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An Outer Membrane Protein a Family Outer Membrane Protein Is Required for Disease Symptom Development and Colonization o...

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Title: An Outer Membrane Protein a Family Outer Membrane Protein Is Required for Disease Symptom Development and Colonization of Sugarcane by Xanthomonas Albilineans
Physical Description: 1 online resource (63 p.)
Language: english
Creator: Fleites, Laura Ashley
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: albilineans -- membrane -- mopb -- ompa -- outer -- protein -- xanthomonas
Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Xanthomonas albilineans (Xa) is a systemic, xylem-invading pathogen that causes sugarcane leaf scald. Xa produces albicidin, an antibiotic and phytotoxin which blocks chloroplast differentiation, causing the foliar symptoms of leaf scald, including long, thin chlorotic "pencil-line" streaks that are diagnostic of the disease. Albicidin is the only known pathogenicity factor in Xa. In an attempt to identify additional pathogenicity factors, 1,216 independent Tn5 insertions in Xa strain XaFL07-1 were screened for reduced pathogenic symptoms and reduced capacity to multiply in stalks by inoculation onto sugarcane cultivar CP80-1743. Following such screening, 61 mutants were recovered and each insertion site determined by sequencing. Five (8.2%) of the Tn5 insertions were found in XaompA1 (XALc_0557), which is predicted to encode an OmpA family outer membrane protein. One mutant, M768, was able to consistently colonize stalk tissue but at severely reduced levels. Additional phenotypic studies showed that these mutants 1) produced albicidin, 2) were less motile (except M768), 3) were unable to grow in the presence of SDS (except M768) and 4) were slower growing than the wild type Xa in vitro. The divergent phenotype of M768 may be due to a translational fusion of the Tn5 insertion with an intact C-terminal domain of XaOmpA1. Three different complementation constructs were created to verify the pleiotropic phenotypes of ompA mutants were due to Tn5 insertional inactivation of XaompA1. One construct, designed to recreate the translational fusion identified in M768, restored SDS resistance and symptoms in planta to M1152, but did not restore motility. A second construct, carrying the entire XaompA1 gene, fully complemented M468, including restoring SDS resistance, motility and symptoms in planta. A third construct, pRK-mopB, carrying an ompA homolog from X.campestris pv. campestris was unstable in Xa. However, in plate assays for SDS resistance and motility, where antibiotic selection could be used to maintain the plasmid, limited complementation was observed. Taken together, this work demonstrates that ompA is required for both disease symptom development and colonization of sugarcane by Xa.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Laura Ashley Fleites.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Gabriel, Dean W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043886:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043886/00001

Material Information

Title: An Outer Membrane Protein a Family Outer Membrane Protein Is Required for Disease Symptom Development and Colonization of Sugarcane by Xanthomonas Albilineans
Physical Description: 1 online resource (63 p.)
Language: english
Creator: Fleites, Laura Ashley
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: albilineans -- membrane -- mopb -- ompa -- outer -- protein -- xanthomonas
Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Xanthomonas albilineans (Xa) is a systemic, xylem-invading pathogen that causes sugarcane leaf scald. Xa produces albicidin, an antibiotic and phytotoxin which blocks chloroplast differentiation, causing the foliar symptoms of leaf scald, including long, thin chlorotic "pencil-line" streaks that are diagnostic of the disease. Albicidin is the only known pathogenicity factor in Xa. In an attempt to identify additional pathogenicity factors, 1,216 independent Tn5 insertions in Xa strain XaFL07-1 were screened for reduced pathogenic symptoms and reduced capacity to multiply in stalks by inoculation onto sugarcane cultivar CP80-1743. Following such screening, 61 mutants were recovered and each insertion site determined by sequencing. Five (8.2%) of the Tn5 insertions were found in XaompA1 (XALc_0557), which is predicted to encode an OmpA family outer membrane protein. One mutant, M768, was able to consistently colonize stalk tissue but at severely reduced levels. Additional phenotypic studies showed that these mutants 1) produced albicidin, 2) were less motile (except M768), 3) were unable to grow in the presence of SDS (except M768) and 4) were slower growing than the wild type Xa in vitro. The divergent phenotype of M768 may be due to a translational fusion of the Tn5 insertion with an intact C-terminal domain of XaOmpA1. Three different complementation constructs were created to verify the pleiotropic phenotypes of ompA mutants were due to Tn5 insertional inactivation of XaompA1. One construct, designed to recreate the translational fusion identified in M768, restored SDS resistance and symptoms in planta to M1152, but did not restore motility. A second construct, carrying the entire XaompA1 gene, fully complemented M468, including restoring SDS resistance, motility and symptoms in planta. A third construct, pRK-mopB, carrying an ompA homolog from X.campestris pv. campestris was unstable in Xa. However, in plate assays for SDS resistance and motility, where antibiotic selection could be used to maintain the plasmid, limited complementation was observed. Taken together, this work demonstrates that ompA is required for both disease symptom development and colonization of sugarcane by Xa.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Laura Ashley Fleites.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Gabriel, Dean W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043886:00001


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1 AN OUTER MEMBRANE PROTEIN A FAMILY OUTER MEMBRANE PROTEIN IS REQUIRED FOR DISEASE SYMPTOM DEVELOPMENT AND COLONIZATION OF SUGARCANE BY XANTHOMONAS ALBILINEANS By LAURA ASHLEY FLEITES A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011

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2 2011 Laura Ashley Fleites

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3 This work is dedicated to my family.

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4 ACKNOWLEDGMENTS I would like to acknowledge my m ajor p rofessor, Dr. Dean W. Gabriel, for giving me the opportunity to pursue graduate studies in plant pathology, and for his patience and guidance throughout the course of my research. What I knew upon begin ning graduate school pales in comparison to what I have learned working with Dr. Gabriel. I would also like to acknowledge Dr. Philippe Rott, who also taught me so much during molecular biology, and because of his example I am a more meticulous and careful researcher. I thank Dr. K. T. Shanmugam and Dr. Jeff Jones, members of my committee, for their guidance and advice. Many thanks go to Patricia Rayside for her assistance wi th plant assays and for lending an ear when I was discouraged. I would also like to thank Dr. Shujian Zhang for acknowledge Dr. Zomary Flores Cruz and Dr. Pranjib Ch akrabarty, who were both There are many people at the Division of Plant Industry who gave me a chance to gain experience in plant pathology and who believed in me: Dr. Ru Nguyen, Darlene Ge orge Hill, Minjin Hao, Dr. Xiaoan Sun, Debbie Jones, Dr. Angela Vincent Jurick, Lisa Jones, Dr. Yong Ping Duan, Dr. Carlyle Baker, Carol Scoates, and Mark Gooch. Finally I would like to thank my family: mom for her constant support and for talking me thro ugh difficulties and frustrations, dad for believing in me (and for helping me clean up the sugarcane greenhouse during his vacati on!), and my brothers for their support and advice.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 13 Introduction ................................ ................................ ................................ ............. 13 Sugarcane Leaf Scald Disease ................................ ................................ ............... 14 The Tn 5 Transposon ................................ ................................ ............................... 18 Outer Membrane Proteins ................................ ................................ ....................... 20 Outer Membrane Protein A (OmpA) ................................ ................................ ....... 23 Objectives of this Study ................................ ................................ .......................... 24 2 MATERIALS AND METHODS ................................ ................................ ................ 25 Bacterial Strains and Culture Media ................................ ................................ ....... 25 Electrocompetent X. albilineans Cells ................................ ................................ ..... 26 Electroporation of X. albilineans ................................ ................................ ............. 26 Construction of Complementing Plasmid pLF004 ................................ ................... 26 Construction of Complementing Plasmid pCT47.3 ................................ ................. 27 Recovery of Empty Vector from pRK mopB ................................ ............................ 28 Methylation of pLF004 ................................ ................................ ............................ 29 Sodium Dodecyl Sulfate (SDS) Tolerance Assay ................................ ................... 29 Motility Assay ................................ ................................ ................................ .......... 30 Albicidin Assay ................................ ................................ ................................ ........ 30 Assessment o f Growth Rates ................................ ................................ ................. 3 0 Inoculation of Sugarcane ................................ ................................ ........................ 31 Symptom Assessments ................................ ................................ .......................... 31 Assessment of Leaf Colonization ................................ ................................ ............ 31 Assessment of Stalk Colonization ................................ ................................ ........... 32 Measurement of Stalk Elongation ................................ ................................ ........... 32 DNA Sequence Analysis ................................ ................................ ......................... 33 3 RESULTS ................................ ................................ ................................ ............... 34 Transposon Mutagen esis ................................ ................................ ........................ 34 Phenotypes of XaTn5 ompA Mutants: in planta ................................ ...................... 36 Phenotypes of XaTn 5 ompA Mutants: in vitro ................................ ......................... 39

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6 Construction and Assessment of Complementing Clone pLF004 ........................... 45 Assessment of Complementing Clone pCT47.3 ................................ ..................... 49 Assessment of Complementing Clone pRK mopB ................................ ................. 51 4 DISCUSSION ................................ ................................ ................................ ......... 54 LIST OF REFERENCES ................................ ................................ ............................... 58 BIOGRAPHIC AL SKETCH ................................ ................................ ............................ 63

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7 LIST OF TABLES Table page 1 1 The top 15 sugarcane producers, according to the United Nations Food and Agriculture Organization, as of 2009 ................................ ................................ .. 15 3 1 Summary of the pathogenicity data of the Tn 5 mutants and w ild type strains. ... 37 3 2 Assessment of leaf colonization of Tn 5 mutants and the wild type strain. .......... 38 3 3 Motility assay for Tn 5 mutants and the wild type strain. ................................ ..... 41 3 4 Motility assay of M468 containing complementation construct pLF004. ............. 47 3 5 Motility assay of M1152 containing complementation construct pCT47.3. ......... 50 3 6 Motility assay of M468 containing complementation construct pRK mopB ( non selective media). ................................ ................................ ........................ 52 3 7 Motility assay of M468 containing complementation construct pRK mopB (selective media). ................................ ................................ ............................... 52 3 8 Motility assay of M468 containing complementation construct pRK mopB (nonselective media). ................................ ................................ ......................... 53

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8 LIST OF FIGURES Figure page 1 1 Phylogenetic tree based on the concatenated nucleotide sequences of seven housekeeping genes ................................ ................................ .......................... 15 1 2 Symptoms of infection by X. albilineans. ................................ ............................ 16 1 3 Red vascular bundles in infected stalk. ................................ .............................. 17 1 4 Map of the wild type prokaryotic transposon Tn 5 ................................ .............. 19 1 5 Comparison of gram negative and gram positive cellular walls. ......................... 20 1 6 barrel tertiary protein structure. ................................ ................................ ................................ ............ 21 1 7 Drawing of the six known secretion systems from the genus Xanthomonas ...... 22 3 1 Map of the EZ Tn 5 KAN 2 transposon ................................ ............................... 34 3 2 Schematic of the location of XaompA1 and adjacent genes.. ............................. 35 3 3 Location and directionality of Tn5 insertions within and adjacent to XaompA1 .. 35 3 4 Symptoms two months post inoculation. ................................ ............................ 36 3 5 Comparison of stalk elongation and girdling around inoculation zone.. .............. 38 3 6 Albicidin production by XaTn 5 ompA mutants and wild type strain. ................... 39 3 7 Growth curves of Xa strains in liquid media in two separate experiments .......... 40 3 8 Growth of Xa strains ten days post inoculation in motility medium ..................... 41 3 9 Growth of Xa strains on SDS media three days post inoculation. ....................... 42 3 10 Location of p rimers in the context of M768. ................................ ........................ 43 3 11 PCR to check for reversion of M768. ................................ ................................ .. 43 3 12 The complete DNA sequence of Tn 5 Transposon. ................................ ............. 44 3 13 Depiction of the translational fusion created by the Tn 5 insertion in M768. ........ 45 3 14 Verification of profile of pLF004. ................................ ................................ ......... 46

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9 3 15 gentamycin. ................................ ................................ ................................ ........ 47 3 16 Symptoms in sugarcane inoculated with M468 containing complementation construct pLF004 ................................ ................................ ................................ 48 3 17 ............................. 49 3 18 Symptoms in sugarcane inoculated with M1152 containing complementation construct pCT47.3 ................................ ................................ .............................. 50 3 19 tetracycline. ................................ ................................ ................................ ........ 51

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10 LIS T OF ABBREVIATIONS bp base pair IM inner membrane MW OM outer membrane OMP outer membrane protein ORF open reading frame SD Shine Dalgarno SDS sodium dodecyl sulfate SLSD Leaf Scald Disease Tn 5 prokaryotic transposon Tn 5 Xa Xanthonomas albilineans Xcc Xanthomonas campestris pv. campestris Xf Xylella fastidiosa

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science AN O UTER MEMBRANE PROTEIN A FAMILY OUTER MEMBRANE PROTEIN IS REQUIRED FOR DISEASE SYMPTOM DEVELOPMENT AND COLONIZATION OF SUGARCANE BY XANTHOMONAS ALBILINEANS By Laura Ashley Fleites December 2011 Chair: Dean Gabriel Major: Plant Pathology Xanthomonas albilineans (Xa) is a systemic, xylem invading pathogen that causes sugarcane leaf scald. Xa produces albicidin, a n antibiotic and phytotoxin which blocks chloroplast differentiation, causing the foliar symptoms of leaf scald, including long, thin chloroti Albicidin is the only known pathogenicity factor in Xa In an attempt to identify additional pathogenicity factors, 1,216 independent Tn 5 insertions in Xa strain XaFL07 1 were screened for reduce d pathogenic symptoms and reduced capacity to multiply in stalks by inoculation onto sugarcane cultivar CP80 1743. Following such screening 61 mutants were recovered and each insertion site determined by sequencing Five (8. 2 %) of the Tn 5 insertions were found in XaompA1 ( XALc_0557 ) which is predicted to encode an OmpA family outer membrane protein. One mutant, M768, was able to consistently colonize stalk tissue but at severely reduced levels. Additional phenotypic st udies showed that these mutants 1 ) produced albicidin, 2) were less motile (except M768), 3) were unable to grow in the presence of SDS (except M768) and 4) were slower growing

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12 than the wild type Xa in vitr o. The divergent phenotype of M768 may be due to a translational fusion of the Tn 5 insertion with an intact C termin al domain of Xa O mpA1. Three different complementation constructs were created to verify the pleiotropic phenotypes of ompA mutants were due to Tn 5 insertional inactivation of XaompA1 One construct, designed to recreate th e translational fusion identified in M768 restore d SDS resistance and symptoms in planta to M1152, but did not restore motility. A second construct, carrying the entire Xa ompA1 gene, fully complemented M468 including restoring SDS resistance, motility an d symptoms in planta A third construct, pRK mopB, c arrying an ompA homolog from X campestris pv campestris was unstable in Xa. However, in plate assays for SDS resistance and motility where antibiotic selection could be used to maintain the plasmid, lim ited complementation was observed. Taken together, this work demonstrates that ompA is required for both disease symptom development and colonization of sugarcane by Xa.

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13 CHAPTER 1 LITERATURE REVIEW Introduction The search for genes affecting pathogenicity has been accelerated by the decreased cost and increased efficiency of automated DNA sequencing. When the entire genomic DNA sequence of a n organism is known, it becomes possible to apply reverse genetics to study the biological function of specific genes of potential interest, identified by homology searches and in some cases position in the genome. Investigators can create mutations in the gene of interest, study the corresponding phenotypes of the mutant, and complement the mutation to verify restoration of the wild type phenotype. Reverse genetics is a much faster approach to understanding the function of a specific gene than forward gene tics, the classic approach to functional genomics. With forward genetics, a mutagen is applied, mutants are screened for a specific phenotype, and the mutation is mapped with in the chromosome. Regardless of the approach, the availability of sequence inform ation has greatly facilitated biological research. Pieretti, et al. (2009) published the complete genome sequence of Xanthomonas albilineans the causal agent of sugarcane leaf scald. The only pathogenicity factor known prior to sequencing and annotation of the genome was albicidin, a phytotoxin and antibiotic produced only in this bacterial species. Albicidin is a moderately well characterized DNA gyrase inhibitor that blocks chloroplast differentiation and causes leaf scald, which includes pencil line sy mptoms in sugarcane leaves that are diagnostic of the disease (Birch and Patil 1985 and 1987; Hashimi et al. 2007). At least 22 genes in three regions of the genome were found to be needed for X. albilineans to produce

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14 albicidin (Vivien et al. 2005). As mutational studies of the albicidin genes progressed, it became apparent that albicidin was not the only pathogenicity factor contributing to symptom development and colonization of the sugarcane plant (Rott et al. 1996 and 2010). Understanding the genetic s behind the virulence and pathogenicity of an organism is useful for understanding host pathogen interactions and for developing better tools for disease control. Sugarcane Leaf Scald Disease Sugarcane Leaf Scald Disease ( S LSD) is caused by Xanthomonas albilineans X. albilineans is a G ram negative, xylem invading phytopathogen. It is a rod shaped bacterium with a single polar flagellum. Colonies are shiny, slow growing, non mucoid, honey yellow in color and often show dimorphism in size. X. albilineans is quite distinct from other members of the genus, and is closest in phylogeny to Xylella ( F igure 1 1 ; Pieretti et al. 2009). Like Xylella and unlike all other pathogenic xan thomonads, it does not have the h ypersensitive r esponse and p athogenicity ( hrp ) secretion system. Furthermore, X. albilineans is the only known x anthomona d that does not produce xanthan gum, produces the unique phytotoxin albicidin and has a comparatively small genome of 3,768,695 bp (Pieretti et al. 2009). The first reported sugar cane leaf scald disease ( S LSD) outbreak was in Indonesia in the 1920s (Pan 1997; Ricaud and Ryan 1989). The pathogen was detected in the continental United States in 1967 (Koike 1968), and is present in at least 66 countries worldwide, including most of th e major sugarcane producing countries ( T able 1 1 ; Rott and Davis 2000; Champoiseau et al. 2006).

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15 Table 1 1. The top 15 sugarcane producers, according to the United Nations Food and Agriculture Organization, as of 2009 ( http://faostat.fao.org ) Country Production (tons) Xa present* Brazil 671395000 Y India 285029000 Y China 116251272 Y Thailand 66816400 Y Pakistan 50045400 Y Colombia 38500000 Y Australia 31456900 Y Argentina 29950000 Y USA 27456000 Y Indonesia 26500000 Y Philippines 22932800 Y South Africa 20500000 Y Guatemala 18391700 Y Egypt 17000000 N Vietnam 15246400 Y *Y=yes; N=no Figure 1 1 Phylogenetic tree based on the concatenated nucleotide sequences of seven housekeeping genes ( gyrB atpD dnaK efp groEL glnA and recA ) (from P ieretti et al 2009).

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16 SLSD is described as having latent, chronic and acute phases. In latency, sugarcane is colonized by the pathogen but no symptoms are present. This phase can last for years, allowing the disease to spread in fields undetected. The chronic form is characterized by the classic pencil line symptoms: white, sharply defined lines running parallel to the midvein for the entire length of the leaf blade (F igure 1 2 ). The pencil lines turn into a burnt reddish color and the leaf becomes necrotic from the apex inwards as the disease progresses. Vascular bundles in infected stal k tissues turn red (F igure 1 3 ). Side buds may proliferate earlier in infected plants. The infection can lead to death of the plant. The acute form is the most severe, where the plant wilts and dies often without expression of the symptoms associated with the chronic form (Rott and Davis 2000). Figure 1 2. Symptoms of infection by X. albilineans Arrow points to a pencil line. Note necrotic tissue on other side of the midrib The host range of X. albilineans is limited to members of the Poaceae family. The major host affected by the pathogen is sugarcane, Saccharum spp. hybrids. Corn grown in proximity to infected sugarcane can become infected as well (Autrey et al. 1995; Birch 2001). Other natural hosts include Brachiaria piligera Imperata cylindrica

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17 Figure 1 3. Red vascular bundles in infected stalk. (Cogongrass), Panicum maximum (Guineagrass), Pa s p alum spp., Pennisetum purpureum (Elephant grass) and Rottboellia cochinchinensis (Itchgrass) (Bi rch 2001). Disease symptoms have been reported on all of the aforementioned alternative hosts (Leyns et al. 1984). Because many of these alternative hosts grow in the same areas as sugarcane, and the disease may remain latent for extended periods of time, these grasses can serve as reservoirs for the pathogen. It should also be noted that some of these grasses are grown for grain, fodder, and biofuels, and therefore the reservoirs of this pathogen can be extensive. X. albilineans is spread mechanically, v ia infected propagat ed material, aerially and through contaminated soil (Daugrois et al. 2003; Birch 2001). Disease control can be achieved by ensuring plant ing material is clean. Disinfect ion of tools can minimize the spread of SLSD ; long term control is achieved by breeding for resistance (Rott and Davis 2000).

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18 The Tn 5 Transposon Transposable elements are mobile genetic elements first discovered in maize by Barbara McClintock in 1948. Thought to be ancient in origin, transposable e lements are found in both prokaryotic and eukaryotic cells, and are implicated as a driving force of and include a wide array of elements from viral and nonviral retroposons to DNA transposons, to mobile genomic islands (such as pathogenicity islands), to transposable prophages (Roberts et al. 2008). Sometimes referred to as jumping genes or molecular p arasites, these segments of DNA are capable of moving from one place on a genome to another by nonhomologous ( recA independent) recombination, which can result in mutations at both sites (Kleckner 1977). Transposition can be beneficial, neutral, deleteriou s or lethal in effect, depending on where the insertion occurs within a genome. Transposons fall into many classes based on the mechanism of transposition. Some copy themselves irectly excised. Likewise, some are pasted into random target sequence while in other types a copy is inserted into a target sequence. Transposons can also contain one or more accessory genes, including those encoding resistance to antibiotics (Roberts 200 8). The Tn 5 transposon is a bacterial transposon that inserts semi randomly into chromosomes or plasmids, and has been widely used for gene discovery in bacterial genetics (Vizvaryova and Valkova 2004). It is a composite transposon, which is a transposa ble element that contains one or more antibiotic resistance genes flanked by insertion sequence (IS) elements (Roberts 2008). The IS elements of Tn 5 are IS50L

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19 and IS50R; between the IS elements lie three antibiotic resistance genes. IS50R encodes a transpo sase (Tnp) and an inhibitor of transposition (Inh) (Reznikoff 2008). The IS elements themselves are each bracketed by 19 bp sequences called inside end (IE) and outside end (OE) sequences (F igure 1 4 ) Figure 1 4. Map of the wild type prokaryotic trans poson Tn 5 str r streptomycin resistance ; ble r bleomycin resistance; kan r kanamycin resistance (from Naumann and Reznikoff 2000). Transposons have proven to be useful tools for studying gene function for decades (Mills 1985). Early studies using various transposons to create random insertional knockouts in Agrobacterium tumefaciens causal agent of crown gall disease, helped elucidat e the role of the tumor inducing (Ti) plasmid in oncogenicity (Bevan 1982). The development of an in vitro system for transposition of the Tn 5 transposon using a n exogenously supplied and hyperactive transposase has significantly increased the frequency o f transposition compared to th at of the wild type transpos on which has very low levels of transposition (Goryshin et al. 1998; Reznikoff 2008). Since this breakthrough, many commercial kits have been developed that exploit the efficiency of adding exogeno us, hyperactive transposase to the system. The EZ Tn 5 insertion kit from Epicentre Biotechnologies, for example, has been used to generate libraries of random transposon insertion mutants in many genera of bacteria and has facilitated the search fo r genes involved in pathogenicity. From 10 6 to 10 9 mutants can

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20 be obtained in a single reaction, and because the Tn 5 transposon inserts itself in a largely random manner one can obtain better coverage of a given genome if enough mutants are screened. For example, for an X. albilineans genome of 3.8Mb size, and assuming an average gene size of 1 kb, it would require 7.6 10 6 insertion events to obtain two Tn 5 insertions into each gene. Outer Membrane Proteins Gram negative bacteria are characterized by the presence of an inner membrane (IM) comprising a lipid bilayer, an outer membrane (OM) and a layer of pe ptidoglycan in between (F igure 1 5 ; Dirienzo et al. 1978). The IM and OM are functionally and compositionally different. The OM of G ram negative bact eria serves as an external barrier which protects bacteria from the surrounding environment (Koebnik et al. 2000), and contain s a mosaic of lipopolysaccharides and proteins arranged in the fluid phospholipid bilayer (Osborn and Wu 1980). Oute r membrane proteins (OMPs) are major component s of the OM and account for approximately 50% of the total mass (Koebnik et al. 2000; Lin et al. 2002). Figure 1 5. Comparison of Gram negative and G ram positive cellular walls. A, cytoplasmic membrane. B, peptidogly can layer. C, outer membrane. The proteins of the OM can be divided into a two basic categories: 1) integral membrane proteins, which are highly expressed and permanently bound to the OM, and

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21 2) minor proteins that are expressed as needed. Most OMPs are barrel structures, as opposed to the inner membrane proteins which tend to have helical structure (F igure 1 6 ; Koebnik et al. 2000). Figure 1 barrel tertiary protein structur e. Gray shading represents the phospholipid bilayer. OMPs serve many important roles in a variety of cellular processes. In addition to stabilizing the OM via linkages to the peptidoglycan layer, OMPs may have enzymatic activity, serve as receptors for bac teriophages, take part in signal transduction or play a role in horizontal gene transfer. A large subset serves as pores for cellular uptake of nutrients and other molecules. Some, such as OmpC, D and F, act as general porins, which allow for the nonspecif ic and passive diffusion of small hydrophilic solutes into the cell. Other porins are substrate specific, such as LamB of E. coli which allows the passage of maltose or malodextrins, and FhuA and FepA of E. coli which are essential for uptake of large i ron siderophore complexes (Koebnik et al. 2000; Lin et al. 2002).

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2 2 Other OMPs are involved in secretion and efflux of pathogenicity factors, antibiotics, detergents and other substances. While the general secretory pathway is sufficient for translocation of substanc es into extracellular space of G ram positive bacteria, this system is only able to transport substances into the periplasm of G ram negative bacteria because of the presence of the double membrane. Thus, systems involving multiple proteins evolved that coordinate the translocation of substances into the extracellular milieu, and in the case of the Type III Secretion System, directly into the host cell, in a one or multistep process. There are six kno wn secretion systems in G ram negative bacteria (F igure 1 7 ; Buttner and Bonas 2009; Tseng 2009). These secreted effectors perform a range of functions for the cell. Some examples of secreted molecules include phytotoxins (albicidin, Xanthomonas albilineans ; Birch and Patil 1987; Bostock et al. 2006), adhesins involved in biofilm formation (XadA1 and XadA2, Xylella fastidiosa ; Caserta et al. 2010) and nucleic acids (T DNA, Agrobacterium tumefaciens ; Matthysse and Stump 1976). Figure 1 7 Drawing of the si x known secretion systems from the genus Xanthomonas (from Buttner and Bonas 2009).

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23 Outer Membrane Protein A (OmpA) The role of OmpA in animal pathogens has been studied extensively. In several bacterial species, this protein has been shown to play a criti cal role in virulence. For example, in the enteric pathogen Cronobacter sakazakii OmpA mutants were 87% less invasive than the wild type strain in INT 407, a human cell line (Mohan Nair and Venkitanarayanan 2007). Similarly, OmpA mutants of Escherichia coli were less able to adhere and invade C6 glioma cells (Wu et al. 2009). In another study, OmpA mutants of Escherichia coli were less invasive in brain microvascular endothelial cells and less able to penetrate the blood brain barrier than their wild type counterparts (Wang and Kim 2002). OmpA mutants of Escherichia coli have also been shown to be significantly decreased in biofilm formation (Barrios et al. 2005). MopB of Xylella fastidiosa (Xf) a homolog of XaOmpA1, was associated with the abil ity of Xf to elicit chloro sis in Chenopodium quinoa ( Bruening et al ., 2001 ) MopB was confirmed to be a major OMP of Xf and may be involved in xylem colonization, since purified protein adhered to xylem rich balsa wood ( Bruening et al 2001) Several attem pts to clone the gene with its native promoter in E. coli failed, but a construct containing the mopB open reading frame driven by an inducible bacteriophage T7 promoter was successfully created, and when expression was induced by IPTG, low levels of MopB were detected. A chimeric construct comprising the N terminal region of Xf M opB fused to the C terminal region of E. coli O mpA peptide of O mpA of E. coli was also created. wanted to exploit the exposed elements of the MopB protein to find a high affinity binding protein to inactivate MopB (Bruening et al. 2005). Ultimately, it was concluded that EF Tu (elongation factor

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24 temperature unstable), and not M opB, was responsible f or the chlorosis inducing activity (Bruening et al. 2007). Recently, the first evidence that a major OMP might be a pathogenicity factor in phytopathogenic bacteria was published (Chen et al 2010) A knockout mutant of an OmpA homolog, MopB from Xanth omon as campestris pv campestris was created. MopB mutants were no longer pathogenic in planta and in vitro cells aggregated abnormally, were more sensitive to higher temperatures, SDS in the media, and alkaline pH, and were deficient in EPS production, adh esion and motility (Chen et al. 2010). Objectives of this Study The first objective of this study is to locate the precise insertion site s of the Tn 5 transposon in the five ompA mutants. Next, the phenotypes of the five mutants in vitro and in planta will be assessed and compared to th e wild type strain. Finally the mutation must be complemented using the endogenous XaompA1 gene to verify the ascertained phenotypes are due the insertional inactivation of the gene by the Tn 5 transposon.

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25 CHAPTER 2 MATERIALS AND METHOD S Bacterial Strains and Culture Media Xanthomonas albilineans strain XaFL07 1 isolated in 2007 from sugarcane sampled in Canal Point, Florida, was used in all experiments XaTn 5 ompA mutants 227, 468, 573, 768 and 1152 were created by electroporation of XaFL07 1 with 20 ng of the transposase Tn 5 DNA synaptic complex (Epicentre Biotechnologies, Madison, WI, U.S.A.; for detailed methodology see Rott et al. 2011). Bacteria were routinely cultured on Modified Wilbrinks (MW) medium (10 g suc rose, 5 g peptone, 0.50 g K 2 HPO 4 2 O, 0.25 g MgSO 4 2 SO 3 15 g agar, 1 L deionized water at a pH of 6.8 to 7.0) at 28 to 30 C Transposon Tn 5 insertion mutants were grown on MW agar supplemented with 20 mg/l kanamycin. All strains were store d at 80 C as turbid cell suspensions in sterile distilled water. E. coli (Sambrook et al. 1989) or PYGM (5 g peptone, 3 g yeast extract, 40 ml 50% glycerol, 15 g agar in 900 ml deionized water, pH 7.4, after autoclaving, 100 ml MOPS buffer was added; Defeyter et al 1990) at 37 C. Antibiotics were used as needed at the follow ing concentrations (in g/ml): ampicillin 40 kanamycin 50, tetracycline 10, chlo ramphenicol 25 and gentamycin 3 Chemically competent E. coli was transformed using 10 to 150 ng DNA in 50l cells. After adding DNA, cells were incubated for 10 minutes on ice, heat shocked at 42 C for 30 seconds and 250 l room temperature SOC medium (Sambrook et al. 1989) was immediate ly added. The mixture was then incubated at 37 C for one hour and plated on appropriate selective media.

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26 Electrocompetent X. albilineans Cells Liquid cultures of XaTn 5 ompA mutants in a total volume of 25 ml MW medium were grown at 28 C for two to three d ays (to OD 600 = 0.4 to 0.7) shaking at 125rpm in a rot ary waterbath shaker Cultures were chilled in ice wa ter for 15 to 30 minutes and centrifuged at 4 C at 3200 x g for 15 minutes. Cells were washed first in 50 ml and then in 25 ml ice cold distill ed wat er and centrifuged at 4 C at 3200 x g for 15 minutes. Pellets were resuspended in 1 ml sterile distilled water and stored as 50 l aliquots at 80 C. Electroporation of X. albilineans Electrocompetent X. albilineans cells were thawed on ice. Plasmid DNA or ligation mixes (1 to 5 l or approximately 50 to 150 ng) were added to pre chilled cuvettes with 1 mm gaps and put on ice. 40 l competent cells were pipetted into the cuvettes and incubated on ice for 30 seconds. Cells were electroporated at 1800kV using an Eppendorf 2510 electroporator (Westbury, NY, USA). Typically, time constants were between 5.6 and 6.2. Occasionally time constants below 3 were obtained apparently due to arcing of the current; these electropor ations were repeated to obtain a higher time constant. Immediately after electroporating, 900 l MW medium was added and cells were transferred to 14 ml Falcon tubes and allowed to recover at 28 C for three to four hours. Transformed cells were plated on MW medium with appropriate antibiotics (gentamycin 3mg/l for pUFR047 constructs and tetracycline 10mg/l for pRK415 constructs). Construction of Complementing Plasmid pLF004 An 1156 bp fragment comprising the full length XALc_0557 predicted open reading fra me, annotated as Xa o mpA1 and including the native Shine Dalgarno region,

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27 was amplified by polymerase chain reaction (PCR) from strain XaFL07 1 Accuprime Taq High Fidelity polymerase (Invitrogen), 2X Failsafe Buffer D (Epicentre), primer Bam HI Hin dIII site were used. PCR products were cloned into TOPO2.1 (Invitrogen) and transformed into E. coli Mach 1 cells. Plasmid DNA was extracted from 30 colonies and sequenced. Because no single PCR product cloned was a perfect DNA sequence match to XALc_0557, one clone carrying an Age I Sac I segment containing a n error was corrected by swapping the fragment with an error free Ag e I Sac I section from another XALc_0557 plasmid clone, and transformed into E. coli Mach 1. After the sequence was verified, this construct was given the name pLF003. The plasmid was digested sequentially with Bam HI and Hin d III and the resulting fragment wa s gel purified and ligated into a stable, broad host range, repW shuttle vector pUFR047 (DeFeyter et al. 1990) also digested with Bam HI and Hin d III. Ligation mix was transformed into E. coli Mach 1 and transformants were selected on LB agar medium with amp icillin. The resulting plasmid, pLF004, was digested with Bgl II to verify the profile. Construction of C omplementing P lasmid pCT4 7.3 A 351 bp fragment comprising the C Terminal end of XaOmpA1 from XALc_0557 was amplified by PCR from XaFL07 1 Accuprime T aq High Fidelity polymerase Eco RI site and Shine Hin d III site were used. The construct was digested

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28 sequentially, first with Eco RI and then with Hin d III and the resulting fragment was gel purified and liga ted into pUFR047 digested with Eco RI and Hin d III. Ligation mix was directly transformed into electrocompetent XaTn 5 ompA M1152. Ten colonies were obtained. All colonies were screened using several primer sets to verify the presence of the gene within the vector. Seven of the ten colonies were PCR positive. Four of the seven colonies were inoculated in 10 ml liquid cultures in MW medium, grown for three days and plasmids were extracted and sequenced. Only one plasmid extracted from colony 3 had a sequence that was 100% identical to the expected XALc_0557 sequence, and this construct was given the name pCT47.3 (for C terminus in pUFR047 colony 3). Recovery of Empty Vector from pRK mopB Plasmid pRK mopB was kindly provided by Yi Hsiung Tseng of Institute of M icrobiology, Immunology and Molecular Medicine, Tzu Chi Unive rsity, Hualien 907, Taiwan, ROC. In order to compare the phenotypes of XaTn 5 ompA mutants transformed with this plasmid to the wild type, the wild type must contain the empty vector The mutants must also contain the empty vector. Otherwise, effects of the vector itself might be confused with effects of the gene. To release the empty vector, pRK mopB was digested with Hin dIII and Xba I. DNA was digested next with Age I to further cut the insert DNA, reducing the chance of contamination of the vector with the insert. The 10666 bp band representing the empty vector was gel purified and blunted using DNA polymerase I Large ( Klenow ) fragment. The DNA was then purified using QIAEXII gel extraction kit fo llowing the protocol for desalting and concentrating DNA solutions (Qiagen, Valencia, CA, U.S.A.). DNA was ligated to itself and transformed into E. coli TOP10 chemically competent cells. Plasmid DNA from f our blue colonies w as cut with

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29 Hin dIII (which shou ld not cut if the site was eliminated via Klenow end filling) and in a separate reaction Eco RI (which should linearize the plasmid). All four showed the correct restriction profiles and consequently plasmid extracts from two colonies were sent to be sequen ced. The plasmid extracted from c olony 2 had the predicted sequence. This plasmid was given the name pRK415K.2. Methylation of pLF 004 and pRK mopB E. coli Xa, a plasmid containing the methylase gene cluster from X. albilineans strain GPE PC73 (Champoiseau et al. 2006), was inoculated into 1ml LB broth supplemented with 25 g/ml chloramphen icol. Cells were cultured at 37 C for 16 hours. A 50 l aliquot was inoculated in 10 ml LB supplemented with 25 g/ml chlora mphenicol and inc ubated at 37 C for 2 hours. Cells were transferred to a 40 ml Oakridge tube and pelleted at room temperature. The cell pellet was resuspended in 700 l cold 0.1M CaCl 2 solution and 200 l aliquots of cells were transferred to four sterile 16 x 100 mm glass tubes and placed on ice. Approximately 250 ng plasmid DNA was added to the cells and incubated on ice for 20 minutes. Tubes were transferred to 42 C for one minute and incubated on ice for an additional 10 minutes. A volume of 1.8 ml LB medium was added t o each tube and cells were in cubated without shaking at 37 C for 2 hours. Aliquots of 100 l were plated on PYGM agar supplemented with gentamycin and chloramphenicol. Methlyation was verified by comparing the Hin cII restriction profile of methylated and u nmethylated pLF004 or pRK mopB extracts. Sodium Dodecyl Sulfate (SDS) Tolerance Assay MW agar medium was prepared as described above, autoclaved and filtered SDS (10%) was added to a final concentration of 25ppm Two to three day old cultures of Xa

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30 wild t ype and mutant cultures were streaked onto these plates, incubated at 30 C for four days, and relative growth rates were compared. Motility Assay Sucrose peptone agar (SPA) medium (20 g sucrose, 5 g peptone, 1 L deionized water, pH 6.8 7.0; Hayward 1960) was prepared with 0.25% (wt/vol) agar. Media was allowed to solidify for one day. Two to three day old cultures were stab inoculated into the center of the Petri dishes and in cubated (agar side down) at 28 C for 6 to 10 days. Albicidin Assay Albicidin as says were performed as described by Rott et al. (1996) with some modifications. Basically, three day old cultures of Xa were suspended in sterile distilled water, standardized to OD 600 = 0.450 0.010, diluted with sterile distilled water to 10 6 and 40 l aliquots were spread onto at least 3 plates of SPA medium Plates were incubated at 30 C until colonies were ~1 mm in diameter. Colonies were then overlaid with 4ml of a mix containing 2 ml of 1.5% Noble (Difco) agar (wt/vol) and a 2 ml suspension of E. coli 7 CFU/ml sterile distilled water) and incubated at 37 C for 24 to 48 hours. Albicidin production was quantified by measuring the width of the E. coli growth inhibition ring (GIR): (D d)/2 where D = diameter of the E. coli growth inhibition r ing and d = diameter of the X. albilineans colony. Assessment of Growth Rates Two day old cultures were transferred from solid media to sterile distilled water, and suspensions were standardized to a starting OD 600 = 0.300 0.010 Aliquots of 5 l were i mmediately transferred into 25 ml MW medium in 250 ml Nephelo flasks. Cultures were i ncubated at 28 C and OD 600 was measured in four hour intervals for 12 days.

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31 Inoculation of Sugarcane Suspensions of Xa strains to be tested for pathogenicity were standardized to OD 600 = 0.300 0.025 in sterile distilled water. Sugarcane cultivar CP80 1743 with at least 3 developed stalk nodes was inoculated by the decapitation method as described by Rott et al. (1997) in greenhouse conditions. Briefly, using steri le pruning shears, the plant was pruned below the third dewlap and 300 to 600 l cell suspension was immediately added to the exposed leaf whorl. Each strain was inoculated into at least five plants per assay. Symptom Assessments One month post inoculatio n, visual observations of qualitatively assessed leaf symptoms were recorded for at least three emerging leaves per plant. The symptoms were scored as follows: 0 = no symptoms, 1 = one to five pencil lines, 2 = six to ten pencil lines, 3 = more than 10 pen cil lines, 4 = leaf chlorosis or less than 10% necrosis, 5 = 10 50% leaf necrosis, and 6 = more than 50% necrosis. Assessment of Leaf Colonization One month post inoculation, leaves were sampled using scissors sterilized with 95% ethanol Leaves were cut w ith a scalpel into ~2 inch sections and weighed. Leaf tissue was sterilized by submersion in 95% ethanol and flaming. Sterilized leaf tissue was then chopped into small pieces using a sterile scalpel and forceps in plastic Petri dishes and 1 ml TBS buffer was pipetted onto the chopped leaf fragments. After 2 hours incubation at room temperature, homogenates were serially diluted and plated in triplicate on WCNCB medium (= MW medium supplemented with 25 mg/l cephalexin, 30 mg/l novobiocin, 50 mg/l cyclohexim ide and 12.5 mg/l benomyl; Rott el at. 2011) and

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32 WKNCB medium (= MW medium supplemented with 20 mg/l kanamycin, 30 mg/l novobiocin, 50 mg/l cycloheximide and 12.5 mg/l benomyl; Rott et al. 2011). Plates were incubated for three to five days at 30 C. Assessment of Stalk Colonization Two months post inoculation, leaves were removed and stalks were cut at the soil level by sterile pruning shears. The rind of the stalk was cleaned with 95% ethanol and paper towels. It was sprayed again with 95% ethanol an d flame sterilized. Using sterilized pruning shears, the stalk was cut in between nodes and the cut section was pressed onto WCNCB and WKNCB for Tn 5 mutants or WGNCB for Tn 5 mutants carrying plasmid constructs with gentamycin resistance (= MW medium supple mented with 3 mg/l gentamycin, 30 mg/l novobiocin, 50 mg/l cycloheximide and 12.5 mg/l benomyl) or WTNCB for plasmid constructs with tetracycline resistance (= MW medium supplemented with 10 mg/l tetracycline 30 mg/l novobiocin, 50 mg/l cycloheximide and 12.5 mg/l benomyl ). Stalk colonization was assessed in 10 locations: I 4 (representing four internodes below the point of inoculation) through I +5 (representing five internodes above the point of inoculation). Stalk colonization was quantified with the fol lowing scoring system: 0 = no bacterial colony in the stalk imprint, 1 = 1 to 10 colonies in the stalk imprint, 2 = more than 10 colonies or confluent growth of bacteria in less than 25% of the stalk imprint, 3 = confluent growth of bacteria in 25 to 75% o f the stalk imprint, 4 = confluent growth of bacteria in more than 75% of the stalk imprint. M easurement of Stalk Elongation Two months post inoculation, the elongation of harvested stalks was assessed. The length of the stalk was measured with a tape meas 0 to I + 8 ).

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33 DNA Sequence Analysis ORFs were predicted using pDRAW32 by AcaClone Software ( http://www.acaclone.com ) Tn 5 insertions within the Xa genome were located using the iANT (integrated ANnotation Tool) platform at INRA Toulouse (France). Conserved OmpA domains were identified using BLASTp and homologues of XaompA1 were identified using the BLASTn algorithms from NCBI ( http://blast.ncbi.nlm.nih.gov/ )

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34 CHAPTER 3 RESULTS Transposon Mutagenesis A total of 1216 independently derived EZ Tn 5 KAN 2 Tn 5 insertions in strain XaFL07 1 were screened in sugarcane for reduced pathogenicity (reduced ability to caus e disease symptoms and/or reduced ability to colonize the sugarcane stalk). Sequence analysis of t he region s flanking the Tn 5 insertion sites in first 10 mutants obtained indicated that the transposon integrated randomly into the genome. Of the 1216 Tn 5 mutants screened in sugarcane, 61 mutants affected pathogenicity. These mutants were analyzed to determine the precise location of the Tn 5 insertion site (Rott et al. 2011) Genomic DNA from the mutants was digested with Eco RI which is not found in the T n 5 T he mixture of fragments was cloned into a TA vector transformed into E. coli and transformants containing the Tn 5 were selected for with kanamycin Primers that read off of the ends of the Tn 5 were used for sequencing ( F igure 3 1). Figure 3 1 Map of the EZ Tn 5 KAN 2 transposon, showing location of sequencing primers KAN 2 FP 1 and KAN 2 RP 1.

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35 Surprisingly a total of five insertions were found within XaompA1 (F igure 3 2) and these became the focus of the remainder of this study. Figure 3 2. Schematic of the location of XaompA1 and adjac ent genes. Not drawn to scale. Locus tags indicating genes predicted to be transcribed left to right are shown above the horizontal line; t he gene indicated below the ho rizontal line is predicted to be transcri bed right to left. Predicted rho independent transcriptional terminator regions are indicated by lollipops in both directions. Again, surprisingly, a ll insertions were in the same direction. In M573, the Tn 5 was inserted in the promoter region upstream o f the Shine Dalgarno sequence. M227 and M468 had insertions in the Surface Antigen 2 (SAG 2) domain. In M1152 the transposon was inserted in the OmpA domain. Lastly, in M768 the transposon inserted in b etween the two domains ( F igure 3 3 ). Figure 3 3 Location and directionality of Tn5 insertions within and adjacent to XaompA1 (XALc_0557) The Sag and OmpA domain regions are indicated by blue and pink, respectively.

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36 Phenotypes of XaTn5 ompA Mutants: in planta All ompA mutants were significantly reduced in ability to cause symptoms of SLSD (F igure 3 4 ). Whereas the typical disease severity of wild type Xa was above 5 (on a scale of 6), none of the XaTn 5 ompA mutant s tested exhibited disease symptoms anywhere near the severity of wil d type Xa (T able 3 1) The highest disease severity obtained was with XaTn 5 ompA M768 with a disease severity of 0.4 in one experiment. Sugarcane inoculated with any of the ompA mutants was usually completely asymptomatic up to two months post inoculation, when stalks were harvested to measure the extent of stalk colonization. Figure 3 4 Symptoms two months post inoculation. A, M227. B, M468. C, M573. D, M768. E, M1152. F, XaWT. G, H 2 O. A B D F E G C

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37 All ompA mutants were significantly reduced in ability to colonize sugarcane stalks (T able 3 1) Across multiple independent plant inoculation assays, the wild type strain exhibited a minimum extent of stalk colonization (ESC) of 82% and maximum ESC of 99%. By contrast, in one experiment M227 colonized 14% and M573 colonized 6% of the stalk tissue assayed, but neither strain was detected again in stalk tissue in independent plant inoculation assays. The most consistent colonization was seen with M768, which colonized 5% of the stalk in one assay and 60% in a separate assay. M468 and M1152 were never recovered from sampled stalk tissue. Table 3 1. Summary of the pathogenicity data of the Tn 5 mutants and wild type strains. Disease severity values are on a 0 6 scale and extent of stalk colonization values are on a 0 100 scale. Strain Disease severity on leaves Extent of stalk colonization XaFL07 1 4.6 90 M227 0.12 4.7 M468 0.02 0 M573 0 3 M768 0.2 32.5 M1152 0.08 0 The mechanical damage inflicted by inoculation of sugarcane using the decapitation method result ed in stunted growth of stalk tissue around the point of inoculation; nonetheless, the plant s quickly recovered and normal growth patterns return ed The elongat ion of sugarcane stalk tissue inoculated with the wild type strain was not only impeded to an extent surpassing the effects of the injury but the diameter of the stalk contracts as well. This stunting and girdling was not observed for any XaTn 5 ompA mutant (F igure 3 5 ).

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38 Figure 3 5 Comparison of stalk elongation and girdling around inoculation zone. Lines 2 O. B, XaWT. C, M468. Leaf colonization was assayed for all ompA mutants at least twice; bacteria were recovered at least once for all XaTn 5 mutants (T able 3 2 ). Table 3 2. Assessment of leaf colonization of Tn 5 mutants and the wild type strain Isolation 1 (01 09) Isolation 2 (10 09) Isolation 3 (06 10) Isolation 4 (12 10) WT + + + + 227 + + 468 + ND + + 573 + ND 768 ND ND + + 1152 ND ND + + = Xa recovered ; = Xa not recovered ND = not determined A C B

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39 Phenotypes of XaTn 5 ompA Mutants: in vitro Despite the lack of symptoms in the plant, albicidin production was detected in all three XaTn 5 ompA mutants assayed. M227, M468 and M573 all showed visible growth inhibition rings on media overlain with albicidin sensitive E. coli DH 5 F igure 3 6 ). There was no reason to expect ompA was involved in albicid in production. Figure 3 6 Albicidin production by XaTn 5 ompA mutants and wild type strain, as evidenced by visible clearing of media surrounding single colonies of Xa. All mutants tested showed significantly reduced growth rates in liquid media. Mutants reached the exponential growth stage later and overall cell yield was lower tha n the wild type strain (F igure 3 7; data shown are from two experiments ).

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40 Figure 3 7 Growth curve s of Xa strains in liquid MW in two separate experiments 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0 2 4 6 8 10 12 OD 600 Days Post Inoculation Growth Curve of X. albilineans and ompA mutants XaWTSC 1 M227-1 M468-1 M768-1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0 2 4 6 8 10 12 OD 600 Days Post Inoculation Growth Curve of X. albilineans WT and ompA mutants XaWTSC 2 M227-2 M468-2 M573-2 M768-2

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41 XaTn 5 ompA mutants were variably affected in an assay designed to assess swimming motility. M768, notably, was more motile than the wild type strain. All other mutants displayed some degree of attenuated motility (T able 3 3 and F igure 3 8 ). Table 3 3 Motility assay for Tn 5 mutants and the wild type strain Diameter of bacterial growth (DBG) in mm in plate # Mean SD Strain 1 2 3 4 5 DBG DBG XaFL07 1 WT 21 23 26 26 19 23 3 M227 10 6 12 9 9 9.2 2 M468 17 15 10 10 8 12 4 M573 10 10 10 11 8 9.8 1 M768 29 30 36 29 28 30.4 3 M1152 19 15 12 11 10 13.4 4 Figure 3 8. Growth of Xa strains ten days post inoculation in motility medium Sodium dodecyl sulfate (SDS) is a detergent that has been used to detect perturbations in bacterial membranes. The wild type strain was able to grow in media

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42 supplemented with 25ppm SDS, but was unable to grow at the next highest concentration tested, 100p pm. All XaTn 5 ompA mutants, except M768, were unable to grow in media suppleme nted with SDS at 25ppm ( F igure 3 9 ). Figure 3 9 Growth of Xa strains on SDS medium three days post inoculation. Because of the unexpected growth of M768 on medium containing SDS, cells were tested by PCR to verify that this strain was not a revertant and the transposon was still intact. Three different primer sets were used: 1/ Kan2FP + ompAR1, 2/ Kan2RP + ompAF2, and 3/ ompAF2 + ompAR1 ( F ig ure 3 10 ). Using M768 as PCR substr ate, primer set 1 should amplify a 437bp band, set 2 a 1379bp band and set 3 a 2884bp band. The wild type strain should display a 1663bp amplicon with primer set 3. Colony touch PCR was performed directly off the plates containing SDS as well as from medi a without SDS. The predicted amplicon was obtain ed for all primer sets ( F igure 3 11 ).

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43 Figure 3 10 Location of primers in the context of M768. Figure 3 11 PCR to check for reversion of M768. Lanes 1 and 14, 1kb ladder (New England Biolabs); Lanes 2 5 = primer set 1; Lanes 6 9 = primer set 2; Lanes 10 13 = primer set 3; Lane 2, 6 and 10: M768 from SDS media; Lane 3, 7 and 11: M768 from plain media; Lane 4, 8 and 12: XaWT; Lane 5, 9 and 13: H 2 0 The inconsistent phenotype observed with M768 (motility comparable to the wild type strain, SDS resistance, and more consistent stalk colonization) was unexpected. A closer look at the Tn 5 transposon sequence showed a weak predicted Shine Dalgarno (SD) be ginning at the 1196 th base and an ATG start site 6 bp immediately downstream, which could initiate an ORF and form a translational fusion with an existing ORF if the EZ Tn 5 KAN 2 transposon inserted in frame. Importantly, no known transcriptional 1 2 3 4 5 6 7 8 9 10 11 12 13 14 4 3 2 1.5 1 0.5

PAGE 44

44 terminato r is known to be downstream from the k an2 gene on the transposon, which could allow formation of an operon driven by the Kan 2 promoter and including the t ranslational fusion (F igure 3 12 ). All ompA mutants were checked for possible transcriptional/translational fusions with the Tn 5 transposon. Figure 3 12 The complete DNA sequence of Tn 5 Transposo n. The start codon and stop codon of the k an 2 gene are indicated by the green arrow and red box, res pectively. The putative SD and methionine start site are indicated by the purple box and blue box, respectively.

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45 When the sequence of M768 was analyzed in detail, th e transcriptional and translational fusion of the entire ompA domain of XaompA1 was predicted Three separate ORFs were predicted and the ompA domain wa s functionally and cleanly separated from the SAG 2 domain with the M768 insertion ( F igure 3 13 ). No translational (in frame) fusions were created by insertion s in the four other ompA muta nts obtained in this study Figure 3 13 Depiction of the translational fusion created by the Tn 5 insertion in M768. The arrows pointing to the right within the colored bar represent ORFs. Construction and Assessment of Complemen ting Clone pLF004 Several unsuccessful attempts were made to clone the full length (1101 bp) Xao mpA 1 gene with its native promoter, but a ll clones contained sequence errors in different locations. Similarly, cloning of Xao mpA1 w ithout its native promoter was initially unsuccessful. A DNA fragment swapping strategy was then employed in which error free fragment s from different construct s were used to construct the full length XaompA1 gene with 100% sequence identity to the native gene in vector TOPO2.1 Once this construct, pLF003, was made, the gene was subcloned into broad host range vector pUFR047 resulting in pLF004 and transformed into E. coli The DNA was digested with Bgl II and the correc t profile was observed ( F igure 3 14 ). T his construct was moved by electroporation into M2 27. One colony was obtained. SAG 2 domain ORF 1 ORF 3 ORF 2

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46 Figure 3 14 Verification of profile of pLF004. Lane 1, 1kb ladder (New England Biolabs). Lane 2, Bgl II profile of pLF004 extracted from E. coli Expected profile = 4113bp, 3129bp and 2514bp The plasmid, pLF004, was then extracted from M227 and digested with Bgl II to check for rearrangement. The expected profile was not observed. X. albilineans is known to carry a restriction modification (RM) system (Philippe Rott, unpublished data). RM systems can interfere with transformations using double stranded DNA plasmids. Therefore a plasmid carrying the methylation gene cluster (as a 10599 bp insert) cloned in pCNS (pSU18 derived; Bartolome et al 1991) was used in E. coli in an attempt to methylate pLF004 prior to extraction and use in electroporation. The (methylated) pLF004 plasmid DNA extracted from E. coli carrying pMetXa was transformed by electroporation into M468. Many colonies (thousands) were obtained. Potential complementation was first checked by plating on media amended with SDS. Resistan ce to SDS was restored ( F igure 3 1 5 ).

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47 Figure 3 1 5 gentamycin. A, M468 + pUFR047; B, M468 + pLF004; C, XaWT + pUFR047 Complementation appeared to be confirmed when m otility was also restored for M468 containing pLF004 in two independent a ssays ( T able 3 4 ). Table 3 4 Motility assay of M468 containing complementation construct pLF004 Diameter of bacterial growth (DBG) in mm in plate # Mean SD Strain 1 2 3 4 5 6 7 8 9 DBG DBG WT/ pUFR047 43 37 45 40 39 38 44 42 41.0 3 M468/ pUFR047 32 32 35 30 30 30 32 35 30 31.8 2 M468/ pLF004.4Met 37 39 39 39 36 39 36 38 34 37.4 2 Finally, c omplementation was confirmed in planta Symptoms in sugarcane plants inoculated with M468 containing pLF004 although not as severe as the wild type strain were restored. M468/p LF004 exhibited diagnostic pencil lines and necrosis typical of SLSD (F igure 3 16). These results confirmed that the Tn5 insertion in M468 was C B A

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48 responsible for the mutant phenotypes, including loss of pathogenicity ( F igure 3 4), loss of motility ( F igure 3 8), and sensitivity to SDS ( F igure 3 9) Figure 3 16 Symptoms in sugarcane inoculated with M468 containing complementation construct pLF004 ( two weeks post inoculation ) M468 + pUFR047 M468 + pLF004.4Met WT + pUFR047 Water control

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49 Assessment of Complementing Clone pCT47.3 P lasmid pCT47.3 was constructed in an attempt to recreate the aberrant phenotype of M768 To this end, the translational fusion created by the Tn 5 insertion just upstream of the ompA domain of XaompA1 XaompA1 from the wild type strain using primers with an added SD and methionine start site, into pUFR047. The methionine start site of the forward primer is followed by the first codon of the ompA domain of X aompA1 The weak SD of the Tn 5 was replaced by the native SD from XaompA1 however. When transformed into M1152, pCT47.3 restored resistance to SDS ( F igure 3 17) as well as modest levels of pathogenicity ( F igure 3 18). However motility was not restored ( T able 3 5 ). Figure 3 17 A, M768 + pUFR047 ; B, M1152 + pUFR047 ; C, XaWT + pUFR047 ; D, M1152 + pCT47.3 col. S25 ; E M1152 + pCT47.3 col. S26 ; F M1152 + pCT47.3 col. S27 ; F M1152 + pCT47.3 col. S28 ; G M1152 + pCT47.3 col. S29 H C D E F A B G

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50 Tabl e 3 5 Motility assay of M1152 containing complementation construct p CT 4 7.3 Diameter of bacterial growth (DBG) in mm in plate # Mean SD Strain 1 2 3 4 5 6 7 8 9 DBG DBG WT/ pUFR047 42 50 45 c* 51 51 45 44 46.9 4 M1152/ pUFR047 33 34 31 28 33 30 30 32 31.4 2 M1152/ pCT47.3 S6 28 26 26 31 24 24 29 22 22 25.8 3 M1152/ pCT47.3 S21 32 25 20 23 21 22 22 23 21 23.2 4 contaminated Symptoms are not as severe as the wild type strain or M468 containing the compleme nti ng construct pLF004 (F igure 3 18 ). Figure 3 18 Symptoms in sugarcane inoculated with M1152 containing complementation construct pCT47.3 (two weeks post inoculation) A, M1152 + pUFR047; B, M1152 + pCT47.3 S21; C, XaWT + pUFR047; D, H 2 O B A C D

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51 Assessment of Complementing Clone pRK mopB The plasmid pRK mopB (kindly provided by Yi Hsiung Tseng of Tzu Chi University, Taiwan, ROC) contains mopB cloned from Xanthomonas campestris pv. campe stris in vector pRK415 (Chen et al. 2010). This gene is 83% identical at the nucleotide level to XaompA1 When this (methylated) plasmid was electroporated into M468, resistance to SDS in s olid media was restored provided antibiotic selection pressure (ie ., tetracycline in the medium) for the plasmid was maintained (F igure 3 1 9 ). Figure 3 1 9 Growth of strains in MW medium amended with SDS and 10g/ml tetracycline. A, M468 + pRK415K; B, M468 + pRK mopB Met colony 1; C, XaWT + pRK415K However, when selection pressure was dropped in motility assays, inconsistent results were obtained In the first assay, three colonies that originally were positive by A C B

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52 PCR for the insert (colony 1, 3 and 9) and one colony that was negative by PCR (colony 2 ) were tested. The PCR positive colonies were more motile than the PCR negative colony, but the standard deviations were too large to be statistica lly significant (T able 3 6 ). Table 3 6 Motility assay of M 468 containing complementation construct pRK mopB Met (non selective media) Diameter of bacterial growth (DBG) Mean SD Strain 1 2 3 4 5 6 DBG DBG WT 45 63 60 55 51 43 52.8 8 M468 48 50 44 43 46 32 43.8 6 M468/ pRK mopB Met c .1 (PCR +) 62 37 36 44 40 46 44.2 10 M468/ pRK mopB Met c 2 (PCR ) 34 47 32 37 38 37.6 6 M468/ pRK mopB Met c 3 (PCR + ) 41 52 50 43 50 54 48.3 5 M468/ pRK mopB Met c 9 (PCR +) 55 51 51 49 48 50 50.7 2 Bacteria from the edge of every plate (23 plates) were transferred to MW agar with 10g/ml tetracycline to check for loss of the plasmid pRK mopB. There was no growth for any strain indicating that this plasmid was lost in Xa in the absence of tetracycline selection A second motility assay was performed with and without antibiotic selection In t hese assay s there was no evidence of complement ation of this phenotype (T able s 3 7 and 3 8 ). Table 3 7 Motility assay of M468 containing complementation construct pRK mopB Met (selective media) Diameter of bacterial growth (DBG) in mm plate # Mean SD Strain 1 2 3 4 5 6 7 8 9 DBG DBG WT/ pRK415k.2 c.l 26 29 27 28 28 29 26 28 27.6 1 M468 / pRK 415K.2 c. 1 23 24 22 21 20 22 19 20 20 21.2 2 M468 / pRK mopB Met c. 1 21 19 22 22 20 25 20 21 21.3 2

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53 Table 3 8 Motility assay of M468 containing complementation construct pRK mopB Met ( non selective media) Diameter of bacterial growth (DBG) in mm Mean SD Strain 1 2 3 4 5 6 7 8 9 DBG DBG WT/ pRK415K.2 col.1 38 32 34 37 34 35 41 31 35.3 3 M468/ pRK415K.2 col.1 35 34 42 34 36 33 41 37 36 36.4 3 M468/ pRK mopBMet col.1 37 37 37 32 37 41 41 35 34 36.8 3 Despite the apparent instability of this plasmid, plants were inoculated to assess the phenotype in sugarcane. As e xpected, plants inoculated with M468 containing pRK mopB Met remained completely asymptomatic and no bacteria were recovered from stalk tissue.

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54 CHAPTER 4 DISCUSSION In the present study, five independent Tn 5 insertions out of 61 tested that affected Xa pathogenicity and growth were found within gene X aompA1 Comp lementation was achieved with two of the Tn 5 mutants tested These results demonstrate that X aompA1 is required for full pathogenicity of Xa in sugarcane. Interestingly, X aompA strains were unable to colonize or cause the characteristic pencil line streak s and chlorosis in leaf tissue despite the ir ability to produce albicidin when grown on agar plates A lbicidin was previously identified as the only known pathogenicity factor in Xa (Birch and Patil 1985 and 1987; Hashimi et al 2007). Furthermore, it was also shown that some albicidin deficient mutants are still able to cause severe leaf symptoms in sugarcane, demonstrating that undescribed mechanisms are involved in pathogenicity of Xa (Rott et al ., 2011). Although albicidin producti on involves at least 22 genes in three regions of the genome ( Vivien et al 2005 and 2007 ), X aompA1 is not among them. Since X aompA1 affects both symptoms and growth in planta while albicidin affects symptoms but not growth, X aompA1 represents a new and independent Xa pathogenicity factor XaompA1 may play at least an indirect role in the efficiency of albicidin export and secretion from Xa cells in planta Even when leaf populations of X aompa1 mutants were determined to be similar to the wild type str ain, the leaves of these plants remained asymptomatic. It is also possible that Xa o mpA1 may play a regulatory role or is part of a signal transduction pathway that regulates albicidin synthesis or other molecules in the plant. For example, outer membrane protein FecA serves as a signal receiver and transmitter (Braun et al. 2006). The fact that X aompa1 mutants are sensitive to SDS strongly indicates that the integrity of the outer membrane is affected

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55 in these mutants, and this in turn may cause missasse mbly or loss of function of the albicidin efflux pump (Pieretti et al 2009 ). The sensitivity to SDS of the mutants also strongly indicates that the barrier function of the outer membrane has been compromised, which in several plant/bacterial systems (Kin gsley et al 1993, Balsanelli et al 2010 ) has been shown to allow host defense compounds, including phytoalexins or reactive oxygen species, to be much more effective against these mutants. Interruption of a gene can cause polar effects downstream if th e given gene is XaompA1 is predicted to be followed by a rho independent terminator; the closest gene upstream is r ead in the opposite direction (F igure 3 1). Therefore, no polar effects of the Tn 5 insertions on downstream g enes were predicted Evidence linking small non coding RNAs to regulation of ompA in Vibrio cholerae (Song et al. 2008) opens up a different area that might be investigated in the Xa pathosystem. The findings of Song et al. (2008) show that vrrA the gene that encodes the regulatory sRNA, represses ompA translation and is correlated with increased vesicle formation. This is especially intriguing because the X aompA1 mutants show hyperproduction of outer membrane vesicles (Rott, unpublished data). This research confirms and extends recent work done by Chen et al (2010) in Xanthomonas campestris pv. c ampestris (Xcc), the causal agent of black rot of crucifers. In that study, it was shown that mopB a gene with close homology to X aompA1 is required for disease symptom development in cabbage. It was also shown that Xcc mopB mutants were less motile and more sensitive to SDS than the wild type strain consistent with what was found for X aompA1 In cross complementation studies,

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56 the Xcc mopB gene restor ed resistance to SDS in the X aompA1 mutants as long as selection pressure was maintained In the absence of selection pressure, no restoration of motility was observed, and even in the presence of selection, only partial and inconsistent resto ration of mo tility was observed. These data were statistically insignificant. Because of the instability of this plasmid in Xa, complementation in planta could not be assessed. The outer membrane proteins X aompA1 and Xcc mopB a ppear to represent critical pathogenicity factors in both Xa and Xcc. It seems surprising that mutations affecting these genes in other plant pathogenic bacteria were not previously reported Both of these xanthomonads are systemic xylem invaders, and it is possible that these proteins are more critical for xylem invaders tha n for mesophilic xanthomonads. The decreased motility in vitro might explain the lack of colonization in the sugarcane stalk, because the mutant strains were inoculated into the lea f t issue and not into the stalk. However, mutants of the rpf quorum sensing system in Xa, which are highly affected in motility in vitro are still able to spread in the sugarcane stalk like the wild type strain (Rott et al unpublished data). Alternativel y, the xylem of leaf tissue might be easier to traverse for the X aompA1 mutants, possibly due to structural differences or differences in tonicity. The difficulty in cloning XaompA1 in E. coli might be due to toxicity of the hydrophobic domains on the host cell (Laage and Langosch 2000). Alternatively if XaompA1 functions as a porin, the full length gene might allow solutes into the cell that are not tolerated by E. coli O ther reports of diffi culty or inability to clone an OMP in E. coli abound ( omp1 of Fusobacterium nucleatum Bolstad and Jensen 1993 ; ompU of

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57 Vibrio cholerae Sperandio et al. 1996 ; ompH of Pasteurella multocida Lee et al 2004 ; mopB of Xylella fastidiosa Bruening et al 2005 and 2007 ) Notably, low copy vectors were used in both the work reported here and by Chen et al (2010). The predicted X a O mpA1 protein appears to play a role in disease symptom development and colonization of the sugarcane stalk tissue T he localization of outer membrane proteins on the outer surface of the cell makes them vulnerable to proteins or other molecules that can bind or otherwise d eactivate the protein. B ecause this gene is well conserved among all xanthomonads, the applicability of such research is potentially wide. The Xanthomonadaceae family contains many economically important plant pathogens, including Xanthomonas citri (citrus canker) and Xylella fastidiosa The high degree of conservation a nd critical role of pathogenesis makes XaOmpA1 a potential target for the development of new control methods such as the development of transgenic plants producing molecules inhibiting XaOmpA1 not only in Xa but also in other bacterial plant pathogens tha t possess homologues of this protein.

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58 LIST OF REFERENCES Autrey, L. J. C., Saumtally, S., and Dookun, A. 1995. Studies on variation in the leaf cald pathogen, Xanthomonas albilineans Proc. Int. Soc. Sugar Cane Technol. 21:485 497. Balsanelli, E., Serrato, R. V., de Baura, V. A., Sassaki, G., Yates, M. G., Rigo, L. U., Pedrosa, F. O., de Souza, E. M., and Monteiro, R.2010. Herbaspirillum seropedicae rfbB and rfbC genes are required for maize colonization. Env. Microbiol. 12(8):2233 2 244. Bartolome, B., Jubete, Y., Martinez, E., and de la Cruz, F. 1991. Construction and properties of a family of pACYC184 derived cloning vectors compatible with pBR322 and its derivatives. Gene 102(1):75 78. Bevan, M. W., and Chilton, M. D. 1982. T DNA o f the Agrobacterium Ti and Ri plasmids. Annu. Rev. Genet. 16:357 384. Birch, R. G., and Patil, S. S. 1985. Preliminary characterization of an antibiotic produced by Xanthomonas albilineans which inhibits DNA synthesis in Escherichia coli J. Gen. Microbiol 131:1069 1075. Birch, R. G., and Patil, S. S. 1987. Evidence that an albicidin like phytotoxin induces chlorosis in sugarcane leaf scald disease by blocking plastid DNA replication. Physiol. Mol. Plant Pathol. 30:207 214. Birch, R. G. 2001. Xanthomonas a lbilineans and the antipathogenesis approach to disease control. Mol. Plant Pathol. 2:1 11. Bolstad, A. I. and Jensen, H. B. 1993. Complete sequence of omp1 the structural gene encoding the 40 kDa outer membrane protein of Fusobacterium nucleatum strain F ev1. Gene 132:107 112. Bostock, J. M., Huang, G., Hashimi, S. M., Zhang, L., and Birch, R. G. 2006. A DHA14 drug efflux gene from Xanthomonas albilineans confers high level albicidin a ntibiotic resistance in Escherichia coli J. Appl. Microbiol. 101:151 16 0. Braun, V., Mahren, S., and Sauter, A. 2006. Gene regulation by transmembrane signaling. BioMetals. 19:103 113. Bruening, G., and E. L. Civerolo. 2005. Exploiting Xylella fastidiosa proteins for Pierce's disease control, pp. 221 224. In M.A. Tariq, S. Oswalt, P. Blincoe, A. Ba, T. Lorick and T. Esser [eds.], Pierce's Disease Control Program Symposium. California Department of Food and Agriculture, San Diego, CA.

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59 Bruening, G., E. L. C iverolo, and P. A. Feldstein. 2007. Exploiting Xylella fastidiosa p roteins for Pierce's disease control, pp. 173 176, Pierce's Disease Research Symposium. California Department of Food and Agriculture, Sacramento, CA, T. Esser, editor, San Diego, CA. Butt ner, D., and Bonas, U. 2009. Regulation and secretion of Xanthomonas virulence factors. FEMS Microbiol. Rev. 34(2):107 133. Caserta, R., Takita, M. A., Targon, M. L., Rosselli Murai, L. K., de Souza, A. P., Peroni, L., Stach Machado, D. R., Andrade, A., La bate, C. A., Kitajima, E. W., Machado, M. A., and de Souza, A. A. 2010. Expression of Xylella fastidiosa fimbrial and afimbrial proteins during biofilm formation. Appl. Env. Microbiol. 76:4250 4259. Champoiseau, P., Daugrois, J. H., Girard, J. C., Royer, M ., and Rot t, P. 2006. Variation in albicidin biosynthesis genes and in pathogenicity of Xanthomonas albilineans the Sugarcane Leaf Scald Pathogen. Phytopathology 96:33 45. Champoiseau, P., Daugrois, J. H., Pieretti, I., Cociancich, S., Royer, M., and Rott P. 2006. High variation in pathogenicity of genetically closely related strains of Xanthomonas albilineans the sugarcane leaf scald pathogen, in Guadeloupe. Phytopathology 96:1081 1091. Chen, Y. Y., Wu, C. H., Lin, J. W., Weng, S. F., and Tseng, Y. H. 2 010. Mutation of the gene encoding a major outer membrane protein in Xanthomonas campestris pv. cam pestris causes pleiotropic effects, including loss of pathogenicity. Microbiology 156:2842 2854. Daugrois, J. H., Dumont, V., Champoiseau, P., Costet, L., Boisne Noc, R., and Rott, P. 2003. Aerial contamination of sugarcane in Guadeloupe by two strains of Xanthomonas albilineans Eur. J. Plant Pathol. 109:445 458. Defeyter, R., C. I. Kado, and D. W. Gabriel. 1990. Small, stable shuttle vectors for use i n Xanthomonas Gene 88:65 72. Dirienzo, J. M., Nakamura, K., and Inouye, M. 1978. The outer membrane proteins of G ram negative bacteria : biosynthesis, assembly, and functions. Ann. Rev. Biochem. 47:481 532. Gonzalez Barrios, A. F., Zuo, R., Ren, D., and W ood, T. K. 2005. Hha, YbaJ, and OmpA regulate Escherichia coli K12 biofilm formation and conjugation plasmids abolish motility. Biotechnol. Bioeng. 93:188 200. Goryshin, I. Y., and Reznikoff W. S. 1998. Tn 5 in vitro transposition. J. Biol. Chem 273:7367 7374. Hashimi, S. M., Wall, M. K., Smith, A. B., Maxwell, A., and Birch, R. G. 2007. The phytotoxin albicidin is a novel inhibitor of DNA gyrase. Antimicrob. Agents Chemother. 51:181 187.

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60 Hayward, A. C. 1960. A method for characterizing Pseudomonas solana cearum Nature 186:405 406. Kleckner, N. 1977. Translocatable elements in procaryotes. Cell 11:11 23. Koebnik, R., Locher, K. P., and Van Gelder, P. 2000. Structure and function of bacterial outer membrane proteins: barrels in a nutshell. Mol. Microbiol. 3 7:239 253. Koike, H. 1968. Leaf scald of sugarcane in continental United States a first report. Plant Dis. Rep. 52:646 649. Laage, R., and Langosch, D. Strategies for prokaryotic expression of eukaryotic membrane proteins. 2001. Traffic 2:99 104. Lee, J. Kang, S., Park, S. I., Woo, H. J., and Kwon, W. 2004. Molecular cloning and characterization of the gene for outer membrane protein H in a Pasteurella multocida (D:4) isolate from pigs with atrophic rhinitis symptoms in Korea. J. Microbiol. Biotechnol. 1 4(6):1343 1349. Leyns, F., De Cleene, M., Swings, J. G., and De Ley, J. The host range of the genus Xanthomonas Bot. Rev. 50(3):308 356. Lin, J., Huang, S., and Zhang, Q. 2002. Outer membrane proteins: key players for bacterial adaptation to host niches. Microb. Infect. 4:325 331. Matthysse, A. G., and Stump, A. J. 1976. The presence of Agrobacterium tumefaciens plasmid DNA in crown gall tumour cells. J. Gen. Microbiol. 95:9 16. McClintock, B. 1948. Mutable loci in maize. Carnegie Inst. Wash. Year Book, Le o Baeck Institute. 47:155. Mills, D. 1985. Transposon mutagenesis and its potential for studying virulence genes in plant pathogens. Annu. Rev. Phytopath. 23:297 320. Mohan Nair, M. K., and Venkitanarayanan, K. 2007. Role of bacterial OmpA and host cytoske leton in the invasion of human intestinal epithelial cells by Enterobacter sakazakii Pediatr. Res. 62:664 669. Osborn, M. J., and Wu, H. C. P. 1980. Pro teins of the outer membrane of G ram negative bacteria. Annu. Rev. Microbiol. 34:369 422. Pan, Y. B., Gr isham, M. P., and Burner, D. M. 1997. A polymerase chain reaction protocol for the detection of Xanthomonas albilineans the causal agent of sugarcane leaf scald disease. Plant Dis. 81:189 194. pDRAW32 by AcaClone Software (http://www.acaclone.com)

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61 Pieretti, I., Royer, M., Barbe, V., Carrere, S., Koebnik, R., Cociancich, S., Couloux, A., Darrasse, A., Gouzy, J., Jacques, M. A., Lauber, E., Manceau, C., Mangenot, S., Poussier, S., Segurens, B., Szurek, B., Verdier, V., Arlat, M., and Rott, P. 2009. Th e complete genome of Xanthomonas albilineans provides new insights into the reductive genome evolution of the xylem limited Xanthomonadaceae BMC Genomics 10:616. Ricaud, C., and Ryan, C. C. 1989. Leaf Scald. Pages 39 58 in: Diseases of Sugarcane, Major Di seases. Ricaud, C., Egan, B. T., Gillaspie Jr., A. G., and Hughes, C. G. Elsevier, Amsterdam, The Netherlands. Reznikoff, W. S. 1993. The Tn 5 transposon. Annu. Rev. Microbiol. 47:945 963. Reznikoff, W. S. 2008. Transposon Tn 5 Annu. Rev. Genet. 42:269 286. Roberts, A. P., Chandler, M., Courvalin, P., Guedon, G., Mullany, P., Pembroke, T., Rood, J. I., Smith, C. J., Summers, A. O., Tsuda, M., and Berg, D. 2008. Revised nomenclature for transposable genetic elements. Plasmid 60:167 173. Rott, P. C., Costet, L ., Davis, M. J., Frutos, R., and Gabriel, D. W.1996. At least two separate gene clusters are involved in albicidin production by Xanthomonas albilineans J. Bacteriol. 178:4590 4596. Rott, P., and Davis, M. J. 2000. Leaf Scald. Pages 38 44 in: A Guide to S ugarcane Diseases. P. Rott, R. A. Bailey, J. C. Comstock, B. J. Croft, and A. S. Saumtally, eds. La Librairie du Cirad, Montpellier, France. Rott, P., Fleites, L., Marlow, G., Royer, M., and Gabriel, D. W. 2011. Identification of new candidate pathogenici ty factors in the xylem invading pathogen Xanthomonas albilineans by transposon mutagenesis. Mol. Plant Microbe Interact. 24:594 605. Sambrook, J. E., E. F. Fritsh, and T. A. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbo r Laboratory, Cold Spring Harbor, NY. Song, T., Mika, F., Lindmark, B., Liu, Z., Schild, S., Bishop, A., Zhu, J., Camilli, A., Johansson, J., Vogel, J. and Wai, S. N. 2008. A new Vibrio cholerae sRNA modulates colonization and affects release of outer memb rane vesicles. Mol. Microbiol. 70(1):100 111. Sperandio, V., Bailey, C., Giron, J. A., DiRita, V. J., Silveira, W. D., Vettore, A. L., and Kaper, J. B. 1996. Cloning and characterization of the gene encoding the OmpU outer membrane protein of Vibrio choler ae Infect. Immun. 64(12):5406 5409. Tseng, T. T., Tyler, B. M., and Setubal, J. C. 2009. Protein secretion systems in b acterial host associations, and their description in the gene ontology. BMC Microbiol. 9(Suppl 1):S2.

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62 Vieira, J., and Messing, J. New pU C derived cloning vectors with different selectable markers and DNA replication origins. Gene 100:189 194. Vivien, E., Megissier, S., Pieretti, I., Cociancich, S., Frutos, R., Gabriel, D. W., Rott, P.C., and Royer, M. 2005. Xanthomonas albilineans HtpG is required for biosynthesis of the antibiotic and phytotoxin albicidin. FEMS Microbiol. Lett. 251:81 89. Vivien, E., Pitorre, D., Cociancich, S., Pieretti, I.,Gabriel, D. W., Rott, P. C., and Royer, M. 2007. Heterologous production of albicidin: a promising approach to overproducing and characterizing this potent inhibitor of DNA gyrase. Antimicrob. Agents Chemother 51(4):1549 1552. Vizvaryova, M., Valkoa, D. 2004. Transposons the useful genetic tools. Biologia, Bratislava. 59(3):309 318. Wang, Y., and Kim K. S. 2002. Role of OmpA and IbeB in Escherichia coli K1 invasion of brain microvascular endothelial cells in vitro and in vivo Pediatr. Res. 51:559 563. Wu, H. H., Yang, Y. Y., Hsieh, W. S., Lee, C. H., Leu, S. J., and Chen, M. R. 2009. OmpA is the critical component for Escherichia coli invasion induced astrocyte activation. J. Neuropathol. Exp. Neurol. 68:677 690.

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63 BIOGRAPHICAL SKETCH Laura Ashley Fleites was born in Miami, Florida in 1983. Sh e at tended the School for Advanced S tudies, a high school full time dual enrollment program, which allowed her to enter the University of Florida as a ju nior. She obtained her Bachelor of Science in a dvertising wit h a minor in p sychology in 2004. Shortly b efore graduating, she finally realized that advertising was not for her. Her first job after graduating was at the Florida Department of Agriculture and Consumer Services, Division of Plant Industry. She initially thought of this job as temporary until she could decide what direction to go in, but unbeknownst to her, she already found it. When the citrus she maintained exhibited strange, corky lesions, she as immediately intrigued. She told a coworker that she wanted to work in plant pathology, but was discouraged when the coworker said a degree in Xiao an Sun could see the genuine interest and excitement in her when they discussed a position that opened up in the citrus diagnostic lab, because he hired her on the spot. She was on her way. a technician, and in 2009 the great opportunity to attend meetings of the American Phytopathological Society, was involved in the Plant Pathology Graduate Student Associa tion, helped produce the departmental newsletter, and made lifelong friends.