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Pine chitinase gene structure, expression and regulation

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Pine chitinase gene structure, expression and regulation analysis in pine cells and in heterologous systems
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Wu, Haiguo, 1967-
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English
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xii, 139 leaves : ill. ; 29 cm.

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Bombardment ( jstor )
Cells ( jstor )
Complementary DNA ( jstor )
Infections ( jstor )
Leaves ( jstor )
Pathogens ( jstor )
Plant cells ( jstor )
Pollen ( jstor )
Proteins ( jstor )
Species ( jstor )
Dissertations, Academic -- Plant Molecular and Cellular Biology -- UF ( lcsh )
Pine -- Genetics ( lcsh )
Plant Molecular and Cellular Biology thesis, Ph. D ( lcsh )
Plant cell walls ( lcsh )
Plant cells and tissues ( lcsh )
Plant molecular biology ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1996.
Bibliography:
Includes bibliographical references (leaves 117-138).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Haiguo Wu.

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PINE CHITINASE GENE STRUCTURE, EXPRESSION AND REGULATION:
ANALYSIS IN PINE CELLS AND IN HETEROLOGOUS SYSTEMS











By

HAIGUO WU


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


1996




PINE CHITINASE GENE STRUCTURE, EXPRESSION AND REGULATION
ANALYSIS IN PINE CELLS AND IN HETEROLOGOUS SYSTEMS
By
HAIGUO WU
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
1996


This dissertation is dedicated to my Lord and Savior Jesus Christ, and to my
wife, Yang.


ACKNOWLEDGMENTS
I would like to express my sincere appreciation to the members of my
graduate committee: Dr. John Davis, Dr. Ken Cline, Dr. Bill Gurley, Dr. Corby Kistler
and Dr. James Preston for their critical advice, suggestions and discussions, which
have greatly improved this dissertation. In particular, Dr. Gurley has provided me
with constant and enthusiastic support over the years.
I am also indebted to Dr. Don McCarty for allowing me to access his particle
gun, to Dr. Chien-Yuan Kao and Dr. Eva Czarnecka-Verner for teaching me howto
use this gun for bombardment experiments, and to Dr. Rosie Simmen for access to
the automated luminor for luciferase assays. I wish to extend my thanks to Dr.
Craig Echt for sequencing the initial genomic clone and providing part of the DNA
primers for this study, to Don Baldwin for helpful suggestions and discussions about
primer extension experiment.
I would like to thank all the past and present members of Dr. John Davis'
laboratory. My thanks go to Dr. Mark Lesney for helpful discussions and assistance
with the cell cultures, to Dr. Mick Popp, Dr. Yong Qian and Buddy Tignor for their
assistance and friendship. I would like to recognize the excellent technical support
of Tess Korhnak and Thea Edwards, whose collective contributions to this work
ensured the smooth and efficient running of the program.


My most special thanks go to my advisor, Dr. John Davis, for his invaluable
advice and guidance, for his encouragement and generosity, for his tremendous
support and unending confidence in me, for all his efforts on my behalf throughout
the course of this study. Working under his supervision has been a wonderful
experience in my life.
Finally, I would like to thank my wife, Yang, for her continual love,
encouragement and support, for without her and my faith in God, this work would
not have been completed.
IV


TABLE OF CONTENTS
ACKNOWLEDGMENTS iii
LIST OF TABLES vii
LIST OF FIGURES viii
LIST OF ABBREVIATIONS x
ABSTRACT xi
LITERATURE REVIEW 1
General Features of Plant Defense Responses 1
Chitosan, General Elicitors and Specific Elicitors 16
Pathogenesis-Related Proteins 20
Chitinase Structure, Function and Regulation 27
INTRODUCTION 35
MATERIALS AND METHODS 41
Plant Materials 41
Sequence and Sequence Analysis 42
Plant Transformation 43
Elicitor Treatment and RNA Isolation 44
Primer Extension 47
Southern and Northern Analysis 47
Cloning of the Pschi4 cDNA 49
cDNA Expression and Generation of Antibody 51
Protein Isolation and Western Blotting Analysis 52
Particle Bombardment and Transient Expression 55
Histochemical Assays in Transgenic Tobacco 60
v


RESULTS
62
Pschi4 Gene Structure 62
Pschi4 cDNA Cloning and Expression in Bacteria 66
Pschi4 Expression 73
Transient Assay of Pschi4 Promoter-GUS Constructs 81
GUS Expression in Stably Transformed Tobacco Plants 89
Developmental Regulation of Psch¡4 Expression 96
DISCUSSION 101
SUMMARY AND FUTURE DIRECTIONS 110
APPENDIX 114
REFERENCES 117
BIOGRAPHICAL SKETCH 139
VI


LIST OF TABLES
Table page
1 Chitosan-inducted mRNA accumulation in transgenic tobacco plants . 75
2 Summary of WP-GUS tobacco plants 90
vii


LIST OF FIGURES
Figure page
1 Gene-for-gene interactions specify plant disease
resistance or susceptibility 5
2 Different actions between endochitinases and exochitinases 28
3 Domain structure of three classes of chitinases 30
4 Overall structure of the pine genomic
subclones gPschU and gPschi4 38
5 Sites within Pschi4 that were used to design
oligonucleotide primers for this study 48
6 Plasmid constructs 56
7 Partial nucleotide sequence and translation product
encoded by the genomic clone containing Pschi4 63
8 Primer extension analysis to reveal the putative
transcription start site(s) 64
9 Domain structure of the putative Pschi4 protein from pine
with class I and II chitinase from tobacco 67
10 Sequence alignment of Pschi4 with tobacco chitinases 68
11 Genomic Southern blot analysis of DNA from three pine species 69
12 Cloning of Pschi4 cDNA by RT-PCR 70
13 Pschi4 cDNA expression in bacteria 72
viii


14 Transcripts accumulation in chitosan-treated pine cells 74
15 Northern blot showing expression of Pschi4 in a
transgenic tobacco plant 76
16 Pschi4 protein expression in pine suspension cells 78
17 Chitosan-induced Pschi4 protein expression
in pine suspension cells 80
18 Western blot analysis in tobacco suspension cells 82
19 No chitosan-induction in transient assays in onion cells 84
20 Promoter activity in onion cells 85
21 Promoter comparison in maize and in pine cells 87
22 Promoter activity in pine cells 88
23 Particle bombardment perse induces promoter activity 93
24 GUS activity was not induced by chitosan in
stably transformed tobacco plants 94
25 Mechanical wounding induced promoter activity 95
26 Phosphate induced WP-GUS expression in transgenic tobacco 97
27 X-gluc staining of tobacco pollen 98
28 Pschi4 protein expression in tobacco pollen 100
IX


LIST OF ABBREVIATIONS
2,4-D
2,4-dichlorophenoxyacetic acid
2-iP
N6-2-isopentenyl-adenine
BAP
6-benzyl-aminopurine
BCIP
5-bromo-4-chloro-3-indolyl phosphate
DMF
dimethyl formamide
DTT
dithiothreitol
EDTA
ethylenediamine tetraacetate (disodium salt)
GUS
p-glucuronidase
IPTG
isopropyl p-D-thiogalacto-pyranoside
LB
Luria-Bertani medium
MS
Murashige-Skoog medium
MUG
4-methylumbelliferyl p-D-glucuronide
NAA
a-naphthalene acetic acid
NBT
nitro blue tetrazolium
PVPP
polyvinyl-polypyrrolidone
SDS
sodium dodecyl sulfate
X-gluc
5-bromo-4-chloro-3-indolyl-p-D-glucuronide
x


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
PINE CHITINASE GENE STRUCTURE, EXPRESSION AND REGULATION:
ANALYSIS IN PINE CELLS AND IN HETEROLOGOUS SYSTEMS
By
Haiguo Wu
December 1996
Chairman: Dr. John M. Davis
Major Department: Plant Molecular and Cellular Biology
Chitinases are plant enzymes that hydrolyze chitin, which is the major
component of the cell walls of many pathogenic fungi but absent in higher plants.
Chitinases belong to Group III PR-proteins which are believed to play important
roles during plant-pathogen interactions. The present study describes the
structures of several genomic clones from pine trees that appear to encode
extracellular class II chitinase, and examines the expression of these genes in pine
cells as well as in transgenic tobacco plants. One of the genes, Pschi4, potentially
encodes a protein that shares 62% amino acid sequence identity through the
catalytic domain with class II chitinase from tobacco. The corresponding Pschi4
cDNA was cloned by RT-PCR. Nucleotide sequence analysis indicated that the
Pschi4 coding sequence is composed of three exons interrupted by two introns at
locations identical to those found in other chitinase genes that possess introns. In
contrast, PschU contains a stop codon in the first exon and may be a pseudogene.
XI


Pschi4 genes are conserved in several species of pine, and appear to comprise a
small multigene family. Treatment of pine cell suspension cultures with the general
elicitor chitosan induced Pschi4 expression at both mRNA and protein levels. The
regulatory sequences associated with the Pschi4 gene were sufficient to direct
chitosan- and wound-inducible expression of Pschi4 in transgenic tobacco plants,
which indicated that Pschi4 is an actively expressed member of the multigene
family. The region 5' to the putative transcription start site of Pschi4 was fused to
the GUS reporter gene to further analyze these inducible regulatory elements. The
-200 bp 5'-upstream sequence of Pschi4 was demonstrated to contain active
promoter sequences capable of wound-induced expression in transient assays
(particle bombardment) as well as in stably transformed tobacco plants. This region
was also sufficient to induce transcription in pollen of transgenic tobacco. The
putative pine promoter showed higher promoter activity than the widely used CaMV
35S in the transient expression in pine cells, which implies that the Pschi4 promoter
could be a good candidate to regulate transcription of other genes in transgenic
pine cells. The observation that the Pschi4 gene from pine (a gymnosperm) was
appropriately regulated by chitosan in tobacco (an angiosperm) suggests that the
signaling pathways that mediate chitosan-induced transcription are highly
conserved in the plant kingdom.
XII


LITERATURE REVIEW
General Features of Plant Defense Responses
Plants are frequently challenged by pathogens that cause tissue damage and
disease. Resistance to these pathogenic organisms results from the rapid
deployment of a multicomponent defense response (Dixon et al., 1994). The
individual components of this response may include increased production of
antimicrobial phytoalexins (Dixon et al., 1983), hydrolytic enzymes (Stintzi et al.,
1993), lignin (Vance etal., 1980), hydroxyproline-rich glycoproteins (Varner and Lin,
1989), and the development of a hypersensitive response (HR) around the infection
sites (Keen, 1990). Some of these components can also be induced in uninfected
tissue, at sites remote from pathogen infection. This physiologically acquired
resistance is termed systemic acquired resistance (SAR). This systemically induced
state can result in reduced severity of disease, detected when the tissues are
reinfected with any type of pathogen (bacterial, viral or fungal). Signals for
activation of host defense responses are thought to be initiated in recognition of
pathogen elicitors by the plant (Dixon et al., 1994).
1


2
The outcome (resistance or disease) of a plant-pathogen interaction depends
on both the plant and the pathogen. To be successful in infecting a host plant, the
pathogen must possess genes that function in pathogenicity. On the plant side,
some preformed antimicrobial compounds, termed phytoanticipin (Van Etten eta!.,
1994), can serve as constitutive resistance factors (Osbourn, 1996). More
importantly, plants have the ability to develop an activated resistance, also called
an induced response. There are two relevant aspects in this induced process
(Alexander et ai, 1994). The first critical factor is the timing of the development of
the plant's active defense systems. If established quickly enough, these active
responses are usually effective in restricting the pathogen, resulting in disease
resistance. If initiated slowly or not at all, the pathogen may be successful in
infection, leading to disease. The second factor is the relevancy of the activated
responses for a particular pathogen. If the induced reactions have a deleterious
effect on the pathogen, the infection may be limited. If the elicited responses are
not relevant for the pathogen, or if the pathogen can use an alternative strategy to
escape the active responses, disease occurs.
Basic Incompatibility and Susceptibility
Basic incompatibility describes the failure of a fungus to cause disease on
any member of a plant species. This is a non-host, general resistance (Keen and
Dawson, 1992), and is proposed to result from the plant's ability to recognize the
general features of potential pathogens. The recognition of general elicitors, such


3
as chitin and chitosan, will be discussed later. In general, resistance is the rule and
susceptibility is the exception in the plant world (Staskawicz et al., 1995).
Basic susceptibility results in plant disease. Genetically, it is determined by
the pathogen's genes functioning in pathogenicity and host recognition (Keen and
Dawson, 1992). Some secondary metabolites from the pathogen act as host-
selective toxins (HSTs) which are low molecular weight compounds and are positive
agents of pathogenicity (Walton, 1996). Most known HSTs are made by fungi
(Walton, 1996). Successful infection by a fungal pathogen involves four steps: (1)
attachment; (2) germination of the fungal spores; (3) penetration and (4)
colonization of host tissues (Schafer, 1994). During the process, the pathogen may
detoxify host defense compounds such as phytoalexins and suppress defense
responses by modifying molecules in the signal transduction pathway (Keen and
Dawson, 1992).
Hypersensitive Response (HR1
Resistance in fungal-, bacterial- and viral-plant interactions is often
associated with the HR, in which a small number of cells that are at or near the site
of pathogen infection die rapidly. The protective cell suicide is considered as a very
strong defense response induced in plants by the pathogen itself (Stintzi et al.,
1993). The necrotic lesion which is formed around the infection site perhaps
depletes nutrients for the pathogen, and subsequently a very intense response is
induced in this region which confines the spread of pathogen (Lamb et al., 1989).


4
In many cases in which resistance occurs via an HR, the plant and the
pathogen have an apparent "gene-for-gene" relationship (Flor, 1942). In his classic
work, Flor defined the basic elements of gene-for-gene complementarity wherein
single plant disease resistance genes (R) are paired with single complementary
avirulence genes (Avr) in the pathogen resulting in the HR (Flor, 1942). The
functional alleles are proposed to be dominant and involved in recognition
(Ellingboe, 1981). As shown in Fig. 1, resistance occurs only when the plant
resistance gene (R) matches the pathogen avirulence gene (Avr). If either partner
lacks a functional dominant allele, recognition and resistance do not occur and the
plant becomes diseased (Fig. 1). However, a single plant may contain many
different resistance genes directed to a particular pathogenic species. Therefore,
a pathogen biotype must possess recessive alleles for all of the relevant avirulence
genes to successfully escape surveillance (Keen, 1990). A number of pathogen
avirulence genes were isolated in the 1980s (Keen, 1992). Some Avr genes are
proposed to be involved in the production of specific elicitors which will be
discussed later.
Since 1993, many plant resistance genes have also been cloned by
transposon tagging or positional mapping. For example, the tomato PTO gene
(Martin etal., 1993) and PRF gene (Salmern etal., 1996), Arabidopsis RPS2 gene
(Mindrinos etal., 1994), and rice Xa21 gene (Song et al., 1995) were all isolated by
map-based cloning. Other resistance genes such as the tobacco N gene (Whitham
etal., 1994), tomato Cf-9 gene (Jones et al., 1994) and flax L6gene (Lawrence et


5
Plant host cell
c
O)
O
TO
CL
AA
or
Aa
aa
RR or Rr rr
HR
(resistance)
disease
disease
disease
Figure 1. Gene-for-gene interactions specify plant disease resistance or
susceptibility. R denotes the dominant plant disease resistance (R) gene
and A indicates the corresponding pathogen avirulence (Avr) gene.
Resistance occurs only when both dominant alleles, R and Avr, are present in
plant and invading pathogen, respectively. All other combinations lead to
inability of recognition by plant host cells and result in disease.


6
al., 1995) were identified by transposon tagging. With the cloning of more Avr and
R genes, the basic tenet of specific recogntion in the gene-for-gene hypothesis can
be directly tested. A consistent theme is that the R genes that have been cloned
appear to function in signal transduction pathways where they may rapidly activate
plant defense responses after pathogen recognition.
In the case of the hypersensitive response of tobacco to tobacco mosaic
virus (TMV), the areas of highly-induced responses can be easily detected under
UV light as rings, since cells surrounding the necrotic lesions exhibit bright blue
fluorescence. These cells have accumulated compounds of the phenylpropanoid
pathway (Legrand eta!., 1976), some of which are fluorescent.
Phytoalexins
In association with the HR, plant cells produce many compounds that have
direct antimicrobial activity. One of these responses is the production of
phytoalexins. Phytoalexins are low-molecular-weight antimicrobial compounds
synthesized by plants in response to attempted infection by pathogens, exposure
to elicitor molecules, or other biotic and abiotic stresses (Dixon et al, 1983). More
than 350 phytoalexins have been chemically characterized from approximately 30
plant families (Kuc, 1995). Most of them have been isolated from dicots, but they
have also been isolated from monocots such as barley, corn, onion, rice, sorghum
and wheat (Kuc, 1995), and from pines (Lange et al., 1994). Phytoalexins are
isoflavonoid, terpenoid or other compounds of low molecular weight. Similarities


7
have been observed between phytoalexins from the same plant species, while
differences usually exist between phytoalexins from different genera and families
(Kuc and Rush, 1985). For example, isoflavonoid compounds are the major
phytoalexins in the Leguminosae and are rarely found in other plant species
(Dewick, 1988). On the other hand, terpenoid phytoalexins derived from the
isoprenoid pathway are the most abundant in Solanaceae such as tobacco, but
have not been reported in Leguminosae (Kuc, 1982a; Kuc, 1995).
Phytoalexins are synthesized de novo in response to infection as they are
usually not detected prior to infection (Kuc and Rush, 1985). The phytoalexin
precursors are produced from three major biosynthetic pathways in all plants:
shikimate, acetate-malonate and acetate-mevalonate pathways (Kuc and Rush,
1985; Kuc, 1995). Genes encoding individual enzymes in these pathways, such as
phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and 3-hydroxyl-3-
methylglutaryl Coenzyme A reductase (HMGR), have been cloned from several
plant species, and their expression is regulated by environmental factors, including
pathogen infection (Choi et at., 1992; Fritze et al., 1991; Liang et al., 1989; Ohl et
a!., 1990; Stermer et al., 1990). The phytoalexins reported to date are not very
stable in plants and they are eventually degraded by the host plant and/or the
pathogen (Van Etten etal., 1982; Yoshikawa etal., 1979). The detailed mechanism
by which phytoalexin biosynthesis and turnover is controlled remains somewhat
unclear.


8
Cruickshank proposed earlier that in incompatible interactions,
accumulation of phytoalexins halts pathogen growth and thus confers resistance
(Cruickshank, 1963). In compatible interactions, the pathogen can either tolerate
the host phytoalexin, detoxify it, suppress its accumulation, or prevent the initial
elicitation (Cruickshank, 1963). Some studies have supported this proposal
(Kessmann and Barz, 1986; Kuc, 1995; Yamada et al., 1989).
Other Biochemical Responses
In addition to rapid cell death and production of phytoalexins at and in cells
surrounding infection sites, many other changes occur in the same cells (Lamb et
at., 1989). The most obvious observation is cell wall thickening and reinforcement
by deposition of various macromolecules such as callse, hydroxyproline-rich
glycoproteins (Varner and Lin, 1989), lignin (Lesney, 1989; Vance etal., 1980) and
cell wall bound phenolic compounds (Matern and Kneusel, 1988). These
compounds presumably serve as a physical barrier to prevent the pathogen from
spreading.
Another important response at or near the infection site is the accumulation
of numerous pathogenesis-related (PR)-proteins. A few days after TMV infection,
PR-proteins may account for 10% of the total soluble proteins in tobacco leaves
(Jamet et at., 1985; Pierpoint, 1986). More details about PR-proteins will be
discussed later.


9
The above responses (HR, production of phytoalexins, cell wall lignification
and high concentrations of PR-proteins) are generally local responses and are very
effective in limiting pathogen growth.
Systemic Acquired Resistance (SAR)
Besides the local responses, many plants respond to the necrotizing
pathogen with a more thorough protection, the so-called systemic acquired
resistance (SAR). If part of a plant has already responded to an initial inoculation
hypersensitively, the uninoculated parts of this plant develop an increased state of
resistance evidenced by smaller lesions and greater restriction of the pathogen
upon subsequent infection by the same or even unrelated pathogens (McIntyre et
al., 1981; Ross, 1961; Ye etal., 1989). This type of plant immunity has been well
documented in tobacco (Ross, 1961) and cucumber (Kuc, 1982b). Systemic
acquired resistance can be detected a few days after inoculation and can last for
weeks to months (Lawton et al., 1993). Although the cellular intensity of the
systemic response is much lower than the local response, it still represents a
tremendous amplification in the plant defense response as it concerns the whole
plant.
A number of genes are associated with the appearance of SAR, which are
sometimes called SAR genes. Since PR-protein expression parallels the onset of
SAR (Bol and Van Kan, 1988), these genes are believed to be actively involved in
the development of SAR. PR genes have become sensitive markers in the search


10
for signals that are transmitted from necrotic lesions to distant parts. A more
detailed classification of PR-proteins will be discussed later.
Salicylic acid (SA1 and SAR. Exogenously applied SA or acetylsalicylic acid
(aspirin) can induce SAR and the production of at least some PR-proteins in plants
(Ward et al., 1991; White, 1979). This was first discovered in Xanthi-nc tobacco
(Nicotiana tabacum) (White, 1979) which contains a dominant resistance gene (N)
that confers the host HR to specific races of tobacco mosaic virus (TMV). Injection
of solutions containing SA or aspirin into tobacco leaves prior to the inoculation of
TMV caused a dramatic reduction in lesion size (Wieringa-Brants and Schets, 1988)
and number (White, 1979). However, SA was found to be an endogenous signal
molecule only recently. The SA level increases transiently in the phloem of plants
just before the onset of SAR (Malamyef a/., 1990; Metraux et al., 1990). The hybrid
of N. glutinosa X N. debneyi has been shown to contain a high constitutive level of
SA (in the absence of pathogens) (Yalpani et al., 1993b). This tobacco hybrid also
exhibits constitutive SAR and PR-protein expression, and is highly resistant to TMV
(Ahl Goy et al., 1992).
The role of SA in the SAR signaling pathway has been an active area of
research in the past few years. The direct evidence for SA involvement in SAR
induction comes from a transgenic experiment. Salicylate hydroxylase encoded by
a bacterial NahG gene catalyzes the hydroxylation of SA to catechol which does not
induce SAR. Transgenic tobacco plants harboring the NahG gene are unable to
accumulate SA and are defective in SAR induction upon TMV infection (Gaffney et


11
al., 1993). This apparent relationship supports the hypothesis that SA is involved
in the SAR signal transduction pathway. However, SA perse is probably not the
mobile signal from the infection site to other parts of the plant (Vernooij et al., 1994).
This is suggested by the observation that the removal of an infected leaf before any
measurable SA accumulation in the cucumber phloem still results in induction of
SAR genes in distant tissues (Rasmussen et al., 1991). This conclusion was
supported further by grafting experiments from the same group who did the NahG
transgenic experiment. When wild-type scion was grafted on top of NahG rootstock,
the top scion was able to develop SAR in response to TMV inoculation to the
(NahG) rootstock (Vernooij et al., 1994). Despite their inability to accumulate SA,
NahG tissues are fully capable of producing the transmittable mobile signals for
SAR. However, Raskin's group used 180 labelling and obtained evidence that SA
could be a mobile messenger in tobacco (Shulaev et al., 1995). They argued that
the SAR signaling mechanism in TMV-infected tobacco could be different from that
in Pseudomonas syrlngae-inoculated cucumber. Also, expression of salicylate
hydroxylase in NahG rootstock (Vernooij etal., 1994) does not completely block SA
accumulation. Small amounts of SA, above background levels, could escape and
be exported to phloem, and then transported to the upper wild-type scion. While no
consistent conclusion has been made about SAR signaling, the search for real
mobile signal(s) is still an active area of study.
The pathway of SA biosynthesis has been proposed in tobacco, in which SA
is derived from benzoic acid (Yalpani et al., 1993a). The last step is catalyzed by


12
benzoic acid-inducible benzoic acid 2-hydroxylase (BA2H). Activation of BA2H
leads to SA synthesis in tobacco. The level of benzoic acid increases dramatically
in TMV-inoculated tobacco tissue (Yalpani et al., 1993a). Benzoic acid
accumulation has also been observed in pine injured by dothistromin, a toxin from
Dothistroma pini (Franich et al., 1986).
To elucidate the mode of action of SA, Chen et al. identified an SA-binding
protein in tobacco (Chen and Klessig, 1991; Chen et al., 1993a) which turned out
to be a catalase (Chen et al., 1993b). SA may increase H202 levels by inhibiting
catalase activity which normally converts H202 to H20 and 02 (Chen et al., 1993b).
The active oxygen species (AOS), including H202, are thought to be important
molecules during SAR induction.
Active oxygen species (AOS). Active oxygen species (AOS) are generated
in pathogen-infected tissues and are widely involved in host defense responses
(Baker et al., 1993; Legendre etal., 1993; Orlandi etal., 1992). They are potentially
toxic intermediates to pathogens. The term "oxidative burst" describes the rapid
release of AOS during HR formation. This phenomenon has been observed in
many plant species such as potato (Doke, 1983), tomato (Vera-Estrella etal., 1992),
tobacco (Baker et al., 1993; Glazener et al., 1996; Keppler and Baker, 1989),
soybean (Apostol et al., 1989; Levine et al., 1994) and spruce (Schwacke and
Hager, 1992). The predominant species detected in plant-pathogen interactions
include superoxide anion (02), hydrogen peroxide (H202), and hydroxyl radical
(OH ) (Mehdy, 1994). Injection of H202 into tobacco leaves induces the expression


13
of PR-1 genes locally, as is true with the treatment of leaves with glycolate and
paraquat, two substances that promote generation of H202 (Chen et a/., 1993b).
Exogenously applied H202 (8 mM) induced hypersensitive cell death in soybean
suspension cells (Levine et al., 1994; Levine et at., 1996). However, recent
evidence has shown that the relatively low amount of H202 (4-6 pM) generated
during the incompatible interactions between the plant and pathogen is not sufficient
to cause hypersensitive cell death (Glazener etal., 1996). More recently, Naton et
al. provided cytochemical evidence that intracellular accumulation of AOS in
infected parsley cells is related to rapid cell death (Naton etal., 1996). They argued
that intracellular AOS may be more important as possible mediators of rapid cell
death than extracellular AOS, which were measured previously by many
researchers including Glazener et al. (Glazener et al., 1996). Nevertheless, the
extracellular AOS may be involved in many processes associated with plant disease
resistance, e.g., crosslinking of cell wall proteins (Bradley et al., 1992), increased
lignification of cell walls at infection sites, or they may function as direct
antimicrobial compounds and as signal transducers leading to gene activation
(Sutherland, 1991).
The evidence suggests that H202 is involved in HR, that SA can inactivate
a catalase, and that SA is definitely involved in the signal transduction pathway
leading to SAR. However, recent experiments demonstrate that H202 is not a
second messenger downstream from SA and the inactivation of catalase by SA
does not result in SAR (Bi et al., 1995; Neuenschwander et al., 1995). These data


14
suggest that H202 could be an important molecule Involved in local responses, but
it is not the translocated signal and is not sufficient to induce SAR alone. The chain
of events in the SAR signal transduction pathway is still an active area of research.
Wounding Responses
Wounding damage to plant leaves results in activation of a variety of defense
genes locally as well as systemically (Brown and Ryan, 1984; Parsons et al., 1989;
Pena-Cortes etal., 1988). Considerable research has been conducted on induction
of PI genes, especially in tomato (Pearce etal., 1991; Ryan, 1990). These PI gene
products strongly inhibit the serine proteases found in insect gut tissue and their
expression is systemically induced by wounding. This suggests that Pis may serve
as a defense against insect feeding (Green and Ryan, 1972). Proteinase inhibitors
have also been found in tobacco and they are pathogen-inducible as well as wound-
inducible (Linthorst et al., 1993). In contrast to tomato, the tobacco PI genes can
only be induced locally (not systemically) by wounding or exposure to pathogens
(Linthorst et al., 1993). Some plant-derived chemicals such as oligosaccharides
and jasmonic acid have been found to be involved in the induction of these wound-
inducible PI genes (Farmer and Ryan, 1990; Farmer et al., 1992; Ryan, 1988;
Walker-Simmons, etal., 1983).
Jasmonic acid (JAL Jasmonic acid (JA) and its methyl ester (MJ) have been
hypothesized to be a possible key component of intracellular signaling in response
to wounding (Farmer and Ryan, 1990; Farmer et al., 1992). Direct evidence of


15
jasmonate involvement in wound-induction came from the observation that JA levels
increase immediately and transiently after wounding (Albrecht et al., 1993). The
biosynthetic pathway for JA has been elucidated (Vick and Zimmerman, 1984). The
pathway starts from linolenic acid (LA) which is a component of the plasma
membrane. LA is converted to 13-S-hydroperoxylinolenic acid (13-S-HPLA) by
lipoxygenase (LOX). Then hydration and cyclization lead to the formation of 12-oxy-
phytodienoic acid (12-oxy-PDA), followed by three steps of (3-oxidation. It has been
demonstrated that three of the octadecanoid precursors of JA, i.e., LA, 13-S-HPLA
and 12-oxy-PDA, can activate the synthesis of Pis in tomato leaves when applied
to leaf surfaces (Farmer and Ryan, 1992). Inhibitors of LOX activity, such as
salicylhydroxamic acid, reduce JA biosynthesis (Staswick et al., 1991). It is
interesting that salicylic acid (SA), a key molecule in systemic acquired resistance,
has been shown to be an inhibitor of wound-induced PI accumulation (Doares etal.,
1995a; Pena-Cortes et al., 1993). The results suggest that SA could inhibit JA
synthesis by inhibiting the formation of 12-oxy-PDA (Pena-Cortes etal., 1993) or SA
could block JA action by inhibiting an as-yet-undefined step between JA and
transcriptional activation of PI genes (Doares et al., 1995a).
The wound-induced accumulation of Pis in tomato is proposed to be initiated
by the release of pectic polysaccharides from the plant cell walls (Bishop et al.,
1981). It has also been found that chitosan, a polymer of [3-1,4-glucosamine found
in fungal and insect cell walls, is a strong inducer of both JA and PI syntheses in
tomato leaves (Doares et al., 1995b; Walker-Simmons et al., 1983).


16
Chitosan. General Elicitors and Specific Elicitors
Elicitors
In searching for signal molecules that may be involved in the ability of plants
to recognize pathogens, efforts have focused on "elicitor" molecules. The term
"elicitor" was originally used to describe agents that induce the synthesis and
accumulation of antimicrobial compounds (phytoalexins) in plant cells (Keen, 1975),
but is now widely used to refer to molecules that stimulate any plant defense
response, from cellular changes such as the HR to molecular changes such as
transcriptional activation of defense-responsive genes (Dixon et al., 1994).
Elicitors have been divided into two groups: specific elicitors and general
elicitors (non-specific elicitors), based on whether the elicitor exerts similar effects
on all members of a plant species (general) or only on specific genotypes of a plant
species (specific).
Specific Elicitors
Specific elicitors are those involved in the interaction of a certain pathogen
biotype to a particular plant species, and usually refer to the avirulence gene
products (direct or indirect) that are produced in the gene-for-gene interactions. A
well-characterized specific elicitor is a small peptide of 28 amino acids which is


17
produced only by Cladosporium fulvum carrying the Avr9 avirulence gene. This
elicitor induces a defense response only in tomato plants containing the Cf-9
resistance gene (de Wit, 1992). The biological function of these specific elicitors in
pathogens is not clear. Do they reside in the pathogen only to elicit the plant
defense system against itself when it invades the plant? This doesn't seem to be
logical. It is most likely that specific elicitors do have biological functions in the
pathogen, as yet unknown, but the plant developed a defense system later by
recognizing so-called specific elicitors from the pathogen in order to survive. On the
other hand, the pathogen does not intend to produce "elicitors" against itself and
keeps modifying/changing the "elicitor'-related avirulence genes to escape the plant
recognition. Experimental evidence for this dynamic interaction is the Avr4 and
Avr9 genes in the fungus Cladosporium fulvum (de Wit et a/., 1994 and refs,
therein). C. fulvum has exploited at least two different mechanisms to avoid specific
recognition by the host plant (tomato). In the case of Avr9, mutations from
avirulence to virulence involve the deletion of this gene in order to escape
recognition by Cf-9. In the case of the Avr4 gene, virulent races still contain
essentially the same gene except for a single point mutation, resulting in an amino
acid change from cysteine to tyrosine, which is sufficient to avoid Cf-4 recognition.
This may explain why there are so many pairs of Avr-R genes in a single pathogen
biotype and a single plant species. It has been found that the coat protein of TMV
serves as a specific elicitor to tobacco plants carrying the N' resistance gene (Culver
and Dawson, 1989), and the TMV replicase is specifically recognized by tobacco


18
plants carrying the N gene (Whitham et al., 1994). This supports the view that
specific elicitors do have biological functions in the pathogen and have been
exploited by the plant.
General Elicitors and Chitosan
General elicitors. Different from specific elicitors, general elicitors are
involved in the general resistance of plants to broad ranges of pathogens or
potential pathogens. Disease is the exception, not the rule (Staskawicz et al.,
1995). Most plants recognize general features of potential pathogens and can
prevent disease due to an ability to recognize these elicitors. Biotic elicitors include
some oligosaccharides (Hahn et al., 1993), proteins (Ricci et al., 1993) and lipids
(Bostock et al., 1981), and some known abiotic elicitors include heavy metals and
UV radiation. Abiotic elicitors are thought to result in the release of biotic elicitors
from the plant cell walls (Hahn et al., 1993). Biotic elicitors could be of either
pathogen or plant origin, although in most cases, they are pathogen components.
General elicitors are usually present on or near the cell surface of the pathogen and
often serve as cell wall components. Structurally characterized fungal elicitors
include: glucan elicitors, oligogalacturonide elicitors, chitin and chitosan,
glycopeptides, and ergosterol (Granado et al., 1995; Hahn et al., 1993; Keen and
Dawson, 1992).
Chitosan. Chitosan is a deacetylated form of chitin which is a polysaccharide
composed of (3-1,4-linked N-acetylglucosamine. Both chitin and chitosan are cell


19
wall components of many fungi (Bartnicki-Garcia, 1968). Many studies have
demonstrated that oligosaccharides derived from chitin and chitosan elicit defense
responses in various plants. Chitosan and its derived fragments elicit the
accumulation of phytoalexins in pea pods (Hadwiger and Beckman, 1980; Walker-
Simmons eta!., 1983), suspension-cultured soybean cells (Kohle et al., 1984) and
parsley cells (Conrath et al., 1989). Relatively low concentrations of chitosan (as
low as 8 pg/ml) directly inhibit the growth of certain fungal pathogens of pea
(Kendra et al., 1989). Chitosan oligomers with a degree of polymerization (DP)
between 6 and 11 are most active as phytoalexin elicitors in pea. Chitosan-derived
oligosaccharides are also capable of inducing the accumulation of proteinase
inhibitors in both tomato and potato leaves (Pena-Cortes et al., 1988; Walker-
Simmons et al., 1983; Walker-Simmons and Ryan, 1984) and the synthesis of
callse in suspension-cultured soybean (Kohle et al., 1985), parsley (Conrath et al.,
1989), tomato (Grosskopf et al., 1991) and Catharanthus roseus (Kauss et al.,
1989) cells. Oligosaccharide fragments of both chitosan and chitin have both been
shown to induce defense-related lignification of the walls of suspension-cultured
slash pine cells (Lesney 1989, 1990). Chitosan treatment induces pine cells to
synthesize hydrolytic enzymes including chitinase and glucanase (Popp et al.,
1996). Chitin oligomers with a degree of polymerization of 4 to 6 elicited lignification
in wounded wheat leaves (Barber et al., 1989). The deposition of both callse and
lignin are thought to enhance plant defense by strengthening the plant cell walls.


20
Active chitosan and chitin oligomers are thought to be released during plant-
pathogen interactions by plant enzymes such as chitinases. Chitinases belong to
a group of PR proteins (see below) whose synthesis is induced by pathogen
infection and also by chitosan elicitors. It is possible that low levels of constitutive
plant chitinases release active chitin and chitosan oligomers from invading fungi,
which in turn induce a defense response including accumulation of chitinases. This
reaction could serve as an "amplification" of signals and re-enforce the initial
defense response.
Pathoaenesis-Related Proteins
Pathogenesis-related (PR) proteins are highly induced proteins during plant-
pathogen interactions. Expressed both locally and systemically, PR proteins
represent the major quantitative changes in soluble proteins during defense
responses. PR proteins have very distinct physicochemical properties, some of
which enable them to survive the harsh environment where they occur: (1) at acidic
pH (as low as pH 2.8), they are quite stable and soluble, whereas most other plant
proteins are denatured at this pH; (2) they are relatively resistant to proteolytic
enzymes of both endogenous and exogenous origin; (3) they are targeted to
compartments such as the vacuole, the cell wall or apoplast; (4) most of them are
monomers with low molecular weight (8-50 kD).


21
PR proteins were first reported by Van Loon and Van Kammen in the early
1970s. They detected four de novo synthesized proteins in tobacco plants reacting
hypersensitively to infection with TMV (Van Loon and Van Kammen, 1970). Since
then, many other proteins with similar physicochemical (see above) and induction
properties have been identified from tobacco and other plant species (Kombrink et
al., 1988; Stintzi et al., 1993; Vogeli et a!., 1988). The tobacco (Nicotiana tabacum
Samsun)-TMV interaction leading to an HR is still the model for the study of PR
proteins and from which the highest number of PR proteins have been
characterized. PR proteins are induced in response to infection by pathogens of
viral, viroid, bacterial or fungal origins (Van Loon, 1985). However, some members
of PR families have been found to be induced by chemical treatment (elicitors),
stress or wounding where pathogens are not involved. The reason they are still
called "pathogenesis-related" is that they still represent a wide array of pathogen
defense-related proteins in a particular plant species. Furthermore, many of the
chemicals that induce PR proteins mimic natural compounds involved in pathogen-
plant interactions and/or the transduction pathways that are associated with
pathogen-plant signaling (Stintzi et al., 1993).
Classification of PR Proteins
By using different biochemical approaches, more than 30 PR proteins have
been isolated from TMV-infected tobacco plants (Stintzi et al., 1993). Based on
amino acid sequence similarity and serological properties, these tobacco PRs are


22
classified into five groups (Stintzi et al., 1993; Alexander et al., 1994). PR proteins
from other plant species can be put into each group, although there are exceptions.
Group PR-1. Tobacco PR-1 proteins represent most of the earlier work.
Three acidic isoforms of tobacco PR-1, i.e., PR-1 a, PR-1b and PR-1c, were the first
purified PRs (Jamet and Fritig, 1986) and are serologically related to each other in
tobacco and to PRs from other plant species (Nassuth and Sanger, 1986). The
amino acid sequence of the three PRs, deduced from the cloned cDNA (Cutt et al.,
1988), share more than 90% identity. Another clone, which was identified from a
cDNA library of TMV-infected tobacco, appeared to encode a basic isoform of PR-1
protein (Cornelissen et al., 1987). The corresponding protein was actually purified
later and named PR-1g. All of the four PR-1 proteins from tobacco are localized
extracellularly. Tobacco PR-1 a and PR-1b, as well as three members of PR-1 from
tomato, have been shown to contain direct antifungal activities in in vitro assays
(Niderman et al., 1993; Stintzi et al., 1993), although the molecular mechanism
involved needs further investigation.
Group PR-2. The tobacco PR-2 proteins have been found to contain endo-
1,3-p-glucanase activity (Kauffmann et al., 1987) which produces oligomers of 2-6
glucose units from 1,3-p-glucans. Many 1,3-p-glucanases have been purified
(Boiler, 1988), and many genes encoding glucanases have also been cloned (Meins
et al., 1992). Glucanases are usually monomers with a molecular weight of 25-35
kD. Of the five members of PR-2 from tobacco, four of them (PR protein 2, N, O,
Q') are acidic proteins and are targeted to extracellular space (Kauffmann et al.,


23
1987); one (glue b) is basic and is localized in the vacuole (Van den Bulcke et al.,
1989). The specific activities of these glucanases are strikingly different towards a
particular substrate (Stintzi et al., 1993).
Group PR-3. Tobacco PR-3 proteins are chitinases. Chitinase structure,
function and regulation will be discussed in more detail later.
Group PR-4. Tobacco PR-4 group includes four proteins: r1, r2, s1, s2
(Kauffmann et al., 1990) that are all acidic and targeted to the extracellular space
(apoplast). They are small proteins (13-14.5 kD) and are serologically related to
each other. The biological function/activity of PR-4 proteins is not known.
Group PR-5. Two slightly acidic proteins (R & S) and two basic proteins (n-
osmotin and osmotin) from tobacco are included in PR-5 group. The acidic and
basic isoforms are localized in apoplastic space (extracellular) and vacuolar
compartment, respectively (Dore et al., 1991). Genes encoding tobacco PR-5
proteins have been isolated (Van Kan et al., 1989) and their corresponding cDNAs
have also been cloned (Payne et al., 1988). Sequence comparisons have shown
that there are no introns within these genes. The deduced amino acid sequences
of PR-5 proteins are approximately 60% similar to thaumatin, a protein isolated from
Thaumatococcus danielli. Therefore, PR-5 proteins are also called thaumatin-like
PRs (Stintzi etal., 1993). Thaumatin-like proteins have been characterized in many
other plant species such as maize (Richardson et al., 1987), tomato (King et al.,
1988), barley (Bryngelsson and Green, 1989) and potato (Pierpoint et al., 1990).


24
Other groups of PR Proteins. Some PR proteins from tobacco and other
plant species cannot be put into the five major groups. To accommodate these
proteins and novel proteins identified in the future, scientists of the Commission on
Plant Gene Nomenclature (CPGN) have proposed a new Y category which
generally represents plant genes whose sequences are clearly conserved but
whose designations are not based on function. Thus, PR genes can be collectively
designated by Ypr followed by a number (Van Loon et al., 1994). However, for PR
genes whose functions are known such as glucanases and chitinases, suggested
designations Glu or Chi are preferred to Ypr2 or Ypr3.
Regulation of PR Protein Expression
PR gene expression is generally induced by pathogens (fungal, bacterial or
viral), as is obvious from the definition; however, some elicitors and various stresses
can also induce PR gene expression. PR genes are differentially expressed in
response to various stress conditions and during different developmental stages.
Spraying plants with a salicylic acid solution induces expression of some PR genes
including PR-1 (acidic and basic), acidic PR-2 and basic PR-3 (Bol etal., 1990; Van
de Rhee et al., 1993). The tobacco basic PR proteins are highly induced by
wounding and ethephon, which produces ethylene in vivo, whereas acidic PRs are
not (Brederode et al., 1991). Moreover, the basic PRs are constitutively expressed
in roots and lower leaves of healthy plants in contrast to their acidic counterparts
(Memelink et al., 1990; Neale et al., 1990; Van de Rhee et al., 1993). On the other


25
hand, tobacco acidic PRs are systemically elicited by TMV infection, while no or little
induction of basic PRs occurs in non-inoculated leaves (Brederode et al., 1991).
To identify the putative cis-acting elements responsible for PR gene
induction, efforts have been focused on making promoter deletions fused with the
reporter gene (uidA) which encodes bacterial p-glucuronidase (GUS), and testing
the relative GUS activities. The data on PR-3 proteins (chitinases) will be discussed
later. The analysis of upstream sequences of genes encoding tobacco PR-1, PR-2
(glucanase) and PR-5 indicates that PR promoters contain multiple cis-acting
elements (Albrecht et al., 1992; Van de Rhee and Bol, 1993; Van de Rhee et al.,
1993). A recent experiment has shown that the PR-1 a gene promoter of tobacco
contains several elements that can bind GT-1-like nuclear factors (GT-1 factor is
necessary for light-responsive expression of the pea rbcS-3A gene) (Buchel et al.,
1996), but yet, no common sequence motif has been identified from these assays.
However, a 10-bp element which is repeated four times in the 5'-non-translated
region of a barley p-1,3-glucanase gene has been found to be present in the non-
translated regions of over 30 stress- and pathogen-inducible promoters
(Goldsbrough et al., 1993). Gel mobility shift assays have provided preliminary
evidence that this element, known as the TCA motif (TCATCTTCTT), specifically
binds a tobacco nuclear protein, and this binding activity was greatly increased
when tobacco plants were pre-treated with salicylic acid (Goldsbrough et al., 1993).
It is suggested that the TCA motif could be important for induced expression. A 116
bp fragment between -168 and -52 of the parsley PR-2 promoter, which was shown


26
to be necessary for elicitor-mediated expression (Van de Locht etal., 1990), actually
contains a TCA element.
A 61-bp element of the tobacco [3-1,3-glucanase B gene has been shown to
be an enhancer whose activity is independent of orientation. Analysis of point
mutations has identified the sequence AGCCGCC, named the AGC box, which is
essential for the enhancer activity (Hart et a/., 1993). Nuclear extracts from tobacco
leaves contain one or more factors that can interact with this element specifically.
This binding activity is higher in nuclear extracts from ethylene-treated plants than
control plants which correlate with its postulated role in the regulation of the (3-1,3-
glucanase gene (Hart et al., 1993). Whether the same or a similar set of regulatory
proteins are involved in all PR gene induction, or each group of acidic/basic PR
genes are controlled by a unique set of factors, is not clear. It seems that at least
some factors are commonly involved in induction of certain PR proteins, such as
chitinases and glucanases. The purification of these nuclear factors and cloning of
their corresponding genes/cDNAs will help to understand the induction mechanisms
and to give some clues as to the signal transduction pathways involved in the
induced responses.


27
Chitinase Structure. Function and Regulation
Chitinase Structure and Function
Chitinases (PR-3 proteins) are enzymes that hydrolyze chitin, a linear
homopolymer of N-acetylglucosamine. Chitin is the major component of cell walls
of many fungi, but is not present in higher plants. On the other hand, chitinases
have been reported to be present in a variety of higher plants (Boiler et al., 1983;
Broglie and Broglie, 1993). It seems that there is no endogenous substrate for
chitinases in higher plants; therefore, it has been proposed that chitinases in higher
plants may play a role in protecting plants against chitin-containing fungi (Boiler,
1988).
There are two types of chitinases in plants: endochitinases and
exochitinases. Endochitinases randomly hydrolyze internal p-1,4-linkages of chitin,
whereas exochitinases digest chitin from the non-reducing end of the polymer (Fig.
2). Therefore, the smallest substrate for endochitinases is a tetramer of N-
acetylglucosamine and that for exochitinases is a dimer (Fig. 2). Most of the
characterized plant chitinases are endochitinases (EC 3.2.1.14) (Boiler et al. 1983;
Molano et al., 1979); however, exochitinases have also been purified from melon
(Roby and Esquerre-Tugaye, 1987) and carrot (Kurosaki et al., 1987).
Endo-type chitinases have been characterized from many plant species
including barley (Jacobsen et al., 1989; Kragh et al., 1991), bean (Boiler et al.,


28
A.
chitin
endochitinases
I
exochitinases
B.
endochitinases
exochitinases
O N-acetylglucosamine residue
Figure 2. Different substrate specificities for endochitinases and
exochitinases. (A). Endochitinases hydrolyze the internal (3-1,4-bond of
chitin, whereas exochitinases digest the bond from the non-reducing
end. (B). The smallest substrate for endochitinases and exochitinases
are tetramer and dimer of N-acetylglucosamine, respectively.


29
1983), cucumber (Boiler and Metraux, 1988), maize (Nasser et al., 1990), pea
(Mauch et al., 1988a), potato (Kombrink et al., 1988), tobacco (Legrand et al.,
1987), tomato (Joosten and de Wit, 1989) and wheat (Ride and Barber, 1990). In
general, plant chitinases are proteins of 25-35 kD molecular weight which occur as
monomers and have either a high or low isoelectric point (basic or acidic chitinases)
(Boiler, 1988). Three classes of plant chitinases have been proposed based on the
primary structures (Fig. 3) (Broglie and Broglie, 1993; Collinge eta!., 1993; Shinshi
et al., 1990). Class I chitinases are composed of an N-terminal signal sequence,
a cysteine-rich domain of approximately 40 amino acids, a variable length hinge
region, a highly conserved main structure (catalytic domain) and a C-terminal
vacuolar targeting sequence (usually 7 amino acids). Class II chitinases have a
high amino acid sequence identity to the main structure of class I chitinases, but
lack the cysteine-rich domain, the hinge region and C-terminal domain. Class III
chitinases show no sequence similarity to enzymes in class I or class II. The C-
terminal seven amino acids (GLLVDTM) of tobacco class I chitinase are necessary
and sufficient to direct the protein to the vacuole (Neuhaus et al., 1991). In addition
to the difference in domain structure, class I and class II chitinases have other
distinctive properties and are located in separate cell compartments. Class I
chitinases are targeted to the vacuole, whereas class II chitinases are secreted into
the extracellular space. All identified class II chitinases are acidic proteins, but class
I and class III chitinases have been found to be either basic or acidic (Collinge et
al., 1993; Davis et al., 1991; Lawton et al., 1992).


30
cysteine-rich
Class I
signal peptide
1
wmz
hinge
v jr
m
catalytic domain
vacuolar targeting
1
V
Class II
Class III
Figure 3. Domain structure of three classes of chitinases. From left to right, the
domains in class I chitinases are: signal peptide, cysteine-rich, hinge, catalytic,
and vacuolar targeting. Class II chitinases share high homology with class I
chitinases in the catalytic domain, but lack the cysteine-rich, hinge and vacuolar
targeting regions. The catalytic domain of class I chitinases also contain a short
stretch of amino acids not found in class II chitinases. The catalytic domain of
class III chitinases are not related to either class I or class II chitinases.


31
Genes and/or cDNAs encoding chitinases have been cloned from a variety
of plant species, such as Arabidopsis (Samac et al., 1990), bean (Broglie et al.,
1986), cucumber (Metraux et al., 1989), poplar (Davis et al., 1991), potato
(Laflamme and Roxby, 1989), rice (Xu et al., 1996; Zhu and Lamb, 1991) and
tobacco (Payne etal., 1990; Shinshi etal., 1990). Constitutive expression (directed
by the CaMV 35S promoter) of a bean chitinase gene in transgenic tobacco plants
showed increased disease resistance against certain fungi (Broglie et al., 1991).
It was speculated that chitinases may inhibit fungal growth by direct lysis of hyphal
tips, particularly in combination with glucanases (Schlumbaum et al., 1986; Mauch
etal., 1988b; Sela-Buurlage et al., 1993). Chitinases could also function to amplify
defense responses in cells surrounding a site of infection by liberating chitin and
chitosan oligomers from fungal cell walls which may serve as general elicitors to
induce the expression of several defense-related genes (Boiler et al., 1983; Mauch
and Staehelin, 1989). In addition to potential roles in defense, chitinases may play
important roles during early embryo development (de Jong et al., 1992) and other
developmental processes (Neale et al., 1990). This is presumably because plant
cells contain substrates for chitinase that are not chitin perse, but may resemble
chitin structurally (Fisher and Long, 1992; Collinge et al., 1993).
Regulation of Chitinase Gene Expression
Environmental regulation. Chitinase gene expression is regulated by many
factors including pathogen invasion, treatment with elicitors and plant hormones


32
(ethylene), and mechanical wounding. Chitinase enzyme activity, protein and
mRNA levels have been shown to be elevated when the plant is under these
stresses (Boiler, 1988; Collinge et al., 1993; Joosten and de Wit, 1989). Induced
expression of chitinase is often co-ordinated with other PR proteins such as (3-1,3-
glucanases (Joosten and de Wit, 1989; Kombrink etal., 1988; Shinshi et al., 1987;
Vogeli et al., 1988). Individual chitinase isoforms have been reported to be
differentially regulated in barley, pea and tobacco. In barley leaves and grain,
several chitinase isozymes have been found, but only one is induced in response
to pathogen infection (Kragh et al., 1990). In pea, at least two chitinases are
differentially regulated upon fungal infection (Mauch et al., 1988a). In tobacco, the
basic class I and acidic class II chitinases have been shown to be differentially
induced by various stresses such as virus infection, UV light and wounding
(Brederode et al, 1991; Memelink et al., 1990).
Developmental regulation. In addition to stress-induced regulation,
chitinases are also under developmental regulation in healthy Arabldopsis, rice and
tobacco plants. In normal tobacco, chitinase expression was found in roots,
developing flowers and lower older leaves (Memelink et al., 1990; Neale et al.,
1990; Shinshi et al., 1987). In Arabidopsis and rice, constitutive expression of
chitinase was found in roots (Samac et al., 1990; Zhu and Lamb, 1991). The
developmental regulation of chitinase in tobacco was proposed to be controlled by
auxin and cytokinin gradients within the plant (Shinshi et al., 1987). The finding that
chitinases accumulate in a tissue-specific manner during different developmental


33
stages has led to the view that chitinases may serve other functions in plants in
addition to their role in the defense response.
Promoter studies. The regulatory sequences that direct the expression of
chitinase have been studied in a few plant species. The first characterized promoter
is the bean chitinase 5B promoter (CH5B) which contains element(s) for ethylene
induction (Broglie etal., 1986). The ethylene-responsive element was first identified
between positions -422 and -44 (Broglie et al., 1989) by analysis of deleted
chitinase genes in transgenic tobacco plants and was confirmed in a bean
protoplast system (Roby et al., 1991). This region has been further narrowed down
to sequences between nucleotides -305 and -236 by promoter-GUS deletion
analysis in transient expression assays (Broglie and Broglie, 1993). In addition, a
nuclear protein has been observed to bind to this DNA sequence in gel mobility shift
and DNase I protection assays (Broglie and Broglie, 1993). Similar research has
been conducted for Arabidopsis and tobacco chitinase genes (Fukuda and Shinshi,
1994; Samac and Shah, 1991). An acidic class III Arabidopsis chitinase promoter
was fused to the GUS reporter gene and transformed into Arabidopsis. Promoter
activity (GUS expression) was detected in roots, leaf vascular tissue, hydathodes,
guard cells, and anthers in healthy plants, which indicates its developmental
regulation. Induced expression was observed in mesophyll cells surrounding
lesions caused by fungal infection. Promoter deletion analysis demonstrated that
the region 192 bp upstream of the transcription start site is capable of both
developmental and induced expression (Samac and Shah, 1991). In tobacco, the


34
5'-upstream sequence from tobacco class I chitinase was fused to GUS and
introduced into tobacco. Promoter deletion analysis revealed that the region
between nucleotides -574 and -476 is sufficient for inducibility by a fungal elicitor.
Gel mobility shift assays further identified a sequence of 22 bp between -539 to -
518 specifically that binds to a nuclear protein from elicitor-treated cells, but not
from control cells. This 22 bp sequence contains a direct repeat of GTCAG
separated by three nucleotides (Fukuda and Shinshi, 1994).
From the above discussions, it is clear that angiosperm PR genes have been
well documented in the literature. Their gene products and promoter analyses have
been studied in detail. Flowever, little information is available on defense responses
in gymnosperms. In this research, I chose chitinase gene as a model to study pine
defense responses, since chitinase structure seems to be conserved in
angiosperms, and previous work has shown that chitinase activity in pine
suspension cells increases upon chitosan treatment (Popp et al., in press).


INTRODUCTION
Plants are constantly confronted by microbes and other pests that can cause
tissue damage. The cell walls of many of these microbes share common structural
features. Compounds like chitin and chitosan are found in the cell walls of many
fungi, as well as the exoskeleton of arthropods, but these compounds are not found
in plants. In most plants, a defense response is induced when they are treated with
chitin or chitosan (Kohle et al., 1984, 1985; Fritensky et al., 1985; Kombrink and
Hahlbrock, 1986; Lesney, 1989). One component of the defense response to
elicitors is the transcriptional activation of genes that encode pathogenesis-related
(PR) proteins. One class of PR proteins includes chitinases. Chitinases hydrolyze
chitin, a linear homopolymer of N-acetylglucosamine. Most of the characterized
plant chitinases are endochitinases (EC 3.2.1.14) which randomly cleave internal
G-1,4 linkages in chitin and consequently release oligomers of N-acetylglucosamine
(Boiler et al., 1983; Molano et al., 1979). Chitinase alone, or in combination with
glucanase, can directly inhibit fungal growth by causing lysis of hyphal tips (Mauch
et al., 1988b; Schlumbaum etal., 1986; Sela-Buurlage et al., 1993). Chitinase also
seems to amplify plant defense responses by releasing chitin and chitosan elicitors
from fungal cell walls (Boiler et al., 1983; Mauch and Staehelin, 1989). In addition
to potential roles in defense, chitinase may also serve other functions during
35


36
development (de Jong et al., 1992; Neale et al., 1990). Chitinase gene expression
is controlled at the transcriptional level and can be induced by general elicitors and
other stresses. Therefore, chitinase genes can be viewed as "reporters" that can
be used to study defense mechanisms as well as to define components of signal
transduction pathways involved in defense responses.
Pinus is an ancient as well as economically important genus in the plant
kingdom. Pine cells exhibit a pronounced defense response to chitin, chitosan, and
live pathogens (Lesney, 1989; Popp, 1993), and this defense response is
accompanied by secretion of chitinase (Popp et al., In press). To obtain a better
understanding of defense responses and gene regulation in conifers, a chitinase
gene was chosen as a model system for this study. As a first step toward this
overall goal, a chitinase gene from pine trees was isolated and characterized.
Alignment of class I and class II chitinase sequences from a number of
different plant species revealed the presence of highly conserved regions within the
catalytic domain. This feature was exploited as a strategy for cloning related
sequences from pines using PCR. A pair of primers were designed to anneal to
conserved regions of the chitinase catalytic domain. The upstream primer, 5'-
ATAAGCT[CA(AG)AC(ACGT)(AT)(CG)(ACGT)CA(ACGT)GA(AG)AC-3',was1024-
fold degenerate and was expected to anneal to the nucleotide sequence translated
as QTSH(Q)ET. The downstream primer, 5'-ATGGTACCCATCCA(AG 1AACCAAC
GT)A(AGT)(ACGT)GC-3', was 96-fold degenerate and designed to anneal to the
region encoding AI(LM)WFWM. Degenerate positions are shown in parentheses,


37
and restriction sites that were introduced into the primers for directional cloning are
underlined. Fragments about 400 bp in length were amplified from white pine,
cloned, sequenced, and found to show sequence similarity to known chitinase
genes in the expected region of the catalytic domain. The cloned PCR product was
used to screen a genomic library of eastern white pine. Six plaques were selected
due to their hybridization to the probe in duplicate plaque lifts. The resulting
recombinant phage were designated gPschi1-6 (for genomic Pinus strobus
cMinase 1 through 6). Two of these genomic clones, gPschU and gPschi4, were
selected for detailed sequence analysis. Recombinant phage DNA was digested
with Sacl and the insert fragments were subcloned into pBlueScript. Physical
mapping and partial DNA sequence analysis suggested that gPschU and gPschi4
were likely to contain lengthy 5' flanking sequences and intact coding regions (Fig.
4). Most of the above work was accomplished by Dr. John M. Davis, part was done
by Dr. Michael P. Popp (SFRC, University of Florida). DNA sequencing was
performed by Dr. Craig S. Echt (USDA-Forest Service, Rhinelander, Wl).
The first goal of my study was to identify a functional gene from the cloned
chitinase fragments, and to study its inducibility of expression by general elicitors.
The hypothesis to be tested was that pines possess defense-related genes that are
regulated at the transcriptional level. To begin, I introduced the entire Pschi4 gene,
including 4.5 kb of 5' upstream sequence, 0.9 kb of coding sequence and 1.5 kb of
3' downstream sequence, into tobacco via Agrobacterium-mediated transformation.
This gene was confirmed to be transcribed in transgenic tobacco plants by northern


38
o
o
o
CO
CD
CD
CO
CO
CO
g PschH rnrr[>

X
X
o
CD
E
o
E
CD
O
CO
CD
CD
CD
-Q
CD
QQ
CO
OQ
><
CO
gPschi4
IHEj>
sequenced region
1 exon
D intron
1 kb
Figure 4. Overall structure of the pine genomic subclones gPschil and gPschi4.
The transcriptional orientation and putative coding regions, including intron and
exon sequences, are indicated by arrows. The asterisk denotes the stop codon
within exon #1 of gPschH. Restriction endonuclease cleavage sites that were
used for subcloning are presented. PschH was not mapped using SamHI and
Xba\, only with Sad, so lacking of these ssites in PschH does not imply
polymorphism between PschH and Pschi4. The 668 bp Sacl-SamHI fragment in
the gPschi4 coding region was used as hybridization probe for the blots shown in
figures 14 and 15.


39
blot analysis. Accumulation of its mRNA was induced by the general elicitor
chitosan in both pine suspension cells and transgenic tobacco leaves, as well as by
mechanical wounding in transgenic tobacco plants. This suggests that at least part
of the signaling mechanism by which chitosan induces gene expression is
conserved in gymnosperms and angiosperms. A better understanding of the
structure and regulation of these chitinase genes should provide useful insights into
the evolution of defense responses in plants.
My second goal was to identify a functional chitinase promoter and dissect
the promoter for its chitosan/wound-responsive element(s). It has been found that
most transcriptional control elements reside within the 5'-upstream region of the
coding sequence in higher plants. However, this seems not to be necessarily true
for pine genes (Loopstra et al., 1995). Reports have indicated that the regulatory
sequences of pine genes could lie in the coding region or 3'-untranslated portion of
the gene (Loopstra et al., 1995; Loopstra, personal communication). Since the
widely used CaMV 35S promoter and a number of other promoters from
angiosperms do not promote high levels of transcription in conifers (Ellis, 1994 and
refs, cited therein), efforts have been focused on identifying an inducible coniferous
promoter to study gene expression and regulation in conifers, and on the
development of strategies to introduce foreign genes into conifers. The hypothesis
to be tested was that the regulatory elements of a pine gene could be identified
using assays for gene expression in pine cells and in angiosperm cells. The cloned


40
pine chitinase gene, Pschi4, seemed to be a good candidate to test this hypothesis
and to identify a functional promoter region of an inducible gene from pine.
In association with these goals, the Pschi4 cDNA was cloned and expressed
in E.coli. Antibody was then made against the purified recombinant protein. Pschi4
protein expression was monitored in pine suspension cells as well as in transgenic
tobacco plants. Also in association with these goals, a series of Pschi4 promoter-
GUS fusion constructs were made and tested in transient assays as well as in
stably transformed tobacco plants. Collectively, this research represents the first
studies on gene expression, regulation and promoter dissection of a defense-
related gene in pine trees.


MATERIALS AND METHODS
Plant Materials
Pine materials. Seeds obtained by self-pollination of eastern white pine
(Pinus strobus) genotype P-18 were a gift from Dr. Don Riemenschneider, USDA-
Forest Service, Rhinelander, Wl. Seeds were surface-sterilized in a 20% solution
of commercial bleach for 10 min, rinsed in sterile distilled water, stratified in the
refrigerator for one month, and then sown in commercial potting mix. After the
cotyledons had expanded fully, the above-ground portion of the seedlings was used
as a source of DNA for Southern blots. Needle tissue from loblolly pine (P. taeda)
genotype 7-56 was a gift from Dr. Les Pearson (Westvaco Corp., Summerville, SC).
Cell cultures of loblolly pine were derived from an individual seedling from family 10-
38. Cell cultures of slash pine (P. elliottii var. elliottii) genotype 52-56 were initiated
and maintained according to previously described methods (Lesney, 1989). The
starting material for the cell cultures was provided by Greg Powell and Dr. Tim
White (Cooperative Forest Genetics Research Program, University of Florida).
Tobacco materials. Tobacco plants (Nicotiana tabacum var. Turkish and
Nicotiana tabacum var. Samsun; gifts from Dr. F. Zettler and Dr. C. Kao, University
41


42
of Florida, respectively) were grown from seed on agar-solidified MS medium
(Sigma) and maintained in aseptic cultures. These tobacco plants were used as an
explant source for transformation.
Others. White onions were purchased from local grocery stores. Maize
suspension cell line PC-5 was kindly provided by Dr. L. C. Hannah (University of
Florida). These onion and maize cells as well as pine cells were used for particle
bombardment.
Sequencing and Sequence Analysis
After the insert from the recombinant phage DNA was subcloned into the
Sacl site of pBluescript, sequencing reactions were carried out from both ends. The
identified putative coding region, along with ~750 bp of upstream sequence and
-350 bp of downstream sequence, were also sequenced by using an automated
DNA sequencer (ABI 373) with dye terminator chemistry (Dr. Craig Echt, USDA-
Forest Service, Rhinelander, Wl). DNA and protein sequence analysis was
performed using the BLAST search algorithm (Altschul et a/., 1990) and GCG
sequence analysis software (Genetics Computer Group, Madison, Wl).


43
Plant Transformation
Plasmid construction. Tobacco plants were transformed with the putative
chitinase gene located in gPschi4. The two Sacl fragments containing different
regions of Pschi4 were subcloned separately into pBlueScript, then ligated together
as BamHI-SacI and Sacl-Xbal fragments, respectively, into pBlueScript. The
BamHI-SacI fragment contained the putative promoter region and extended -200
bp into the 5' end of the coding region, and the Sacl-Xbal fragment contained the
rest of the coding region and 3' flanking sequence (Fig. 4). The entire 7 kb insert
was excised from pBlueScript by digestion with Kpnl and Xbal and subcloned into
the Agrobacterium binary vector pCIBIO (Rothstein et al., 1987). The resulting
plasmid, pWC4KX18.9, was introduced into Agrobacterium LBA4404 by the freeze-
thaw method (An et al., 1988).
Transformation of tobacco plants. Infection of tobacco (var. turkish) leaf
disks by Agrobacterium, cocultivation and subsequent regeneration were carried out
using standard methods (Rogers et al., 1986) with some modifications. A single
transformed Agrobacterium colony was picked from an LB-agar plate and
transferred to 3-5 ml of LB medium with appropriate antibiotics and incubated at
28C for 24 to 48 hr. Cells were harvested and resuspended in 3-5 ml of sterile MS
salts (Sigma M-5519 plus 3% sucrose). Two ml of the freshly suspended
Agrobacterium was added to 25 ml of MS salts containing tobacco leaf disks in a
sterile 50-ml tissue culture tube and incubated with shaking for one hr. Leaf disks


44
were blotted dry on autoclaved paper towels, placed on MS-agar plates for 12-24
hr (co-cultivation), then transferred to fresh CIM plates (Callus-Inducing-Medium:
MS-agar containing 50 pg/ml Timentin, 100 pg/ml Kanamycin, 5 pM BAP, 0.5 pM
NAA). Leaf disks were transferred to fresh CIM every 4-5 days for the first 2 weeks
and transferred weekly thereafter. After callus was well-formed, NAA was
eliminated to promote shoot regeneration. If shoots were generated, they were
transferred to MS agar without hormones for rooting. Sixteen independent
kanamycin resistant plants were regenerated, and nine were selected at random to
test the expression of the transgene in response to chitosan treatment.
Elicitor Treatment and RNA Extraction
Treatment of transgenic tobacco leaves. Chitosan (Sigma) was dissolved in
0.1 N HCI with heating and the pH adjusted to 5.0 with NaOH prior to autoclaving
(Popp, 1993). Young leaves were excised from transgenic tobacco plants grown
on MS agar in culture vessels (Magenta GA-7), and incubated in a culture dish
(Fisher Scientific) containing a solution of 50 mM KCI or 50 mM KCI plus 60 pg/ml
chitosan for 24 hr in constant light before harvesting. A plant that contained a single
copy of the pine transgene, based on 3:1 segregation of kanamycin resistance in
its progeny, was selected for further analysis and designated as Chi4 tobacco. A
single fully expanded leaf was divided into four sections. One section was placed
in a petri dish lacking chitosan (50 mM KCI), a second section was placed in a


45
culture dish containing chitosan (50 mM KCI + 60 (jg/ml chitosan), and both were
harvested after 24 hr incubation in constant light. A third section was immediately
placed in liquid nitrogen, and the fourth remained attached to the plant and was
mechanically wounded around its margin. Wounding was performed at 0, 2, and
4 hr, and the leaf was collected at 7 hr. The same experiment was conducted on
the control tobacco plant containing pCIBIO vector alone (designated as CIB10
tobacco).
RNA isolation from tobacco leaves. Total RNA was extracted from all
samples using previously described methods for poplar leaves (Davis et a!., 1991)
with minor modifications. Briefly, ground tissue was transferred to 1 volume
extraction buffer (100 mM Tris-pH 8.0, 500 mM NaCI, 20 mM EDTA, 0.5% SDS,
0.5% (3-mercaptoethanol, 0.1% PVPP) (1 ml buffer per gram tissue) and 1 volume
buffered phenol:chloroform. After thoroughly mixing and incubation on ice, the
mixture was centrifuged. The aqueous phase was re-extracted once with
phenol:chloroform (1:1), and RNA was precipitated by adding 1/5 volume of cold 10
M LiCI and incubating on ice overnight (at least 12 hrs). The pellet was
resuspended in 400 pi of RNase-free water and re-extracted with phenohchloroform
in a microfuge tube. The RNA in the aqueous phase was precipitated by addition
of ethanol.
Treatment of pine suspension cells. Pine cultures (both slash pine 52-56 and
loblolly pine 10-38) were maintained on a 7 day subculture interval. Two days after
transfer, sterile chitosan was added to the flasks to a final concentration of 60 pg/ml.


46
After 24 hr of incubation, cells were harvested by suction filtration and stored in
liquid nitrogen. Adjacent flasks that received no chitosan were designated as
controls.
A time course study was performed on slash pine 52-56 suspension cells.
One day after normal transfer, eight flasks of suspension cells were combined and
then re-distributed into 8 flasks to generate a uniform population of cells. After
another day, sterile chitosan was added to the flasks to a final concentration of 60
pg/ml and incubated for various periods of time (0, 0.5, 1.5, 3, 5, 8, 12, 24 hr). At
each time point, one flask of cells was harvested by suction filtration and stored in
liquid nitrogen.
RNA isolation from pine cells. Total RNA was extracted from pine
suspension cells using previously described methods (Schneiderbaueref a/., 1991)
with some modifications. Pine cells were ground to a fine powder in liquid nitrogen
in a mortar, and 20 ml of cold acetone was added directly to the mortar to inactivate
RNase activity and extract phenolic compounds which are abundant in pine cells.
The mixture was transferred to a 50-ml falcon tube and centrifuged. The pellet was
saved and washed again with cold acetone until the solution became clear, instead
of green. The pellet was then dissolved in 12 ml of TNE buffer (100 mM Tris-CI, 10
mM NaCI, 10 mM EDTA, pH 8.0, add 0.1% Triton X-100 and 15 mM DTTjust before
use) and extracted with phenol/chloroform. The supernatant was precipitated by
adding 1/5 of 10 M LiCI and incubating overnight on ice. Following centrifugation,


47
RNA pellet was re-dissolved in water, extracted with phenol/chloroform twice, and
precipitated by addition of ethanol.
Primer Extension
A synthetic oligonucleotide primer (5'-GCCAATAGCAACCTCATCGACATC
ATTC-3') complementary to positions 793 to 820 (Fig. 5, underlined and italicized)
of Pschi4 was end-labeled by T4 polynucleotide kinase and y-32P-ATP. Poly-A+ RNA
isolated from transgenic tobacco plants containing or lacking Pschi4 was hybridized
with the end-labeled primer, and extended with M-MLV reverse transcriptase. Equal
quantities of radioactivity were loaded in each lane, and the products were analyzed
on a 9% polyacrylamide sequencing gel. A sequencing reaction (Sanger et al.,
1977) of the genomic clone was performed using the same end-labeled primer and
run on the same gel.
Southern and Northern Analysis
Genomic DNAs from white pine P-18, loblolly pine 7-56 and slash pine 52-56
were digested with restriction enzymes, fractionated by agarose gel electrophoresis
and transferred to Hybond N+ nylon membrane (Amersham) in a vacuum blotter
(Hoefer) with 0.2 N NaOH as the transfer solution. The Southern membrane was
hybridized with a 729 bp Pschi4 cDNA fragment (see below for the cloning of Pschi4


48
701 rcAAATArTnrTATAAAArfACinflfTTTGCAGCCTGGGATCATCACCACAATTTGCGTTGGCAGCCTAAAGATGGCGTACACGAATATGAAGAGAArGArG
MAYTNMKRMM
801 TGGATGAGGTTGCTATTGGCrCTCACCGCAGTGGCGATAATGAGTTCTTTGTGTTGTTATGTTTCTGCACAACAAGGAGTCGCATCCATCATAAGTGAAG
SMRLLLALTAVAIMSSLCCYVSAQQGVASIISED
901 ATGTTTTCCATCAATTTTTGAAGCACAGAAACGATGACGCGTGCTCGGCGAAAGGCTTCTACACCTACAGCGCCTTCATTGCGGCAGCTAATAGTTTCCC
VFHQFLKHRNDDACSAKGFYTYSAFIAAANSFP
1001 AGACTTCGGCAACATCGGCGATCAAGATAGTCGCAAGAGAGAGCTCGCAGCTTTCTTTGGTCACACGTCGCAGGAGACCACAGg tattattaatttataa
DFGNIGDQDSRKRELAAFFGHTSQETTG
1101 gcttcctctaactcttctgcctccctgccatgccttaaatgttattaatcggattaggatgtatgggtttttacagGCGGGTGGCCAACGGCCCCAGACG
GWPTAPDG
1201 GTCCATATGCGTGGGGTTACTGCTTCAAAGATCAGGTGAATAGCACAGACAGATACCGCGGACGAGGACCTATTCAGCTAACCGGgtagg111 tg11aa t
PYAWGYCFKDQVNSTDRYRGRGPIQLTG
1301 ccgcttcgatttctagcaatagatatggaaaaaatcgaatgaatttcaagcctaatacacttaccgctctgtgggagcagGGACTACAACTACAAAGCTG
D Y N Y K A A
14 01 CGGGGAATGCGTTAGGTTACGATCTCATAAACAATCCGGATCTCGTGGCGACCGATGCCACGGTGTCGTTTAAGACGGCGGTTTGGTTCTGGATGACGGC
GNALGYDLINNPDLVATDATVSFKTAVWFWMTA
1501 GCAGTCTCCGAAGCCTTCGTGCCACGACGTGATTTTGGGAAGATTGACTCCGTCAGTTACCGATACCGCTGCTGGCAGAGTGGCGGGATATGGAATGTTG
QSPKPSCHDVILGRLTPSVTDTAAGRVAGYGML
1601 ACGGACATCATAAACGGTGGGCCGGAATGCGGCACAGGCACAATAAGCGACGTGCAGCAGGGGCGCATCGGGTTCTACCAGAGATACTGTAAGATGCTGG
TDIINGGPECGTGTISDVQQGRIGFYQRYCKMLG
1701 GGGTGGACGTGGGATCCAACCTCGACTACAAAAACCAGAAGCCTTACGGAACTTAATGTCTACGCTACCAACCCATCCAATCGACTACTACTGTTATGGT
VDVGSNLDYKNQKPYGT*
Figure 5. Sites within Pschi4 that were used to design oligonucleotide primers for
this study. The primer used for primer extension is complementary to the underlined,
italicized region. The primer sites used for RT-PCR to clone the Pschi4 cDNA are
shown in underlined boldface. A primer complementary to the double-underlined
sequence was used for cloning of the 5'-upstream sequence of Pschi4.


49
cDNA). Total RNA samples were quantified spectrophotometrically, fractionated on
formaldehyde agarose gels (Sambrook etal., 1989) and vacuum-blotted to Hybond-
N+ membrane with 50 mM NaOH as the transfer solution. Equal loading was
confirmed by ethidium bromide staining of the RNA prior to blotting. The northern
blot was hybridized with a 668 bp Sacl-SamHI fragment that is part of the coding
region of Pschi4 (Fig. 1). Hybridization and high stringency (65C; 1 mM EDTA, 1%
SDS, 40 mM NaHP04 buffer, pH 7.2) washing were performed using reagents and
conditions that were previously described (Church and Gilbert, 1984).
Cloning of the Pschi4 cDNA
RNA template isolation. Leaves of the Chi4 tobacco plant were incubated
with 50 mM KCI + 60 pg/ml chitosan for 24 hr. Total RNA was isolated as
previously described (Davis et a/., 1991). RNA concentration was determined by
both spectrophotometry and confirmed by ethidium bromide staining after
electrophoresis through an agarose gel.
Reverse transcription (RT1-PCR. One pig of total RNA from chitosan-treated
leaves of the Chi4 tobacco plant was reverse transcribed as follows. Total RNA
was heated to 65C for 10 min in the presence of 1 pg of oligo dT and 2 pi of 5 x
AMV RT buffer. The sample was then placed on ice. A mixture of 80 units RNasin
(RNase inhibitor; Promega), 2 pi 10 mM dNTPs and 19 units AMV reverse


50
transcriptase was added to yield a final volume of 10 pi. After 40 min incubation
at 42C, the RT reaction was heated to 98C for 8 min to denature the cDNA-RNA
hybrids, briefly centrifuged and chilled on ice. This fresh RT-cDNA was used as
template in a PCR without further purification. Primers (Forward: 5'-TCTGCACAAC
AAGGAGTCGCATCC-3'and Reverse: 5'-GATTGGATGGGTTGGTAAGCGTAG-3'),
corresponding to nucleotide 864-887 and 1760-1782 in Fig. 5 (underlined and in
boldface), were synthesized by Craig Echt (USDA Forest Service, Rhinelander,
Wl). RT-cDNA template was mixed with a solution containing primers (1 pg each),
10 X PCR buffer, MgCI2, dNTPs to a final volume of 49 pi. The mixture was
denatured at 94C for 2 min, and quickly cooled on ice. After a brief spin, 1 pi of
Taq polymerase (5 units) was added. The mixture was overlaid with one drop of
mineral oil prior to initiation of cycling. The PCR reaction was carried out as follows
in a Coy themocycler (model 50): 94C, 20 sec; 55C, 20 sec; 72C, 1 min 20 sec,
30 cycles with an additional extension of 5 min at 72C before cooling down to 4C.
Cloning of RT-PCR product. The initial RT-PCR product was polished by
adding 10 u of T4 DNA polymerase directly and incubating at 37C for 2 hr. The
product was electrophoresed in a 0.8% TAE agarose gel (Sambrook et a!., 1989).
The DNA fragment of the expected size was excised from the gel and purified by
QIAEX II (Qiagen Inc., CA) according to the manufacturers instructions. The
purified DNA was ligated with Smal-linearized pUC19 and transformed into E. coli.


51
TB1 cells. Positive clones were identified by restriction enzyme digestion and
further confirmed by DNA sequencing.
cDNA Expression and Generation of Antibody
Pschi4 cDNA expression in E. coli. The cloned cDNA was then subcloned
into the BamHI-EcoRI sites of expression vector pET24d (Novagen, Madison, Wl).
Expression was performed in bacterial host BL21 (DE3) pLysS cells according to
the manufacturer's protocol. Briefly, a single colony was inoculated in 3 ml of LB
containing kanamycin (50 pg/ml) and chloramphenicol (34 pg/ml), and grown at
37C until the OD600 reached 0.6 to 1.0. Cells were collected and resuspended in
fresh medium containing antibiotics, and grown at 37C until the OD600 reached 0.6.
At this point, IPTG was added to a final concentration of 1 mM to induce target gene
expression by further incubation for 3 hr. Cells were then harvested and
resuspended in buffer A (20 mM Tris-CI, pH 7.5, 20% sucrose, 1 mM EDTA). After
centrifugation, cells were stored at -80C overnight. The recombinant protein was
expressed as an inclusion body in E.coli. To purify inclusion bodies from other
cellular proteins, frozen cells were resuspended in PBS (1 L of PBS contains 8 g
NaCI, 0.2 g KCI, 1.44 g Na2HP04, 0.24 g KH2P04), and then sequentially incubated
with lysozyme and 1% Triton to lyse cells and DNase I to degrade DNA. After
centrifugation, the pellet was washed with PBS plus 1% Triton and then suspended
in water.


52
Protein purification. Overexpressed Pschi4 protein present in the inclusion
body was purified by using the electro-eluter method (Bio-Rad laboratories,
Richmond, CA). Briefly, the resuspended insoluble pellet was mixed with SDS
loading dye, boiled for 5 min and resolved by SDS-polyacrylamide gel
electrophoresis. After separation, the gel was stained with Coomassie blue in the
absence of acetic acid. The expected protein band was cut out and loaded onto the
electro-eluter (Bio-Rad, Model 422) according to the manufacturer's instructions.
The purity of protein was examined on a 12% SDS-PAGE gel and further confirmed
by sequencing the N-terminal 26 amino acids (ICBR protein core laboratory,
University of Florida).
Rabbit anti-Pschi4 antiserum. Antiserum against r-Pschi4 protein was
prepared by Cocalico Biologicals Inc. (Reamstown, PA). One hundred pg of purified
recombinant Pschi4 proteins were mixed with complete Freund's adjuvant to inject
each of two New Zealand white rabbits. Four booster injections (each time with 50
|Kj antigen and incomplete Freund's adjuvant) were administered at biweekly
intervals to obtain a high titer antiserum.
Protein Isolation and Western Blotting Analysis
Protein Isolation from Pine and Tobacco Suspension Cells
Pine suspension cultures were established and maintained as described
previously (Lesney, 1989). Tobacco suspension cultures were initiated from Chi4


53
and CIB10 tobacco leaves. Specifically, tobacco leaf disks (diameter ca. 4 mm)
were induced to form callus on MS-agar plates containing the hormone 2, 4-D prior
to initiation of suspension cultures.
Pine and transgenic tobacco suspension cells were maintained in LM (Verma
etal., 1982) and M1 (for 1 L: 4.4 g Sigmas M-5524, 30 g sucrose, 1 mg thiamine,
1 mg pyridoxine, 1 mg pantothenic acid, 0.01 mg biotin, 1 mg nicotinic acid, 1 mg
L-cysteine, 0.2 g L-glutamine, 100 mg inositol, 10 pM NAA, 5 pM 2-iP) medium,
respectively. They were transferred to fresh media at seven day intervals. Two
days after transfer, chitosan was added to a 250-ml culture flask containing 50 ml
of medium to a final concentration of 60 pg/ml. After 24 hr of incubation, cells were
harvested by suction filtration. Both cells and supernatant were saved. Cells were
ground in 20 mM of NaOAc (pH 5.2) and centrifuged. Proteins were concentrated
by dialyzing against solid sucrose and the concentrations were determined by using
Sigma's bicinchoninic acid protein assay kit according to the manufacturer's
instructions. Adjacent flasks that received no chitosan were designated as controls.
In a separate dose-effect experiment, pine cells were combined one day after
normal transfer to form a uniform population, and 0.6 ml of cells was incubated in
1 ml, 2 ml or 8 ml of LM medium with chitosan at different concentrations (0, 20, 30,
40, 60, 90, 120, 180, 240 pg/ml) for 23 hr. Media, which contain extracellular
proteins, were then collected. Proteins were quantified and dot-blotted onto a
nitrocellulose membrane.


54
Protein Isolation from Pollen of Pine and Tobacco
Pollen from pine trees was collected in late January and early February.
Pollen from transgenic tobacco plants (Chi4 and CIB10 tobacco was collected
fresh from flowers on plants. Pollen was ground with a micropestle in solubilizing
buffer (22.5% p-mercaptoethanol, 9% SDS, 22.5% glycerol, 0.125 M Tris-CI, pH
6.8) in microcentrifuge tubes, frozen in liquid nitrogen, boiled in 100C water and
ground again. This cycle was repeated for at least three times to release proteins
from pollen grains. The mixture was centrifuged and the supernatant was saved.
Western Blotting Analysis
Equal amounts of protein were loaded onto a 12% SDS-PAGE gel and
electro-transferred to a nitrocellulose membrane by using a Genie electrophoretic
blotter (Idea Scientific Co., Minnesota) according to the manufacturers instructions.
The membranes were probed to anti-Psch¡4 antibody by standard western
blot procedures. Specifically, the membrane was incubated in blocking reagent
(TBST + 5% dried milk; TBST = 20 mM Tris-CI, pH 7.5, 150 mM NaCI, 0.5% Tween
20) for 30 min and then incubated with primary antibody (1:50,000) for 6-12 hr.
After three washes with TBST, the membrane was incubated with goat anti-rabbit
AP (alkaline phosphatase)-conjugated antibody (1:5000 dilution) for 30 min. The
membrane was washed three times with TBST and once with TBS (same as TBST
except TBS lacks Tween 20). Color was developed in 25 ml of solution containing


55
2.5 ml Tris-CI, pH 9.5, 500 nlof5MNaCI, 125 pil of1 M MgCI2, 165 ^NBT(100 mg
dissolved in 2 ml of 70% DMF) and 65 \x\ BCIP (100 mg dissolved in 2 ml of 100%
DMF). Preimmune serum was used as a control (1:50,000 dilution).
Particle Bombardment and Transient Expression
Plasmid Construction
The 5'-flanking region of Pschi4 was amplified from pWC4Sac6, which
contained a 6 kb Sad fragment of gPschi4 (Fig. 4 and Fig. 6), by PCR using a pair
of primers (Forward: Universal primer F; Reverse: JOD5 = 5-GCCAACGCAAATTG
TGGTGATGATCCC-3' complementary to the positions 735 to 761 in Fig. 5, double-
underlined). After T4 DNA polymerase treatment (to make blunt ends), the PCR
fragment was digested with BamHI, purified and cloned into pBlueScript BamH\-
EcoRV sites. The plasmid was then digested with C/al, filled by Klenow Fragment
of E. coli DNA polymerase I, digested with Xba\, and subcloned into pB1101.1
(Clontech) or pGUS (/-//ndlll-EcoRI fragment of pBI101.1 subcloned in pUC19)Xbal-
Smal sites (Fig. 6). The resulting constructs were named pWP16.7 and pWP-
GUS9.3 (white pine promoter-GUS, the number denotes the total size of plasmid
DNA), respectively.
Plasmid DNA of pWP-GUS9.3 was digested with Pst\ and Sa/I and treated
with exonuclease III at 37C for various periods of time (25, 50, 75, 100, 125 sec).


56
O
co
Co
Uni F
X
5
CO
DQ
pWC4Sac6
CO
CO
O
CO
CO
JOD5
TTT^-
pWP16.7
pWP13.4
pWP13.0
pWP12.8
pWP12.4
pB1101
-4.5 kb I
1.2 kb 1
-0.8 kb 1
-0.6 kb 1
-0.5 kb 1
-0.4 kb 1
-0.2 kb |
0 kb | GUS
pWP-GUS9.3
PWP-GUS6.0
pWP-GUS5.8
PWP-GUS5.6
PWP-GUS5.5
PWP-GUS5.2
PWP-GUS5.0
pGUS
t
(cloned in dBI1011
pB1101
GUS nos ter
t
(cloned in dUC191
pGUS
Internal Control
ubiquitin promoter LUC nos ter
Figure 6 Plasmid constructs. These constructs were used for particle
bombardment (labeled as pWP-GUS#) and stable transformation of tobacco
(labeled as pWP#). Constructs were named according to the total size (in kb)
of each plasmid DNA. A nopine synthase terminator was fused to the 3 end
of both GUS and LUC.


57
At each time point, reactions were stopped by adding an equal volume of 2 x exo
III stop buffer (0.3 N NaCI, 7.5 mM EDTA) and incubated at 70C. Following
treatment with Klenow Fragment, the DNAs were self-ligated and transformed into
E. coli strain TB1 cells. This created a nested series of Pschi4 putative promoter-
GUS constructs in pUC19 (Fig. 6) with sizes of 1.2, 0.8, 0.6, 0.5, 0.4 and 0.2 kb.
These constructs, including pWP-GUS9.3, were tested by particle bombardment in
transient expression assays. These constructs were subcloned into the pBI101.3
Hind\\\ site, except pWP-GUS9.3 which was subcloned differently (see above
paragraph) for stable transformation of tobacco.
To normalize for transformation efficiency in the transient assay, a Ubiquitin-
Luciferase construct (kindly provided by Dr. C. Kao in Dr. Don McCarty's laboratory)
was included in each bombardment mixture. Thus, expression data are expressed
as GUS/Ubi-LUC ratios.
Tissue Preparation
White onions were purchased from local grocery stores. The inner epidermis
of an outer layer of onion were peeled using a pair of fine forceps and placed on the
center of MS-agar plates which were to be used for particle bombardment.
Slash pine suspension cultures were maintained as described previously
(Lesney, 1989) and transferred to fresh medium at 7 day intervals. Three days after
transfer, cells from several flasks were pooled to create an experimental population.
Approximately one ml of cells was placed on the filter paper on MS-agar plate and


58
dried for 3-10 min. This procedure varied from plate to plate and needed to be
adjusted empirically. If cells were too wet, gold particles did not penetrate the
aqueous layer, which reduced the transformation efficiency; if cells were too dry,
their viability was reduced.
Particle Bombardment
Particle bombardment was performed essentially as described previously
(Taylor and Vasil, 1991) using a DuPont PDS-100 particle gun. Briefly, 37 pi of a
40 mg/ml gold stock solution was mixed with 5 pg of internal control plasmid DNA
(Ubi-LUC) and 5 pg of testing DNA (WP-GUS) in a total volume of 72 pi in a 1.5-ml
microcentrifuge tube and vortexed briefly. Twenty pi of 100 mM of free base
spermidine and 50 pi of 2.5 M CaCI2 were placed in separate drops on the side of
the tube to avoid pre-mixing of either solution with the gold/DNA solution. The tube
was then mixed immediately by vortexing for 20 sec, which allowed plasmid DNAs
to attach to the gold particle. The tube was centrifuged for 5 sec and supernatant
was removed. Two hundred (200) pi of 100% ethanol was added and sonicated
briefly. After centrifugation and removal of supernatant, 60 pi of 100% ethanol was
added. The tube was placed on ice until all samples were ready for bombardment.
Four pi of gold/DNA solution (sonicated again just before use) was used for each
individual bombardment shot. Each experiment represented 3-6 replicates.


59
Incubation and Extraction of Proteins
Following bombardment, petri dish plates were incubated at room
temperature in constant light for 24 hr. Bombarded onion or pine cells were ground
with mortar and pestle aided by addition of glass beads in 200-800 pi of GUS/LUC
extraction buffer (0.1 M potassium phosphate (pH 7.8), 2 mM EDTA (pH 8.0), 2 mM
DTT, 5% glycerol). The homogenates were centrifuged and supernatants were
transferred to clean tubes.
Quantification of Transient Expression
Quantitative measurement of GUS activities was performed essentially as
described by Jefferson etai (1987), except that the substrate MUG was dissolved
in the extraction buffer described above. For the luciferase assay, an automated
luminometer (AutoLumat model #LB953, Wallac Inc., MA) and Promega's
Luciferase Assay Kit were used. Briefly, 10 pi aliquot of each extract was placed
in each disposable culture tube (Fisher brand, cat.# 14-961-26) and all tubes were
loaded into the luminometer. The instrument automatically injects 100 pi of
substrate luciferin dissolved in its assay buffer (prepared according to Promega's
instruction manual), counts the emitted photons for 60 sec and moves to the next
sample. The unit of measurement is the Relative Light Unit (RLU).


60
Analysis of Promoter Deletion Constructs in Stably Transformed Tobacco
Deletion constructs of the Pschi4 promoter were made and subcloned into
binary vector pB1101 as described above. These constructs were introduced into
tobacco (var. Samsun) using Agrobacterium-mediatedi transformation as described
above. Primary transgenic plants were initially used for chitosan and wounding
assays. In order to minimize the large variation in transgene expression and
inducibility, plants were allowed to flower and set seed. The seeds were placed on
MS-agar plates containing kanamycin (50 pg/ml) to determine transgene copy
number, and to generate a uniform population of plants for use in GUS assays.
Chitosan and wounding treatments were described previously (see above).
Histochemical Assays in Transgenic Tobacco
Histochemical GUS assays were performed essentially as described
previously (Jefferson etai, 1987). Tissue sections were floated in X-gluc solution
(0.5 mg/ml X-gluc in 50 mM of NaHP04 pH 7.0) for 16 to 24 hr at 37C. Tissues
were then fixed in 5% formaldehyde, 5% acetic acid, 20% ethanol and washed in
80% ethanol. Tissue sections from transgenic tobacco containing the full-length
promoter-GUS (pWP16.7) were analyzed first. Untreated leaves, stems, and
reproductive organs (corolla, stigma, ovary, pollen) from pWP16.7 tobacco plants


61
were used. For other transgenic tobacco plants containing shorter promoter-GUS
constructs, only pollen grains were stained in X-gluc and examined microscopically.


RESULTS
Pschi4 Gene Structure
Nucleotide sequence analysis of Pschi4 revealed a complete coding
sequence. In contrast, PschU contained a premature stop codon in the first exon
due to a T residue at nucleotide 1071 (Fig. 7). Pschi4 and PschU are 90% identical
through the putative coding region (not including introns) and 83% identical through
the 5'-flanking sequence. In Pschi4, a putative TATA box is located at nucleotide
711 (Fig. 7). Primer extension analysis using RNA from transgenic tobacco
revealed two major bands in cells containing Pschi4 transcripts that were not
detected in cells lacking Pschi4 transcripts (Fig. 8). The nucleotide G at position
719 (Fig. 7) was considered to be the major putative transcription start site because
it was a longer and more abundant product. Primer extension products were not
seen when slash pine mRNA was used as template (data not shown), probably due
to poor annealing of the primer which was designed from the white pine (P. strobus)
sequence.
Pschi4 contains several possible translation initiation sites. The ATG at
nucleotide 771 (Fig. 7) was considered to be the likely translation start site because
it is the first ATG downstream of the putative start of transcription, and it is flanked
62


63
-140 -131
501 CAGAAGTCAAGATTATGAAGAACAGGAGAGGAGCAGGTATAATGGCCATCGAAATCAAGGACCCACGTTACGCAGGAAIIACCIICTACCTTCGCAGAAA
6 01 TATCGCTAGGAATGGTGGGGCTTGTAGGTTCGACCACAAACACACTATATTCCACGGAGGGGTAGAAAGTTTCACCACCACACGTTATCTCAGTGCTGCG
+ 1
701 GAAATACTGCTATAAAACGAGGGGTTTGCAGCCTGGGATCATCACCACAATTTGCGTTGGCAGCCTAAAGATGGCGTACACGAATATGAAGAGAATGATG
MAYTNMKRMM
801 TCGATGAGGTTGCTATTGGCCCTCACCGCAGTGGCGATAATGAGTTCTTTGTGTTGTTATGTTTCTGCACAACAAGGAGTCGCATCCATCATAAGTGAAG
SMRLLLALTAVAIMSSLCCYVSAQQGVASIISED
901 ATGTTTTCCATCAATTTTTGAAGCACAGAAACGATGACGCGTGCTCGGCGAAAGGCTTCTACACCTACAGCGCCTTCATTGCGGCAGCTAATAGTTTCCC
VFHQFLKHRNDDACSAKGFYTYSAFIAAANSFP
1001 AGACTTCGGCAACATCGGCGATCAAGATAGTCGCAAGAGAGAGCTCGCAGCTTTCTTTGGTCACACGTCGCAGGAGACCACAGgtat tat taat t tataa
DFGNIGDQDSRKRELAAFFGHTSQETTG
1101 gcttcctctaactcttctgcctccctgccatgccttaaatgttattaatcggattaggatgtatgggtttttacagGCGGGTGGCCAACGGCCCCAGACG
GWPTAPDG
1201 GTCCATATGCGTGGGGTTACTGCTTCAAAGATCAGGTGAATAGCACAGACAGATACCGCGGACGAGGACCTATTCAGCTAACCGGgtaggttttgttaat
PYAWGYCFKDQVNSTDRYRGRGPIQLTG
1301 ccgcttcgatttctagcaatagatatggaaaaaatcgaatgaatttcaagcctaatacacttaccgctctgtgggagcagGGACTACAACTACAAAGCTG
D Y N Y K A A
1401 CGGGGAATGCGTTAGGTTACGATCTCATAAACAATCCGGATCTCGTGGCGACCGATGCCACGGTGTCGTTTAAGACGGCGGTTTGGTTCTGGATGACGGC
GNALGYDLINNPDLVATDATVSFKTAVWFWMTA
1501 GCAGTCTCCGAAGCCTTCGTGCCACGACGTGATTTTGGGAAGATTGACTCCGTCAGTTACCGATACCGCTGCTGGCAGAGTGGCGGGATATGGAATGTTG
QSPKPSCHDVILGRLTPSVTDTAAGRVAGYGML
1601 ACGGACATCATAAACGGTGGGCCGGAATGCGGCACAGGCACAATAAGCGACGTGCAGCAGGGGCGCATCGGGTTCTACCAGAGATACTGTAAGATGCTGG
TDIINGGPECGTGTISDVQQGRIGFYQRYCKMLG
1701 GCGTGGACGTGGGATCCAACCTCGACTACAAAAACCAGAAGCCTTACGGAACTTAATGTCTACGCTACCAACCCATCCAATCGACTACTACTGTTATGGT
VDVGSNLDYKNQKPYGT*
1801 CAGCACATAGTCTAATAAATAAATAAATAAAATGAGAATTGCGATAAGTGGTGAGCTTCACTCAGTGGATGGGCTCCCCTCCTAGAAATAGAAAGGGTAA
1901 GCATGGTAGATTAATTATTCATACCTGTACTGTCACTCGTGTTTTTTCACMIAMGAAGAAATAACGCGTATCCACCTTGGCAAAGGTAGCCGAATATT
2001 TCTAAATATTTTCCGCAAATGTGGAAAGTCTGGCGTTTCTTCATATCACTCGCAGGATATATGACTAAATTTGAGCAAAATAAAATAAATGT 2 092
Figure 7. Partial nucleotide sequence and translation product encoded by the
genomic clone containing Pschi4. The putative TATA box is double underlined, as
is the putative transcription start site. The likely signal peptidase cleavage site is
indicated (A). The annealing sites for the degenerate oligonucleotide primers are
shown in italicized boldface, and the position of the T in PschU that results in a stop
codon is overlined. Introns are shown in lower cases. A potential glycosylation site
is shaded. Potential polyadenylation signals are underlined. The TCA-like cis-
element is underlined and in boldface. The complete sequence of PschU and
Pschi4 that were deposited in the Genbank database are shown in the appendix.


64
Figure 8. Primer extension analysis to reveal the putative transcription
start site(s). A primer complementary to positions 793 to 820 (Fig. 5)
was end-labeled, hybridized to poly-A+ RNA from transgenic tobacco
containing Pschi4 or pCIBIO vector alone, and extended by M-MLV
reverse transcriptase. A sequencingg reaction using the same end-
labeled primer was run by side on a 9% polyacrylamide gel. The major
products are indicated by arrows and the nucleotides at which
transcription is initiated are marked with an asterisk.


65
by sequences that are the most similar to the consensus sequence for translation
initiation in eukaryotes (Kozak, 1991). In the 3' flanking region there are seven
potential polyadenylation signals (AATAAA).
A sequence was found between -140 and -131 (Fig. 7, underlined boldface)
that is similar in composition (TTACCTTCTA, identities underlined) and relative
location to a cis element implicated in wound and elicitor responsiveness of
proteinase inhibitor genes (GTACCTTGCC; Palm et al., 1990; Balandin et al.,
1995). This sequence (TTACCTTCTA. identities underlined) is also similar to the
10-bp TCA motif present in more than 30 pathogen-inducible promoters (TCA motif
consensus = TCATCTTCTT; Goldsbrough et al., 1993).
The chitinase coding region is divided into three exons of 313, 109 and 373
bp by two small introns of 93 and 96 bp (Fig. 1 and 7). The presence of introns was
first inferred by AT-richness (64.5% and 61.5% AT, respectively), orthodox
sequences at the putative splicing sites (G|GT ... AG|G), and conserved location in
other plant chitinase genes that possess introns. This was then confirmed by
cloning of the corresponding cDNA (see below). The deduced protein contains an
N-terminal 33 amino acids having the structure expected for a signal peptide, with
several positively charged amino acids near the N-terminus and an internal
hydrophobic region (Chrispeels, 1991). The predicted mature protein has a
molecular mass of 25.3 kD, assuming removal of the putative signal peptide. The
Pschi4 protein is expected to be acidic, with a predicted isoelectric point of 6.1.


66
The amino acid sequence of translated Pschi4 was aligned with tobacco
class I (Shinshi et al., 1990) and class II chitinases (Payne et al., 1990). The
catalytic domain of translated Pschi4 shares 64% and 62% amino acid sequence
identity with the catalytic domains of tobacco class I and II chitinases, respectively
(Fig. 9 and 10). Translated Pschi4 lacked the C-terminal sequence (GLLVDTM)
(Fig. 10) which is sufficient to target tobacco class I chitinase to the vacuole
(Neuhaus et al., 1991).
Genomic Southern blots were used to investigate the number of Pschi4
genes in pine and to assess their presence in different species of pines (Fig. 11).
All three species showed from two to four restriction fragments that accounted for
most of the hybridization to the Pschi4 cDNA probe. The slash and loblolly pine
patterns were more similar to one another than either was to white pine. A 2.5 kb
Sacl fragment, and a 5.5 kb BamH\ fragment were predicted to be present in white
pine (Fig. 1), and were in fact observed (Fig. 11).
Pschi4 cDNA Cloning and Expression in Bacteria
RT-PCR technique was used to clone the Pschi4 cDNA. A 729-bp fragment
was predicted based on the sequence analysis (Fig. 5 between the two primers -
underlined boldface), assuming the two putative introns were spliced; otherwise, it
would be 917 bp. The 729-bp fragment was in fact amplified by RT-PCR from the
RNA isolated from Chi4 tobacco (Fig. 12). As a control, plasmid DNA containing the


67
1% S%
Psch¡4
TobE
100 100
62 79
Figure 9. Domain structure of the putative Pschi4 protein from pine with class I
and II chitinase from tobacco. Class I chitinase from tobacco is vacuolar
(TobV; Shinshi et at., 1990), whereas class II chitinase from tobacco is
extracellular (TobE; Payne et at., 1990). Percent identity (l%) and similarity
(S%) values were calculated by comparing the amino acid sequence in the
catalytic domains (see Fig. 10 for detailed sequence alignment). From the left,
the domains in TobV are: signal peptide, cysteine-rich, hinge, catalytic, and
vacuolar targeting. Pschi4 and TobE lack the cysteine-rich, hinge, and
vacuolar targeting regions. The catalytic domain of TobV also contains a short
stretch of amino acids not found in Pschi4 or TobE.


68
1
50
Pschi4
...MAYTNMK
RMMSMRLLLA
LTAVAIMS..
SLC
CYV
Tob2
MEFS
GS
PMA-F
-C-FFLF...
Tobl
MRLCKF-ALS
SLLFSL S
AS-EQCG-QA
GGARCPSG--
-SKFGWCGNT
51
100
Pschi4
....SAQQGV
ASIISEDVFH
QFLKHRNDDA
Tob2
.LTG-LA--I
G--VTS-L-N
EM- -N GR
Tobl
NDYCGPGNCQ
SQCPGGPTPT
PPTPPGGGDL
G SSM-D
-M N-
101
150
Pschi4
CSAKGFYTYS
AFIAAANSFP
DFGNIGDQDS
RKRELAAFFG
HTSQETTGGW
Tob2
-P-N D

G--TT--DTA
-RK-I
Q--H S
Tobl
-QG S-N
N--R
G--TS--TTA
1 A
Q--H
151
200
Pschi4
PTGPDGPYAW
GYCFKDQVNS
TD
RYR
GRGPIQLTGD
Tob2
LSA..E-FTG
VR-NDQ
S-
- Y
NR
Tobl
A-A
WLREQG-
PGDYCTPSGQ
WPCAPGRK-F
ISHN
201
250
Pschi4
YNYKAAGNAL
GYDLINNPDL
VATDATVSFK
TAVWFWMTAQ
SPKPSCHDVI
Tob2
N--EK--T-I
-QE-V
1
- -1 P-
DN S
Tobl
GPC-R-I
-V--L
PVI
S-L P-

251
300
Pschi4
LGRLTPSVTD
TAAGRVAGYG
MLTDIINGGP
ECGTGTISDV
QQGRIGFYQR
Tob2
I--W AA-
Q N P
VI -N 1
RNDA-
.ED Y-R-
Tobl
I--WQ--AG-
R--N-LP-F-
VI -N L
R--D-R-
. -D R-
301
330
Pschi4
YCKMLGVDVG
SNLDYKNQKP
YGT
Tob2
--G--N-AP-
E CY--RN
F-QG
Tobl
--SI SP-
D CG--RS
F-NGLLVDTM
Figure 10. Sequence alignment of Pschi4 with tobacco chitinases. The deduced
amino acid sequence of Pschi4 was aligned with tobacco class I and class II
chitinase sequences by using the GAP program of the GCG package. Identical
amino acids are indicated by hyphens. Dots represent gaps introduced to optimize
sequence alignment.


69
Sac I BamH I
WP SP LP WP SP LP
Figure 11. Genomic Sothern blot analysis of DNA from three pine species.
Fifteen pg of genomic DNA from white pine (WP), loblolly pine (LP) and slash
pine (SP) were digested with the restriction enzymes Sacl or BamHI,
fractionated, blotted, hybridized with 32P-labeled Pschi4 cDNA probe, and
washed at high stringency. The predicted 2.5 kb Sacl and 5.5 kb BamHI
fragments from white pine are indicated by arrows.


70
Figure 12. Cloning of Psch¡4 cDNA by RT-PCR. One pg of oligo dT was
annealed to 1 pg of total RNA isolated from chitosan-treated Chi4 tobacco. The
first strand cDNA was synthesized by AMV reverse transcriptase, which was
used as the template for subsequent PCR, with a pair of primers spanning the
putative coding region (Fig. 5 underlined boldface). A DNA template of the
Pschi4 genomic clone was also used for PCR as a control.


71
Pschi4 genomic subclone was used as template for conventional PCR with the
same pair of primers. As expected, a 917-bp fragment was amplified (Fig. 12). The
amplified 729-bp fragment was cloned and its nucleotide sequence was determined.
Sequence analysis showed that the cloned fragment was identical to the original
genomic subclone except that the predicted two introns were correctly spliced (see
Fig. 5). Therefore, the 729-bp fragment was considered to be Pschi4 cDNA.
The cDNA was subcloned into expression vector pET24d, and
overexpressed in E.coli. As shown in Fig. 13A, more than 90% of the bacterial
protein was the target protein in the presence of IPTG, while this protein band was
weak or not detected in the absence of IPTG (Fig. 13A, control lane 1 and 2). The
overexpressed protein was further purified to higher homogeneity (Fig. 13B). Based
on the predicted amino acid sequence, the recombinant Pschi4 (r-Pschi4) has a
molecular weight of 26.9 kD, and this was actually observed in the SDS-PAGE gel
(Fig. 13 A&B). The N-terminal 26 amino acids of the purified protein were
determined by the protein sequencing core laboratory (ICBR, University of Florida)
to confirm that the protein was Pschi4. The result is shown in Fig. 13C and it
proved to be correct. Thus, the purified protein was used to generate anti-Pschi4
antibody.


72
C. MASMTGGQQMGRDPNSSDVPSAQQGV
Figure 13. Pschi4 cDNA expression in bacteria. (A). Crude extract of inclusion
body from E. coli. Lane 1. control #1: bacteria were taken out of shaker before
IPTG addition. Lane 2. control #2: bacteria were left in the shaker without IPTG
and grown for 3 h as lane 3-5. Lane 3-5: IPTG was added to these three flasks
and incubated for additional 3 h. (B). Purified recombinant Pschi4 protein from
bacteria. One, 4 or 8 pi of proteins were loaded onto a 12% SDS-PAGE gel and
stained with Commassie blue. (C). The N-terminal sequence of r-Psch¡4. The
underlined amino acids were from Pschi4 (see Fig. 5) and others were from the
vector sequence. The sequence was determined by the ICBR protein
sequencing core lab at University of Florida.


73
Pschi4 Expression
Chitosan-lnduced Expression of Pschi4 at mRNA Level
In the absence of chitosan, Pschi4 transcripts were present at low or
undetectable levels in pine cells. In both slash and loblolly pine suspension cells,
transcripts related to Pschi4 accumulated after treatment with chitosan (Fig. 14).
A time course study was conducted in slash pine suspension cells. Psc/j/4-related
transcripts increased to detectable levels 3 h after chitosan treatment and remained
elevated up to 24 hr (Fig. 14B and data not shown). In parallel with transcript
accumulation, cell browning was observed after 2.5 hr, which is an indication of
lignification of cell walls (Lesney, 1989). Transcript induction was not observed at
0, 3, or 24 hr in the absence of chitosan (data not shown). The observed transcript
size is approximately 0.9 kb, which is consistent with the prediction from sequence
analysis (Fig. 7).
Pschi4 was introduced into tobacco under the control of its own regulatory
sequences by Agrobacterium-mediated transformation. Sixteen independent
transformants, designated as Pschi4A through Pschi4AQ, were generated based
on kanamycin selection. Nine transgenic individuals were randomly selected to test
transgene expression. Chitosan-induced expression of Pschi4 was observed in
seven of these transgenic plants (Table 1). Fig. 15 shows a typical result of an
experiment in which the steady state level of mRNA increased in response to


74
- + chitosan
B.
0 0.5 1.5 3
5 8 12 hr
Pschi4
EtBr
Figure 14. Transcript accumulation in chitosan-treated pine cells. (A).
Loblolly pine 10-38 suspension cells were treated with or without chitosan for
24 h, and (B). Slash pine 52-56 suspension cells were treated with chitosan
for different time points as indicated before total RNA was isolated. Equal
amounts of RNA were subjected to denaturing gel electrophoresis, vacuum-
blotted to a nylon membrane, hybridized with the 668 bp Sacl-BamHI
fragment of Pschi4 (Fig. 4) and washed at high stringency. The bottom panel
shows the ethidium bromide-stained gel prior to blotting.


75
Table 1. Chitosan-induced mRNA accumulation in transgenic tobacco plants.
Nine individual transformants of Pschi4 were randomly selected fortesting chitosan-
induced expression by northern blot analysis.
Transgenic
lines
CIB
10
4.2
4.6
4.7
4.8
4.9
4.10
4.11
4.12
4.14
chitosan-
induction
no
yes
not
sure
yes
yes
not
sure
yes
yes
yes
yes
no no induction (actually no signal was detected in CIB10 tobacco plants)
yes chitosan-induction was observed
not sure inducibility is not sure because of unequal loading of RNA
* One transgenic line (4.10), specifically designated as Chi4 tobacco, was used
as a source for various studies (Fig. 8, Fig. 12, Fig. 15, Fig. 18 and Fig. 28).


76
vector only vector+Pschi4
Figure 15. Northern blot showing expression of Pschi4 in a transgenic tobacco
plant. A single leaf from tobacco transformed with pCIBIO alone (vector only)
or tobacco containing a genomic subclone with Pschi4 (vector + Pschi4) was
divided into four sections, and harvested immediately (untreated), or incubated
in 50 mM KCI (KCI), or 50 mM KCI plus 60 pg/ml chitosan (chitosan), or left on
the plant and mechanically wounded (wounded). Approximately 10 pg of total
RNA was loaded in each lane. Ethidium bromide staining indicated the last
lane (wounded) was underloaded.


77
chitosan treatment and mechanical wounding. This single tobacco plant (4.10
tobacco in Table 1) was used as sources for several experiments in the present
study (such as primer extension, cDNA cloning, initiation of suspension culture and
expression in pollen) and specifically designated as Chi4 tobacco. The pine
chitinase probe did not hybridize to any mRNA transcripts in tobacco transformed
with the pCIBIO vector alone (CIB10 tobacco). The probe appeared to be specific
for the pine chitinase transcripts and did not hybridize to endogenous tobacco
transcripts, since there is only 55% nucleotide sequence identity between Pschi4
and tobacco class II chitinase mRNA (Payne et al., 1990).
Pschi4 Protein Expression
Pschi4 protein expression in pine suspension cells. The expression of
Pschi4 protein in pine suspension cells was examined by western blot analysis.
Two cell lines of pine, slash pine genotype 52-56 and loblolly pine genotype 10-38,
were used. Sequence analysis predicted that Pschi4 would encode an extracellular
protein with an apparent molecular weight of 25.3 kD, assuming removal of the N-
terminal signal sequences. Fig. 16 shows that the anti-Psch¡4 antibody could detect
Pschi4-related proteins in the supernatant (containing extracellular proteins) of both
slash and loblolly pine cells with the size slightly higher probably due to
glycosylation, since a potential glycosylation site (NST) was found in the sequence
(Fig. 7, shaded). Interestingly, this antibody could also recognize a protein
approximately of 32 kD in the cellular fraction of loblolly pine 10-38 cell line, but not


78
slash pine loblolly pine
-1'
c:
Q.
%
c
CL
c
Q.
c
CL
o
CO
o
CO
o
(0
o
(0
J
A
A
A
A
A
A
o
O
o
O
o
O
o
o
Q
Q)
N
(/>
UF81
Figure 16. Pschi4 protein expression in pine suspension cells. Slash pine
52-56 and loblolly pine 10-38 suspension cells were treated with (ch) or
without (ck) chitosan. Total proteins were then isolated from media
(supernatant spnt) or cells (cytosol cyt), fractionated on a 12% SDS-PAGE
gel, and transferred to a nitrocellulose membrane. Duplicate membranes
were incubated with anti-Pschi4 (UF81) or preimmune (PI). The
overexpressed bacterial protein (not purified) was used as a positive control.


79
in slash pine cells (Fig. 16). This size is consistent with other vacuolar chitinases
reported (Broglie et al., 1986; Samac et al., 1990; Shinshi et al., 1987; Zhu and
Lamb, 1991). It is also of interest to note that chitosan did not show much induction
at the protein level in this blot.
However, slash pine cells did show a chitosan response at the protein level
in a separate study (Fig. 17). Equal amounts of suspension cells (0.6 ml) were
incubated in different volume of medium with various concentrations of chitosan.
Within the 1-ml incubation, Pschi4-related protein was low or not detected at
chitosan concentrations of 0, 180 and 240 pg/ml; but accumulated to detectable
levels starting from 20 up to 120 pg/ml chitosan (Fig. 17). Cell browning, from light
to dark, was correlated with the chitosan concentrations from 20 to 240 pg/ml. In
fact, when chitosan was higher than 180 pg/ml in the small volume (1 ml) of cells,
cell cultures became very dark-brown, a phenomenon similar to the hypersensitive
cell death observed in soybean suspension cells exposed to high concentrations of
H202 (Levine etal., 1994; Levine et al., 1996). In the 8-ml incubation, cells became
dark-brown at the concentration of 60 pg/ml in the solution, and probably because
the ratio of net chitosan over cell numbers was much higher.
In early studies, proteins from extracellular and cellular fractions of loblolly
pine suspension cells were applied to a rotofor cell, an apparatus that separates
proteins based on isoelectric focusing (IEF). Extracellular proteins showed higher


80
1 ml
2 ml
0 0 20 40 60 120 180 240 30 60 90 (pg/ml chitosan)
1 pi

4 pi
4 pi
m m ...
#
16 pi
8 pi
'§ # %
m
32 pi
16 pi
# # '
t,
8 pi
32 pi
m m
32 pi
64 pi
120,
2 ml
60,
8 ml
Figure 17. Chitosan-induced Pschi4 protein expression in pine
suspension cells. Slash pine cells (0.6 ml of each) were incubated in 1
ml, 2 ml or 8 ml of LM medium with chitosan at the concentration (pg/ml)
indicated. Proteins were isolated from the media, quantified and adjusted
to same concentration. From 1 to 64 pi of proteins were loaded on to a
nitrocellulose membrane and incubated with the anti-Pschi4 antibody.


81
chitinase activity in the acidic area (pH 4.6 to pH 6.4) after chitosan treatment (data
not shown).
Pschi4 protein expression in transgenic tobacco suspension cells. Tobacco
suspension culture was initiated from transgenic tobacco (Chi4 and CIB10 tobacco)
leaf disks. The protein level of Pschi4 in tobacco suspension cells was also
examined by western blot analysis. Fig. 18 shows that Pschi4 protein was present
in the supernatant (extracellular) fraction of the Chi4 tobacco suspension cells but
not in the cytosolic fraction. The size was approximately 25 kD, indicating it may not
be glycosylated as in pine. In contrast, nothing was detected in the CIB10 tobacco
suspension cells (Fig. 18). It is worthwhile to note that no chitosan responses were
observed at both the protein (Fig. 18) and mRNA levels (data not shown) in tobacco
suspension cells.
Transient Assay of Pschi4 Promoter-GUS Constructs
Plasmid Construction
The 5'-flanking sequence, putative promoter region, of Pschi4 was subcloned
and fused with the reporter gene uidA from bacteria which encodes p-glucuronidase
(GUS). A series of promoter deletion-GUS constructs were also made available by
time-dependent digestion by exonuclease III (Fig. 6). These constructs were made
in the vector pUC19 for transient assays and some of them were subcloned into the


82
CIB10 Ch¡4
anti-Pschi4
c
, .
c
c
, .
Q.
>
CL
>%
CL
o
?
U)
o
X
X
o
O
o
o
o
o
kD
31.0
21.5
preimmune
Figure 18. Western blot analysis in tobacco suspension cells. Tobacco
suspension cell lines were initiated from leaves of transgenic tobacco
containing the Pschi4 or pCIBIO vector. Chi4 cells were treated with (CH) or
without (CK) chitosan (60 pg/ml) for 24 h. Total proteins were isolated from
both medium (supernatant spnt) and cells (cytosol cyt). Western procedures
were the same as described in Fig. 16.


83
binary vector pBI101 for stable transformation into tobacco. All of them were named
according to the total size (kb) of the plasmid (Fig. 6).
Transient Expression in Onion Epidermis Cells
I was interested in identifying a functional pine promoter as well as defining
chitosan-responsive c/s-element(s), if such element(s) exist. Putative promoter-
GUS constructs were first tested by transient assays in onion cells (Fig. 19 and 20).
All constructs showed high promoter activity compared with the promoterless control
(Fig. 19A). Chitosan was included in the incubation, but did not induce higher
activity. Fig. 19 represents two typical results of these experiments. Since the
results from particle bombardment experiments showed large variations in onion,
both GUS and luciferase (LUX) activities are only comparable within a single
experiment. In the experiment shown in Fig. 20, more constructs were used and no
significant differences in activity were seen between constructs (Fig. 20), which
indicates that the smallest -200 bp promoter construct contained sufficient
regulatory elements to direct activated transcription in onion cells by transient
assays.
Promoter Comparison in Maize and Pine Suspension Cells
The commercially available CaMV 35S promoter (35S) has been
demonstrated to be highly expressed in angiosperms, however, there are conflicting
data regarding the efficiency of the 35S promoter in gymnosperms (Ellis, 1994).


A.
no chitosan | + chitosan
no chitosan | + chitosan
Figure 19. No chitosan-induction in transient assays in onion cells.
Onion epidermis cells were bombarded with 5 pg of indicated WP-
GUS (see Fig. 6) and 5 pg of Ubi-LUC, and cultured post
bombardment with or without chitosan. (A) and (B) represent two
separate experiments. Data represent mean (+ SE) of five
replicates.


85
constructs
Figure 20. Promoter activity in onion cells. Onion cells were transformed by
particle bombardment as described in Fig. 19 and cultured in the absence of
chitosan. Data represent mean (+ SE) of six replicates.


86
Constructs containing either 35S-GUS or the white pine chitinase promoter (WP)-
GUS were bombarded into maize and pine suspension cells (Fig. 21). Both 35S
and WP showed significant promoter activity in maize cells, although 35S was much
higher than WP (Fig. 21A&C). In contrast, WP showed higher promoter activity
than 35S in pine cells (Fig. 21B&D). The overall GUS activity detected in pine cells
was lower than in maize (Fig. 21 C&D; scales are different). In fact, the internal
control, Ubi-LUC, also showed much lower activity in pine (average = 3.7 x 104
RLU) than in maize (average = 2.6 x 106 RLU). This is why the ratio of GUS/LUX
was higher in pine (Fig. 21B) than in maize (Fig. 21 A).
Transient Assay in Pine Suspension Cells
Since no chitosan-induction was observed in onion cells (Fig. 19), it was
possible that some components that are present in pine could be missing in onion
cells. To test this idea, I repeatedly tested particle bombardment in pine suspension
cells. After optimization of the technique, GUS activity levels were sufficiently high
to obtain interpretable results (Fig. 21), but chitosan-induction was not observed
(Fig. 22A). Interestingly, the expression pattern was a little bit different in pine cells
from that in onion. The pWP-GUS9.3, containing the 4.5-kb WP promoter, showed
higher promoter activity than any of the others in pine cells (Fig. 22), while all
constructs showed similar activity in onion cells (Fig. 20). One small construct, -136
GUS, did not show any activity in pine cells compared with the -200 construct and
promoterless control (Fig. 22B).


87
A.
B.
C.
D.
Figure 21. Promoter comparison in maize and in pine cells. Maize and pine
suspension cells were bombarded with pBI221 (35S promoter) or pWP-
GUS9.3 (WP; 4.5 kb white pine chitinase promoter, see Fig. 6). Ubi-LUC
was an internal control in all cases. Data in (A) and (B) are expressed as
ratio of GUS activity (pmole MU) over luciferase activity (RLU relative light
unit). Data in (C) and (D) are expressed as GUS activity. All data represent
mean of two to five replicates.


Full Text

PAGE 1

PINE CHITINASE GENE STRUCTURE, EXPRESSION AND REGULATION : ANALYSIS IN PINE CELLS AND IN HETEROLOGOUS SYSTEMS By HAIGUOWU 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 1996

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This dissertation is dedicated to my Lord and Savior Jesus Christ, and to my wife, Yang.

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ACKNOWLEDGMENTS I would like to express my sincere appreciation to the members of my graduate committee: Dr. John Davis, Dr. Ken Cline Dr. Bill Gurley Dr. Corby Kistler and Dr. James Preston for their critical advice, suggestions and discussions, which have greatly improved this dissertation. In particular Dr. Gurley has provided me with constant and enthusiastic support over the years I am also indebted to Dr. Don McCarty for allowing me to access his particle gun to Dr Chien-Yuan Kao and Dr. Eva Czarnecka-Verner for teaching me how to use this gun for bombardment experiments, and to Dr Rosie Simmen for access to the automated luminor for luciferase assays I wish to extend my thanks to Dr Craig Echt for sequencing the initial genomic clone and providing part of the DNA primers for this study to Don Baldwin for helpful suggestions and discussions about primer extension experiment. I would like to thank all the past and present members of Dr. John Davis' laboratory. My thanks go to Dr. Mark Lesney for helpful discussions and assistance with the cell cultures to Dr. Mick Popp Dr Yong Qian and Buddy Tignor for their assistance and friendship I would like to recognize the excellent technical support of Tess Korhnak and Thea Edwards, whose collective contributions to this work ensured the smooth and efficient running of the program iii

PAGE 4

My most special thanks go to my advisor Dr. John Davis for his invaluable advice and guidance, for his encouragement and generosity for his tremendous support and unending confidence in me, for all his efforts on my behalf throughout the course of this study. Working under his superv i sion has been a wonderful experience in my life. Finally I would like to thank my wife Yang for her continual love encouragement and support, for without her and my faith in God this work would not have been completed iv

PAGE 5

TABLE OF CONTENTS ACKNOWLEDGMENTS ... . ...... ..................... . ........ iii LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . vii LIST OF FIGURES .... . .... .................... .............. viii LIST OF ABBREVIATIONS . ............................. ......... x ABSTRACT ..... ..... .......... ........ ........ ..... . ..... ... xi LITERATURE REVIEW ..... . . . ... ..................... ....... 1 General Features of Plant Defense Responses ..... .... .... .... 1 Chitosan General Elicitors and Specific Elicitors ................ 16 Pathogenesis-Related Proteins .... ............. ............. 20 Chitinase Structure Function and Regulation . .... ......... .... 27 INTRODUCTION ......................... . ............... ..... 35 MATERIALS AND METHODS . ......... . . .... . ......... .... . 41 Plant Materials ... ..... ....... ..... .............. ......... 41 Sequence and Sequence Analysis ........................ ... 42 Plant Transformation ........ .............................. 43 Elicitor Treatment and RNA Isolation ......... ......... . ..... 44 Primer Extension . . . . . . . . . . . . . . . . . . . . . 4 7 Southern and Northern Analysis . . . . . . . . . . . . . . . 4 7 Cloning of the Pschi4 cDNA ................................. 49 cDNA Expression and Generation of Antibody ................... 51 Protein Isolation and Western Blotting Analysis . . . . . . . . . 52 Particle Bombardment and Transient Expression ........... .... . 55 Histochemical Assays in Transgenic Tobacco .......... . . . ... 60 V

PAGE 6

RESULTS ............................... ............. ........ 62 Pschi4 Gene Structure ..... ................................. 62 Pschi4 cDNA Cloning and Expression in Bacteria ....... .......... 66 Pschi4 Expression ................................... . .... 73 Transient Assay of Pschi4 Promoter-GUS Constructs .... ......... 81 GUS Expression in Stably Transformed Tobacco Plants ........ .... 89 Developmental Regulation of Pschi4 Expression . . . . . . . . 96 DISCUSSION .............. ..................... .............. 101 SUMMARY AND FUTURE DIRECTIONS ..... ............. ........ 110 APPENDIX ... ... ............................................. 114 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . 117 BIOGRAPHICAL SKETCH .... . . ............................... 139 vi

PAGE 7

LIST OF TABLES Table 1 Chitosan-inducted mRNA accumulation in transgenic tobacco plants .. 75 2 Summary of WP-GUS tobacco plants ......................... 90 vii

PAGE 8

LIST OF FIGURES Figure 1 Gene-for-gene interactions specify plant disease resistance or susceptibility . . . . . . . . . . . . . . . . . . . 5 2 Different actions between endochitinases and exochitinases ........ 28 3 Domain structure of three classes of chitinases ................... 30 4 Overall structure of the pine genomic subclones gPschi1 and gPschi4 . .................... ....... 38 5 Sites within Pschi4 that were used to design oligonucleotide primers for this study ......................... 48 6 Plasmid constructs . . . . . . . . . . . . . . . . . . . . 56 7 Partial nucleotide sequence and translation product encoded by the genomic clone containing Pschi4 ............ ... 63 8 Primer extension analysis to reveal the putative transcription start site(s) . ...................... .......... 64 9 Domain structure of the putative Pschi4 protein from pine with class I and II chitinase from tobacco .................... . 67 1 O Sequence alignment of Pschi4 with tobacco chitinases ............ 68 11 Genomic Southern blot analysis of DNA from three pine species .... 69 12 Cloning of Pschi4 cDNA by RT-PCR . ...... .................. 70 13 Pschi4 cDNA expression in bacteria . . . . . . . . . . . . . 72 viii

PAGE 9

14 Transcripts accumulation in chitosan-treated pine cells ............ 74 15 Northern blot showing expression of Pschi4 in a transgenic tobacco plant . . . . . . . . . . . . . . . . . 76 16 Pschi4 protein expression in pine suspension cells . . . . . . . . 78 17 Chitosan-induced Pschi4 protein expression in pine suspension cells . . . . . . . . . . . . . . . . . 80 18 Western blot analysis in tobacco suspension cells . . . . . . . . 82 19 No chitosan-induction in transient assays in onion cells ........... 84 20 Promoter activity in onion cells .... .......................... 85 21 Promoter comparison in maize and in pine cells ............ ... .. 87 22 Promoter activity in pine cells . . . . . . . . . . . . . . . . 88 23 Particle bombardment per se induces promoter activity ............ 93 24 GUS activity was not induced by chitosan in stably transformed tobacco plants . . . . . . . . . . . . . 94 25 Mechanical wounding induced promoter activity ............ ..... 95 26 Phosphate induced WP-GUS expression in transgenic tobacco ...... 97 27 X-gluc staining of tobacco pollen .............................. 98 28 Pschi4 protein expression in tobacco pollen .................... 100 ix

PAGE 10

2 ,4-0 2-iP BAP BCIP OMF OTT EOTA GUS IPTG LB MS MUG NAA NBT PVPP sos X-gluc LIST OF ABBREVIATIONS 2,4-dichlorophenoxyacetic acid N6-2-isopentenyl-adenine 6-benzyl-aminopurine 5-bromo-4-chloro-3-indolyl phosphate dimethyl formamide dithiothreitol ethylenediamine tetraacetate (disodium salt) p-glucuronidase isopropyl p-0-thiogalacto-pyranoside Luria-Bertani medium Murashige-Skoog medium 4-methylumbelliferyl P-0-glucuronide a-naphthalene acetic acid nitro blue tetrazolium polyvinyl-polypyrrolidone sodium dodecyl sulfate 5-bromo-4-chloro-3-indolyl-P-O-glucuronide X

PAGE 11

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 PINE CHITINASE GENE STRUCTURE, EXPRESSION AND REGULATION: ANALYSIS IN PINE CELLS AND IN HETEROLOGOUS SYSTEMS By Haiguo Wu December 1996 Chairman : Dr John M Davis Major Department: Plant Molecular and Cellular Biology Chitinases are plant enzymes that hydrolyze chitin, which is the major component of the cell walls of many pathogenic fungi but absent in higher plants Chitinases belong to Group Ill PR-proteins which are believed to play important roles during plant-pathogen interactions The present study describes the structures of several genomic clones from pine trees that appear to encode extracellular class II chitinase, and examines the expression of these genes in pine cells as well as in transgenic tobacco plants One of the genes Pschi4 potentially encodes a protein that shares 62% amino acid sequence identity through the catalytic domain with class II chitinase from tobacco The corresponding Pschi4 cDNA was cloned by RT-PCR. Nucleotide sequence analysis indicated that the Pschi4 coding sequence is composed of three exons interrupted by two intrans at locations identical to those found in other chitinase genes that possess intrans In contrast, Pschi1 contains a stop codon in the first exon and may be a pseudogene xi

PAGE 12

Pschi4 genes are conserved in several species of pine and appear to comprise a small multigene family Treatment of pine cell suspension cultures with the general elicitor chitosan induced Pschi4 expression at both mRNA and protein levels. The regulatory sequences associated with the Pschi4 gene were sufficient to direct chitosanand wound-inducible expression of Pschi4 in transgen i c tobacco plants which indicated that Pschi4 is an actively expressed member of the multigene family The region 5' to the putative transcription start site of Pschi4 was fused to the GUS reporter gene to further analyze these i nducible regulatory elements. The -200 bp 5'-upstream sequence of Pschi4 was demonstrated to contain active promoter sequences capable of wound-induced expression in transient assays (particle bombardment) as well as in stably transformed tobacco plants This region was also sufficient to induce transcription in pollen of transgenic tobacco The putative pine promoter showed higher promoter activity than the widely used CaMV 35S in the transient expression in pine cells which implies that the Pschi4 promoter could be a good candidate to regulate transcription of other genes in transgenic pine cells. The observation that the Pschi4 gene from pine (a gymnosperm) was appropriately regulated by chitosan in tobacco (an angiosperm) suggests that the signal ing pathways that mediate chitosan induced transcription are highly conserved in the plant kingdom xii

PAGE 13

LITERATURE REVIEW General Features of Plant Defense Responses Plants are frequently challenged by pathogens that cause tissue damage and disease. Resistance to these pathogenic organisms results from the rapid deployment of a multicomponent defense response (Dixon et al., 1994) The individual components of this response may include increased production of antimicrobial phytoalexins (Dixon et al., 1983), hydrolytic enzymes (Stintzi et al. 1993) lignin (Vance et al., 1980) hydroxyproline-rich glycoproteins (Varner and Lin 1989) and the development of a hypersensitive response (HR) around the infection sites (Keen, 1990). Some of these components can also be induced in uninfected tissue, at sites remote from pathogen infection This physiologically acquired resistance is termed systemic acquired resistance (SAR) This systemically induced state can result in reduced severity of disease detected when the tissues are reinfected with any type of pathogen (bacterial viral or fungal) Signals for activation of host defense responses are thought to be initiated in recognition of pathogen elicitors by the plant (Dixon et al. 1994). 1

PAGE 14

2 The outcome (resistance or disease) of a plant-pathogen interaction depends on both the plant and the pathogen. To be successful in infecting a host plant the pathogen must possess genes that function in pathogenicity On the plant side some preformed antimicrobial compounds termed phytoanticipin (Van Etten et al. 1994) can serve as constitutive resistance factors (Osbourn 1996) More importantly, plants have the ability to develop an activated resistance also called an induced response There are two relevant aspects in this induced process (Alexander et al., 1994 ) The first critical factor is the timing of the development of the plant s active defense systems If established quickly enough these active responses are usually effective in restricting the pathogen resulting in disease resistance If initiated slowly or not at all the pathogen may be successful in infect i on leading to disease The second factor is the relevancy of the activated responses for a particular pathogen If the induced reactions have a deleterious effect on the pathogen the infection may be limited. If the elicited responses are not relevant for the pathogen or if the pathogen can use an alternative strategy to escape the active responses disease occurs Bas i c Incompatibility and Susceptibility Basic incompatibility describes the failure of a fungus to cause disease on any member of a plant species This is a non host general resistance (Keen and Dawson 1992) and i s proposed to result from the plant's ability to recognize th e general features of potential pathogens The recognition of general elicitors such

PAGE 15

3 as chitin and chitosan, will be discussed later In general, resistance is the rule and susceptibility is the exception in the plant world (Staskawicz et al., 1995) Basic susceptibility results in plant disease Genetically it is determined by the pathogen's genes functioning in pathogenicity and host recognition (Keen and Dawson, 1992). Some secondary metabolites from the pathogen act as host selective toxins (HSTs) which are low molecular weight compounds and are positive agents of pathogenicity (Walton 1996). Most known HSTs are made by fungi (Walton, 1996). Successful infection by a fungal pathogen involves four steps: (1) attachment; (2) germination of the fungal spores ; (3) penetration and (4) colonization of host tissues (Schafer, 1994) During the process, the pathogen may detoxify host defense compounds such as phytoalexins and suppress defense responses by modifying molecules in the signal transduction pathway (Keen and Dawson, 1992). Hypersensitive Response (HR) Resistance in fungal-, bacterialand viral-plant interactions is often associated with the HR in which a small number of cells that are at or near the site of pathogen infection die rapidly. The protective cell suicide is considered as a very strong defense response induced in plants by the pathogen itself (Stintzi et al., 1993). The necrotic lesion which is formed around the infection site perhaps depletes nutrients for the pathogen, and subsequently a very intense response is induced in this region which confines the spread of pathogen (Lamb et al., 1989)

PAGE 16

4 In many cases in which resistance occurs via an HR, the plant and the pathogen have an apparent "gene-for-gene" relationship (Flor, 1942). In his classic work Flor defined the basic elements of gene-for-gene complementarity wherein single plant disease resistance genes (R) are paired with single complementary avirulence genes (Avr) in the pathogen resulting in the HR (Flor, 1942). The functional alleles are proposed to be dominant and involved in recognition (Ellingboe 1981 ) As shown in Fig. 1, resistance occurs only when the plant resistance gene (R) matches the pathogen avirulence gene (Avr). If either partner lacks a functional dominant allele, recognition and resistance do not occur and the plant becomes diseased (Fig. 1 ) However, a single plant may contain many different resistance genes directed to a particular pathogenic species Therefore a pathogen biotype must possess recessive alleles for all of the relevant avirulence genes to successfully escape surveillance (Keen 1990) A number of pathogen avirulence genes were isolated in the 1980s (Keen, 1992). Some Avr genes are proposed to be involved in the production of specific elicitors which will be discussed later. Since 1993 many plant resistance genes have also been cloned by transposon tagging or positional mapping For example the tomato PTO gene (Martin et al., 1993) and PRF gene (Salmeron et al. 1996), Arabidopsis RPS2 gene (Mindrinos et al. 1994), and rice Xa21 gene (Song et al., 1995) were all isolated by map-based cloning Other resistance genes such as the tobacco N gene (Whitham et al., 1994), tomato Cf-9 gene (Jones et al., 1994) and flax L6 gene (Lawrence et

PAGE 17

Plant host cell RR or Rr rr AA HR or Aa (resistance) disease C Q) C) 0 .c -t'O a.. aa disease disease Figure 1 Gene for-gene interactions specify plant disease resistance or susceptibility. R denotes the dominant plant disease resistance (R) gene and A indicates the corresponding pathogen avirulence (Avr) gene Resistance occurs only when both dominant alleles R and Avr, are present in plant and invading pathogen respectively Al i other combinations lead to inabiltity of recognition by plant host cells and result in disease. 5

PAGE 18

6 a/., 1995) were identified by transposon tagging. With the cloning of more Avr and R genes the basic tenet of specific recogntion in the gene-for-gene hypothesis can be directly tested. A consistent theme is that the R genes that have been cloned appear to function in signal transduction pathways where they may rapidly activate plant defense responses after pathogen recognition In the case of the hypersensitive response of tobacco to tobacco mosaic virus (TMV) the areas of highly-induced responses can be easily detected under UV light as rings, since cells surrounding the necrotic lesions exhibit bright blue fluorescence. These cells have accumulated compounds of the phenylpropanoid pathway (Legrand et al., 1976), some of which are fluorescent. Phytoalexins In association with the HR, plant cells produce many compounds that have direct antimicrobial activity One of these responses is the production of phytoalexins. Phytoalexins are low-molecular-weight antimicrobial compounds synthesized by plants in response to attempted infection by pathogens exposure to elicitor molecules or other biotic and abiotic stresses (Dixon et al, 1983). More than 350 phytoalexins have been chemically characterized from approximately 30 plant families (Kuc 1995). Most of them have been isolated from dicots, but they have also been isolated from monocots such as barley corn onion, rice sorghum and wheat (Kuc, 1995), and from pines (Lange et al., 1994). Phytoalexins are isoflavonoid terpenoid or other compounds of low molecular weight. Similarities

PAGE 19

7 have been observed between phytoalexins from the same plant species, while differences usually exist between phytoalexins from different genera and families (Kuc and Rush 1985) For example, isoflavonoid compounds are the major phytoalexins in the Leguminosae and are rarely found in other plant species (Dewick, 1988) On the other hand, terpenoid phytoalexins derived from the isoprenoid pathway are the most abundant in Solanaceae such as tobacco, but have not been reported in Leguminosae (Kuc, 1982a ; Kuc 1995) Phytoalexins are synthesized de novo in response to infection as they are usually not detected prior to infection (Kuc and Rush, 1985) The phytoalexin precursors are produced from three major biosynthetic pathways in all plants : shikimate acetate-malonate and acetate-mevalonate pathways (Kuc and Rush 1985; Kuc, 1995) Genes encoding individual enzymes in these pathways such as phenylalanine ammonia-lyase (PAL) chalcone synthase (CHS) and 3-hydroxyl-3methylglutaryl Coenzyme A reductase (HMGR), have been cloned from several plant species, and their expression is regulated by environmental factors, including pathogen infection (Choi et al., 1992 ; Fritze et al., 1991; Liang et al., 1989 ; Ohl et al., 1990; Stermer et al., 1990) The phytoalexins reported to date are not very stable in plants and they are eventually degraded by the host plant and/or the pathogen (Van Etten et al., 1982 ; Yoshikawa et al., 1979) The detailed mechanism by which phytoalexin biosynthesis and turnover is controlled remains somewhat unclear

PAGE 20

8 Cruickshank proposed earlier that in incompatible interactions accumulation of phytoalexins halts pathogen growth and thus confers resistance (Cruickshank 1963) In compatible interactions the pathogen can either tolerate the host phytoalexin detoxify it suppress its accumulation or prevent the initial elicitation (Cruickshank, 1963). Some studies have supported this proposal (Kessmann and Barz 1986 ; Kuc, 1995 ; Yamada et al. 1989). Other Biochemical Responses In addition to rapid cell death and production o f phytoalexins at and in cells surrounding infection sites, many other changes occur in the same cells (Lamb et a/., 1989) The most obvious observation is cell wall thickening and reinforcement by deposition of various macromolecules such as callose hydroxyproline-rich glycoproteins (Varner and Lin 1989), lignin (Lesney 1989 ; Vance et al., 1980) and cell wall bound phenolic compounds (Matern and Kneusel 1988) These compounds presumably serve as a physical barrier to prevent the pathogen from spreading Another important response at or near the infection site is the accumulation of numerous pathogenesis-related (PR)-proteins A few days after TMV infection PR proteins may account for 10% of the total soluble proteins in tobacco leaves (Jamet et al. 1985 ; Pierpoint 1986) More details about PR-proteins will be discussed later.

PAGE 21

9 The above responses (HR, production of phytoalexins, cell wall lignification and high concentrations of PR-proteins) are generally local responses and are very effective in limiting pathogen growth. Systemic Acquired Resistance (SAR) Besides the local responses, many plants respond to the necrotizing pathogen with a more thorough protection, the so-called systemic acquired resistance (SAR) If part of a plant has already responded to an initial inoculation hypersensitively the uninoculated parts of this plant develop an increased state of resistance evidenced by smaller lesions and greater restriction of the pathogen upon subsequent infection by the same or even unrelated pathogens (McIntyre et a/., 1981; Ross 1961 ; Ye et al., 1989) This type of plant immunity has been well documented in tobacco (Ross, 1961) and cucumber (Kuc 1982b). Systemic acquired resistance can be detected a few days after inoculation and can last for weeks to months (Lawton et al., 1993). Although the cellular intensity of the systemic response is much lower than the local response it still represents a tremendous amplification in the plant defense response as it concerns the whole plant. A number of genes are associated with the appearance of SAR which are sometimes called SAR genes. Since PR-protein expression parallels the onset of SAR (Bol and Van Kan 1988) these genes are believed to be actively involved in the development of SAR. PR genes have become sensitive markers in the search

PAGE 22

10 for signals that are transmitted from necrotic lesions to distant parts. A more detailed classification of PR-proteins will be discussed later. Salicylic acid (SA) and SAR Exogenously applied SA or acetylsalicylic acid (aspirin) can induce SAR and the production of at least some PR-proteins in plants (Ward et al. 1991 ; White, 1979). This was first discovered in Xanthi-nc tobacco (Nicotiana tabacum) (White, 1979) which contains a dominant resistance gene (N) that confers the host HR to specific races of tobacco mosaic virus (TMV). Injection of solutions containing SA or aspirin into tobacco leaves prior to the inoculation of TMV caused a dramatic reduction in lesion size (Wieringa-Brants and Schets 1988) and number (White 1979). However SA was found to be an endogenous signal molecule only recently The SA level increases transiently in the phloem of plants just before the onset of SAR (Malamy et al., 1990 ; Metraux et al. 1990). The hybrid of N. glutinosa X N debneyi has been shown to contain a high constitutive level of SA (in the absence of pathogens) (Yalpani et al., 1993b). This tobacco hybrid also exhibits constitutive SAR and PR-protein expression and is highly resistant to TMV (Ahl Goy et al., 1992) The role of SA in the SAR signaling pathway has been an active area of research in the past few years. The direct evidence for SA involvement in SAR induction comes from a transgenic experiment. Salicylate hydroxylase encoded by a bacterial NahG gene catalyzes the hydroxylation of SA to catechol which does not induce SAR Transgenic tobacco plants harboring the NahG gene are unable to accumulate SA and are defective in SAR induction upon TMV infection (Gaffney et

PAGE 23

11 al., 1993). This apparent relationship supports the hypothesis that SA is involved in the SAR signal transduction pathway. However, SA per se is probably not the mobile signal from the infection site to other parts of the plant (Vernooij et al., 1994) This is suggested by the observation that the removal of an infected leaf before any measurable SA accumulation in the cucumber phloem still results in induction of SAR genes in distant tissues (Rasmussen et al., 1991). This conclusion was supported further by grafting experiments from the same group who did the NahG transgenic experiment. When wild-type scion was grafted on top of NahG rootstock the top scion was able to develop SAR in response to TMV inoculation to the (NahG) rootstock (Vernooij et al., 1994). Despite their inability to accumulate SA NahG tissues are fully capable of producing the transmittable mobile signals for SAR. However Raskin's group used 180 labelling and obtained evidence that SA could be a mobile messenger in tobacco (Shulaev et al., 1995). They argued that the SAR signaling mechanism in TMV-infected tobacco could be different from that in Pseudomonas syringae-inoculated cucumber. Also, expression of salicylate hydroxylase in NahG rootstock (Vernooij et al., 1994) does not completely block SA accumulation Small amounts of SA, above background levels could escape and be exported to phloem, and then transported to the upper wild-type scion While no consistent conclusion has been made about SAR signaling, the search for real mobile signal(s) is still an active area of study. The pathway of SA biosynthesis has been proposed in tobacco in which SA is derived from benzoic acid (Yalpani et al. 1993a) The last step is catalyzed by

PAGE 24

12 benzoic acid-induc i ble benzoic acid 2-hydroxylase (BA2H) Activation of BA2H leads to SA synthesis in tobacco. The level of benzoic acid increases dramatically in TMV-inoculated tobacco tissue (Yalpani et al. 1993a) Benzoic acid accumulation has a l so been observed in pine injured by dothistromin a toxin from Dothistroma pini (Franich et al. 1986). To elucidate the mode of action of SA Chen et al. identified an SA binding protein in tobacco (Chen and Klessig 1991 ; Chen eta/., 1993a) which turned out to be a catalase (Chen et al., 1993b) SA may increase H202 leve l s by inhibiting catalase activity which normally converts H202 to H20 and 02 (Chen et al., 1993b) The active oxygen species (AOS), including H202 are thought to be important molecules during SAR induction Active oxygen species (AOS) Active oxygen species (AOS) are generated in pathogen-infected tissues and are widely involved in host defense responses (Baker et al 1993 ; Legendre et al., 1993; Orlandi et al., 1992) They are potentially toxic intermediates to pathogens The term "oxidative burst" describes the rapid release of AOS during HR formation. This phenomenon has been observed in many plant species such as potato (Doke 1983) tomato (Vera-Estrella et al., 1992) tobacco (Baker et al., 1993 ; Glazener et al., 1996 ; Keppler and Baker 1989) soybean (Apostol et al., 1989 ; Levine et al. 1994) and spruce (Schwacke and Hager 1992) The predominant species detected in plant-pathogen i nte r act i ons include superoxide anion (021 hydrogen peroxide (H2 02 ) and hydroxyl radical (OH) (Mehdy 1994). Injection of H202 into tobacco leaves induces the expression

PAGE 25

13 of PR-1 genes locally, as is true with the treatment of leaves with glycolate and paraquat, two substances that promote generation of H2O2 (Chen et al. 1993b) Exogenously applied H2O2 (8 mM) induced hypersensitive cell death in soybean suspension cells (Levine et al., 1994; Levine et al., 1996) However recent evidence has shown that the relatively low amount of H2O2 (4-6 M) generated during the incompatible interactions between the plant and pathogen is not sufficient to cause hypersensitive cell death (Glazener et al., 1996). More recently Naton et a/. provided cytochemical evidence that intracellular accumulation of AOS in infected parsley cells is related to rapid cell death (Naton et al., 1996). They argued that intracellular AOS may be more important as possible mediators of rapid cell death than extracellular AOS, which were measured previously by many researchers i ncluding Glazener et al. (Glazener et al. 1996) Nevertheless the extracellular AOS may be involved in many processes associated with plant disease resistance e.g. crosslinking of cell wall proteins (Bradley et al., 1992) increased lignification of cell walls at infection sites or they may function as d i rect antimicrobial compounds and as signal transducers leading to gene activation (Sutherland 1991 ) The evidence suggests that H2O2 is involved in HR that SA can inact i vate a catalase and that SA is definitely involved in the signal transduction pathway leading to SAR. However, recent experiments demonstrate that H2O2 is not a second messenger downstream from SA and the inactivation of catalase by SA does not result in SAR (Bi et al., 1995 ; Neuenschwander et al., 1995) These data

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14 suggest that H202 could be an important molecule involved in local responses but it is not the translocated signal and is not sufficient to induce SAR alone. The chain of events in the SAR signal transduction pathway is still an active area of research. Wounding Responses Wounding damage to plant leaves results in activation of a variety of defense genes locally as well as systemically (Brown and Ryan 1984 ; Parsons et al., 1989 ; Pena-Cortes et al., 1988). Considerable research has been conducted on induction of Pl genes, espec i ally in tomato (Pearce et al., 1991; Ryan, 1990). These Pl gene products strongly inhibit the serine proteases found in insect gut tissue and their expression is systemically induced by wounding This suggests that Pis may serve as a defense against insect feeding (Green and Ryan 1972) Proteinase inhibitors have also been found in tobacco and they are pathogen-inducible as well as wound inducible (Linthorst et al., 1993). In contrast to tomato the t obacco Pl genes can only be induced locally (not systemically) by wounding or exposure to pathogens (Linthorst et al. 1993). Some plant-derived chemicals such as oligosaccharides and jasmonic acid have been found to be involved in the induction of these wound inducible Pl genes (Farmer and Ryan, 1990 ; Farmer et al., 1992 ; Ryan 1988 ; Walker Simmons et al. 1983) Jasmonic acid (JA) Jasmonic acid (JA) and i ts methyl ester (MJ) have been hypothesized to be a possible key component of intracellula r signaling in response to wounding (Farmer and Ryan 1990 ; Farmer et al., 1992) Direct ev i dence of

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15 jasmonate involvement in wound-induction came from the observation that JA levels increase immediately and transiently after wounding (Albrecht et al., 1993). The biosynthetic pathway for JA has been elucidated (Vick and Zimmerman, 1984). The pathway starts from linolenic acid (LA) which is a component of the plasma membrane. LA is converted to 13-S-hydroperoxylinolenic acid (13-S-HPLA) by lipoxygenase (LOX). Then hydration and cyclization lead to the formation of 12-oxy phytodienoic acid (12-oxy-PDA), followed by three steps of p-oxidation. It has been demonstrated that three of the octadecanoid precursors of JA, i.e., LA, 13-S-HPLA and 12-oxy-PDA, can activate the synthesis of Pis in tomato leaves when applied to leaf surfaces (Farmer and Ryan, 1992) Inhibitors of LOX activity, such as salicylhydroxamic acid, reduce JA biosynthesis (Staswick et al., 1991). It is interesting that salicylic acid (SA), a key molecule in systemic acquired resistance has been shown to be an inhibitor of wound-induced Pl accumulation (Doares et al., 1995a; Pena-Cortes et al., 1993). The results suggest that SA could inhibit JA synthesis by inhibiting the formation of 12-oxy-PDA (Pena-Cortes et al., 1993) or SA could block JA action by inhibiting an as-yet-undefined step between JA and transcriptional activation of Pl genes (Doares et al., 1995a) The wound-induced accumulation of Pis in tomato is proposed to be initiated by the release of pectic polysaccharides from the plant cell walls (Bishop et al. 1981). It has also been found that chitosan, a polymer of P-1,4-glucosamine found in fungal and insect cell walls, is a strong inducer of both JA and Pl syntheses in tomato leaves (Doares et al., 1995b; Walker-Simmons et al., 1983)

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16 Chitosan, General Elicitors and Specific Elicitors Elicitors In searching for signal molecules that may be involved in the ability of plants to recognize pathogens efforts have focused on "elicitor" molecules The term "elicitor" was originally used to describe agents that induce the synthesis and accumulation of antimicrobial compounds (phytoalexins) in plant cells (Keen, 1975) but is now widely used to refer to molecules that stimulate any plant defense response, from cellular changes such as the HR to molecular changes such as transcriptional activation of defense-responsive genes (Dixon et al., 1994). Elicitors have been divided into two groups : specific elicitors and general elicitors (non-specific elicitors), based on whether the elicitor exerts similar effects on all members of a plant species (general) or only on specific genotypes of a plant species (specific). Specific Elicitors Specific elicitors are those involved in the interaction of a certain pathogen biotype to a particular plant species, and usually refer to the avirulence gene products (direct or indirect) that are produced in the gene-for-gene interactions. A well-characterized specific elicitor is a small peptide of 28 amino acids which is

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17 produced only by Cladosporium fulvum carrying the Avr9 avirulence gene. This elicitor induces a defense response only in tomato plants containing the Cf-9 resistance gene (de Wit, 1992). The biological function of these specific elicitors in pathogens is not clear. Do they reside in the pathogen only to elicit the plant defense system against itself when it invades the plant? This doesn't seem to be logical. It is most likely that specific elicitors do have biological functions in the pathogen, as yet unknown, but the plant developed a defense system later by recognizing so-called specific elicitors from the pathogen in order to survive. On the other hand, the pathogen does not intend to produce "elicitors" against itself and keeps modifying/changing the "elicitor"-related avirulence genes to escape the plant recognition. Experimental evidence for this dynamic interaction is the Avr4 and Avr9 genes in the fungus Cladosporium fulvum (de Wit et al., 1994 and refs therein). C. fulvum has exploited at least two different mechanisms to avoid specific recognition by the host plant (tomato) In the case of Avr9, mutations from avirulence to virulence involve the deletion of this gene in order to escape recognition by Cf-9 In the case of the Avr4 gene, virulent races still contain essentially the same gene except for a single point mutation, resulting in an amino acid change from cysteine to tyrosine, which is sufficient to avoid Cf-4 recognition. This may explain why there are so many pairs of Avr-R genes in a single pathogen biotype and a single plant species It has been found that the coat protein of TMV serves as a specific elicitor to tobacco plants carrying the N' resistance gene (Culver and Dawson 1989), and the TMV replicase is specifically recognized by tobacco

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18 plants carrying the N gene (Whitham et al. 1994). This supports the view that specific elicitors do have biological functions in the pathogen and have been exploited by the plant. General Elicitors and Chitosan General elicitors Different from specific elicitors, general elicitors are involved in the general resistance of plants to broad ranges of pathogens or potential pathogens Disease is the exception not the rule (Staskawicz et al. 1995) Most plants recognize general features of potentia l pathogens and can prevent d i sease due to an ability to recognize these elicitors. Biotic elicitors include some oligosaccharides (Hahn et al 1993) proteins (Ricci et al., 1993) and lip i ds (Bostock et al., 1981) and some known abiotic elicitors include heavy metals and UV radiation Abiotic elicitors are thought to result in the release of biotic elicitors from the plant cell walls (Hahn et al. 1993) Biotic elicitors could be of either pathogen or plant origin a l though in most cases, they are pathogen components General elicitors are usually present on or near the cell surface of the pathogen and often serve as cell wall components Structurally characterized fungal elicitors include : glucan elicitors oligogalacturonide elicitors chitin and chitosan glycopeptides, and ergosterol (Granado et al., 1 995 ; Hahn et al., 1993 ; Keen and Dawson 1992) Chitosan Chitosan is a deacetylated form of chitin which is a polysaccharide composed of ~-1 ,4-linked N-acetylglucosamine Both chi tin and chitosan are cell

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19 wall components of many fungi (Bartnicki-Garcia, 1968) Many studies have demonstrated that oligosaccharides derived from chitin and chitosan elicit defense responses in various plants. Chitosan and its derived fragments elicit the accumulation of phytoalexins in pea pods (Hadwiger and Beckman 1980 ; Walker Simmons et al., 1983) suspension-cultured soybean cells (Ko hie et al., 1984) and parsley cells (Conrath et al., 1989). Relatively low concentrations of chitosan (as low as 8 g/ml) directly inhibit the growth of certain fungal pathogens of pea (Kendra et al., 1989). Chitosan oligomers with a degree of polymerization (DP) between 6 and 11 are most active as phytoalexin elicitors in pea Chitosan-derived oligosaccharides are also capable of inducing the accumulation of proteinase inhibitors in both tomato and potato leaves (Pena-Cortes et al., 1988 ; Walker Simmons et al., 1983 ; Walker-Simmons and Ryan, 1984) and the synthesis of callose in suspension-cultured soybean (Kohle et al., 1985) parsley (Conrath et al., 1989), tomato (Grosskopf et al., 1991) and Catharanthus roseus (Kauss et al., 1989) cells. Oligosaccharide fragments of both chitosan and chitin have both been shown to induce defense-related lignification of the walls of suspension-cultured slash pine cells (Lesney 1989, 1990) Chitosan treatment induces pine cells to synthesize hydrolytic enzymes including chitinase and glucanase (Popp et al., 1996). Chitin oligomers with a degree of polymerization of 4 to 6 elicited lignification in wounded wheat leaves (Barber et al., 1989). The deposition of both callose and lignin are thought to enhance plant defense by strengthening the plant cell walls

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20 Active chitosan and chitin oligomers are thought to be released during plant pathogen interactions by plant enzymes such as chitinases. Chitinases belong to a group of PR proteins (see below) whose synthesis is induced by pathogen infection and also by chitosan elicitors. It is possible that low levels of constitutive plant chitinases release active chitin and chitosan oligomers from invading fungi which in turn induce a defense response including accumulation of chitinases This reaction could serve as an "amplification" of signals and re-enforce the initial defense response Pathogenesis-Related Proteins Pathogenesis-related (PR) proteins are highly induced proteins during plant pathogen interactions. Expressed both locally and systemically PR proteins represent the major quantitative changes in soluble proteins during defense responses PR proteins have very distinct physicochemical properties some of which enable them to survive the harsh environment where they occur : (1) at acidic pH (as low as pH 2 .8), they are quite stable and soluble whereas most other plant proteins are denatured at this pH; (2) they are relatively resistant to proteolytic enzymes of both endogenous and exogenous origin ; (3) they are targeted to compartments such as the vacuole, the cell wall or apoplast ; (4) most of them are monomers with low molecular weight (8 50 kD)

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21 PR prote i ns were first reported by Van Loon and Van Kammen in the early 1970s They detected four de nova synthesized proteins in tobacco plants reacting hypersensitively to infection with TMV (Van Loon and Van Kammen 1970). Since then many other proteins with similar physicochemical (see above) and induction properties have been identified from tobacco and other plant species (Kombrink et al., 1988; Stintzi et al., 1993 ; Vogeli et al., 1988) The tobacco (Nicotiana tabacum Samsun)-TMV interaction leading to an HR is still the model for the study of PR proteins and from which the highest number of PR proteins have been characterized. PR proteins are induced in response to infection by pathogens of viral viroid bacterial or fungal origins (Van Loon, 1985) However some members of PR families have been found to be induced by chemical treatment (elicitors) stress or wounding where pathogens are not involved The reason they are still called "pathogenesis-related" is that they still represent a wide array of pathogen defense related proteins in a particular plant species Furthermore many of the chemicals that induce PR proteins mimic natural compounds involved in pathogen plant interactions and/or the transduction pathways that are associated with pathogen-plant signaling (Stintzi et al., 1993). Classification of PR Proteins By us ing different biochemical approaches more than 30 PR proteins have been isolated from TMV infected tobacco plants (Stintz i et al. 1993) Based on amino acid sequence similarity and serological properties these tobacco PRs are

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22 classified into five groups (Stintzi et al., 1993; Alexander et al., 1994). PR proteins from other plant species can be put into each group although there are exceptions Group PR-1. Tobacco PR1 proteins represent most of the earl ier work Three acidic isoforms of tobacco PR-1 i.e. PR-1 a, PR-1 b and PR-1 c were the first purified PRs (Jamet and Fritig 1986) and are serologically related to each other in tobacco and to PRs from other plant species (Nassuth and Sanger, 1986) The amino acid sequence of the three PRs deduced from the cloned cDNA (Cutt et al., 1988) share more than 90% identity Another clone which was identified from a cDNA library of TMVinfected tobacco, appeared to encode a basic isoform of PR 1 protein (Cornelissen et al., 1987) The corresponding protein was actually puri fied later and named PR-1g All of the four PR-1 proteins from tobacco are localized extracellularly Tobacco PR-1a and PR 1b as well as three members of PR-1 from tomato have been shown to contain direct antifungal activ i ties i n in vitro assays (Niderman et al., 1993 ; Stintzi et al. 1993), although the molecular mechanism involved needs further investigation Group PR 2. The tobacco PR 2 proteins have been found to contain endo 1 3-p-glucanase activity (Kauffmann et al., 1987) which produces ol i gomers of 2-6 glucose units from 1 3-p glucans Many 1 3-p glucanases have been purified (Boller 1988) and many genes encoding glucanases have also been cloned (Meins et al., 1992) Glucanases are usually monomers with a molecula r weight of 25-35 kD Of the five members of PR-2 from tobacco four of them (PR protein 2 N 0 Q') are acidic proteins and are targeted to extracellular space (Kauffmann e t al.,

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23 1987) ; one (glue b) is basic and is localized in the vacuole (Van den Bulcke et al., 1989) The specific activities of these glucanases are strikingly different towards a particular substrate (Stintzi et al. 1993) Group PR-3. Tobacco PR-3 proteins are chitinases Chitinase structure function and regulation will be discussed in more detail later Group PR-4 Tobacco PR-4 group includes four proteins : r1, r2, s1, s2 (Kauffmann et al. 1990) that are all acidic and targeted to the extracellular space (apoplast) They are small proteins (13-14 5 kD) and are serologically related to each other The biological function/activity of PR-4 proteins is not known. Group PR-5 Two slightly acidic proteins (R & S) and two basic proteins (n osmotin and osmotin) from tobacco are included in PR-5 group. The acidic and basic isoforms are localized in apoplastic space (extracellular) and vacuolar compartment respectively (Dore et al., 1991) Genes encoding tobacco PR-5 proteins have been isolated (Van Kan et al., 1989) and their corresponding cDNAs have also been cloned (Payne et al., 1988). Sequence comparisons have shown that there are no intrans within these genes. The deduced amino acid sequences of PR-5 proteins are approximately 60% similar to thaumatin a protein isolated from Thaumatococcus danielli Therefore PR-5 proteins are also called thaumatin-like PRs (Stintzi et al., 1993) Thaumatin-like proteins have been characterized in many other plant species such as maize (Richardson et al. 1987) tomato (K ing et al. 1988) barley (Bryngelsson and Green, 1989) and potato (Pierpoint et al. 1990)

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24 Other groups of PR Proteins Some PR proteins from tobacco and other plant species cannot be put into the five major groups To accommodate these proteins and novel proteins identified in the future, scientists of the Commission on Plant Gene Nomenclature (CPGN) have proposed a new Y category which generally represents plant genes whose sequences are clearly conserved but whose designations are not based on function. Thus, PR genes can be collectively designated by Yprfollowed by a number (Van Loon et al., 1994) However for PR genes whose functions are known such as glucanases and chitinases suggested designations Glu or Chi are preferred to Ypr2 or Ypr3 Regulation of PR Protein Expression PR gene expression is generally induced by pathogens (fungal, bacterial or viral), as is obvious from the definition ; however some elicitors and various stresses can also induce PR gene expression PR genes are differentially expressed in response to various stress conditions and during different developmental stages Spraying plants with a salicylic acid solution induces expression of some PR genes including PR-1 (acidic and basic) acidic PR-2 and basic PR-3 (Bol et al. 1990 ; Van de Rhee et al. 1993) The tobacco basic PR proteins are highly induced by wounding and ethephon which produces ethylene in vivo whereas acidic PRs are not (Brederode et al., 1991) Moreover, the basic PRs are constitutively expressed in roots and lower leaves of healthy plants i n contrast to the i r acidic counterparts (Memelink et al. 1990; Neale et al., 1990 ; Van de Rhee et al. 1993). On the other

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25 hand, tobacco acidic PRs are systemically elicited by TMV infection while no or little induction of basic PRs occurs in non-inoculated leaves (Brederode et al. 1991) To identify the putative cis-acting elements responsible for PR gene induction efforts have been focused on making promoter deletions fused with the reporter gene (uidA) which encodes bacterial p-glucuronidase (GUS) and testing the relative GUS activities. The data on PR-3 proteins (chitinases) will be discussed later The analys i s of upstream sequences of genes encoding tobacco PR-1, PR-2 (glucanase) and PR5 indicates that PR promoters contain multiple cis-acting elements (Albrecht et al., 1992 ; Van de Rhee and Bol 1993 ; Van de Rhee et al. 1993) A recent experiment has shown that the PR-1a gene promoter of t obacco contains several elements that can bind GT1-like nuclear factors (GT-1 factor is necessary for light responsive expression of the pea rbcS-3A gene) ( Buchel e t al., 1996) but yet no common sequence motif has been identified from these assays However a 10 bp element which is repeated four times in the 5' non-translated region of a barley P-1, 3-glucanase gene has been found to be present in t he non translated regions of over 30 stressand pathogen-inducible promoters (Goldsbrough et al. 1993) Gel mobility shift assays have provided prelim i nary evidence that this element known as the TCA motif (TCATCTTCTT) specifically binds a tobacco nuclear protein and this binding activity was great l y increased when tobacco plants were pre-treated with salicylic acid ( Goldsbrough et al., 1993) It is suggested that the TCA motif could be important for induced expression A 116 bp fragment between 168 and -52 of the parsley PR-2 promoter wh ich was shown

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26 to be necessary for elicitor-mediated expression (Van de Locht et al. 1990) actually contains a TCA element. A 61-bp element of the tobacco P-1 3-g lucanase 8 gene has been shown to be an enhancer whose activity is independent of orientation. Analysis of point mutations has identified the sequence AGCCGCC, named the AGC box which is essential for the enhancer activity (Hart et al. 1993) Nuclear extracts from tobacco leaves contain one or more factors that can interact with this element specifically This binding act i vity is higher in nuclear extracts from ethylene treated plants than contro l plants which correlate with its postulated role in the regulation of the P-1, 3 glucanase gene (Hart et al. 1993) Whether the same or a similar set of regulatory proteins are involved in all PR gene induction or each group of acidic/basic PR genes are controlled by a unique set of factors is not clear It seems that at least some factors are commonly involved in induction of certain PR proteins such as chitinases and glucanases The purification of these nuclear factors and cloning of their corresponding genes/cDNAs will help to understand the induction mechanisms and to give some clues as to the signal transduction pathways involved in the induced responses

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27 Chitinase Structure, Function and Regulation Chitinase Structure and Function Chit i nases (PR-3 proteins) are enzymes that hydrolyze chitin a linear homopolymer of N acetylglucosamine. Chitin is the major component of cell walls of many fungi but is not present in higher plants. On the other hand chitinases have been reported to be present in a variety of higher plants (Boller et al. 1983 ; Broglie and Broglie, 1993). It seems that there is no endogenous substrate for chitinases in higher plants; therefore it has been proposed that chitinases in higher plants may play a role in protecting plants against chitin-containing fungi (Boller 1988). There are two types of chitinases in plants: endochitinases and exochitinases Endochitinases randomly hydrolyze internal P 1 ,4-linkages of chitin whereas exochitinases digest chitin from the non-reducing end of the polymer (Fig 2). Therefore the smallest substrate for endochitinases is a tetramer of N acetylglucosam i ne and that for exochit i nases is a dimer (Fig 2) Most of the characterized plant chitinases are endochitinases (EC 3 2 1 14) (Boller et al. 1983 ; Molano et al., 1979) ; however exochitinases have also been purified from melon (Roby and Esquerre-Tugaye 1987) and carrot (Kurosaki et al. 1987) Endo-type chitinases have been characterized from many plant species i ncluding barley (Jacobsen et al. 1989 ; Kragh et al. 1991) bean (Bolle r et al.,

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A. chitin B endochitinases endochitinases exochitinases exochitinases e N-acetylglucosamine residue Figure 2 Different substrate specificities for endochitinases and exochitinases (A). Endochitinases hydrolyze the internal ~-1,4-bond of chitin, whereas exochitinases digest the bond from the non-reducing end (B). The smallest substrate for endochitinases and exochitinases are tetramer and dimer of N-acetylglucosamine, respectively 28

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29 1983) cucumber (Boller and Metraux, 1988) maize (Nasser et al., 1990), pea (Mauch et al. 1988a) potato (Kombrink et al., 1988) tobacco (Legrand et al., 1987) tomato (Joosten and de Wit 1989) and wheat (Ride and Barber 1990) In general plant chitinases are proteins of 25-35 kD molecular weight which occur as monomers and have either a high or low isoelectric point (bas i c or acidic chitinases) (Boller 1988). Three classes of plant chitinases have been proposed based on the pr i mary structures (Fig. 3) (Broglie and Broglie 1993 ; Collinge et al., 1993 ; Shinshi et al 1990) Class I chitinases are composed of an N-terminal signal sequence a cysteine-rich domain of approximately 40 amino acids a variable length h i nge region a highly conserved main structure (catalytic domain) and a C-terminal vacuolar targeting sequence (usually 7 amino acids) Class II chitinases have a high amino acid sequence identity to the main structure of class I chitinases but lack the cysteine-rich domain, the hinge region and C terminal domain Class Ill chitinases show no sequence similarity to enzymes in class I or class II. The C terminal seven amino acids (GLLVDTM) of tobacco class I chitinase are necessary and sufficient to direct the protein to the vacuole (Neuhaus et al 1991 ). In addition to the difference in domain structure class I and class II chitinases have other distinctive properties and are located in separate cell compartments. Class I chitinases are targeted to the vacuole whereas class II chitinases are secre t ed into the extracellular space All identified class II chitinases are acidic proteins but class I and class Ill chitinases have been found to be either basic or acidic (Collinge et al., 1993 ; Davis et al., 1991 ; Lawton et al., 1992)

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cysteine-rich l signal peptide t hinge t catal vacuolar targeting t Class I Class II Class Ill ----Figure 3. Domain structure of three classes of chitinases From left to right the domains in class I chitinases are: signal peptide, cysteine-rich hinge, catalytic and vacuolar targeting. Class II chitinases share high homology with class I chitinases in the catalytic domain, but lack the cysteine-rich hinge and vacuolar targeting regions The catalytic domain of class I chitinases also contain a short stretch of amino acids not found in class II chitinases The catalytic domain of class Ill chitinases are not related to either class I or class II chitinases 30

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31 Genes and/or cDNAs encoding chitinases have been cloned from a variety of plant species such as Arabidopsis (Samac et al. 1990) bean (Broglie et al., 1986), cucumber (Metraux et al. 1989) poplar (Davis et al., 1991) potato (Laflamme and Roxby 1989) rice (Xu et al., 1996 ; Zhu and Lamb 1991) and tobacco (Payne et al., 1990 ; Shinshi et al., 1990) Constitutive expression (directed by the CaMV 35S promoter) of a bean chitinase gene in transgenic tobacco plants showed increased disease resistance against certain fungi (Broglie et al., 1991). It was speculated that chitinases may inhibit fungal growth by direct lysis of hyphal tips particularly in combination with glucanases (Schlumbaum et al. 1986 ; Mauch et al., 1988b ; Sela-Buurlage et al., 1993). Chitinases could also function to amplify defense responses in cells surrounding a site of infection by liberating chitin and chitosan oligomers from fungal cell walls which may serve as general elicitors to induce the expression of several defense-related genes (Boller et al., 1983 ; Mauch and Staehelin 1989). In addition to potential roles in defense chitinases may play important roles during early embryo development (de Jong et al. 1992) and other developmental processes (Neale et al., 1990) This i s presumably because plant cells contain substrates for chitinase that are not chitin per se but may resemble chitin structurally (Fisher and Long 1992 ; Collinge et al. 1993) Regulation of Chitinase Gene Expression Environmental regulation Chitinase gene expression i s regulated by many factors including pathogen invasion treatment with elicitors and plant hormones

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32 (ethylene), and mechanical wounding. Chitinase enzyme activity, protein and mRNA levels have been shown to be elevated when the plant is under these stresses (Boller 1988; Collinge et al. 1993 ; Joosten and de Wit 1989) Induced expression of chitinase is often co-ordinated with other PR proteins such as P-1,3glucanases (Joosten and de Wit, 1989; Kombrink et al. 1988; Shinshi et al. 1987 ; Vogeli et al. 1988). Individual chitinase isoforms have been reported to be differentially regulated in barley, pea and tobacco. In barley leaves and grain several chitinase isozymes have been found, but only one is induced in response to pathogen infection (Kragh et al., 1990) In pea at least two chitinases are differentially regulated upon fungal infection (Mauch et al., 1988a) In tobacco, the basic class I and acidic class II chitinases have been shown to be differentially induced by var i ous stresses such as virus infection UV light and wounding (Brederode et al, 1991; Memelink et al. 1990). Developmental regulation In addition to stress-induced regulation chitinases are also under developmental regulation i n healthy Arabidopsis rice and tobacco plants In normal tobacco chitinase expression was found in roots developing flowers and lower older leaves (Memelink et al., 1990 ; Neale et al. 1990 ; Shinshi et al., 1987) In Arabidopsis and rice constitutive expression of chitinase was found in roots (Samac et al. 1990 ; Zhu and Lamb 1991) The developmental regulation of chi tinase in tobacco was proposed to be controlled by auxin and cytokinin gradients within the plant (Shinshi et al., 1987) The finding that chitinases accumulate in a tissue-specific manner during different developmental

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33 stages has led to the view that chitinases may serve other functions in plants in addition to their role in the defense response. Promoter studies The regulatory sequences that direct the expression of chitinase have been studied in a few plant species The first characterized promoter is the bean chitinase 58 promoter (CH58) which contains element(s) for ethylene induction (Broglie et al., 1986) The ethylene-responsive element was first identified between positions -422 and -44 (Broglie et al., 1989) by analysis of deleted chitinase genes in transgenic tobacco plants and was confirmed in a bean protoplast system (Roby et al., 1991). This region has been further narrowed down to sequences between nucleotides -305 and -236 by promoter-GUS deletion analysis in transient expression assays (Broglie and Broglie 1993) In addition a nuclear protein has been observed to bind to this DNA sequence in gel mobility shift and DNase I protection assays (Broglie and Broglie 1993) Similar research has been conducted for Arabidopsis and tobacco chitinase genes (Fukuda and Shinshi 1994 ; Samac and Shah 1991). An acidic class Ill Arabidopsis chitinase promoter was ft,1sed to the GUS reporter gene and transformed into Arabidopsis Promoter activity (GUS expression) was detected in roots leaf vascular tissue hydathodes guard cells and anthers in healthy plants which indicates its developmental regulation Induced expression was observed in mesophyll cells surrounding lesions caused by fungal infection Promoter deletion analysis demonstrated that the region 192 bp upstream of the transcription start site is capable of both developmental and induced expression (Samac and Shah 1991 ) In tobacco the

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34 5'-upstream sequence from tobacco class I chitinase was fused to GUS and introduced into tobacco Promoter deletion analysis revealed that the region between nucleotides -574 and -476 is sufficient for inducibility by a fungal elicitor. Gel mobility shift assays further identified a sequence of 22 bp between -539 to 518 specifically that binds to a nuclear protein from elicitor-treated cells but not from control cells This 22 bp sequence contains a direct repeat of GTCAG separated by three nucleotides (Fukuda and Shinshi 1994). From the above discussions it is clear that angiosperm PR genes have been well documented in the literature. Their gene products and promoter analyses have been studied in detail. However, little information is available on defense responses in gymnosperms. In this research, I chose chitinase gene as a model to study pine defense responses since chitinase structure seems to be conserved in angiosperms and previous work has shown that chitinase activity in pine suspension cells increases upon chitosan treatment (Popp et al. in press).

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INTRODUCTION Plants are constantly confronted by microbes and other pests that can cause tissue damage. The cell walls of many of these microbes share common structural features Compounds like chitin and chitosan are found in the cell walls of many fungi, as well as the exoskeleton of arthropods, but these compounds are not found in plants In most plants a defense response is induced when they are treated with chitin or chitosan (Kohle et al. 1984, 1985; Fritensky et al. 1985; Kombrink and Hahlbrock 1986; Lesney, 1989). One component of the defense response to elicitors is the transcriptional activation of genes that encode pathogenesis-related (PR) proteins. One class of PR proteins includes chitinases. Chitinases hydrolyze chitin, a linear homopolymer of N-acetylglucosamine. Most of the characterized plant chitinases are endochitinases (EC 3 2.1.14) which randomly cleave internal ~-1,4 linkages in chitin and consequently release oligomers of N-acetylglucosamine (Boller et al 1983 ; Molano et al 1979) Chitinase alone, or in combination with glucanase can directly inhibit fungal growth by causing lysis of hyphal tips (Mauch et al. 1988b; Schlumbaum et al., 1986; Sela-Buurlage et al., 1993) Chitinase also seems to amplify plant defense responses by releasing chitin and chitosan elicitors from fungal cell walls (Boller et al. 1983; Mauch and Staehelin 1989) In addition to potential roles in defense chitinase may also serve other functions during 35

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36 development (de Jong et al., 1992; Neale et al. 1990). Chitinase gene expression is controlled at the transcriptional level and can be induced by general elicitors and other stresses. Therefore, chitinase genes can be viewed as "reporters" that can be used to study defense mechanisms as well as to define components of signal transduction pathways involved in defense responses Pinus is an ancient as well as economically important genus in the plant kingdom Pine cells exhibit a pronounced defense response to chitin chitosan and live pathogens (Lesney 1989; Popp, 1993) and this defense response is accompanied by secretion of chitinase (Popp et al., In press). To obtain a better understanding of defense responses and gene regulation in conifers, a chitinase gene was chosen as a model system for this study As a first step toward this overall goal a chitinase gene from pine trees was i solated and characterized. Alignment of class I and class II chitinase sequences from a number of different plant species revealed the presence of highly conserved regions within the catalytic domain This feature was exploited as a strategy for cloning related sequences from pines using PCR. A pair of primers were designed to anneal to conserved regions of the chitinase catalytic domain The upstream primer 5' AT AAGCTTCA(AG)AC(ACGT)(AT)(CG)(ACGT)CA(ACGT)GA(AG)AC -3' was1024 fold degenerate and was expected to anneal to the nucleotide sequence translated as QTSH(Q)ET. The downstream primer 5'-ATGGTACCCATCCA(AG)AACCA(AC GT)A(AGT)(ACGT)GC 3' was 96-fold degenerate and designed to anneal to the region encoding Al(LM)WFWM Degenerate positions are shown in parentheses

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37 and restriction sites that were introduced into the primers for directional cloning are underlined Fragments about 400 bp in length were amplified from white pine cloned, sequenced and found to show sequence similarity to known chitinase genes in the expected region of the catalytic domain. The cloned PCR product was used to screen a genomic library of eastern white pine Six plaques were selected due to their hybrid i zation to the probe in duplicate plaque lifts. The resulting recombinant phage were designated gPschi1-6 (for genomic Pinus strobus chitinase 1 through 6). Two of these genomic clones, gPschi1 and gPschi4, were selected for detailed sequence analysis Recombinant phage DNA was digested with Sacl and the insert fragments were subcloned into pBlueScript. Physical mapping and partial DNA sequence analysis suggested that gPschi1 and gPschi4 were likely to contain lengthy 5' flanking sequences and intact coding regions (Fig 4) Most of the above work was accomplished by Dr. John M Davis part was done by Dr. Michael P Popp (SFRC University of Florida) DNA sequencing was performed by Dr Craig S Echt (USDA-Forest Service Rhinelander WI) The first goal of my study was to identify a funct i onal gene from the cloned chitinase fragments and to study its inducibility of expression by general elicitors The hypothesis to be tested was that pines possess defense related genes that are regulated at the transcriptional level. To begin I introduced the entire Pschi4 gene including 4.5 kb of 5' upstream sequence, 0.9 kb of coding sequence and 1.5 kb of 3' downstream sequence into tobacco via Agrobacterium-mediated transformation This gene was confirmed to be transcribed in transgenic tobacco plants by northern

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38 gPschi1 (.) I I C1l E c3 E C1l c3 Cl) C1l C1l C1l C1l co Cl) co Cl) I I I I gPschi4 sequenced region I exon D intron 1 kb Figure 4 Overall structure of the pine genomic subclones gPschi1 and gPschi4 The transcriptional orientation and putat i ve coding regions including intron and exon sequences are indicated by arrows. The asterisk denotes the stop codon within exon #1 of gPschi1 Restriction endonuclease cleavage sites that were used for subcloning are presented Pschi1 was not mapped using BamHI and Xbal only with S acl, so lacking of these ssites in Pschi1 does not imply polymorphism between Pschi1 and Pschi4 The 668 bp Sacl-B a mHI fragment in the gPschi4 coding region was used as hybridization probe for the blots shown in figures 14 and 15

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39 blot analysis. Accumulation of its mRNA was induced by the general elicitor chitosan in both pine suspension cells and transgenic tobacco leaves as well as by mechanical wounding in transgenic tobacco plants. This suggests that at least part of the signaling mechanism by which chitosan induces gene expression is conserved in gymnosperms and angiosperms A better understanding of the structure and regulation of these chitinase genes should provide useful insights into the evolution of defense responses in plants My second goal was to identify a functional chitinase promoter and dissect the promoter for its chitosan/wound-respons i ve element(s) It has been found that most transcriptional control elements reside within the 5'-upstream region of the coding sequence in higher plants However, this seems not to be necessarily true for pine genes (Loopstra et al., 1995) Reports have indicated that t he regulatory sequences of pine genes could lie in the coding region or 3'-untranslated portion of the gene (Loopstra et al. 1995; Loopstra personal communication) Since the widely used CaMV 35S promoter and a number of other promoters from angiosperms do not promote high levels of transcription i n conifers (Ellis 1994 and refs cited therein) efforts have been focused on identifying an inducible coniferous promoter to study gene expression and regulation in conifers and on the development of strategies to introduce foreign genes into conifers The hypothesis to be tested was that the regulatory elements of a p i ne gene could be identified using assays for gene express ion in pine cells and i n angiosperm cells The cloned

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40 pine chitinase gene Pschi4 seemed to be a good candidate to test this hypothesis and to identify a functional promoter region of an inducible gene from pine. In association with these goals the Pschi4 cDNA was cloned and expressed in E.coli. Antibody was then made against the purified recombinant protein Pschi4 prote i n expression was monitored in pine suspension cells as well as in transgenic tobacco plants Also in association with these goals a series of Pschi4 promoter GUS fusion constructs were made and tested in transient assays as well as i n stably transformed tobacco plants Collectively this research represents the first studies on gene expression regulation and promoter dissect i on of a defense related gene in pine trees

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MATERIALS AND METHODS Plant Materials Pine materials. Seeds obtained by self-pollination of eastern white pine (Pinus strobus) genotype P-18 were a gift from Dr Don Riemenschneider USDA Forest Service Rhinelander WI. Seeds were surface-sterilized in a 20% solution of commercial bleach for 10 min rinsed in sterile distilled water stratified in the refrigerator for one month and then sown in commercial potting mix. After the cotyledons had expanded fully the above-ground portion of the seedlings was used as a source of DNA for Southern blots Needle t i ssue from loblolly pine (P. taeda ) genotype 7-56 was a gift from Dr Les Pearson (Westvaco Corp Summerville SC) Cell cultures of loblolly pine were der i ved from an individual seedling from family 10 -38. Cell cultures of slash pine (P. elliottii var. elliottil) genotype 52 56 were initiated and maintained according to previously described methods (Lesney 1989) The starting material for the cell cultures was provided by Greg Powell and Dr Tim White (Cooperative Forest Genetics Research Program University of Florida) Tobacco materials Tobacco plants (Nicotiana tabacum var Turkish and Nicotiana tabacum var Samsun ; gifts from Dr F Zettler and Dr C. Kao University 41

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42 of Florida respectively) were grown from seed on agar-solidified MS medium (Sigma) and maintained in aseptic cultures. These tobacco plants were used as an explant source for transformation Others. White onions were purchased from local grocery stores. Maize suspension cell line PC-5 was kindly provided by Dr. L. C. Hannah (University of Florida). These onion and maize cells as well as pine cells were used for particle bombardment. Sequencing and Sequence Analysis After the insert from the recombinant phage DNA was subcloned into the Sacl site of pBluescript sequencing reactions were carried out from both ends. The identified putative coding region, along with -750 bp of upstream sequence and -350 bp of downstream sequence, were also sequenced by using an automated DNA sequencer (ABI 373) with dye terminator chemistry (Dr Craig Echt USDA Forest Service Rhinelander, WI). DNA and protein sequence analysis was performed using the BLAST search algorithm (Altschul et al., 1990) and GCG sequence analysis software (Genetics Computer Group, Madison, WI)

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43 Plant Transformation Plasmid construction Tobacco plants were transformed with the putative chitinase gene located in gPschi4. The two Sacl fragments containing different regions of Pschi4 were subcloned separately into pBlueScript, then ligated together as BamHI-Sacl and Sacl-Xbal fragments, respectively, into pBlueScript. The BamHI-Sacl fragment contained the putative promoter region and extended -200 bp into the 5' end of the coding region, and the Sacl-Xbal fragment contained the rest of the coding region and 3' flanking sequence (Fig. 4). The entire 7 kb insert was excised from pBlueScript by digestion with Kpnl and Xbal and subcloned into the Agrobacterium binary vector pCIB10 (Rothstein et al. 1987). The resulting plasmid pWC4KX18.9 was introduced into Agrobacterium LBA4404 by the freeze thaw method (An et al. 1988) Transformation of tobacco plants Infection of tobacco (var turkish) leaf disks by Agrobacterium cocultivation and subsequent regeneration were carried out using standard methods (Rogers et al. 1986) with some modifications. A single transformed Agrobacterium colony was picked from an LB-agar plate and transferred to 3-5 ml of LB medium with appropriate antibiotics and incubated at 28C for 24 to 48 hr Cells were harvested and resuspended in 3-5 ml of sterile MS salts (Sigma M-5519 plus 3% sucrose) Two ml of the freshly suspended Agrobacterium was added to 25 ml of MS salts containing tobacco leaf disks in a sterile 50-ml tissue culture tube and incubated with shaking for one hr Leaf disks

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44 were blotted dry on autoclaved paper towels placed on MS-agar plates for 1224 hr (co-cultivation) then transferred to fresh CIM plates (Callus-Inducing-Medium : MS-agar containing 50 g/ml Timentin 100 g/ml Kanamycin 5 M BAP 0.5 M NAA). Leaf disks were transferred to fresh CIM every 4-5 days for the first 2 weeks and transferred weekly thereafter After callus was well-formed NAA was eliminated to promote shoot regeneration If shoots were generated they were transferred to MS agar without hormones for root i ng. Sixteen independent kanamycin resistant plants were regenerated and nine were selected at random to test the express i on of the transgene in response to chitosan treatment. Elicitor Treatment and RNA Extraction Treatment of transgenic tobacco leaves Chitosan (Sigma) was dissolved i n 0 1 N HCI with heating and the pH adjusted to 5 0 with NaOH prior to autoclaving (Popp 1993) Young leaves were excised from transgenic tobacco plants grown on MS agar in culture vessels (Magenta GA-7) and incubated in a culture dish (Fisher Scientific) conta i ning a solution of 50 mM KCI or 50 mM KCI plus 60 g/ml chi tosan for 24 hr in constant light before harvesting A plant that contained a single copy of the pine transgene based on 3 : 1 segregation of kanamycin resistance in its progeny was selected for further analysis and designated as Chi4 tobacco A single fully expanded leaf was div i ded into four sections One section was placed in a petri dish lacking chitosan (50 mM KCI) a second section was placed in a

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45 culture dish containing chitosan (50 mM KCI + 60 g/ml chitosan), and both were harvested after 24 hr incubation in constant light. A third section was immediately placed in liquid nitrogen and the fourth remained attached to the plant and was mechanically wounded around its margin. Wounding was performed at 0 2 and 4 hr and the leaf was collected at 7 hr. The same exper i ment was conducted on the control tobacco plant containing pCIB10 vector alone (designated as CIB10 tobacco) RNA isolation from tobacco leaves. Total RNA was extracted from all samples using prev i ously described methods for poplar leaves (Dav i s et al. 1991) with minor modifications. Briefly ground tissue was transferred to 1 volume extraction buffe r (100 mM Tris pH 8.0, 500 mM NaCl 20 mM EDTA 0.5% SOS, 0 5% ~-mercaptoethanol 0 1 % PVPP) (1 ml buffer per gram tissue) and 1 volume buffered phenol : chloroform After thoroughly mixing and incubation on ice the mixture was centrifuged The aqueous phase was re-extracted once with phenol : chloroform (1 :1), and RNA was precipitated by adding 1/5 volume of cold 10 M LiCI and incubating on ice overnight (at least 12 hrs). The pellet was resuspended in 400 I of RNase-free water and re-extracted with phenol : chloroform in a microfuge tube The RNA in the aqueous phase was precipitated by addition of ethanol. T reatment of pine suspension cells Pine cultures (both slash pine 52-56 and loblolly p i ne 10 38) were maintained on a 7 day subculture interval. Two days after transfer sterile chitosan was added to the flasks to a final concentration of 60 g /ml.

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46 After 24 hr of incubation cells were harvested by suction filtration and stored i n liquid nitrogen Adjacent flasks that received no chitosan were des i gnated as controls A time course study was performed on slash pine 52-56 suspension cells One day after normal transfer eight flasks of suspension cells were combined and then re-distributed into 8 flasks to generate a uniform population of cells After another day, sterile chitosan was added to the flasks to a final concentration of 60 g/ml and incubated for various periods of time (0 0.5 1 5 3 5 8 12 24 hr) At each time point one flask of cells was harvested by suction filtration and stored in liquid nitrogen RNA isolation from pine cells Total RNA was extracted from pine suspension cells using previously described methods (Schneiderbauer et al. 1991) with some modifications. Pine cells were ground to a fine powder in liquid nitrogen in a mortar and 20 ml of cold acetone was added directly to the mortar to inactivate RNase activity and extract phenolic compounds which are abundant in pine cells The mixture was transferred to a 50-ml falcon tube and centrifuged. The pellet was saved and washed again with cold acetone until the solution became clear instead of green The pellet was then dissolved in 12 ml of TNE buffer (100 mM Tris-Cl 10 mM NaCl 10 mM EDTA pH 8 0 add 0.1% Triton X-100 and 15 mM OTT just before use) and extracted with phenol/chloroform The supernatant was prec i pitated by add ing 1/5 of 10 M LiCI and i ncubating overnight on ice Following centrifugation

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47 RNA pellet was re-dissolved in water extracted with phenol/chloroform twice, and precipitated by addition of ethanol. Primer Extension A synthetic oligonucleotide primer (5'-GCCAA TAGCAACCTCATCGACATC ATTC-3') complementary to positions 793 to 820 (Fig. 5 underlined and italicized) of Pschi4 was end-labeled by T4 polynucleotide kinase and y -32P-ATP Poly-A + RNA isolated from transgenic tobacco plants containing or lacking Pschi4 was hybridized with the end-labeled primer and extended with M-MLV reverse transcriptase Equal quantities of radioactivity were loaded in each lane and the products were analyzed on a 9% polyacrylamide sequencing gel. A sequencing reaction (Sanger et al., 1977) of the genomic clone was performed using the same end-labeled primer and run on the same gel. Southern and Northern Analysis Genomic DNAs from white pine P-18 loblolly pine 7-56 and slash pine 52-56 were digested with restriction enzymes, fractionated by agarose gel electrophoresis and transferred to Hybond N+ nylon membrane (Amersham) in a vacuum blotter (Hoefer) with 0.2 N NaOH as the transfer solution The Southern membrane was hybridized with a 729 bp Pschi4 cDNA fragment (see below for the cloning of Pschi4

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48 701 GAAATACTGCTATAAAACGAGGGGTTTGCAGCCTGGGATCATCACCACAATTTGCGTIGGCAGCCTAAAGATGGCGTACACGAATATGAAGAGAATGATG M A Y T N M K R M M 801 TCGATGAGGTTGCTATTGGCCCTCACCGCAGTGGCGATAATGAGTTCTTI'GTGTTGTTATGT'ITCTGCACAACAAGGAGTCGCATCCATCATAAGTGAAG S M R L L L A L T A V A I M S S L C C Y V S A Q Q G V A S I I S E D 901 ATGTTTTCCATCAATTTTTGAAGCACAGAAACGATGACGCGTGCTCGGCGAAAGGCTTCTACACCTACAGCGCCTTCATTGCGGCAGCTAATAGTTTCCC V F H Q F L K H R N D D A C S A K G F Y T Y S A F I A A A N S F P 1001 A G ACTTCGGCAACATCGGCGATCAAG ATAGTCGCAAG A G A G AGCTCGCAGCTTTCTTI'GGTCACACGTCGCAGGA G ACCACAGgtattatta a tttataa D F G N I G D Q D S R K R E L A A F F G H T S Q E T T G 1101 gcttcctctaactcttctgcctccctgccatgccttaaatgttattaatcggattaggatgtatgggtttttacagGCGGGTGGCCAACGGCCCCAGACG G W P T A P D G 1201 GTCCATATGCGTGGGGTTACTGCTTCAAAGATCAGGTGAATAGCACAGACAGATACCGCGGACGAGGACCTATTCAGCTAACCGGgtaggttttgttaat P Y A W G Y C F K D Q V N S T D R Y R G R G P I Q L T G 1301 ccgcttcgatttctagcaatagatatggaaaaaatcgaatgaatttcaagcctaatacacttaccgctctgtgggagcagGGACTACAACTACAAAGCTG D Y N Y K A A 1401 CGGGGAATGCGTTAGGTTACGATCTCATAAACAATCCGGATCTCGTGGCGACCGATGCCACGGTGTCGTTTAAGACGGCGGTTTGGTTCTGGATGACGGC G N A L G Y D L I N N P D L V A T D A T V S F K T A V W F W M T A 1501 GCAGTCTCCGAAGCCTTCGTGCCACGACGTGATTTTGGGAAGATTGACTCCGTCAGTTACCGA T ACCGCTGCTGGCAGAGTGGCGGGATATGGAATGTTG Q S P K P S C H D V I L G R L T P S V T D T A A G R V A G Y G M L 1601 ACGGACATCATAAACGGTGGGCCGGAATGCGGCACAGGCACAATAAGCGACGTGCAGCAGGGGCGCATCGGGTTCTACCAGAGATACTGTAAGATGCTGG T D I I N G G P E C G T G T I S D V Q Q G R I G F Y Q R Y C K M L G 1701 GCGTGGACGTGGGATCCAACCTCGACTACAAAAACCAGAAGCCTTACGGAACTTAATGTCTACGCTACCAACCCATCCAATCGACTACTACTGTTATGGT V D V G S N L D Y K N Q K P Y G T Figure 5 Sites within Pschi4 that were used to design oligonucleotide primers for this study. The primer used for primer extension is complementary to the underlined, italicized region. The primer sites used for RT PCR to clone the Pschi4 cDNA are shown in underlined boldface. A primer complementary to the double-underlined sequence was used for cloning of the 5'-upstream sequence of Pschi4.

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49 cDNA) Total RNA samples were quantified spectrophotometrically fractionated on formaldehyde agarose gels (Sambrook et al., 1989) and vacuum-blotted to Hybond N+ membrane with 50 mM NaOH as the transfer solut i on Equal loading was confirmed by ethidium bromide staining of the RNA prior to blott ing. The northern blot was hybridized with a 668 bp Sacl-BamHI fragment that is part of the coding region of Pschi4 (Fig. 1). Hybridization and high stringency (65 C ; 1 mM EDTA 1% SOS, 40 mM NaHPO4 buffer pH 7 2) washing were performed using reagents and conditions that were previously described (Church and Gilbert 1984 ) Cloning of the Pschi4 cDNA RNA template isolation Leaves of the Chi4 tobacco plant were incubated with 50 mM KCI + 60 g/ml chitosan for 24 hr. Total RNA was isolated as previously described (Davis et al. 1991) RNA concentration was determined by both spectrophotometry and confirmed by ethidium bromide staining after electrophoresis through an agarose gel. Reverse transcript i on (RT) PCR. One g of total RNA from chitosan treated leaves of the Chi4 tobacco plant was reverse transcribed as follows Total RNA was heated to 65 C for 10 min in the presence of 1 g of oligo dT and 2 I of 5 x AMV RT buffer. The sample was then placed on ice A m i xture of 80 uni ts RNas i n (RNase inh i bitor ; Promega) 2 I 10 mM dNTPs and 19 units AMV r everse

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50 transcriptase was added to yield a final volume of 10 I. After 40 min incubation at 42C, the RT reaction was heated to 98C for 8 min to denature the cDNA-RNA hybrids briefly centrifuged and chilled on ice This fresh RT-cDNA was used as template in a PCR without further purification Primers (Forward : 5'TCTGCACAAC AAGGAGTCGCATCC-3' and Reverse : 5'-GA TTGGATGGGTTGGT AAGCGTAG-3') corresponding to nucleotide 864-887 and 1760-1782 in Fig. 5 (underlined and in boldface) were synthesized by Craig Echt (USDA Forest Service Rhinelander WI) RT-cDNA template was mixed with a solution containing primers (1 g each) 10 X PCR buffer MgCl2 dNTPs to a final volume of 49 I. The mixture was denatured at 94 C for 2 min and quickly cooled on ice After a brief spin 1 I of Taq polymerase (5 units) was added The mixture was overlaid with one drop of mineral oil prior to initiation of cycling The PCR reaction was carried out as follows in a Coy themocycler (model 50): 94C 20 sec ; 55C 20 sec ; 72C 1 min 20 sec 30 cycles with an additional extension of 5 min at 72C before cooling down to 4C Cloning of RT-PCR product. The initial RT-PCR product was polished by adding 10 u of T4 DNA polymerase directly and incubating at 37 C for 2 hr. The product was electrophoresed in a 0 8% TAE agarose gel (Sambrook et al., 1989) The DNA fragment of the expected size was excised from the gel and purified by QIAEX II (Qiagen Inc., CA) according to the manufacturer's instructions The purified DNA was ligated with Smal linearized pUC 19 and transformed into E. coli

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51 TB 1 cells Pos i t i ve clones were identified by restriction enzyme digestion and further confirmed by DNA sequencing. cDNA Express i on and Generation of Antibody Pschi4 cDNA expression in E. coli The cloned cDNA was then subcloned into the BamHI-EcoRI sites of expression vector pET24d (Novagen Madison, WI) Expression was performed in bacterial host BL21 (DE3) plysS cells according to the manufacturer s protocol. Briefly a single colony was inoculated in 3 ml of LB containing kanamycin (50 g/ml) and chloramphenicol (34 g/ml) and grown at 37C until the 00600 reached 0.6 to 1.0 Cells were collected and resuspended in fresh medium conta i n ing antibiotics and grown at 37C until the 00600 reached 0 6 At this point IPTG was added to a final concentration of 1 mM to induce target gene expression by further incubation for 3 hr Cells were then harvested and resuspended in buffer A (20 mM Tris -Cl, pH 7 5 20% sucrose 1 mM EDTA) After centrifugation cells were stored at 80 C overnight. The recombinant protein was expressed as an inclus i on body in E.coli To purify inclusion bodies from other cellular proteins frozen cells were resuspended in PBS (1 L of PBS contains 8 g NaCl 0 2 g KCI, 1.44 g Na2HPO4 0 24 g KH2PO4 ) and then sequentially incubated with lysozyme and 1 % T r i ton to lyse cells and DNase I to degrade DNA. After centr i fugation the pellet was washed with PBS plus 1 % Triton and then suspended in water

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52 Protein purification Overexpressed Pschi4 protein present in the inclusion body was purified by using the electro-eluter method (Bio-Rad laboratories Richmond CA). Briefly, the resuspended insoluble pellet was mixed with SOS loading dye boiled for 5 min and resolved by SOS-polyacrylamide gel electrophoresis. After separation, the gel was stained with Coomassie blue in the absence of acetic acid The expected protein band was cut out and loaded onto the electro-eluter (Bio-Rad, Model 422) according to the manufacturer's instructions. The purity of protein was examined on a 12% SOS-PAGE gel and further confirmed by sequencing the N-terminal 26 amino acids (ICBR protein core laboratory University of Florida) Rabbit anti-Pschi4 antiserum. Antiserum against r-Pschi4 protein was prepared by Cocalico Biologicals Inc (Reamstown, PA) One hundred g of purified recombinant Pschi4 proteins were mixed with complete Freund's adjuvant to inject each of two New Zealand white rabbits. Four booster injections (each time with 50 g antigen and incomplete Freund's adjuvant) were administered at biweekly intervals to obtain a high titer antiserum. Protein Isolation and Western Blotting Analysis Protein Isolation from Pine and Tobacco Suspension Cells Pine suspension cultures were established and maintained as described previously (Lesney 1989). Tobacco suspension cultures were initiated from Chi4

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53 and CIB10 tobacco leaves. Specifically, tobacco leaf disks (diameter ca. 4 mm) were induced to form callus on MS-agar plates containing the hormone 2 4-D prior to initiation of suspension cultures Pine and transgenic tobacco suspension cells were maintained in LM (Verma et al., 1982) and M1 (for 1 L : 4.4 g Sigma's M-5524 30 g sucrose, 1 mg thiamine 1 mg pyridoxine 1 mg pantothenic acid, 0 .01 mg biotin, 1 mg nicotinic acid 1 mg L-cysteine, 0.2 g L-glutamine 100 mg inositol 10 M NAA, 5 M 2-iP) medium respectively They were transferred to fresh media at seven day intervals. Two days after transfer chitosan was added to a 250-ml culture flask containing 50 ml of medium to a final concentration of 60 g/ml. After 24 hr of incubation cells were harvested by suction filtration Both cells and supernatant were saved Cells were ground in 20 mM of NaOAc (pH 5.2) and centrifuged Proteins were concentrated by dialyzing against solid sucrose and the concentrations were determined by using Sigma's bicinchoninic acid protein assay kit according to the manufacturer's instructions. Adjacent flasks that received no chitosan were designated as controls. In a separate dose-effect experiment, pine cells were combined one day after normal transfer to form a uniform population, and 0 6 ml of cells was incubated in 1 ml 2 ml or 8 ml of LM medium with chitosan at different concentrations (0, 20 30 40, 60, 90, 120 180 240 g/ml) for 23 hr. Media which contain extracellular proteins were then collected Proteins were quantified and dot-blotted onto a nitrocellulose membrane.

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54 Protein Isolation from Pollen of Pine and Tobacco Pollen from pine trees was collected in late January and early February. Pollen from transgenic tobacco plants (Chi4 and CIB10 tobacco F1 ) was collected fresh from flowers on plants Pollen was ground with a micropestle in solubilizing buffer (22.5% ~-mercaptoethanol, 9% SOS 22.5% glycerol, 0 125 M Tris-Cl pH 6 8) in microcentrifuge tubes, frozen in liquid nitrogen, boiled in 100C water and ground again. This cycle was repeated for at least three times to release proteins from pollen grains The mixture was centrifuged and the supernatant was saved Western Blotting Analysis Equal amounts of protein were loaded onto a 12% SOS-PAGE gel and electro-transferred to a nitrocellulose membrane by using a Genie electrophoretic blotter (Idea Scientific Co., Minnesota) according to the manufacturer s instructions The membranes were probed to anti-Pschi4 antibody by standard western blot procedures. Specifically the membrane was incubated in blocking reagent (TBST + 5% dried milk; TBST = 20 mM Tris-Cl, pH 7.5 150 mM NaCl 0 5% Tween 20) for 30 min and then incubated with primary antibody (1: 50 000) for 6-12 hr After three washes with TBST, the membrane was incubated with goat anti -r abbit AP (alkaline phosphatase)-conjugated antibody (1 : 5000 dilution) for 30 min The membrane was washed three times with TBST and once with TBS (same as TBST except TBS lacks Tween 20) Color was developed in 25 ml of solution containing

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55 2.5 ml Tris-Cl, pH 9 .5, 500 I of 5 M NaCl, 125 I of 1 M MgCl2 165 I NBT (100 mg dissolved in 2 ml of 70% DMF) and 65 I BCIP (100 mg dissolved in 2 ml of 100% DMF). Preimmune serum was used as a control (1 :50,000 dilution) Particle Bombardment and Transient Expression Plasmid Construction The 5'-flanking region of Pschi4 was amplified from pWC4Sac6, which contained a 6 kb Sacl fragment of gPschi4 (Fig 4 and Fig. 6) by PCR using a pair of primers (Forward : Universal primer F; Reverse: JOD5 = 5'-GCCAACGCAAA TTG TGGTGATGATCCC-3' complementary to the positions 735 to 761 in Fig. 5, double underlined) After T4 DNA polymerase treatment (to make blunt ends), the PCR fragment was digested with BamHI, purified and cloned into pBlueScript BamHI EcoRV sites The plasmid was then digested with C/al, filled by Kienow Fragment of E. coli DNA polymerase I digested with Xbal and subcloned into pBl101 1 (Clontech) or pGUS (Hindlll-EcoRI fragment of pBl101.1 subcloned in pUC19) Xbal Smal sites (Fig. 6) The resulting constructs were named pWP16.7 and pWP GUS9.3 (white pine promoter-GUS, the number denotes the total size of plasmid DNA) respectively. Plasmid DNA of pWP-GUS9.3 was digested with Pstl and Sa/I and treated with exonuclease Ill at 37C for various periods of time (25 50 75, 100 125 sec).

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<3 I
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57 At each time point reactions were stopped by adding an equal volume of 2 x exo Ill stop buffer (0 3 N NaCl, 7 5 mM EDTA) and incubated at 70C. Following treatment with Kienow Fragment, the DNAs were self-ligated and transformed into E. coli strain TB1 cells. This created a nested series of Pschi4 putative promoter GUS constructs in pUC19 (Fig. 6) with sizes of 1 .2, 0.8, 0.6 0.5, 0.4 and 0.2 kb These constructs, including pWP-GUS9.3, were tested by particle bombardment in transient expression assays. These constructs were subcloned into the p8I101 3 Hindlll site, except pWP-GUS9 3 which was subcloned differently (see above paragraph) for stable transformation of tobacco To normalize for transformation efficiency in the transient assay, a Ubiquitin Luciferase construct (kindly provided by Dr. C Kao in Dr. Don McCarty's laboratory) was included in each bombardment mixture. Thus expression data are expressed as GUS/Ubi-LUC ratios Tissue Preparation White onions were purchased from local grocery stores. The inner epidermis of an outer layer of onion were peeled using a pair of fine forceps and placed on the center of MS-agar plates which were to be used for particle bombardment. Slash pine suspension cultures were maintained as described previously (Lesney, 1989) and transferred to fresh medium at 7 day intervals. Three days after transfer, cells from several flasks were pooled to create an experimental population. Approximately one ml of cells was placed on the filter paper on MS-agar plate and

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58 dried for 3-10 min. This procedure varied from plate to plate and needed to be adjusted empirically If cells were too wet, gold particles did not penetrate the aqueous layer which reduced the transformation efficiency ; if cells were too dry their viability was reduced. Particle Bombardment Particle bombardment was performed essentially as described previously (Taylor and Vasil, 1991) using a DuPont PDS-100 particle gun Briefly 37 I of a 40 mg/ml gold stock solution was mixed with 5 g of internal control plasmid DNA (Ubi-LUC) and 5 g of testing DNA (WP-GUS) in a total volume of 72 I in a 1 5-ml microcentrifuge tube and vortexed briefly Twenty I of 100 mM of free base spermidine and 50 I of 2.5 M CaCl2 were placed in separate drops on the side of the tube to avoid pre-mixing of either solution with the gold/DNA solution The tube was then mixed immediately by vortexing for 20 sec which allowed plasmid DNAs to attach to the gold particle The tube was centrifuged for 5 sec and supernatant was removed Two hundred (200) I of 100% ethanol was added and sonicated briefly After centrif u gation and removal of supernatant 60 I of 100% ethanol was added. The tube was p l aced on ice until all samples were ready for bombardment. Four I of gold/DNA solution (sonicated again just before use) was used for each individual bombardment shot. Each exper i ment represented 3-6 replicates

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59 Incubation and Extraction of Proteins Following bombardment, petri dish plates were incubated at room temperature i n constant light for 24 hr. Bombarded onion or pine cells were ground with mortar and pestle aided by addition of glass beads in 200-800 I of GUS/LUC extraction buffer (0. 1 M potassium phosphate (pH 7 .8), 2 mM EDTA (pH 8 .0), 2 mM OTT 5% glycerol) The homogenates were centrifuged and supernatants were transferred to clean tubes. Quantification of Transient Expression Quantitative measurement of GUS activities was performed essentially as described by Jefferson et al. (1987) except that the substrate MUG was dissolved in the extraction buffer described above For the luciferase assay an automated luminometer (Autolumat model #LB953, Wallac Inc. MA) and Promega's Luciferase Assay K i t were used Briefly 10 I aliquot of each extract was placed in each disposable culture tube (Fisher brand cat.# 14-961-26) and all tubes were loaded into the luminometer. The instrument automatically injects 100 I of substrate luciferin dissolved in its assay buffer (prepared according to Promega s instruction manual) counts the emitted photons for 60 sec and moves to the next sample The unit of measurement is the Relative Light Unit (RLU)

PAGE 72

60 Analysis of Promoter Deletion Constructs in Stably Transformed Tobacco Deletion constructs of the Pschi4 promoter were made and subcloned into binary vector pBl101 as described above. These constructs were introduced into tobacco (var Samsun) using Agrobacterium-mediated transformation as described above. Primary transgenic plants were initially used for chitosan and wounding assays In order to minimize the large variation in transgene expression and inducibility plants were allowed to flower and set seed. The seeds were placed on MS-agar plates containing kanamycin (50 g/ml) to determine transgene copy number, and to generate a uniform population of plants for use in GUS assays. Chitosan and wounding treatments were described previous l y (see above). Histochemical Assays in Transgenic Tobacco Histochemical GUS assays were performed essentially as described previously (Jefferson et al. 1987). Tissue sections were floated in X-gluc solution (0.5 mg/ml X-gluc in 50 mM of NaHPO4 pH 7 .0) for 16 to 24 hr at 37 C Tissues were then fixed in 5% formaldehyde 5% acetic acid 20% ethanol and washed in 80% ethanol. Tissue sections from transgenic tobacco containing the full-length promoter GUS (pWP16 7) were analyzed first. Untreated leaves stems and reproductive organs (corolla stigma ovary pollen) from pWP16.7 tobacco plants

PAGE 73

61 were used. For other transgenic tobacco plants containing shorter promoter-GUS constructs only pollen grains were stained in X-gluc and examined microscopically

PAGE 74

RESULTS Pschi4 Gene Structure Nucleotide sequence analysis of Pschi4 revealed a complete coding sequence. In contrast, Pschi1 contained a premature stop codon in the first exon due to a T residue at nucleotide 1071 (Fig. 7). Pschi4 and Pschi1 are 90% identical through the putative coding region (not including intrans) and 83% identical through the 5'-flanking sequence In Pschi4 a putative TATA box is located at nucleotide 711 (Fig. 7). Primer extension analysis using RNA from transgenic tobacco revealed two major bands in cells containing Pschi4 transcripts that were not detected in cells lacking Pschi4 transcripts (Fig. 8). The nucleotide G at position 719 (Fig. 7) was considered to be the major putative transcription start site because it was a longer and more abundant product. Primer extension products were not seen when slash pine mRNA was used as template (data not shown) probably due to poor annealing of the primer which was designed from the white pine (P. strobus) sequence. Pschi4 contains several possible translation initiation sites The ATG at nucleotide 771 (Fig 7) was considered to be the likely translation start site because it is the first ATG downstream of the putative start of transcription and it is flanked 62

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63 -140 -131 501 CAGAAGTCAAGATTATGAAGAACAGGAGAGGAGCAGGTATAATGGCCATCGAAATCAAGGACCCACGTTACGCAGGAATTACCTTCTACCTTCGCAGAAA 601 TATCGCTAGGAATGGTGGGGCTTGTAGGTTCGACCACAAACACACTATATTCCACGGAGGGGTAGAAAGTTTCACCACCACACGTTATCTCAGTGCTGCG +l 701 GAAATACTGC'.IAIAAAAC~AGGGGTTTGCAGCCTGGGATCAT~ACCACAATTTGCGTTGGCAGCCTAAAGATGGCGTACACGAATATGAAGAGAATGATG M A Y T N M K R M M 801 TCGATGAGGTTGCTATTGGCCCTCACCGCAGTGGCGATAATGAGTTCTTTGTGTTGTTATGTTTCTGCACAACAAGGAGTCGCATCCATCATAAGTGAAG S M R L L L A L T A V A I M S S L C C Y V S A Q Q G V A S I I S E D 901 ATGTTTTCCATCAATTTTTGAAGCACAGAAACGATGACGCGTGCTCGGCGAAAGGCTTCTACACCTACAGCGCCTTCATTGCGGCAGCTAATAGTTTCCC V F H Q F L K H R N D D A C S A K G F Y T Y S A F I A A A N S F P 1001 AGACTTCGGCAACATCGGCGATCAAGATAGTCGCAAGAGAGAGCTCGCAGCTTTCTTTGGTCACACGTCGCAGGAGACCACAGgtattattaatttataa D F G N I G D Q D S R K R E L A A F F G H T S Q E T T G 1101 gcttcctctaactcttctgcctccctgccatgccttaaatgttattaatcggattaggatgtatgggtttttacagGCGGGTGGCCAACGGCCCCAGACG G W P T A P D G 1201 GTCCATATGCGTGGGGTTACTGCTTCAAAGATCAGGTGAATAGCACAGACAGATACCGCGGACGAGGACCTATTCAGCTAACCGGgtaggttttgttaat P Y A W G Y C F K D Q V N S T D R Y R G R G P I Q L T G 1301 ccgcttcgatttctagcaatagatatggaaaaaatcgaatgaatttcaagcctaatacacttaccgctctgtgggagcagGGACTACAACTACAAAGCTG D Y N Y K A A 1401 CGGGGAATGCGTTAGGTTACGATCTCATAAACAATCCGGATCTCGTGGCGACCGATGCCACGGTGTCGTTTAAGACGG CGGTTTGGTTCTGGAT GACGGC G N A L G Y D L I N N P D L V A T D A T V S F K T A V W F W M T A 1501 GCAGTCTCCGAAGCCTTCGTGCCACGACGTGATTTTGGGAAGATTGACTCCGTCAGTTACCGATACCGCTGCTGGCAGAGTGGCGGGATATGGAATGTTG Q S P K P S C H D V I L G R L T P S V T D T A A G R V A G Y G M L 1601 ACGGACATCATAAACGGTGGGCCGGAATGCGGCACAGGCACAATAAGCGACGTGCAGCAGGGGCGCATCGGGTTCTACCAGAGATACTGTAAGATGCTGG T D I I N G G P E C G T G T I S D V Q Q G R I G F Y Q R Y C K M L G 1701 GCGTGGACGTGGGATCCAACCTCGACTACAAAAACCAGAAGCCTTACGGAACTTAATGTCTACGCTACCAACCCATCCAATCGACTACTACTGTTATGGT V D V G S N L D Y K N Q K P Y G T 1801 CAGCACATAGTCTMTAMTAAATAAATAAAATGAGAATTGCGATAAGTGGTGAGCTTCACTCAGTGGATGGGCTCCCCTCCTAGAAATAGAAAGGGTAA 1901 GCATGGTAGATTAATTATTCATACCTGTACTGTCACTCGTGTTTTTTCACMTAMGAAGAAATAACGCGTATCCACCTTGGCAAAGGTAGCCGAATATT 2001 TCTAAATATTTTCCGCAAATGTGGAAAGTCTGGCGTTTCTTCATATCACTCGCAGGATATATGACTAAATTTGAGCAAMTAMATAAATGT 2092 Figure 7 Partial nucleotide sequence and translation product encoded by the genomic clone containing Pschi4. The putative TATA box is double underlined, as is the putative transcription start site. The likely signal peptidase cleavage site is indicated (") The annealing sites for the degenerate oligonucleotide primers are shown in italicized boldface, and the position of the T in Pschi1 that results in a stop codon is overlined. lntrons are shown in lower cases. A potential glycosylation site is shaded Potential polyadenylation signals are underlined. The TCA -like cis element is underlined and in boldface. The complete sequence of Pschi1 and Pschi4 that were deposited in the Genbank database are shown in the appendix.

PAGE 76

T A A A A C G A G G G G T T T G C A G C C T G G G A T C A T C A CTAG 13 Q + .... 0 t5 Q) > .... 0 t5 Q) > Figure 8 Primer extension analysis to reveal the putative transcription start site(s) A primer complementary to positions 793 to 820 (Fig. 5) was end labeled hybridized to poly A + RNA from transgenic tobacco containing Pschi4 or pCIB10 vector alone and extended by M-MLV reverse transcriptase A sequencingg reaction using the same end labeled primer was run by side on a 9% polyacrylamide gel. The major products are indicated by arrows and the nucleotides at which transcription is initiated are marked with an asterisk. 64

PAGE 77

65 by sequences that are the most similar to the consensus sequence for translation initiation in eukaryotes (Kozak, 1991 ) In the 3' flanking region there are seven potential polyadenylation signals (AATAAA}. A sequence was found between -140 and -131 (Fig. 7, underlined boldface) that is similar in composition (TTACCTTCTA, identities underlined) and relative location to a cis element implicated in wound and elicitor responsiveness of proteinase inhibitor genes (GTACCTTGCC; Palm et al. 1990 ; Balandin et al., 1995). This sequence (ITACCTTCTA, identities underlined) is also similar to the 10-bp TCA motif present in more than 30 pathogen-inducible promoters (TCA motif consensus= TCATCTTCTT; Goldsbrough et al. 1993). The chitinase coding region is divided into three exons of 313, 109 and 373 bp by two small introns of 93 and 96 bp (Fig 1 and 7). The presence of introns was first inferred by AT-richness (64.5% and 61.5% AT, respectively), orthodox sequences at the putative splicing sites (GIGT . AGIG) and conserved location in other plant chitinase genes that possess introns This was then confirmed by cloning of the corresponding cDNA (see below) The deduced protein contains an N-terminal 33 amino acids having the structure expected for a signal peptide with several positively charged amino acids near the N-terminus and an internal hydrophobic region (Chrispeels, 1991 ). The predicted mature protein has a molecular mass of 25.3 kD, assuming removal of the putative signal peptide The Pschi4 protein is expected to be acidic, with a predicted isoelectric point of 6.1.

PAGE 78

66 The amino acid sequence of translated Pschi4 was aligned with tobacco class I (Shinshi et al., 1990) and class II chitinases (Payne et al. 1990) The catalytic domain of translated Pschi4 shares 64% and 62% amino acid sequence identity with the catalytic domains of tobacco class I and II ch i tinases respectively (Fig. 9 and 10) Translated Pschi4 lacked the C-terminal sequence (GLLVDTM) (Fig. 10) which is sufficient to target tobacco class I chitinase to the vacuole (Neuhaus et al., 1991) Genomic Southern blots were used to investigate the number of Pschi4 genes in pine and to assess their presence in different species of pines (Fig 11 ) All three species showed from two to four restriction fragments that accounted for most of the hybridization to the Pschi4 cDNA probe The slash and loblolly pine patterns were more similar to one another than either was to wh i te pine A 2 5 kb Sacl fragment and a 5.5 kb BamHI fragment were predicted to be present in white pine (Fig 1 ) and were in fact observed (Fig. 11 ) Pschi4 cDNA Cloning and Expression in Bacteria RT-PCR technique was used to clone the Pschi4 cDNA. A 729-bp fragment was pred i cted based on the sequence analysis (Fig 5 between the two primers underl i ned boldface) assuming the two putative intrans were spliced ; otherwise i t would be 917 bp. The 729-bp fragment was in fact amplified by RT-PCR from the RNA isolated from Chi4 tobacco (Fig 12) As a control plasmid DNA containing the

PAGE 79

1% S% Pschi4 -----------------~ 100 100 TobE -62 79 1 TobV -' I I f{:I 64 81 Figure 9. Domain structure of the putative Pschi4 protein from pine with class I and II chitinase from tobacco. Class I chitinase from tobacco is vacuolar (TobV; Shinshi et al., 1990) whereas class II chitinase from tobacco is extracellular (TobE ; Payne et al., 1990) Percent identity (1%) and similarity (S%) values were calculated by comparing the amino acid sequence in the catalytic domains (see Fig 10 for detailed sequence alignment) From the left, the domains in TobV are: signal peptide, cysteine-rich, hinge, catalytic, and vacuolar targeting Pschi4 and TobE lack the cysteine-rich hinge and vacuolar targeting regions. The catalytic domain of TobV also contains a short stretch of amino acids not found in Pschi4 or TobE 67

PAGE 80

68 1 50 Pschi4 ... MAYTNMK RMMSMRLLLA LTAVAIMS .. . . . SLC CYV ....... Tob2 .......... ..... M E FS GS ........ . . PMA-F -C-FFL F ... T obl MRL CKF-ALS S LLFSL---S AS-EQCG-QA GGARCPSG---SKFGWCGNT 51 100 Pschi4 . . . . . .......... .... SAQQGV ASIISEDVFH QFLKHRNDDA Tob2 . . . . . .......... .LTG-LA--I G--VTS-L-N EM--N---GR Tobl NDYCGPGNCQ SQCPGGPTPT PPTPPGGGDL G----SSM-D -M------N-101 150 Pschi4 CSAKGFYTYS AFIAAANSFP D FGNIGDQDS RKRELAAFFG HTSQETTGGW Tob2 -P-N-----D ----------G--TT--DTA -RK-I-----Q--H-----S Tobl -QG----S-N ---N--R---G--TS--TTA ----I----A Q--H------151 200 Pschi4 PTGPDGPYAW GYCFKDQVNS TD . ..... ....... RYR GRGPIQLTGD Tob2 LSA .. E-FTG ----VR-NDQ s-........ ....... --Y --------NR Tobl A-A----------WLREQG-PGD YCTPSGQ WPCAPGRK-F ------ISHN 201 250 Pschi4 YNYKAAGNAL GYDLINNPDL VATDATVSFK TAVWFWMTAQ SPKPSCHDVI Tob2 N--EK--T I -QEV ---------I---I----P -DN---8 ----Tobl ---GPC-R-I -V--L--------PVI---S-L-----P-----------251 300 Pschi4 LGRLTPSVTD TAAGRVAGYG MLTDIINGGP ECGTGTISDV QQGRIGFYQR Tob2 I--W---AA-Q--N--P---VI-N-----I ----RNDA-.ED---Y-R-Tobl I --WQ-AGR--N LP F -VIN---L ---R--D R .-D-----R -301 330 Pschi4 YCKMLGVDVG SNLDYKNQKP YGT ....... Tob2 --G--N-AP-E---CY--RN F -QG ..... Tobl --SI---SP-D---CG--RS F-NGLLVDTM F i gur e 10 S e q ue nce alignm ent of Psch i 4 with tobac c o chi tinases The ded u ce d amino acid sequence of Pschi4 was a lign ed with tobacco class I and class II c h itina se sequen c es by using the GAP progr am of t h e GCG p a c k age I den t i cal a mino acids ar e indica ted by hyphens. Do t s r e pres ent g aps i n trodu ce d to o p timize se q uence al i gnm ent.

PAGE 81

12 0 -8 .0-7 .0-6 .0-5.0-4 0 -3 .02.0 Sac I BamH I WP SP LP WP SP LP Figure 1 1 Genomic Sothern blot analysis of DNA from three pine species Fifteen g of genomic DNA from white pine (WP) loblolly pine (LP) and slash pine (SP) were digested with the restr i ction enzymes Sacl or BamHI fractionated blotted hybridized with 32P labeled Pschi4 cDNA probe and washed at high stringency. The predicted 2 5 kb Sacl and 5 5 kb BamHI fragments from white pine are indicated by arrows 69

PAGE 82

.... Q) "O "O m <{ Cl:'.'. z 0 (.) ..c a.. I I..... Cl:'.'. Cl:'.'. (.) a.. _/917bp 729 bp Figure 12. Cloning of Pschi4 cDNA by RT-PCR. One g of oligo dT was annealed to 1 g of total RNA isolated from chitosan-treated Chi4 tobacco The first strand cDNA was synthesized by AMV reverse transcriptase which was used as the template for subsequent PCR with a pair of primers spanning the putative coding region (Fig. 5 underlined boldface). A DNA template of the Pschi4 genomic clone was also used for PCR as a control. 70

PAGE 83

71 Pschi4 genomic subclone was used as template for conventional PCR with the same pair of primers. As expected, a 917-bp fragment was amplified (Fig. 12) The amplified 729-bp fragment was cloned and its nucleotide sequence was determined. Sequence analys i s showed that the cloned fragment was identical to the orig i nal genomic subclone except that the predicted two introns were correctly spliced (see Fig. 5). Therefore the 729-bp fragment was considered to be Pschi4 cDNA. The cDNA was subcloned into expression vector pET24d and overexpressed in E.coli. As shown in Fig 13A more than 90% of the bacteria l protein was the target protein in the presence of IPTG while th i s protein band was weak or not detected in the absence of IPTG (Fig. 13A control lane 1 and 2). The overexpressed protein was further purified to higher homogeneity (Fig 138). Based on the predicted amino acid sequence the recombinant Pschi4 (r-Pschi4) has a molecular weight of 26.9 kD and this was actually observed in the SOS-PAGE gel (Fig 13 A&B) The N terminal 26 amino acids of the purified protein were determined by the protein sequencing core laboratory (ICBR University of Florida ) to confirm that the protein was Pschi4 The result is shown in Fig 13C and it proved to be correct. Thus the purified protein was used to generate ant i -Pschi4 antibody

PAGE 84

A. B. C kD kD 31. 0 21. 5 .... Q) ::it:. .... ro E 1 2 3 4 5 purified r-Pschi4 MASMTGGQQMGRDPNSSDVPSAQQGV 72 Figure 13. Pschi4 cDNA expression in bacteria. (A) Crude extract of inclusion body from E. coli. Lane 1 control #1: bacteria were taken out of shaker before IPTG addition. Lane 2 control #2: bacteria were left in the shaker without IPTG and grown for 3 h as lane 3-5 Lane 3-5: IPTG was added to these three flasks and incubated for additional 3 h. (B) Purified recombinant Pschi4 protein from bacteria One, 4 or 8 I of proteins were loaded onto a 12% SOS-PAGE gel and stained with Commassie blue (C). The N-terminal sequence of r-Pschi4. The underlined amino acids were from Pschi4 (see Fig. 5) and others were from the vector sequence The sequence was determined by the ICBR protein sequencing core lab at University of Florida

PAGE 85

73 Pschi4 Expression Chitosan-lnduced Expression of Pschi4 at mRNA Level In the absence of chitosan, Pschi4 transcripts were present at low or undetectable levels in pine cells. In both slash and loblolly pine suspension cells transcripts related to Pschi4 accumulated after treatment with chitosan (Fig. 14) A time course study was conducted in slash pine suspension cells. Pschi4-related transcripts increased to detectable levels 3 h after chitosan treatment and remained elevated up to 24 hr (Fig. 148 and data not shown). In parallel with transcript accumulation cell browning was observed after 2.5 hr, which is an indication of lignification of cell walls (Lesney, 1989). Transcript induction was not observed at 0, 3 or 24 hr in the absence of chitosan (data not shown) The observed transcript size is approximately 0.9 kb, which is consistent with the prediction from sequence analysis (Fig 7) Pschi4 was introduced into tobacco under the control of its own regulatory sequences by Agrobacterium-mediated transformation. Sixteen independent transformants, designated as Pschi4.1 through Pschi4.16, were generated based on kanamycin selection Nine transgenic individuals were randomly selected to test transgene expression. Chitosan induced expression of Pschi4 was observed in seven of these transgenic plants (Table 1 ). Fig. 15 shows a typical result of an experiment in which the steady state level of mRNA increased in response to

PAGE 86

A. + chitosan B 0 0.5 1 5 3 5 8 12 hr Pschi4 EtBr Figure 14 Transcript accumulation in chitosan-treated pine cells. (A). Lob lolly pine 10-38 suspension cells were treated with or without chitosan for 24 h and (B) Slash pine 52-56 suspension cells were treated with chitosan for different time points as indicated before total RNA was isolated Equal amounts of RNA were subjected to denaturing gel electrophoresis vacuumblotted to a nylon membrane, hybridized with the 668 bp Sacl-BamHI fragment of Pschi4 (Fig 4) and washed at high stringency The bottom panel shows the ethidium bromide-stained gel prior to blotting 74

PAGE 87

75 Table 1. Chitosan-induced mRNA accumulation in transgenic tobacco plants Nine individual transformants of Pschi4 were randomly selected for testing chitosan induced expression by northern blot analysis Transgenic CIB 4.2 4 6 4 7 4 8 4.9 4.10 4 .11 4.12 4.14 lines 10 chitosanno yes not yes yes not yes yes yes yes induction sure sure no no induction (actually no signal was detected in CIB10 tobacco plants) yes chitosan-induction was observed not sure inducibility is not sure because of unequal loading of RNA One transgenic line (4.10), specifically designated as Chi4 tobacco, was used as a source for various studies (Fig. 8, Fig 12, Fig 15, Fig. 18 and Fig 28).

PAGE 88

vector only vector+ Pschi4 Figure 15. Northern blot showing expression of Pschi4 in a transgenic tobacco plant. A single leaf from tobacco transformed with pCIB10 alone (vector only) or tobacco containing a genomic subclone with Pschi4 (vector + Pschi4) was divided into four sections, and harvested immediately (untreated) or incubated in 50 mM KCI (KCI) or 50 mM KCI plus 60 g/ml chitosan (chitosan) or left on the plant and mechanically wounded (wounded) Approximately 10 g of total RNA was loaded in each lane. Ethidium bromide staining indicated the last lane (wounded) was underloaded 76

PAGE 89

77 chitosan treatment and mechanical wounding. This single tobacco plant (4.10 tobacco in Table 1) was used as sources for several experiments in the present study (such as primer extension, cDNA cloning, initiation of suspension culture and expression in pollen) and specifically designated as Chi4 tobacco. The pine chitinase probe did not hybridize to any mRNA transcripts in tobacco transformed with the pCIB10 vector alone (CIB10 tobacco). The probe appeared to be specific for the pine chitinase transcripts and did not hybridize to endogenous tobacco transcripts, since there is only 55% nucleotide sequence identity between Pschi4 and tobacco class II chitinase mRNA (Payne et al., 1990). Pschi4 Protein Expression Pschi4 protein expression in pine suspension cells. The expression of Pschi4 protein in pine suspension cells was examined by western blot analysis Two cell lines of pine, slash pine genotype 52-56 and loblolly pine genotype 10-38, were used. Sequence analysis predicted that Pschi4 would encode an extracellular protein with an apparent molecular weight of 25.3 kD, assuming removal of the N terminal signal sequences Fig. 16 shows that the anti-Pschi4 antibody could detect Pschi4-related proteins in the supernatant (containing extracellular proteins) of both slash and loblolly pine cells with the size slightly higher probably due to glycosylation, since a potential glycosylation site (NST) was found in the sequence (Fig. 7, shaded). Interestingly, this antibody could also recognize a protein approximately of 32 kD in the cellular fraction of loblolly pine 10-38 cell line, but not

PAGE 90

slash pine +-' +-' +-' C +-' C >, a. >, a. (.) en (.) en I I I I .c .c (.) (.) (.) (.) UF81 Pl loblolly pine +-' +-' +-' C +-' C >, a. >, a. (.) en (.) en I I I I .c .c (.) (.) (.) (.) .c (.) en a.. 0 ......., Q) N u; 9 7 4 66. 2 ~l1 40. 0 31. 0 2 1 5 97.4 66. 2 ~l1 4 0 0 3 1 0 21. 5 Figure 16 Pschi4 protein expression in pine suspension cells Slash pine 52-56 and loblolly pine 10 38 suspension cells were treated with (ch) or without (ck) chitosan. Total proteins were then isolated from media (supernatant spnt) or cells (cytosol cyt) fractionated on a 12% SOS-PAGE gel and transferred to a nitrocellulose membrane Duplicate membranes were incubated with anti Pschi4 (UF81) or preimmune (Pl) The overexpressed bacterial protein (not purified) was used as a positive control. 78

PAGE 91

79 in slash pine cells (Fig. 16). This size is consistent with other vacuolar chitinases reported (Broglie et al. 1986; Samac et al. 1990; Shinshi et al., 1987; Zhu and Lamb 1991 ). It is also of interest to note that chitosan did not show much induction at the protein level in this blot. However slash pine cells did show a chitosan response at the protein leve l in a separate study (Fig 17). Equal amounts of suspension cells (0.6 ml) were incubated in different volume of medium with various concentrations of chitosan Within the 1-ml incubation, Pschi4 related protein was low or not detected at chitosan concentrations of 0 180 and 240 g/ml ; but accumulated to detectable levels starting from 20 up to 120 g/ml chitosan (Fig 17) Cell browning from light to dark was correlated with the chitosan concentrations from 20 to 240 g/ml. In fact when chitosan was higher than 180 g/ml in the small volume (1 ml) of cells cell cultures became very dark-brown a phenomenon similar to the hypersensitive cell death observed i n soybean suspension cells exposed to high concentrations of H202 (Levine et al., 1994 ; Levine et al. 1996) In the 8-ml incubation cells became dark brown at the concentration of 60 g/ml in the solution and probably because the ratio of net chitosan over cell numbers was much higher In early studies proteins from extracellular and cellular fractions of loblolly pine suspension cells were applied to a rotato r cell an apparatus that separates proteins based on isoelectric focusing (IEF) Extracellular proteins showed higher

PAGE 92

1 ml 2ml 0 0 20 40 60 120 180 240 30 60 90 (g/ml chitosan) 1 I 4 I 120, 4 I 16 I ()\ 2ml 8 I ,1r,1 32 I 16 I : ,!I; 8 I , 60 32 I :4'\l 32 I 8ml 64 I Figure 17 Chitosan induced Pschi4 protein expression in pine suspension cells. Slash pine cells (0.6 ml of each) were incubated in 1 ml, 2 ml or 8 ml of LM medium with chitosan at the concentration (g/ml) indicated Proteins were isolated from the media quantified and adjusted to same concentration From 1 to 64 I of proteins were loaded on to a nitrocellulose membrane and incubated with the anti-Pschi4 antibody. 80

PAGE 93

81 chitinase activity in the acidic area (pH 4.6 to pH 6.4) after chitosan treatment (data not shown) Pschi4 protein expression in transgenic tobacco suspension cells Tobacco suspension culture was initiated from transgenic tobacco (Chi4 and CIB10 tobacco) leaf disks The protein level of Pschi4 in tobacco suspension cells was also examined by western blot analysis. Fig 18 shows that Pschi4 protein was present in the supernatant (extracellular) fraction of the Chi4 tobacco suspension cells but not in the cytosolic fraction. The size was approximately 25 kD indicating it may not be glycosylated as in pine In contrast nothing was detected in the CIB10 tobacco suspension cells (Fig. 18) It is worthwhile to note that no chitosan responses were observed at both the protein (Fig 18) and mRNA levels (data not shown) in tobacco suspension cells Transient Assay of Pschi4 Promoter GUS Constructs Plasm i d Construction The 5 -flanking sequence, putative promoter reg i on of Pschi4 was subcloned and fused with the reporter gene uidA from bacteria which encodes f3glucuronidase (GUS) A series of promoter deletion GUS constructs were also made avai l able by time dependent digestion by exonuclease Ill (Fig 6) These constructs were made in the vector pUC 19 for transient assays and some of them were subcloned into the

PAGE 94

CIB10 Chi4 -C -C -C -a. >, a. >, a. >, (/) u (/) u (/) u I I I I I I I I kD () () () () () () anti-Pschi4 31.0 21. 5 preimmune 31. 0 21.5 Figure 18. Western blot analysis in tobacco suspension cells. Tobacco suspension cell lines were initiated from leaves of transgenic tobacco containing the Pschi4 or pCIB10 vector Chi4 cells were treated with (CH) or without (CK) chitosan (60 g/ml) for 24 h. Total proteins were isolated from both medium (supernatant spnt) and cells (cytosol cyt). Western procedures were the same as described in Fig. 16. 82

PAGE 95

83 binary vector pBI 101 for stable transformation into tobacco. All of them were named according to the total size (kb) of the plasmid (Fig. 6). Transient Expression in Onion Epidermis Cells I was interested in identifying a functional pine promoter as well as defining chitosan-responsive cis-element(s), if such element(s) exist. Putative promoter GUS constructs were first tested by transient assays in onion cells (Fig 19 and 20). All constructs showed high promoter activity compared with the promoterless control (Fig 19A). Chitosan was included in the incubation, but did not induce higher activity. Fig. 19 represents two typical results of these experiments. Since the results from particle bombardment experiments showed large variations in onion both GUS and luciferase (LUX) activities are only comparable within a single experiment. In the experiment shown in Fig 20, more constructs were used and no significant differences in activity were seen between constructs (Fig. 20) which indicates that the smallest -200 bp promoter construct contained sufficient regulatory elements to direct activated transcription in onion cells by transient assays. Promoter Comparison in Maize and Pine Suspension Cells The commercially available CaMV 35S promoter (35S) has been demonstrated to be highly expressed in angiosperms however, there are conflicting data regarding the efficiency of the 35S promoter in gymnosperms (Ellis 1994)

PAGE 96

A. B. 6000 0 ,g :34500 0 ><~ 3 a.3000 0 '? en o 0 ..-1500 0 0 0 D no chitosan I + chitosan I I -n 1200 800 500 200 0 constructs D no chitosan I +chitosan constructs Figure 19. No chitosan induction in transient assays in onion cells Onion epidermis cells were bombarded with 5 g of indicated WP GUS (see Fig. 6) and 5 g of Ubi-LUC and cultured post bombardment with or without chitosan (A) and (B) represent two separate experiments. Data represent mean (_:!: SE) of five replicates 84

PAGE 97

2500 ~----------------, 2000 o '.2 .;:, 3 0 15 0 .-->< 3 a. -'f 3 0 1000 C) ..500 -4500 -2600 1200 800 600 500 200 constructs Figure 20 Promoter activity in onion cells Onion cells were transformed by particle bombardment as described in Fig. 19 and cultured in the absence of chitosan. Data represent mean (! SE) of six replicates. 85

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86 Constructs containing either 35S-GUS or the white pine chitinase promoter (WP)GUS were bombarded into maize and pine suspension cells (Fig 21) Both 35S and WP showed significant promoter activity in maize cells although 35S was much higher than WP (Fig 21A&C) In contrast, WP showed higher promoter activity than 35S in pine cells (Fig 21 B&D) The overall GUS activity detected in p i ne cells was lower than in maize (Fig 21 C&D; scales are different). In fact the internal control Ubi-LUC a l so showed much lower activity in pine (average = 3 7 x 104 RLU) than in maize (average = 2 6 x 106 RLU) This is why the ratio of GUS/LUX was higher in pine (Fig 21 B) than in maize (Fig 21A). Transient Assay in Pine Suspension Cells Since no chitosan-induction was observed in onion cells (Fig 19) it was possible that some components that are present in pine could be missing in onion cells To test this idea, I repeatedly tested particle bombardment in pine suspension cells After optimization of the technique GUS activity levels were sufficiently high to obtain i nterpretable results (Fig 21 ) but chitosan -i nduction was not observed (Fig 22A). Interestingly, the expression pattern was a little b i t different in pine cells from that in onion. The pWP-GUS9 3 containing the 4 5-kb WP promoter showed higher promoter activ i ty than any of the others in pine cells (Fig 22) while all constructs showed similar activity in onion cells (Fig 20). One small construct -136 GUS did not show any activity in pine cells compared with the 200 construct and promoterless control (Fig 228).

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A. 3600 -,--------, 3000 0 '.c 2400 3 1800 u5 '7 :, 0 1200 ('.) 600 0 ~...___,~--~ 35S WP maize C 9000 ~------, ~ _g 6000 .?: !!! t5 .c ro :3 Cl) :::i: 0 :::i: 3000 -=35S maize B. 9200 -,--------, 6900 4600 2300 35S WP pine D. 220 .0~---~ 165 0 110 0 -------------55 0 0 0 17 35S WP p i ne Figure 21. Promoter comparison in maize and in pine cells. Maize and pine suspension cells were bombarded with p8I221 (35S promoter) or pWP GUS9.3 (WP ; 4.5 kb white pine chitinase promoter see Fig 6) Ubi-LUC was an internal control in all cases Data in (A) and (B) are expressed as ratio of GUS activity (pmole MU) over luciferase activity (RLU relative light unit) Data in (C) and (D) are expressed as GUS activity All data represent mean of two to five replicates 87

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A. B. I +chi tosan 0 no chitosan 5000 ~---------------~ ~ 4000 0 .c ~3 :::!!3000. ::J :::\! ..J a. c7.i '? 2000 ::J 0 C) ..... ~1000. -4500 -1200 700 500 -400 200 constructs 4000 ~---------------~ Q 3000 -:::, :::\! >< :::\! 3 a. 2000 u5 <7 ::J 0 C> 1000 -4500 -200 -136 0 constructs Figure 22 Promoter activity in pine cells (A) Pine suspension cells were bombarded with Ubi-LUC and promoter constructs (Fig. 6) as indicated and cultured in the presence or absence of chitosan (B) Bombarded cells were cultured in the absence of chitosan. Each bar represents mean (~ SE) of four (in panel A) or six (in panel B) replicates. 88

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89 GUS Expression in Stably Transformed Tobacco Plants I was not able to detect chitosan-responses in either onion or pine cells in the transient assays (Fig 19 and 22) The possible reasons could be : (1) The chitosan response element(s) is/are not within the 5'-non-coding region of Pschi4; or (2) the strong wounding effect imposed by particle bombardment may have masked the chitosan-responses since the Pschi4 gene was shown to be strongly induced by wounding (Fig 15). Stably transformed tobacco plants could be used to test these ideas and to study developmental regulation of Pschi4 as well. Some promoter constructs (see Fig. 6) were subcloned into the binary vector p8I101 (Clontech Inc., CA) and introduced into tobacco (Nicotiana tabacum var Samsun) by Agrobacterium-mediated transformation. Independent transformants of each constructs were numbered continuously (for example pWP16 .7-1, pWP16.7-2 etc ) Seeds from primary transgenic plants were germinated on kanamycin-containing MS-agar plates to examine true transgenics. The result is summarized in Table 2 Bombardment-Induced Expression in Tobacco To test if particle bombardment alone could induce WP promoter activity some transgenic tobacco leaves were bombarded with gold particles with no DNA. Results showed that particle bombardment per se significantly induced WP GUS

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90 Table 2 Summary of results of WP-GUS transgenic tobacco construct transgenic line Km (r/s) pollen (blue/yellow) wound-induction pWP16.7-1 s yellow pWP16 7-2 r blue yes pWP16.7-3 r n/d yes 4.5 kb pWP16 7-4 r blue pWP16.7-7 s n/d pWP16.7-8 s n/d pWP16.7-9 r blue yes pWP16 7-10 r n/d yes pWP13.4-1 n/d blue 1 2 kb pWP13 4-2 n/d blue pWP13 .0-1 r blue pWP13 0-2 n/d yellow pWP13 0-3 r blue yes pWP13.0-4 n/d blue 0.8 kb pWP13 0-5 n/d blue pWP13 0-6 n/d blue pWP13.0-7 n/d blue pWP13.0-8 r blue yes (continued on next page)

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(Table 2 continued) construct transgenic line Km (r/s) pollen (blue/yellow) pWP12.8-2 r blue pWP12.8-3 r blue 0.6 kb pWP12.8-4 s yellow pWP12.8-5 r blue pWP12.8-6 r yellow pWP12.8-7 r blue pWP12.4-1 n/d yellow pWP12.4-2 r blue pWP12.4-3 n/d yellow 0 2 kb pWP12.4-4 r n/d pWP12.4-5 n/d blue pWP12.4-6 n/d yellow pWP12.4-7 r blue 0 kb p8I101 r yellow no wild type s yellow 1 The size of p8I101 vector is 12 2 kb All constructs were named by the total plasmid size 2 Km (r/s) kanamyc i n resistance or sensitivity of the progenies 3 pollen incubated with X-gluc blue (GUS expression) or yellow (wild type) 4 n/d not determined 91 wound-induction yes yes yes yes yes

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92 expression in tobacco leaves (Fig. 23). There was no difference between one, two or three shots of bombardment (Fig. 23A). No Chitosan-lnduced GUS Expression in Transgenic Tobacco A number of attempts have been expended to identify the chitosan-induced GUS expression in stably transformed tobacco plants However, the overall data showed that GUS activity was not increased upon chitosan treatment. Fig. 24 represents one typical result of these tests. Similar results are shown in Fig. 238 and 25A. One individual transformant pWP16.7-2 always showed much higher expression than any others (the scales of the right panel are different from the left in Fig. 24 and 25), which was probably due to multi-copies of transgene. Mechanical Wounding-Induced Expression Mechanical wounding was also applied to these transgenic tobacco plants To minimize variations observed during various assays the progenies of primary transgenics were used As expected mechanical wounding induced GUS expression (Fig. 25) Phosphate Induced GUS Expression In a recent paper, Chris Lamb s group reported another defense-related gene, PAL (phenylalanine ammonia lyase), from rice They claimed that the PAL gene was induced by wounding (Zhu et al., 1995). However the wounding they

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25 A. 20 ~'.E C: .l!! 15 0 C. s; Cl 1 0 (ti C: en E ::::) 5 ('.) ::::) 0 KCI only ch i to san 2 3 om a men D KCI alone I ch it o s an i=. bo m bardme nt B 300 0 s 2 50 0 ::::) Q) :::, 2 00 0 (J) C ~ ~ 1 50 0 >, .c ~ ; J:: 1 00 0 ~.e> Cf) ::J E 50 0 ('.) 0 0 1 6 7-2 12 8 5 12 8 7 1 2 .4 -2 12.45 B1101 pla nt# Figure 23 Particle bombardment per se induces promoter activity (A) Five leaf disks (diameter = 1 .5 cm) were cut from a single leaf of a transgenic tobacco WP16.7-2 placed on the center of MS-agar plates and three of them were randomly chosen for no-DNA bombardment for one two or three shots respectively. All five leaf disks were then incubated in 50 mM KCI, except that one of the two that received no bombardment was put in KCI plus 60 g/ml chitosan for 22 h. Total proteins were extracted and used for GUS activity assay (see Materials and Methods for details). Each bar represents the mean of two duplicates (B). Similar experiments were conducted on more transgenic tobacco plants, except that only one shot was used in the bombardment. 93

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D KCI alone I KCI + chitosan 200 ~--------------, 50 o I l B1101 12 4 5 12. 4-7 12 8 2 12. 8 5 12 8-7 16 7-10 plant# 1 000 800 600 400 200 WP16 7 2 Figure 24 GUS activity was not induced by chitosan in stably transformed tobacco plants Two leaf disks (diameter= 1 5 cm) were cut from the same leaf of the indicated transgenic tobacco and incubated in 50 mM KCI, or KCI plus 60 g/ml chitosan at room temperature with light for 22 h Total proteins were extracted and used to monitor GUS activity See also Fig. 238 and Fig. 25A for similar results 94

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A. B. D no treatment [] KCI I chitosan 8 wounding 6-.-----------------------, 5 -------------B1101 12.4-2 12 4-4 12 8-6 13.0-3 13 0-8 plant # o no treatment i;;j wounding 4 5 -,-----------------, 4 -----------3 5 3 ------------2 --------------1 5 B1101 12 4 2 12 4-4 12 8 7 16 7-3 16 7-9 16 7-10 12 4-49 plant# 20 0 -,-----, 18 0 16 0 14 0 12.0 10 0 8 0 6.0 4.0 ru-1 14 12 10 8 6 4 2 0 pWP16 7 2 16 .7-2 Figure 25. Mechanical wounding induced promoter activity. Seeds from primary transgenic tobacco were germinated on kanamycin-containing MS agar plates and then transferred into soil in GA-7 Magenta box. (A) A single leaf from each line was divided into four sections and harvested immediately (no treatment) incubated in 50 mM KCI (KCI), KCI plus 60 g/ml chitosan (chitosan) or left on the plant and mechanically wounded (wounding) All samples were stored in liquid N2 for at least 12 h before total proteins were extracted and assayed for GUS activity. (B). Wounding experiment, with no treatment control, was conducted on more transgenic tobacco lines. 12.4-4g : leaf disks were from the primary transgenic tobacco plant grown in greenhouse. 95

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96 described was a kind of excision wounding", where a portion of a leaf was excised from the plant and incubated in 50 mM phosphate buffer This is similar to the KCI treatment I did and is different from the mechanical wounding described above where the leaf was left on the plant and wounded by pliers for three times around its margin at 2 h intervals. Since the wounding induction was observed only by mechanical wounding (Fig. 25) but not by the excision effect when excised leaf sections were incubated in 50 mM KCI (Fig 15 25A) the previously reported result could be a simple phosphate effect. To test this I incubated the excised leaves in 50 mM phosphate buffer and did a similar GUS activity assay. Interestingly phosphate buffer did induce WP-GUS gene expression (Fig. 26) Developmental Regulation of Pschi4 Expression X gluc Staining of GUS in Transgenic Tobacco Plants The transgenic tobacco plants were also used for developmental studies X gluc is another substrate for GUS and can be converted to a blue precipitate by the GUS enzyme (Jefferson et al., 1987). X-gluc staining was first conducted on untreated leaves stems, and reproductive organs (corolla stigma ovary pollen) of pWP16 7 transgenic tobacco Only pollen grains showed blue staining (Fig 27 ) Thus, pollen gra i ns from tobacco plants containing other constructs were tested in X gluc solutions and the results are summarized in Table 2 In no cases was blue

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20 ~15 C (l) ......, ::J en 10 s; ..... C) u E co -5 CJ) :E :::, :::, c., 0 0 no treatment NaPO4 I NaPO4 + chitosan 1 2 3 pWP16 7 2 4 5 Figure 26. Phosphate induced WP-GUS express ion in transgenic tobacco Seeds of transgenic tobacco (pWP16 7 2) were germinated on MS agar for two weeks and five individual siblings were chosen for this experiment. For each individual plant three young leaves were excised One was frozen in l i quid nitrogen immediately (No treatment) ; the second was i ncubated in 50 mM NaPO4 buffer (pH 7.0) and the third was incubated in phosphate buffe r plus 200 g/ml chitosan for 24 h before harvesting (frozen in liquid nitrogen). Total protein was extracted and GUS activity was measured. 97

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--~--~~ Figure 27 X-gluc staining of tobacco pollen Pollen from transgenic tobacco containing the promoter-GUS (top) or vector pBI 101 alone (bottom) was incubated with X-gluc at 37 C for 16 hr and examined by microscopy 98

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99 staining observed in wild type tobacco (Nicotiana tabacum var Samsun) or tobacco containing p8I101 (promoterless GUS construct) vector alone. Pschi4 Expression in Pollen Since we have observed that GUS was actively expressed in healthy, non treated pollen of tobacco containing WP-GUS constructs, we asked if Pschi4 protein is similarly expressed in pollen of Chi4 tobacco plants. Total proteins were isolated from pollen of Chi4 and CIB10 tobacco plants and subjected to western blot analysis. A distinct band with the predicted size was detected in Chi4 but not CIB10, tobacco pollen (Fig 28). Similar western blotting was conducted on normal slash pine pollen, but no specific band was detected (data not shown).

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anti-Pschi4 Preimmune :,g: .c () 0 co () 0 u UJ kD 31. 0 21.5 31.0 21. 5 Figure 28. Pschi4 protein expression in tobacco pollen. Total protein was isolated from pollens of transgenic tobacco containing Pschi4 (Chi4) or pCIB10 vector alone (CIB10), separated on a 12% SOS-PAGE gel and transferred to a nitrocellulose membrane. Duplicate membranes were incubated with anti-Pschi4 antiserum or preimmune serum. Overexpressed E. coli protein was used as a positive control. The band present in the Chi4 lane but not in the CIB10 lane is indicated by an arrow. 100

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DISCUSSION I have characterized the structure and expression of a gene (Pschi4) from pine trees that appears to encode an extracellular chitinase The gene is induced in pine cells at the mRNA level by the general elicitor chitosan and mechanical wounding and thus provides a useful marker for defense gene expression i n p i nes The putative promoter of this gene was also studied and could be potentially useful to direct gene expression in pines Pschi4 Appears to Encode a Class 11 Chitinase The presumed catalytic domain of translated Pschi4 showed the same amount of sequence similarity to class I and to class II chitinases The assignment of Pschi4 as a class II chitinase was based on its inferred domain structure which was ident i cal to class II chitinase from tobacco (Fig 9 and 10) Like class II chitinase translated Pschi4 has an apparent N terminal signal peptide and a catalytic domain but lacks a cysteine-rich domain a hinge region and a C-terminal vacuolar targeting sequence (Neuhaus et al. 1991 ) The calculated isoelectric point of Pschi4 is 6 1 suggesting it encodes an acidic protein which is also consistent with class II chitinases 101

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102 I present evidence that Pschi4 encodes an extracellular protein as examined by western blot analyses (Fig 16 and 18) The predicted size of mature Psch i 4 protein is 25.3 kD which was actually observed in the culture medium of tobacco suspension cells containing the Pschi4 gene (Fig. 18) The detected Pschi4 protein in the extracellular fraction of both slash and loblolly pine suspension cells was -27 kD (Fig 16) suggesting it might be glycosylated since a putative glycosylation site was found in the primary sequence of Pschi4 Glycosylation has been reported for acidic chitinases from bean and carrot (de Jong et al. 1992 ; Margis-Pinheiro et al. 1991) An alternative explanation is that the class II chitinase isoform in slash and loblolly pine is indeed larger than Pschi4 which is from white pine It is i nteresting to note that ant i -Pschi4 antiserum could also detect a 32 kD protein i n the cellular fraction of loblolly pine but not in slash pine cells. This size is consistent with other class I chitinases reported (Broglie et al., 1986 ; Samac et al., 1990 ; Shinshi et al., 1987 ; Zhu and Lamb 1991 ). This suggests that certain isoforms of class I chitinases that are immunologically related to Pschi4 may be present in loblolly pine but might not ex i st in slash pine or at least they are quite different from Psch i 4 in sequence Class II chitinase accumulation is induced locally and systemically by pathogens in tobacco plants (Legrand et al. 1987 ; Wa r d et al. 1991 ) It is reasonable to speculate that Pschi4 could be s i milarly induced as a defense response in intact pine trees where its extracellular location would facilitate the hydrolysis of chitin in fungal cell walls This activity could directly inhib i t fungal

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103 growth and/or release chitin and chitosan which could amplify PR gene expression (Boller et al. 1983; Mauch and Staehelin, 1989) or elicit other aspects of the host response Pschi4 Represents a Small Family of Chitinase Genes Southern blot analysis indicated that genes similar to Pschi4 were present in several pine species (Fig. 11) as well as in spruce (Dr. John Davis personal communication) The presence of hybridizing DNA fragments in all these species indicates that the coding sequences of Pschi4 chitinase genes have been conserved and that Pschi4 represents a small multigene family The hybridization patterns observed in slash pine and loblolly pine were more similar to one another than either was to white pine This is consistent with the taxonomic relationships among these species since slash and loblolly pines are placed in the subgenus Pinus whereas white pine is in the subgenus Strobus (Harlow et al., 1979). One member of the Pschi gene family Pschi1 is unlikely to encode a functional protein because it contains a premature stop codon in the first exon. This region of Pschi1 was sequenced four times using dye primer and dye terminator methods to confirm the presence of a Tat that pos i tion (nucleotide 1071 in Fig. 7 overlined). It is of interest to note that the remainder of Pschi1 suggests it has all of the sequence structure required for encoding a functional product. An orthodox TATA sequence putative transcription and translation start sites open reading frames in exons 2 and 3 intron-exon splice sites and six potential polyadenylation

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104 signals were all found in gPschi1 (see appendix for complete nucleot i de sequence of Pschi1) We cannot strictly exclude the possibility that the T residue at position 1071 is a cloning artifact that was introduced during construction and screening of the genomic library An alternative explanation is that Pschi1 is a pseudogene If this is true, it would appear that the C > T loss-of-function mutation occurred very recently in genotype P 18 (white pine). Pschi4 Expression in Pine and Tobacco The transcr i pts that are closely related to Pschi4 were detected in pine cells treated with chitosan (Fig 14) In other plant species PR genes are induced at the transcriptional level (Lamb et al. 1989; Dixon and Lamb 1990) and tend to be coordinately regulated (Ward et al., 1991 ). Similarly i n pines chitosan i nduces Pschi4 mRNA accumulation in addition to regulating levels of other mRNA species (Mason 1995) Chitosan probably induces a coordinated defense response in pine cells that includes PR gene transcription one of which appears to encode extracellular chitinase Another aspect of elicitor induced defense is induct ion of the phenylpropanoid pathway which occurs in both herbaceous angiosperms and pines (Campbell and Ellis 1992 ; Jaeck et al. 1992 ; Mason 1995). The availability of cloned genes that a r e specific for different aspects of pine defense responses should provide useful tools for monitoring host responses in intact plants during disease interactions

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105 The presence of multigene families can complicate analysis of gene expression because different members of the family can differ in their tissue specificity and/or regulation by environmental factors. A transgenic approach can be valuable for identifying individual genes or individual promoters that are sufficient to direct transcription (i.e active gene family members) Because of the current technical difficulty in producing transgenic pines, we successfully utilized a heterologous transgenic system to identify Pschi4 as an actively transcribed gene Other groups have successfully exploited tobacco as a host for tree transgenes (Clarke et al., 1994; Hallick and Gordon, 1995). The regulatory sequences associated with the Pschi4 gene that was introduced into tobacco were sufficient to direct chitosan-induced expression in tobacco (Fig 15) Certain cis-elements in pine genes function similarly in pine and angiosperms (Kojima et al., 1994; Yamamoto et al., 1994) whereas others appear to differ (Martinussen et al., 1995). In either case herbaceous angiosperms such as tobacco would be useful "hosts" to compare and contrast cis-elements involved in pine gene regulation in angiosperms and gymnosperms. Northern blot analysis also revealed that the Pschi4 probe was specific and did not hybridize to endogenous tobacco chitinase transcripts (Fig 15). Thus this system simplified the genome complexity and eliminated a great deal of background noise, which ensured the success of primer extension and cDNA cloning in this study. Chitinase activity can increase in plants exposed to a variety of acute stresses including elicitors, pathogens and active oxygen, as well as the

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106 endogenous compounds ethylene and salicylic acid (Collinge et al. 1993 ; Klessig and Malamy 1994). Class I and II chi tinases are often d i fferentially regulated ; Class I genes are induced by wounding (Parsons et al. 1989 ; Chang et al., 1995) or ethylene (Boller et al. 1983; Mauch and Staehelin 1989) whereas the Class II genes are normally pathogen-induced (Stintzi et al., 1993 ; Ward et al. 1991) Pschi4 was induced by mechanical wounding in transgenic tobacco (Fig. 15) suggesting Pschi4 may be regulated more like the tobacco class I gene than the class II gene. Chitosan treatment like mechanical wounding can induce the octadecanoid pathway leading to jasmonic acid synthesis in solanaceous plants (Doares et al. 1995) Jasmonic acid accumulation has been observed in elicitor treated cells of monocots dicots and gymnosperms (Mueller et al. 1993) Pschi4 should prove to be a useful reporter gene for identifying compounds that may act as signal molecules in i nducing pathogen-, chitosan-, and wound responsive gene expression i n gymnosperms Plant responses to chitosan appear to be conserved in that many plants show similar types of defense responses to chitosan treatments (Keen and Dawson 1992 and refs therein) The conserved nature of the response is considered to reflect the ability of most plants to recognize and mount a defense response against the general features of most potential pathogens (chitinous cell walls etc ) Our experiments i ndicate that a specific chitosan-induced gene from pine is also chitosan induced in the genome of an angiosperm This supports the view that all or at least major components of the signaling pathway mediating the chitosan

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107 response are conserved in the plant kingdom The experimental system described here should prove useful to further test that view and ultimately to ident i fy those conserved components and define their functions. Pschi4 Promoter By studying the transient expression of Pschi4 putative promoter-GUS fusions in onion and pine cells as well as studying stable expression in transgenic tobacco plants, I was able to show that the -200 bp upstream region contained sufficient promoter sequences to direct wounding-induced expression. It is reasonable to speculate that one or more wound i ng-induced proteins (transcriptional activators) may bind to a small portion of this 200-bp sequence and induce Pschi4 gene transcription If so one can identify the putative cis element(s) and eventually identify the binding protein(s) In the transient assays in pine cells the putative Pschi4 promoter also showed higher activity than CaMV 35S promoter which is a commercially available and highly expressed promoter in angiosperms Therefore the Pschi4 promoter could be a useful candidate to direct heterologous gene transcription in pine The chitosan-induced expression which was initially observed in transgenic tobacco containing intact Pschi4 genomic clone was not seen later in transgenic tobacco containing the 5' upstream sequence and GUS fusion only However I have observed that chitosan as a general elicitor is not a potent elicitor of tobacco defense responses Chitosan showed relatively lower inducibility than wounding

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108 (Fig 15) even in Chi4 tobacco which contained the intact Pschi4 clone. I was able to detect wounding-induced GUS expression, but the increased level was relatively low (Fig 25) Taken together, it is unclear whether the putative chitosan-response element(s) reside in the 5'-upstream region or not. A more powerful elicitor or pathogen itself such as TMV may be needed to show more significant induction and to study the relevant regulatory sequences On the other hand, one can not completely exclude the possibility that the putative chitosan-response elements might lie in the 3'-flanking sequence or even in the coding region. In fact the promoter of a xylem-specific gene from pine is not within the 5'-upstream region (Loopstra et al., 1995) and has been found to reside in the 3'-downstream sequences (Loopstra personal communication). It might also be possible that such element(s) do not exist; rather some chitosan-induced proteins may help to stabilize certain transcripts of defense-related genes. Developmental Regulation of Pschi4 Interestingly GUS expression was detected in pollen of tobacco containing Pschi4 promoter-GUS fusions by X-gluc staining, but this blue-colored staining was never seen in wild-type tobacco (Nicotiana tabacum var Samsun) or tobacco containing the promoterless GUS (p81101 vector). These results suggested that Pschi4 gene expression is also developmentally regulated. This also implies that the Pschi4 promoter might direct Pschi4 gene expression in pine pollen after

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109 meiosis and Pschi4 chitinase might serve some function in the process of pine pollination Pollination in Pinus has been studied in several pine species, such as P taeda (Brown and Bridgwater 1987) and P. contorta (Owens et al. 1981) In Pinus ovules are flask-shaped and exposed (Owens 1993) Each ovule secretes a large pollination drop at night when conelets are at the fully receptive stage (Brown and Bridgwater 1987) Droplets recede during the day but more ovules exude droplets each successive night. The pollination drops which contain 4.3% glucose and 3 8% sucrose are thought to play a dual role : first to pick up and draw pollen into the micropyle and second to serve as a medium for pollen germination (Owens 1993). Pschi4 chitinase expressed in pine pollen could "sterilize" the pollination drops and prevent potential fungi from growing on the surface of pollination drops

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SUMMARY AND FUTURE DIRECTIONS Summary of Results The major objective of this research was to gather some basic information on a gene (Pschi4) from pine trees, which is potentially important in plant defense responses. The sequence that spans the coding region was analyzed extensively Southern blot assays were conducted to test its conserved nature in several pine species and to estimate the copy number of Pschi4 gene in pine genome. The single genomic clone was introduced into tobacco by Agrobacterium-mediated transformation to study its active expression, and to isolate it from other gene family members that could complicate expression analysis. Basic information about Pschi4 was obtained by using this unique tobacco system: the putative transcription start site was determined by primer extension; Pschi4 cDNA was cloned by RT PCR. Antibody against Pschi4 was made available by overexpressing Pschi4 cDNA in E. coli. The induced expression of Pschi4 in pine suspension cells and in transgenic tobacco plants was studied at mRNA (northern blots) and protein (western blots) levels The 5'-upstream sequence of Pschi4 was fused to GUS reporter gene and series of deletion constructs were transiently expressed in onion 110

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111 and pine cells, some of which were stably transformed into tobacco plants. The putative pine chitinase promoter was also compared with the widely used CaMV 35S promoter for their relative activities in pine and maize cells Based on these studies, several conclusions can be drawn as follows: Pschi4 is an active gene and its high expression is induced by the general fungal elicitor chitosan and by mechanical wounding Pschi4 has one open reading frame and the coding region is interrupted by two intrans which can be correctly spliced in transgenic tobacco plants Pschi4 appears to encode an extracellular protein, belonging to class II chitinases. Pschi4 represents a small multigene family and is conserved in several pine species The 5'-upstream region of Pschi4 contains sufficient sequence to direct activated transcription. The -200 bp upstream region of Pschi4 contains sufficient promoter sequences to direct wound-inducible expression in tobacco plants. The promoter activity of Pschi4 is higher than CaMV 35S promoter in pine cells. Therefore Pschi4 promoter could be useful in directing expression of other genes in pine.

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112 Future Directions While many results have been achieved based on the present study, many interesting questions remain to be answered and merit further investigation. Gel-Shift Assay During both transient and stable assays, no major difference was observed between these promoter deletion constructs However the -200 bp promoter seemed to be fully capable of activated transcription whereas a -136 construct did not show any activity in a transient assay (Fig 228) Sequence analysis also indicated that a putative 10-bp cis-element (between -140 and -131 Fig. 7) is present. To identify the active cis-element gel mobility-shift assay seems to be necessary and should be carried out soon TMV-lnduction in Transgenic Tobacco Plants Chitosan as a general fungal elicitor did not significantly induce expression of WP-GUS constructs in transgenic tobacco It is possible that chitosan is a weak inducer of WP GUS expression in tobacco In most cases researchers like to use a more powerful inducer such as a pathogen to study induced promoter activity of a defense-related gene In considering this it may be reasonable to apply TMV or a more potent elicitor to transgenic tobacco to induce the transgene Preliminary data did show that TMV induced higher GUS activity in pWP16 7-2 tobacco (data not shown) and this needs to be studied more thoroughly

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113 Induction of Pschi4 in Pine Trees Pschi4 expression was induced by chitosan in transgenic tobacco and pine suspension cells Induction of Pschi4 by mechanical wounding was also observed in transgenic tobacco But is Pschi4 induced in pathogen-infected pines? Are salicylate and jasmonate involved in the regulation of Pschi4? If so do they work synergistically or not? These questions can now be addressed in pines and in transgenic tobacco using Pschi4 as a reporter. Pschi4 Expression in Pine Pollen Pschi4 promoter-directed gene expression was seen in tobacco pollen. One can speculate that it would similarly direct gene expression in pine pollen This can be tested at the mRNA and protein levels. Since transgenic conifers currently cannot be easily regenerated through tissue culture pollen has been proposed to be an alternative vector for introducing foreign genes into conifers (Junttila et al., 1993) If a reporter gene is to be driven by the Pschi4 promoter its active expression in pollen would serve as a marker during the early stages of transformation

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APPENDIX Sequence Data The complete nucleotide sequences of Psch i 1 and Pschi4 have been deposited into the Gen bank Database under the accession numbers U57 409 and U57410, respectively For your reference they are listed here as an appendix. 114

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115 Nucleotide Sequence of Pschi4 1 CCCGCAATCA TAAATGTGTG AGACCTATAC CCCCCCAGAA ATTAGCCATA 51 AATCATAAAT GTGTAAATAG CCACTCATAA AGCCATTTAG GGAGAGACAA 101 CAATGCCTCA AATAATTAAT TATAAAAACA CAGAATACAC AATCATTACA 151 AGTGAGAGAG CACACTCTAC CCAATTTTAC AATCATTGAG ATATAATCCA 201 TGCCACTCCT GACCACCATC CTAAAATTAC CCTTAAACCT TTGCTATTTA 251 GAATTGATGT GGCTGAATGA TGGATACACG CTACTTAGAA TTGATGCCGC 301 TGAATGATGG ATACTAGCTA CTTATAATTG AGGTGGCTGA AAGATGGATA 351 CACAATATTG TGGTATGTGT GAAAAGCCCA AAAACAATGA GTTCTTTCCA 401 AAAAGATTAA CTTCCATAAT ATTGTTGTAT GTGTGAAAAG CCGAAAACCA 451 AAGTCTGCCC TCAGAGGAGA AGTGGAGAAG AGGAAGAAGC GAAGGAAGAT 501 CAGAAGTCAA GATTATGAAG AACAGGAGAG GAGCAGGTAT AATGGCCATC 551 GAAATCAAGG ACCCACGTTA CGCAGGAATT ACCTTCTACC TTCGCAGAAA 601 TATCGCTAGG AATGGTGGGG CTTGTAGGTT CGACCACAAA CACACTATAT 651 TCCACGGAGG GGTAGAAAGT TTCACCACCA CACGTTATCT CAGTGCTGCG 701 GAAATACTGC TATAAAACGA GGGGTTTGCA GCCTGGGATC ATCACCACAA 751 TTTGCGTTGG CAGCCTAAAG ATGGCGTACA CGAATATGAA GAGAATGATG 801 TCGATGAGGT TGCTATTGGC CCTCACCGCA GTGGCGATAA TGAGTTCTTT 851 GTGTTGTTAT GTTTCTGCAC AACAAGGAGT CGCATCCATC ATAAGTGAAG 901 ATGTTTTCCA TCAATTTTTG AAGCACAGAA ACGATGACGC GTGCTCGGCG 951 AAAGGCTTCT ACACCTACAG CGCCTTCATT GCGGCAGCTA ATAGTTTCCC 1001 AGACTTCGGC AACATCGGCG ATCAAGATAG TCGCAAGAGA GAGCTCGCAG 1051 CTTTCTTTGG TCACACGTCG CAGGAGACCA CAGGTATTAT TAATTTATAA 1101 GCTTCCTCTA ACTCTTCTGC CTCCCTGCCA TGCCTTAAAT GTTATTAATC 1151 GGATTAGGAT GTATGGGTTT TTACAGGCGG GTGGCCAACG GCCCCAGACG 1201 GTCCATATGC GTGGGGTTAC TGCTTCAAAG ATCAGGTGAA TAGCACAGAC 1251 AGATACCGCG GACGAGGACC TATTCAGCTA ACCGGGTAGG TTTTGTTAAT 1301 CCGCTTCGAT TTCTAGCAAT AGATATGGAA AAAATCGAAT GAATTTCAAG 1351 CCTAATACAC TTACCGCTCT GTGGGAGCAG GGACTACAAC TACAAAGCTG 1401 CGGGGAATGC GTTAGGTTAC GATCTCATAA ACAATCCGGA TCTCGTGGCG 1451 ACCGATGCCA CGGTGTCGTT TAAGACGGCG GTTTGGTTCT GGATGACGGC 1501 GCAGTCTCCG AAGCCTTCGT GCCACGACGT GATTTTGGGA AGATTGACTC 1551 CGTCAGTTAC CGATACCGCT GCTGGCAGAG TGGCGGGATA TGGAATGTTG 1601 ACGGACATCA TAAACGGTGG GCCGGAATGC GGCACAGGCA CAATAAGCGA 1651 CGTGCAGCAG GGGCGCATCG GGTTCTACCA GAGATACTGT AAGATGCTGG 1701 GCGTGGACGT GGGATCCAAC CTCGACTACA AAAACCAGAA GCCTTACGGA 1751 ACTTAATGTC TACGCTACCA ACCCATCCAA TCGACTACTA CTGTTATGGT 1801 CAGCACATAG TCTAATAAAT AAATAAATAA AATGAGAATT GCGATAAGTG 1851 GTGAGCTTCA CTCAGTGGAT GGGCTCCCCT CCTAGAAATA GAAAGGGTAA 1901 GCATGGTAGA TTAATTATTC ATACCTGTAC TGTCACTCGT GTTTTTTCAC 1951 AATAAAGAAG AAATAACGCG TATCCACCTT GGCAAAGGTA GCCGAATATT 2001 TCTAAATATT TTCCGCAAAT GTGGAAAGTC TGGCGTTTCT TCATATCACT 2051 CGCAGGATAT ATGACTAAAT TTGAGCAAAA TAAAATAAAT GT 2092

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116 Nucleotide Sequence of Pschi1 1 CAAACACACC ATTAAAACCT TGCTAGAGGT AGAGATATTA CACCATTGTT 51 CTATCCCATT AAAGCAATTC AGGCGATCTA TCTCTTGCTT GACCGCTTGA 101 CGACAGGGTG GCGCTACCAC GAGGACCTCT TGCTACTTAA AATGGAGTGA 151 ACTCCTCCTT GTCCGTGTGT TTGATAATCT TACATAACCG TCACGTAGCG 201 TAAGTCTAAA CGTTTAAAAA GCTAATCTGT TGACGTTTAA CCAAGTTATA 251 TTTCCTTGAT AGCAACTTAG TTGCCATAAT GTTGAAGTTT GAATAGGGCC 301 AAACACCGAT AATTTTCTTA CCCCCCCCAG CGACAAACAT AGTATTACCA 351 TACTGTTGTA TGTGTGAAAA GGGCAAATCC TAAGTCTGCC CTTCAAAGGA 401 AGAGTGAGAT TGGTCCAGGC GTGGAAGAAG CGAAAGGAAG ATCTGAAGTC 451 AAGATTATGA AGAACAGAGG CTATAAATCG TTTTAACCTG AACCCACGTT 501 ACGCGGCATG TACCTCCTAC GTCCCTTCGC AGAAATATCG TTATGAATGG 551 GGAGCTTTCA GGTTCGACCA TAAGACGTGA ACGCCACGGA ATGGTACAAA 601 GTTTCAACCC CCCATGGCAC GATATCTGAC TCCTGCACAA AATCCAGCTA 651 TAAAACGAGG GGTTTGCAGC CTGGAATCAT CACCACAATT TGCGTTGGCA 701 GCCTAAAGAT GGCCTACACG AATATGAGGA GAATGATGTC GATGAGGTTG 751 CTATTGGCCC TCACCGCAGT AGCGATGAGT ACTTTGTGTT GTTATGTTTC 801 TGCACAATTG GGGGCCGCAT CCATCATAAG TGAAGATGTT TTCAATCAAT 851 TTCTGTTGCA CAGAAACGAC GCCGTATGCC CCGCGAGAGA CTTCTACACC 901 TACAGCGCCT TCATTGCGGC TGCTAATACT TTCCCAGACT TCGGCAACAA 951 CGGCAATCTA GAGAGTCGCA AGAGAGAGCT CGCAGCTTTC TTTGGTCAAA 1001 CGTCGTAGGA GACCACAGGT ATTATTAATT TAGCCTCCTC TAACTCTTCT 1051 GTCTCCCTGC TATTCCTTAT TGTTATTAAT TAATGGCATT AAGATAATTG 1101 GGTTTCTACA GGTGGGTGGG CGACGGCTCG AGACGGACCA TATGCGTGGG 1151 GTTACTGCTT CAAAGAGGAG AGTAGCGGAG ACAGATACCA TGGACGAGGA 1201 CCTATTCAGC TAACAGGGTA ATATATGTCT ATCTATTTCT ATATCTAAAT 1251 TTTGTTAATA GAGATGGAAA AAATCGAATG AATTTCAAGC CTAATACACT 1301 TACCGCTTTC TGTGAGTAGG GACTACAACT ATAAAGATGC GGGGGATGCG 1351 TTAGGCTACG ATCTCATAAA CAATCCAGAT CTCGTGGCGA GCGATGCCAC 1401 GGTATCGTTT AAGACGGCGG TGTGGTTCTG GATGACGGCG CAGTCTCCGA 1451 AGCCTTCGTG CCACGACGTG ATTTTGGGAA GATGGACTCG GTCAGACACC 1501 GATACCGCTA CGGGCAGAGT GGCGGGATAT GGAATGTTGA TGAACATCAT 1551 TAACGGTGGG GTGGAATGCG GCACTGGCAC AATAAGCGAC CCGCAGCAGG 1601 GCCGAATCAG GTTCTACCAG AGATACTGCA GTTTGCTGGG CGTGGACACA 1651 GGATCCAACC TGGACTGCAA AAACCAGAAG CCCTTCGGAA CTTAACCTCT 1701 ACAATTCGAA GGCATCACAT CGACAACTAC TCTCCTGCGT GTTCGATGTT 1751 GTGGTCAGCA CAGAGTCTTC CGTCTCATAA ATAAATAAAT AAAATGAGAA 1801 TTGCAATAAC TGGTGACGTT AGCTCACTGG ATGGGCTCCC CTACCTGACA 1851 GGGATGCAGA ATAGATTAAT TATTTATACC TGTAACATCA GAATAGATTA 1901 GATTAATTAT TTATACCTGT AACTTCACTC ATGTTTTTTT TTACAATAAG 1951 AAAAAATAAA GTGGGATCCA CGTTGGTCAT GGTAGCCAAA TATCTTTTCC 2001 AAATATTTTG AAACAAGTTT GATATTTGTG TAATTGATGC GATGATATTT 2051 GTAATATTT 2059

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138 Xu, Y. Zhu, Q Panbangred, W., Shirasu, K., and Lamb C. J. (1996) Regulation, expression and function of a new basic chitinase gene in rice (Oryza sativa L.) Plant Mol. Biol. 30: 387-401. Yalpani N., Leon J., Lawton, M A., and Raskin, I. (1993a). Pathway of salicylic acid biosynthesis in healthy and virus-inoculated tobacco plant Physiol. 103: 315-321 Yalpani, N., Shulaev V., and Raskin, I. (1993b). Endogenous salicylic acid levels correlate with accumulation of pathogenesis-related proteins and virus resistance in tobacco Phytopathology 83: 702-708. Yamada T., Hashimota, H., Shiraishi, T., and Oker, H. (1989). Suppression of pisatin, phenylalanine ammonia lyase mRNA, and chalcone synthase mRNA accumulation by a putative pathogenicity factor from the fungus Mycosphaerella pinodes. Mol. Plant-Microbe lnterct. 2: 256-261. Yamamoto, N. Tada Y and Fujimura T (1994) The promoter of a pine photosynthetic gene allows expression of a beta-glucuronidase reporter gene in transgenic rice plants in a lightindependent but tissue-specific manner. Plant Cell Physiol. 35: 773-778 Ye, X. S. Pan, S Q., and Kuc, J (1989) Pathogenesis-related proteins and systemic resistance to blue mold and tobacco mosaic virus induced by tobacco mosaic virus, Peronospora tabacina and aspirin. Physiol. Mol. Plant Pathol. 35: 161-175 Yoshikawa M., Yamauchi K., and Masago H (1979) Biosynthesis and biodegradation of glyceollin by soybean hypocotyls infected with Phytophthora megasperma var. sojae Physiol. Plant Pathol. 14 : 157-169. Zhu, Q., Dabi T. Beeche A., Yamamoto, R., Lawton M A. and Lamb, C (1995). Cloning and properties of a rice gene encoding phenylalanine ammonia lyase Plant Mol. Biol. 29 : 535-550 Zhu Q ., and Lamb C J (1991 ) Isolation and characterization of a rice gene encoding a basic chitinase. Mol. Gen Genet. 226: 289-296

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BIOGRAPHICAL SKETCH Haiguo Wu was born in TaiXing JiangSu Province China, on August 16, 1967 He graduated from Nanjing University with a B S degree in Biochemistry in 1989 After three years of graduate study at the Institute of Genetics Chinese Academy of Sciences, Beijing he earned a M. S degree with specialization i n Plant Molecular Genetics in 1992. Then he moved to Gainesville Florida and began his doctoral stud i es in the University of Florida Plant Molecular and Cellular Biology (PMCB) Program in January 1993 139

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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 Pos h John ~-Davis, Ch r Assistant Professor of Plant Molecular and Cellular Biology 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 William B. Gurley Associate Profes or of Plant Molecular and Cellular Biology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation a d is fully adequate in scope and quality as a dissertation for the degree of Doct r of Philosophyj. Ken Professor of Plant Molecular and Cellular Biology 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 Philoso hy )/ c. !l,~L Harold C Kistler Associate Professor of Plant Molecular and Cellular Biology 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 dissertat ion for the degree of Doc~=~~
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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 Phi losophy t .J.7 December, 1996 Dean C lege of Agr i culture Dean Graduate School

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