREDFWHULXP +LGDND HW DO f DQG LQIHFWLRQ RI HSLFRW\O VHJPHQWV ZLWK $JUREDFWHULXP 0RRUH HW DO .DQH\RVKL HW DO 3HD HW DO f 7KHUH DUH WZR UHSRUWV RI FLWUXV WUDQVIRUPDWLRQ XVLQJ WKH &3 JHQH RI &79 *XWLUUH] HW DO 6FKHOO HW DO f 7KH SODQWV REWDLQHG LQ WKHVH H[SHULPHQWV DUH EHLQJ WHVWHG FXUUHQWO\ IRU UHVLVWDQFH WR &79 *$ 0RRUH DQG -: *URVVHU SHUVRQDO FRPPXQLFDWLRQf 7R VWXG\ WKH IHDVLELOLW\ RI XVLQJ VRPH RI WKH QRQ&3 JHQHV RI &79 LQ $JUREDFWHULXPPHGLDWHG SODQW WUDQVIRUPDWLRQ S DQG S JHQHV ZHUH XVHG WR WUDQVIRUP 3LQHDSSOH VZHHW RUDQJH SODQWV 7UDQVJHQLF SODQWOHWV ZHUH VHOHFWHG DIWHU WDUJHW JHQH H[SUHVVLRQ ZDV GHWHFWHG LQ LQIHFWHG FLWUXV WLVVXH

PAGE 77

0DWHULDOV DQG 0HWKRGV 0DWHULDOV &DOI LQWHVWLQDO DONDOLQH SKRVSKDWDVH 3KRWRJHQH 1XFOHLF $FLG 'HWHFWLRQ 6\VWHP 9HUVLRQ DQG ELRWLQG$73 ZHUH IURP *,%&2 %5/ *DLWKHUVEXUJ 0'f 0XUDVKLJXH DQG 6NRRJ WLVVXH FXOWXUH EDVDO VDOW PL[WXUH 06f DQG DQWLELRWLFV ZHUH IURP 6LJPD &KHPLFDO &RPSDQ\ 6W /RXLV 02f 0HIR[LQ ZDV IURP 0HUFN 6KDUS t 'RKPH :HVW 3RLQW 3$f 3ODVPLGV S021 S021 DQG $JUREDFWHULXP $%, ZHUH REWDLQHG IURP 0RQVDQWR 6W /RXLV 02f 3LQHDSSOH VZHHW RUDQJH VHHGV ZHUH IURP :LOOLWV t 1HZFRPE ,QF $UYLQ &$f 7KH EURPRFKORURLQGRO\OM' JOXFXURQLF DFLG F\FORKH[\ODPPRQLXP VDOW ;JOXFf ZDV VXSSOLHG E\ %LRV\QWK %LRFKHPLFD t 6\QWKHWLFD 6WDDG 6ZLW]HUODQGf RU E\ *ROG %LRWHFKQRORJ\ ,QF 6W /RXLV 02f &ORQLQJ RI S ,QWR WKH 7UDQVIRUPDWLRQ 9HFWRU &79 JHQH S ZDV FORQHG LQWR S021 IURP WKH S8&S UHFRPELQDQW SODVPLG &KDSWHU f 7KH JHQH FRQWDLQV WKH FRQVHQVXV VHTXHQFH IRU RSWLPDO FRQWH[W RI WKH $8* VWDUW FRGRQ .R]DN *DOOLH f $SSUR[LPDWHO\ [J RI S021 SODVPLG '1$ DQG cLT RI S8&S ZHUH GLJHVWHG VHSDUDWHO\ ZLWK (FR5, Xf DQG ;EDO Xf LQ P0 7ULV+&O S+ DW r&f P0 0J&O P0 1D&O DW r& IRU K 6DPSOHV ZHUH SXULILHG LQ /03 DJDURVH DQG OLJDWHG DV H[SODLQHG LQ 0DWHULDOV DQG 0HWKRGV &KDSWHU H[FHSW WKDW WKH 7 SRO\QXFOHRWLGH NLQDVH VWHS ZDV RPLWWHG 7KH SODVPLG ZDV

PAGE 78

WUDQVIRUPHG LQWR ( FROL '+D FRPSHWHQW FHOOV DQG SODWHG RQ [ <7 DJDU SODWHV FRQWDLQLQJ JPO RI VSHFWLQRP\FLQ 3ODVPLG '1$ LQFUHDVHG DQG SXULILHG IURP WKH WUDQVIRUPHG FRORQLHV ZDV VFUHHQHG IRU SUHVHQFH RI WKH LQVHUW E\ GLJHVWLRQ ZLWK (FR5, DQG HOHFWURSKRUHVHG LQ D b DJDURVH JHO ZLWK ODPEGD+LQGOOO PROHFXODU ZHLJKW PDUNHUV 7KH\ ZHUH DOVR VFUHHQHG E\ GLJHVWLRQ ZLWK (FR5, DQG ;EDO DQG HOHFWURSKRUHVHG LQ DJDURVH JHOV 7KH JHQH IRU JOXFXURQLGDVH *86f ZDV FORQHG LQWR S021S IURP S021 %RWK SODVPLGV XJ RI '1$ HDFKf ZHUH VHSDUDWHO\ GLJHVWHG ZLWK 1RWO Xf LQ P0 7ULV+&O S+ DW r&f P0 0J&O P0 1D&O P0 '77 DQG SXULILHG LQ /03 DJDURVH /LQHDUL]HG S021S ZDV WUHDWHG ZLWK FDOI LQWHVWLQDO DONDOLQH SKRVSKDWDVH Xf LQ P0 7ULV+&O S+ P0 ('7$ DFFRUGLQJ WR WKH PDQXIDFWXUHUnV LQVWUXFWLRQV DQG WKHQ H[WUDFWHG ZLWK SKHQRO SKHQRO FKORURIRUP FKORURIRUP DQG SUHFLSLWDWHG ZLWK HWKDQRO 6DPEURRN HW DO f /LJDWLRQ RI WKH *86 JHQH LQWR S021S DQG WUDQVIRUPDWLRQ LQWR ( FROL '+D ZDV DV GHVFULEHG DERYH 7R FRQILUP SUHVHQFH DQG RULHQWDWLRQ RI WKH S JHQH WKH LQVHUW ZDV VHJXHQFHG IROORZLQJ WKH SURWRFRO H[SODLQHG LQ 0DWHULDOV DQG 0HWKRGV &KDSWHU 7KLV SODVPLG ZDV GHVLJQDWHG DV S9)O )LJ f &ORQLQJ RI S ,QWR WKH 7UDQVIRUPDWLRQ 9HFWRU 7KH FORQLQJ SURFHGXUH IRU S ZDV VLPLODU WR WKDW GHVFULEHG DERYH EXW WKH *86 JHQH ZDV FORQHG LQ S021 ILUVW 7KH S

PAGE 79

)LJXUH 5HVWULFWLRQ PDS RI WKH S9)O SODVPLG FRQWDLQLQJ S

PAGE 80

JHQH ZDV FORQHG VXEVHTXHQWO\ IURP S8&S XVLQJ ;EDO DQG 6DF, UHVWULFWLRQ VLWHV 7KH QHZ SODVPLG ZDV GHVLJQDWHG DV S9) )LJ f 7UDQVIRUPDWLRQ RI S9) SODVPLGV LQWR $DUREDFWHULXP 7UDQVIRUPDWLRQ RI $JUREDFWHULXP ZLWK WKH S9) SODVPLGV ZDV E\ XVLQJ WKH WULSOH PDWLQJ SURFHGXUH 2YHUQLJKW PO FXOWXUHV LQ /% OLTXLG PHGLXP RI WKH IROORZLQJ EDFWHULD ZHUH XVHG $JUREDFWHULXP $%, VXSSOHPHQWHG ZLWK JPO FKORUDPSKHQLFRO MXJPO NDQDP\FLQf ( FROL 35. SLJPO NDQDP\FLQf DQG ( FROL '+D ZLWK HLWKHU S9)O RU S9) LJPO VSHFWLQRP\FLQf 6DPSOHV RI c[O IURP HDFK FXOWXUH ZHUH PL[HG LQ D PLFURFHQWULIXJH WXEH DQG FHQWULIXJHG IRU VHF DW USP 7KH SHOOHWV ZHUH UHVXVSHQGHG LQ c[O RI P0 0J6 DQG SODFHG RQ VLQJOH /% DJDU SODWHV IUHH RI DQWLELRWLFV DQG ZLWKRXW VSUHDGLQJ WKH EDFWHULD 7KH SODWHV ZHUH LQFXEDWHG DW r& IRU K %DFWHULD ZHUH FROOHFWHG IURP VLQJOH SODWHV DQG UHVXVSHQGHG LQ PO RI P0 0J6 6DPSOHV RI DQG c[O ZHUH SODWHG RQ /% DJDU FRQWDLQLQJ AJPO FKORUDPSKHQLFRO c;JPO NDQDP\FLQ DQG LJPO VSHFWLQRP\FLQ DQG LQFXEDWHG IRU WR GD\V DW r& 6XUYLYLQJ EDFWHULDO FXOWXUHV ZHUH WKHQ NHSW RQ <(3 b \HDVW H[WUDFW b SHSWRQH b 1D&O S+ f DJDU SODWHV FRQWDLQLQJ WKH WKUHH DQWLELRWLFV 6FUHHQLQJ RI 7UDQVIRUPHG %DFWHULD $JUREDFWHULXP FRORQLHV REWDLQHG LQ WKH WUDQVIRUPDWLRQ HYHQW ZHUH VFUHHQHG XVLQJ 3&5 WR GHWHUPLQH WKH SUHVHQFH RI WKH S9) SODVPLGV 6DPSOHV IURP FRORQLHV ZHUH UHVXVSHQGHG LQ LO

PAGE 81

)LJXUH 5HVWULFWLRQ PDS RI WKH S9) FRQWDLQLQJ S SODVPLG

PAGE 82

RI P0 7ULV+&O S+ P0 ('7$ b 7ULWRQ ; ERLOHG IRU PLQ DQG FHQWULIXJHG IRU D IHZ VHFRQGV 3&5 UHDFWLRQV FRQWDLQHG WKH QRUPDO FRQVWLWXHQWV SOXV , RI WKH EDFWHULDO H[WUDFWV DQG WKH DSSURSULDWH SULPHUV IRU S S RU WKH *86 JHQH 7UDQVIRUPDWLRQ 6HOHFWLRQ DQG 5HJHQHUDWLRQ RI &LWUXV 3ODQWV 6HHG JHUPLQDWLRQ 7KH SURFHGXUH IRU VHHG JHUPLQDWLRQ ZDV PRGLILHG IURP 0RRUH HW DO f ,QWDFW VHHGV ZHUH VXUIDFH VWHULOL]HG PLQ LQ b HWKDQRO PLQ LQ b &ORUR[ DQG GURSV RI 7ZHHQ ULQVHG WLPHV ZLWK VWHULOH GLVWLOOHG ZDWHUf DQG JHUPLQDWHG LQGLYLGXDOO\ LQ [ PP FDSSHG WXEHV FRQWDLQLQJ PO RI KDOIVWUHQJWK 06 EDVDO VDOW PHGLXP b VXFURVH b P\RLQRVLWRO b DJDU S+ DQG PDLQWDLQHG DW r& ZLWK K IOXRUHVFHQW OLJKW 6HHGOLQJV ZHUH XVHG WR PRQWKV DIWHU VRZLQJ %DFWHULDO FXOWXUH ,QGLYLGXDO FRORQLHV RI $JUREDFWHULXP ZLWK HLWKHU S9)O RU S9) ZHUH LQFUHDVHG LQ PO IODVNV FRQWDLQLQJ PO <(3 PHGLXP ZLWK WKH WKUHH DQWLELRWLFV DQG LQFXEDWHG RYHUQLJKW DW r& DQG USP WR SRVW ORJ SKDVH 7KH EDFWHULD ZHUH FROOHFWHG E\ FHQWULIXJDWLRQ DW J IRU PLQ DW r& DQG UHVXVSHQGHG LQ PO RI <(3 ZLWKRXW DQWLELRWLFV 7KH VROXWLRQ ZDV NHSW RQ LFH XQWLO LWV XVH IRU WUDQVIRUPDWLRQ 3ODQW WUDQVIRUPDWLRQ 1RGDO LQWHUQRGDO DQG URRW VHJPHQWV IURP 3LQHDSSOH VZHHW RUDQJH VHHGOLQJV ZHUH XVHG DV H[SODQWV IRU WUDQVIRUPDWLRQ 0RRUH HW DO f 7KH LQWHUQRGDO

PAGE 83

DQG URRW SLHFHV FPf ZHUH LQVHUWHG RQ 06 PHGLXP 06 EDVDO VDOW b VXFURVH b P\RLQRVLWRO b DJDU S+ f ZLWK WKH DSLFDO HQG SURWUXGLQJ 1RGDO VHJPHQWV ZHUH SODFHG OHQJWKn ZD\V RQ WKH 06 PHGLXP 7KH H[SODQWV ZHUH LQRFXODWHG ZLWK RQH GURS RI $JUREDFWHULXP XVLQJ D V\ULQJH DQG FRFXOWLYDWHG IRU GD\V DW r& DQG K RI IOXRUHVFHQW OLJKW )LJ $ DQG %f 6HOHFWLRQ $IWHU GD\V WKH H[SODQWV ZHUH WUDQVIHUUHG WR SHWUL SODWHV [ f ZLWK 06 PHGLXP ZLWKRXW JURZWK UHJXODWRUV DQG VXSSOHPHQWHG ZLWK LJPO RI NDQDP\FLQ DQG AJPO RI PHIR[LQ $IWHU WR ZHHNV VKRRWV EHJDQ WR HPHUJH IURP WKH VHJPHQWV 6KRRWV ZHUH KDUYHVWHG DQG H[SODQWV WUDQVIHUUHG WR IUHVK VHOHFWLRQ PHGLXP HYHU\ ZHHNV IRU XS WR WKUHH PRQWKV ZKHQ WKH\ ZHUH GLVFDUGHG )LJ &f $QDO\VLV RI VKRRWV +DUYHVWHG VKRRWV ZHUH DVVD\HG KLVWRFKHPLFDOO\ IRU *86 DFWLYLW\ 6WRPS f 6HJPHQWV H[FLVHG IURP WKH EDVDO HQG RI WKH VKRRWV ZHUH LQFXEDWHG XVLQJ PLFURWLWHU SODWHV LQ A RI 0 1D3 EXIIHU S+ P0 ('7$ b 7ULWRQ ; PJPO ;JOXF DW r& RYHUQLJKW 7UDQVIRUPHG WREDFFR OHDI VHJPHQWV FRQWDLQLQJ WKH *86 JHQH NLQGO\ SURYLGHG E\ 0RRUHf ZHUH LQFOXGHG DV SRVLWLYH FRQWUROV 7KH WLVVXH VDPSOHV ZHUH IL[HG DQG GHVWDLQHG ZLWK cL RI b HWKDQRO DFHWLF DFLG f DW URRP WHPSHUDWXUH IRU DSSUR[LPDWHO\ K EHIRUH H[DPLQDWLRQ XQGHU WKH PLFURVFRSH 3&5 DQDO\VLV 6RPH RI WKH *86 SRVLWLYH *86f WLVVXH VDPSOHV ZHUH IXUWKHU DQDO\]HG E\ 3&5 XVLQJ S RU S VSHFLILF

PAGE 84

)LJXUH 6FKHPDWLF UHSUHVHQWDWLRQ RI WKH WUDQVIRUPDWLRQ SURWRFRO IRU VZHHW RUDQJH SODQWV $f VHHGOLQJV WR PRQWKV ROGf JHUPLQDWHG LQ FXOWXUH WXEHV ZHUH SUXQHG WR HOLPLQDWH OHDYHV 6HJPHQWV RI DSSUR[LPDWHO\ FP ZHUH FXW RXW IURP URRWV DQG VWHPV VHSDUDWLQJ QRGDO DQG LQWHUQRGDO SRUWLRQVf %f H[SODQWV ZHUH SODFHG LQ FXOWXUH SODWHV FRQWDLQLQJ 06 PHGLXP ZLWK WKH DSLFDO HQG SURWUXGLQJ LQWHUQRGHV DQG URRWVf RU OHQJWKZD\V RQ WKH PHGLXP QRGHVf ([SODQWV ZHUH WKHQ LQRFXODWHG ZLWK RQH GURS RI $JUREDFWHULXP $IWHU WKUHH GD\V RI LQFXEDWLRQ WKH H[SODQWV ZHUH WUDQVIHUUHG WR VHOHFWLRQ PHGLXP 06 ZLWK DQWLELRWLFVf &f DSSUR[LPDWHO\ IRXU WR VL[ ZHHNV DIWHU WKH WUDQVIRUPDWLRQ VKRRWV VWDUWHG WR UHJHQHUDWH 7KH VKRRWV ZHUH H[FLVHG IURP WKH H[SODQWV DQG DVVD\HG IRU *86 DFWLYLW\

PAGE 85

F

PAGE 86

SULPHUV &KDSWHU f )L[HG PDWHULDOV ZHUH ZDVKHG WLPHV PLQ HDFKf ZLWK , RI VWHULOH GLVWLOOHG ZDWHU DQG '1$ H[WUDFWHG XVLQJ WKH SURFHGXUH RI 5RJHUV DQG %HQGLFK f 7KH WRWDO XQTXDQWLILHG '1$ H[WUDFWHG IURP HDFK VDPSOH ZDV XVHG LQ D LO 3&5 UHDFWLRQ FRQWDLQLQJ P0 7ULV+&O S+ P0 .&O b 7ULWRQ ; P0 0J&O P0 '77 P0 G173V X 7DT SRO\PHUDVH DQG S0 RI HDFK SULPHU 7KH PL[WXUHV ZHUH JLYHQ F\FOHV RI LQFXEDWLRQ DW r& IRU PLQ r& IRU PLQ r& IRU PLQ DQG D ILQDO LQFXEDWLRQ F\FOH DW r& IRU PLQ r& IRU PLQ DQG r& IRU PLQ $SSUR[LPDWHO\ WLO RI WKH 3&5 SURGXFW ZHUH DQDO\]HG E\ b DJDURVH JHO HOHFWURSKRUHVLV 6RXWKHUQ DQDO\VLV RI WKH 3&5 SURGXFWV [Of ZDV FRQGXFWHG XVLQJ WKH SURFHGXUH GHVFULEHG LQ 6DPEURRN HW DO f 7KH PHPEUDQHV ZHUH SUREHG XVLQJ WKH 3KRWRJHQH V\VWHP %LRWLQ\ODWHG S SUREHV ZHUH SURGXFHG XVLQJ S9)O FORQHV DV WHPSODWHV LQ 3&5 UHDFWLRQV 5DVKWFKLDQ DQG 0DFNH\ 0HUW] HW DO f 5HJHQHUDWLRQ RI VKRRWV *86 SRVLWLYH VKRRWV ZHUH WUDQVIHUUHG WR VWHULOH EDE\ IRRG MDUV FRQWDLQLQJ PO RI VWHULOH VRLO 0HWURPL[ f DQG LUULJDWHG ZLWK PO RI VWHULOH WZRWKLUGV VWUHQJWK 06 EDVDO VDOW PHGLXP $ VROXWLRQ RI AJPO NDQDP\FLQ MXJPO PHIR[LQ DQG 5RR7RQH *UHHQ /LJKW &R 6DQ $QWRQLR 7H[DVf ZDV DSSOLHG WR WKH EDVH RI WKH VKRRWV EHIRUH SODQWLQJ LQWR VRLO 3ODQWV ZHUH PDLQWDLQHG DW r& ZLWK K IOXRUHVFHQW OLJKW

PAGE 87

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f 3&5 ZLWK S S )LJ f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

PAGE 88

)LJXUH 3&5 RI $JUREDFWHULXP $%, FXOWXUHV WR FRQILUP WKH SUHVHQFH RI S9) SODVPLGV /DQH $JUREDFWHULXPS9)O EDFWHULDO H[WUDFW DPSOLILHG XVLQJ S SULPHUV /DQH $JUREDFWHULXPS9) EDFWHULDO H[WUDFW DPSOLILHG XVLQJ S SULPHUV /DQH SXULILHG S9)O SODVPLG '1$ DPSOLILHG ZLWK S SULPHUV /DQH SXULILHG S9) SODVPLG '1$ DPSOLILHG ZLWK S SULPHUV /DQH /DPEGD '1$ GLJHVWHG ZLWK +LQGOOO XVHG DV D 0: PDUNHU

PAGE 89

)LJXUH $GYHQWLWLRXV VKRRW IRUPDWLRQ RQ LQWHUQRGDO VHJPHQWV $f DQG *86 VKRRWV LQ VRLO IRU URRWLQJ %f

PAGE 90

7KH UHVXOWV LQ 7DEOH LQGLFDWH WKDW PRUH DGYHQWLWLRXV VKRRWV ZHUH SURGXFHG IURP URRW VHJPHQWV FRPSDUHG WR QRGHV RU LQWHUQRGHV 1RGHV SURGXFHG WKH ORZHVW DYHUDJH QXPEHU RI DGYHQWLWLRXV VKRRWV 7KHUH ZDV QR GLIIHUHQFH LQ VKRRW SURGXFWLRQ ZKHQ H[SODQWV ZHUH LQRFXODWHG ZLWK S9)O RU S9) +RZHYHU FRPSDULVRQ RI WKH HIILFLHQFLHV RI WUDQVIRUPDWLRQ DV SHUFHQWDJH RI *86 VKRRWV REWDLQHG IURP WKH WRWDOf VKRZHG WKDW LQWHUQRGHV ZHUH PRUH HIILFLHQWO\ WUDQVIRUPHG ZLWK S9)O 7DEOH f WKDQ QRGHV RU URRWV 7KLV LQGLFDWHV WKDW SURSRUWLRQDWHO\ LQWHUQRGHV JHQHUDWHG WZLFH DV PDQ\ *86 )LJ f VKRRWV WKDQ WKH URRW VHJPHQWV ,QWHUHVWLQJO\ WUDQVIRUPDWLRQ ZLWK S9) ZDV PRUH VXFFHVVIXO RQ QRGHV WKDQ RQ LQWHUQRGHV 7DEOH f $JDLQ WDNLQJ UHJHQHUDWLRQ DQG WUDQVIRUPDWLRQ HIILFLHQF\ WRJHWKHU LQWHUQRGH VHJPHQWV SURGXFHG WKUHH WLPHV DV PDQ\ *86 VKRRWV WKDQ QRGHV 7UDQVIRUPDWLRQ HIILFLHQF\ ZDV TXLWH YDULDEOH EHWZHHQ H[SHULPHQWV UDQJLQJ IURP WR b 7KH RYHUDOO HIILFLHQF\ ZDV ORZHU WKDQ GHVLUDEOH 7DNLQJ WKH UHVXOWV IURP DOO WKH WUDQVIRUPDWLRQ H[SHULPHQWV XVLQJ S9)O IURP VKRRWV REWDLQHG bf ZHUH *86 :KHQ S9) ZDV XVHG IURP VKRRWV REWDLQHG bf ZHUH *86 6XUYLYDO RI WKH *86 VKRRWV LQ VRLO 7DEOH f UDQJHG IURP WR b VHYHQ PRQWKV DIWHU WUDQVIHU DOWKRXJK QRQH RI WKHP KDYH URRWHG DW WKH WLPH WKLV LV ZULWWHQ 7KH UHVXOWV IURP WKH VKRRWV REWDLQHG IURP URRW VHJPHQWV DUH QRW LQFOXGHG DV WKH\ KDYH EHHQ LQ VRLO RQO\ WZR PRQWKV

PAGE 91

7DEOH (IIHFWV RI H[SODQW W\SH DQG SODVPLG RQ DGYHQWLWLRXV VKRRW IRUPDWLRQ LQ nf3LQHDSSOH VZHHW RUDQJH (;3/$17 3/$60,' 1R 2) 6(*0(176 6+2276 3(5 6(*0(17 1RGHV S9)O s S9) s 7RWDO s ,QWHUQRGHV S9)O s S9) s 7RWDO s 5RRWV S9)O s

PAGE 92

)LJXUH *86 DVVD\ RQ DGYHQWLWLRXV VKRRWV RI 3LQHDSSOH VZHHW RUDQJH UHJHQHUDWHG IURP $JUREDFWHULXPLQRFXODWHG LQWHUQRGDO VHJPHQWV $f *86 VHJPHQW %f *86 VHJPHQW

PAGE 93

7DEOH 7UDQVIRUPDWLRQ HIILFLHQFLHV DQG VXUYLYDO RI VKRRWV RI 3LQHDSSOH VZHHW RUDQJH XVLQJ S9)O Sf DQG S9) Sf 3/$60,' (;3/$17 1R 2) 6+2276 1R 2) *86 6+2276 b *86 6+2276 1R 6859,9,1* b 6859,9,1* S9)O 1RGHV ,QWHUQRGHV 5RRWV F F S9) 1RGHV ,QWHUQRGHV D S9)O S S9) S E 6XUYLYDO LQ VRLO VHYHQ PRQWKV DIWHU WUDQVIHU F $GYHQWLWLRXV VKRRWV GHULYHG IURP URRW VHJPHQWV KDYH EHHQ LQ VRLO OHVV WKDQ VHYHQ PRQWKV &7

PAGE 94

7R FRQILUP SUHVHQFH RI WKH &79 JHQHV LQ WKH *86 SODQWV VDPSOHV ZHUH DQDO\]HG XVLQJ 3&5 )LJ 7DEOH f )RU WKRVH *86 VKRRWV WUDQVIRUPHG ZLWK S9)O b ZHUH DOVR 3&5 )RU S9) b RI WKH *86 VKRRWV ZHUH DOVR 3&5 7UDQVIRUPHG *86n VHJPHQWV ZHUH XVHG DV QHJDWLYH FRQWUROV WR UXOH RXW WKH SRVVLELOLW\ RI REWDLQLQJ 3&5 SURGXFWV IURP FRQWDPLQDWLQJ $JUREDFWHULXP +\EULGL]DWLRQ RI WKH 3&5 SURGXFWV REWDLQHG IURP WKH SODQWV ZLWK S RU S SUREHV GHPRQVWUDWHG WKH\ ZHUH &79 VSHFLILF VHJXHQFHV )LJ f 'LVFXVVLRQ 7KH QXPEHU RI VKRRWV SURGXFHG E\ WKH LQWHUQRGHV LV FRPSDUDEOH WR ZKDW ZDV REWDLQHG SUHYLRXVO\ IRU &DUUL]R FLWUDQJH >& VLQHQVLV /f 2VE [ 3RQFLUXV WULIROLDWD /f 5DI@ XQGHU VLPLODU H[SHULPHQWDO FRQGLWLRQV 0RRUH HW DO f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b FRQVLGHULQJ DOO H[SHULPHQWV WRJHWKHUf ZHUH *86 (VFDSHV

PAGE 95

0 \ )LJXUH 3&5 DPSOLILFDWLRQ RI '1$ H[WUDFWV IURP 3LQHDSSOH VZHHW RUDQJH *86 VHJPHQWV WUDQVIRUPHG ZLWK S $f RU S %f $f /DQH /DPEGD '1$ GLJHVWHG ZLWK +LQGOOO XVHG DV D 0: PDUNHU /DQHV DQG *86 VHJPHQWV /DQHV WR DQG WR *86 VHJPHQWV /DQH S9)O %f /DQH /DPEGD '1$ GLJHVWHG ZLWK +LQGOOO XVHG DV D 0: PDUNHU /DQH *86 VHJPHQW /DQHV WR *86 VHJPHQWV /DQH S9)

PAGE 96

7DEOH 1XPEHU DQG SHUFHQWDJH RI 3LQHDSSOH VZHHW RUDQJH VKRRWV WKDW WHVWHG SRVLWLYH IRU *86 DQG E\ 3&5 3/$60,' *86 3&5 b 3&5 S9)O S9)

PAGE 97

)LJXUH 6RXWKHUQ DQDO\VLV RI 3&5 SURGXFWV IURP *86 VHJPHQWV RI 3LQHDSSOH VZHHW RUDQJH K\EULGL]HG ZLWK D S VSHFLILF SUREH /DQHV DQG *86 VHJPHQW /DQHV WR DQG *86 VHJPHQWV WUDQVIRUPHG ZLWK S JHQH /DQH /DPEGD '1$ GLJHVWHG ZLWK +LQGOOO /DQH DPSOLILHG S IURP S9)O

PAGE 98

KDYH EHHQ UHSRUWHG SUHYLRXVO\ 0RRUH HW DO 3HD HW DO f DQG FRQVWLWXWH D VHULRXV SUREOHP LQFUHDVLQJ WKH ZRUN QHFHVVDU\ LQ VFUHHQLQJ WKH VKRRWV 7KLV LQHIIHFWLYH .DQDP\FLQ VHOHFWLRQ PD\ EH H[SODLQHG E\ RQH RU VHYHUDO SRVVLELOLWLHV f SURWHFWLRQ RI QRQWUDQVIRUPHG FHOOV E\ WKH VXUURXQGLQJ WUDQVIRUPHG FHOOV f FRQWDPLQDWLRQ ZLWK $JUREDFWHULXP RU f E\ HQGRJHQRXV OHYHOV RI 137 ,, DFWLYLW\ -RUGDQ DQG 0F+XJKHQ 'DQGHNDU HW DO 0RRUH HW DO 3HD HW DO f 7UDQVIRUPDWLRQ HIILFLHQFLHV H[SUHVVHG DV SHUFHQWDJH RI *86 VKRRWV IURP WKH WRWDO DGYHQWLWLRXV VKRRWV SURGXFHG ZHUH YHU\ YDULDEOH EHWZHHQ H[SHULPHQWV DQG LQ JHQHUDO ORZ 9DOXHV UDQJHG IURP WR b ZLWK DQ DYHUDJH RI b 7KHVH UHVXOWV DUH FRPSDUDEOH WR WKRVH REWDLQHG E\ 0RRUH HW DO f EXW ORZHU WKDQ WKH b REWDLQHG ZLWK &DUUL]R FLWUDQJH E\ 3HD HW DO f RU WKH b WR b UHSRUWHG E\ .DQH\RVKL HW DO f ZLWK 3RQFLUXV WULIROLDWD 5DI 2QH RI WKH GLIIHUHQFHV LQ WKH WUDQVIRUPDWLRQ SURWRFRO XVHG KHUH DQG WKH RQHV PHQWLRQHG DERYH LV WKDW WKH VHJPHQWV DUH LQRFXODWHG E\ VXEPHUVLRQ IRU VHYHUDO PLQXWHV WR D IHZ GD\V LQ D VROXWLRQ FRQWDLQLQJ WKH $JUREDFWHULXP $QRWKHU GLIIHUHQFH LV WKDW LQ WKRVH FDVHV WKH H[SODQWV ZHUH REWDLQHG IURP UHODWLYHO\ \RXQJ VHHGOLQJV WR ZHHNV ROGf LQVWHDG RI WR PRQWKV ROG DV UHSRUWHG KHUH )LQDOO\ WKH SURFHGXUH RI .DQH\RVKL HW DO f LQFOXGHV DFHWRV\ULQJRQH LQ WKH LQRFXODWLRQ PHGLD WR LQFUHDVH WKH HIILFLHQF\ RI EDFWHULDO LQIHFWLRQ

PAGE 99

5RRWLQJ RI WKH WUDQVIRUPHG VKRRWV KDV EHHQ GLIILFXOW ZLWK ORZHU VXUYLYDO UDWHV WKDQ UHSRUWHG E\ RWKHU DXWKRUV 0RRUH HW DO .DQH\RVKL HW DO 3HD HW DO f 7KLV PD\ EH GXH WR D GHWULPHQWDO HIIHFW RI WKH DQWLELRWLFV RQ WKH H[SODQWV RU SRVVLEO\ WR WKH H[SUHVVLRQ RI WKH &79 JHQH SURGXFWV LQ WKH VKRRWV &79 LQIHFWLRQ UHGXFHV URRW DQG EXG IRUPDWLRQ RI VKRRWV LQ YLWUR 'XUDQ9LOD f ,W LV SRVVLEOH WKDW WKH FRQVWLWXWLYH H[SUHVVLRQ S RU S FDXVHV WKH UHGXFWLRQ RI URRW IRUPDWLRQ LQ WKH H[SODQWV 3&5 DQDO\VLV RI WKH VDPH VHJPHQWV XVHG LQ WKH KLVWRFKHPLFDO *86 DVVD\ FRQILUPHG WKH SUHVHQFH RI S DQG S LQ WKH SXWDWLYHO\ WUDQVIRUPHG VKRRWV 2QO\ b Sf RU b Sf RI WKH *86 VHJPHQWV ZHUH DOVR 3&5 7KLV PD\ EH GXH WR WUXH IDOVH SRVLWLYHV RU WR GHILFLHQFLHV LQ WKH '1$ H[WUDFWLRQ SURFHGXUH EHFDXVH RI WKH VPDOO DPRXQW RI WLVVXH XVHG +\EULGL]DWLRQ H[SHULPHQWV FRQILUPHG WKH VSHFLILFLW\ RI WKH EDQGV REWDLQHG LQ WKH 3&5 DQDO\VLV GHPRQVWUDWLQJ WKDW WKH\ DUH S DQG S JHQHV

PAGE 100

&+$37(5 6800$5< $1' &21&/86,216 f 7KH H[SUHVVLRQ RI &79 25)V S DQG S ZDV GHWHFWHG LQ YLYR LQ &79LQIHFWHG SODQWV XVLQJ VSHFLILF SRO\FORQDO DQWLERGLHV UDLVHG DJDLQVW WKH UHFRPELQDQW SURWHLQV f &HOO IUDFWLRQDWLRQ DQDO\VLV LQGLFDWHG WKDW S DFFXPXODWHV LQ WKH FHOO ZDOO IUDFWLRQ ZKHUHDV S DFFXPXODWHV LQ WKH VROXEOH SURWHLQ IUDFWLRQ f 7LVVXH EORWV LQGLFDWHG WKDW S LV SUHVHQW LQ WKH SKORHP 7KLV LV LQ DJUHHPHQW ZLWK &79 EHLQJ D SKORHP OLPLWHG YLUXV f 7KH IXQFWLRQ RI S LV VWLOO XQNQRZQ EXW EDVHG RQ VHJXHQFH KRPRORJ\ DQG FHOO IUDFWLRQ VWXGLHV S FRXOG EH LQYROYHG LQ DQ\ RU DOO RI WKUHH IXQFWLRQV YLUXV DVVHPEO\ YLUXV PRYHPHQW DQG DSKLG WUDQVPLVVLRQ f %DVHG RQ VHJXHQFH FRQVHUYDWLRQ WKH DFWLYH VLWH RI S DSSHDUV WR EH ORFDWHG LQ WKH &WHUPLQDO SRUWLRQ RI WKH SURWHLQ

PAGE 101

f 7KH IXQFWLRQ RI S LV DOVR XQNQRZQ ,WV LQ YLYR H[SUHVVLRQ DQG DFFXPXODWLRQ GLG QRW JLYH DQ\ LQGLFDWLRQV DERXW LWV SRVVLEOH UROH LQ WKH &79 OLIH F\FOH f 6HTXHQFH DQG SK\ORJHQHWLF DQDO\VLV RI WKH S JHQH IURP GLIIHUHQW &79 VWUDLQV LQGLFDWHG D UHODWLRQVKLS EHWZHHQ GHGXFHG DPLQR DFLG VHTXHQFH DQG ELRORJLFDO DFWLYLW\ RI WKH VWUDLQV f 66&3 DQDO\VLV SHUPLWWHG WKH GLIIHUHQWLDWLRQ RI &79 VWUDLQV 0RVW PLOG VWUDLQV FRXOG WR EH LGHQWLILHG XVLQJ WKLV PHWKRG f 7KH JHQHV HQFRGLQJ S DQG S ZHUH WUDQVIRUPHG LQWR 3LQHDSSOH VZHHW RUDQJH XVLQJ $JUREDFWHULXP 7UDQVIRUPDWLRQ ZDV FRQILUPHG XVLQJ *86 DQG 3&5 DVVD\V f ,QWHUQRGDO VHJPHQWV IURP VHHGOLQJ HSLFRW\OV ZHUH WKH PRVW HIILFLHQW LQ SURGXFLQJ *86 DGYHQWLWLRXV VKRRWV

PAGE 102

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t 6RQV 1HZ
PAGE 103

%HDFK\ 51 /RHVFK)ULHV 6 DQG 7XUQHU 1( &RDW SURWHLQ PHGLDWHG UHVLVWDQFH DJDLQVW YLUXV LQIHFWLRQ $QQ 5HY 3K\WRSDWKRO %R\NR 93 .DUDVHY $9 $JUDQRYVN\ $$ .RRQLQ (9 DQG 'ROMD 99 &RDW SURWHLQ JHQH GXSOLFDWLRQ LQ D ILODPHQWRXV 51$ YLUXV RI SODQWV 3URF 1DWO $FDG 6FL 86$ %UODQVN\ 5+ /HH 5) DQG *DUQVH\ 60 ,Q VLWX LPPXQRIOXRUHVFHQFH IRU WKH GHWHFWLRQ RI FLWUXV WULVWH]D YLUXV LQFOXVLRQ ERGLHV 3ODQW 'LVHDVH %UODQVN\ 5+ 3HORVL 55 *DUQVH\ *0
PAGE 104

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f :RUNVKRS RQ FLWUXV WULVWH]D YLUXV7R[RSWHUD FLWULFLGD LQ &HQWUDO $PHULFD DQG WKH &DULEEHDQ %DVLQ 7XUULDOED &RVWD 5LFD *DUQVH\ 60 *XPSI '5RLVWDFKHU &1 &LYHUROR (/ /HH 5)
PAGE 105

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
PAGE 106

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f 3URF WK &RQI RI ,QW 2UJDQ &LWUXV 9LURO ,2&9 5LYHUVLGH &$ /HH 5) DQG 5RFKD3HD 0$ &LWUXV WULVWH]D YLUXV 3DJHV LQ .XPDU &KDXEH +6 6LQJK 86 0XNKRSDGK\D\ $1 HGVf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

PAGE 107

0HQGW 5 +LVWRU\ RI &79 LQ 9HQH]XHOD 3DJHV LQ /DVWUD 5 /HH 5 5RFKD3HD 0 1LEOHWW &/ 2FKRD ) *DUQVH\ 60 DQG
PAGE 108

3DSSX +5 .DUDVHY $9 $QGHUVRQ (3DSSX 66 +LOI 0( )HEUHV 9(FNORII 50* 0F&DIIHU\ 0 %R\NR 9 *RZGD 6 'ROMD 99 .RRQLQ (9 *XPSI '&OLQH .& *DUQVH\ 60 'DZVRQ :2 /HH 5) DQG 1LEOHWW &/ 1XFOHRWLGH VHTXHQFH DQG RUJDQL]DWLRQ RI HLJKW n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n QRQ WUDQVODWHG UHJLRQV RI SRW\YLUXVHV $SSOLFDWLRQ WR GDVKHHQ PRVDLF YLUXV 9LURORJLFDO 0HWKRGV 3DVFDO ( *RRGORYH 3( :X & DQG /D]DURZLW] 6* 7UDQVJHQLF WREDFFR SODQWV H[SUHVVLQJ WKH JHPLQLYLUXV %/ SURWHLQ H[KLELWV V\PSWRPV RI YLUDO GLVHDVH 3ODQW &HOO 3HD / &HUYHUD 0 -X£UH] 2UWHJD & 3LQD -$ 'XU£Q9LOD 1 DQG 1DYDUUR / +LJK HIILFLHQF\ $JUREDFWHULXPPHGLDWHG WUDQVIRUPDWLRQ DQG UHJHQHUDWLRQ RI FLWUXV 3ODQW 6FLHQFH 3HUPDU 7 *DUQVH\ 60 *XPSI 'DQG /HH 5) $ PRQRFORQDO DQWLERG\ WKDW GLVFULPLQDWHV VWUDLQV RI FLWUXV WULVWH]D YLUXV 3K\WRSDWKRORJ\ 3RZHOO &$ 3HORVL 55 DQG &RKHQ 0 6XSHULQIHFWLRQ RI RUDQJH WUHHV FRQWDLQLQJ PLOG LVRODWHV RI FLWUXV WULVWH]D YLUXV ZLWK VHYHUH LVRODWHV RI FLWUXV WULVWH]D YLUXV 3ODQW 'LVHDVH

PAGE 109

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n1RYDn W£QJHOR ZLWK WKH FRDW SURWHLQ JHQH RI FLWUXV WULVWH]D FORVWHURYLUXV 3K\WRSDWKRORJ\ 6FKQHLGHU + 7KH DQDWRP\ RI WULVWH]DYLUXVLQIHFWHG FLWUXV 3DJHV LQ :DOODFH -0 HGf &LWUXV 9LUXV 'LVHDVHV 8QLYHUVLW\ RI &DOLIRUQLD %HUNHOH\ &$ 6FRU]D 5 5DYHORQDQGUR 0 &DOODKDQ $0 &RUGWV -0 )XFKV 0 'XQH] DQG *RQVDOYHV 7UDQVJHQLF SOXPV 3UXQXV GRPHVWLFD /f H[SUHVV WKH SOXP SR[ YLUXV FRDW SURWHLQ 3ODQW &HOO 5HSRUWV 6HNL\D 0( /DZUHQFH 6' 0F&DIIHU\ 0 DQG &OLQH 0ROHFXODU FORQLQJ DQG QXFOHRWLGH VHTXHQFLQJ RI WKH FRDW SURWHLQ JHQH RI FLWUXV WULVWH]D YLUXV *HQ 9LURO

PAGE 110

6KHIILHOG 9& %HFN -6 .ZLWHN $( 6DQGVWURP ': DQG 6WRQH (0 7KH VHQVLWLYLW\ RI VLQJOHVWUDQGHG FRQIRUPDWLRQ SRO\PRUSKLVP DQDO\VLV IRU WKH GHWHFWLRQ RI VLQJOHV EDVH VXEVWLWXWLRQV *HQRPLFV 6SLQDUGL / 0D]DUV 5 DQG 7KHLOOHW & 3URWRFROV IRU DQ LPSURYHG GHWHFWLRQ RI SRLQW PXWDWLRQV E\ 66&3 1XFOHLF $FLGV 5HVHDUFK 6WRPS $ 0 +LVWRFKHPLFDO ORFDOL]DWLRQ RI JOXFXURQLGDVH 3DJHV LQ *DOODJKHU 65 HGf *86 3URWRFROV 8VLQJ WKH *86 *HQH DV D 5HSRUWHU RI *HQH ([SUHVVLRQ $FDGHPLF 3UHVV ,QF 6DQ 'LHJR &$ 6WXGLHU ): 5RVHQEHUJ $+ 'XQQ -DQG 'XEHQGRUII -: 8VH RI 7 51$ SRO\PHUDVH WR GLUHFW H[SUHVVLRQ RI FORQHG JHQHV 3DJHV LQ *RHGGHO '9 HGf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

PAGE 111


PAGE 112

%,2*5$3+,&$/ 6.(7&+ 9LFHQWH -RV )HEUHV5RGULJXH] ZDV ERUQ LQ &DUDFDV 9HQH]XHOD LQ +H REWDLQHG D EDFKHORUnV GHJUHH LQ ELRORJ\ LQ IURP WKH 6LPQ %ROLYDU 8QLYHUVLW\ LQ &DUDFDV $IWHU KLV JUDGXDWLRQ KH ZRUNHG DW WKH 9HQH]XHODQ ,QVWLWXWH IRU 6FLHQWLILF 5HVHDUFK ,9,&f XQWLO KH PRYHG WR &RVWD 5LFD LQ WR FRQWLQXH VWXGLHV DW WKH 7URSLFDO $JURQRPLF &HQWHU IRU 5HVHDUFK DQG 7HDFKLQJ &$7,(f LQ 7XUULDOED +H REWDLQHG KLV PDVWHUnV LQ SODQW EUHHGLQJ LQ DQG FRQWLQXHG ZRUNLQJ DW &$7,( DV D FRQVXOWDQW ,Q KH VWDUWHG KLV VWXGLHV IRU D 3K' LQ SODQW SDWKRORJ\ DW WKH 8QLYHUVLW\ RI )ORULGD IURP ZKLFK KH JUDGXDWHG LQ

PAGE 113

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n&FKDLU 3URIHVVRU RI 3ODQW 3DWKRORJ\ FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ ac/&/ bt8f-/ -XGH : *URVVHU 3URIHVVRU RI +RUWLFXOWXUDO 6FLHQFH

PAGE 114

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


76
and root pieces (1 cm) were inserted on MS medium (MS basal
salt, 5% sucrose, 0.01% myo-inositol, 0.8% agar pH 7.5) with
the apical end protruding. Nodal segments were placed length
ways on the MS medium. The explants were inoculated with one
drop of Agrobacterium using a syringe and co-cultivated for 3
days at 27C and 16 h of fluorescent light (Fig. 18, A and B).
Selection. After 3 days, the explants were transferred to
petri plates (20 x 100) with MS medium, without growth
regulators, and supplemented with 100 ig/ml of kanamycin and
200 /^g/ml of mefoxin. After 4 to 6 weeks, shoots began to
emerge from the segments. Shoots were harvested and explants
transferred to fresh selection medium every 4 weeks for up to
three months, when they were discarded (Fig. 18, C).
Analysis of shoots. Harvested shoots were assayed
histochemically for GUS activity (Stomp., 1992). Segments
excised from the basal end of the shoots were incubated, using
microtiter plates, in 25 ^1 of 0.1 M NaP04 buffer, pH 7.0, 10
mM EDTA, 0.1% Triton X-100, 0.25 mg/ml X-gluc at 37C
overnight. Transformed tobacco leaf segments, containing the
GUS gene (kindly provided by G. Moore), were included as
positive controls. The tissue samples were fixed and destained
with 50 ¡i 1 of 95% ethanol: acetic acid (3:1) at room
temperature for approximately 1 h before examination under the
microscope.
PCR analysis. Some of the GUS positive (GUS+) tissue
samples were further analyzed by PCR using p27 or p20 specific


13
mixtures with the template RNA were incubated in an automatic
thermocycler at 42C for 45 min, followed by 40 cycles of
incubations at 94C for 1 min, 45C for 1 min and 72C for 1
min, and a final incubation at 72 C for 10 min. The same
parameters were used for the amplification of all three ORFs.
After the PCR reaction was completed, a 5 jUl aliquot was
electrophoresed in a 0.8% agarose gel in TBE buffer (9 mM
Tris-borate, 2 mM EDTA) to determine the amplification of the
DNA fragment of the correct size. Lambda DNA digested with
Hindlll was used as a molecular weight marker.
Purification of the PCR products. Once the fragments of
interest were detected, the rest of the PCR samples (95 /l)
were electrophoresed in 0.8% LMP agarose in TBE buffer at 4C
and 50 to 75 V for about 1 h. The DNA bands of interest were
excised from the gel with a sterile blade under UV light and
transferred to a microcentrifuge tube. TE buffer was added to
a final volume of 0.6 ml. Samples were then incubated at 65C
for 7 min to melt the agarose, and then extracted with phenol,
phenol: chloroform, and chloroform followed by ethanol
precipitation according to Sambrook at al. (1989). The final
DNA pellet was resuspended in 10 ¡jl 1 of sterile distilled
water.
Initially, the amplified p27, p20 and pl8 genes were
cloned into the Smal site of the pUC118 vector. Restriction
sites for Hindlll and Xbal were included in the sense primers
to facilitate subcloning into the pETH expression vector and


62
1
2 3 4
5
6
7
Figure 15. SSCP analysis of the p27 gene from
other CTV strains. 1, T30; 2, B5; 3, B32; 4, B192;
5, B274; 6, B272; 7, T36; 8, Bll; 9, B37; 10, B28;
11, B148 and 12, T36.


25
1 2 3 4 5 6
7 8 9 10
Figure 2. Western blot analysis of CTV-infected and
uninfected citrus leaf extracts with p27 polyclonal
antiserum (lanes 1-5) and with MCA-13 monoclonal
antibody (lanes 6-10). Lanes 1 and 10, pETH-3b
without insert; Lanes 2 and 9, E. coli-expressed
fusion p27 (lane 9 contains 100 times more protein
than lane 2); Lanes 3 and 8, E. coli-expressed CP;
Lanes 4 and 7, uninfected citrus extracts; Lanes 5
and 6, CTV T36-infected citrus extracts. Molecular
weights in kilodaltons are indicated on the right.