Progress 10/01/08 to 09/30/13
Outputs
Target Audience: Scentific community, citrus growers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? We have trainedsix undergraduate students, four graduate students, six postdocs, four visiting sentisits and students from abroad. How have the results been disseminated to communities of interest? Publications, seminars, posters, interviews with journal reporters from US and Germany, conversions with citrus growers, collaborative development of the patent for application with industry. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Key findings Genome sequencing of 30 Xac related strains, which are different in virulence, host range or copper resistance, has been completed. The post-transcriptional regulator rsmA/csrA activates T3SS by stabilizing the 5' UTR of hrpG, the master regulator of hrp/hrc genes, in Xanthomonas citri. Understand how host immune responses accelerate pathogen evolution using Xac as a model. Identification of biofilm formation inhibiting compounds that enhances control of citrus canker caused by Xac. Identification of susceptibility gene CsLOB1 of citrus to citrus canker disease Impacts We have published 19 peer-reviewed publications with 18 published and one accepted. Among them, three papers are published on high-profile journals, i.e., PLoS Pathogens (Impact factor 9.13, corresponding author), ISME J. (Impact factor 8.9, corresponding author), and PNAS (impact factor 9.7, co-author). Patent for biofilm inhibitors. Leading to research on potential citrus canker resistant citrus by modification of the EBE in the promoter region of the susceptibility gene CsLOB1 in citrus.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2008
Citation:
Trivedi P.**, Wang N. 2013 Host immune responses accelerate pathogen evolution ISME J. (advance online publication, December 5 2013; doi:10.1038/ismej.2013.215)
Yang Hu*, Junli Zhang, Hongge Jia, Davide Sosso, Ting Li, Wolf B. Frommer, Bing Yang, Frank F. White, Nian Wang, and Jeffrey B. Jones Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease Proc Natl Acad Sci U S A. (doi/10.1073/pnas.1313271111).
Jalan N, Kumar D, Andrade MO, Yu F, Jones JB, Graham JH, White FF, Setubal JC, Wang N. 2013 Comparative genomic and transcriptome analyses of pathotypes of Xanthomonas citri subsp. citri provide insights into mechanisms of bacterial virulence and host range. BMC Genomics. doi: 10.1186/1471-2164-14-551.
Jalan N, Kumar D, Yu F, Jones JB, Graham JH, Wang N. Complete Genome Sequence of Xanthomonas citri subsp. citri Strain Aw12879, a Restricted-Host-Range Citrus Canker-Causing Bacterium. Genome Announc. 1(3). doi:pii: e00235-13.
Kogenaru, S.,Yan, Q., Guo, Y., and Wang, N. 2012 RNA-seq and microarray complement each other in transcriptome profiling. BMC Genomics 13:629.
Guo, Y., Zhang, Y., Li, J.L., Wang, N. 2012 DSF-mediated quorum sensing plays a central role in coordinating gene expression of Xanthomonas citri subsp. citri. Molecular Plant-Microbe Interactions 25: 165179.
Yan, Q., and Wang, N. 2012 High-throughput screening and analysis of genes of Xanthomonas citri subsp. citri involved in citrus canker symptom development Molecular Plant-Microbe Interactions 25:69-84.
Guo, Y., Figueiredo, F., Jones, J., and Wang, N. 2011 HrpG, together with HrpX, play global roles in coordinating different virulence traits of Xanthomonas axonopodis pv. citri Molecular Plant-Microbe Interactions 12:381-96.
Yan Q., and Wang, N. 2011a The ColR/ColS two-component system plays multiple roles in the pathogenicity of the citrus canker pathogen Xanthomonas citri subsp. citri Journal of Bacteriology 193:1590-1599.
Li, J. and Wang, N. 2011 The wxacO gene of Xanthomonas citri ssp. citri encodes a protein with a role in lipopolysaccharide biosynthesis, biofilm formation, stress tolerance and virulence Molecular Plant Pathology 12:381-396.
Guo, Y., Sagaram, U.S., Kim, J.S., and Wang, N. 2010. Requirement of the galU gene for polysaccharide production by and pathogenicity and growth in planta of Xanthomonas citri subsp. citri. Applied and Environmental Microbiology 76:2234 2242.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: These findings have been shared to communities through presentations at the APS meeting and citrus canker workshop, and five publications. PARTICIPANTS: Wang, N, Li, J., Guo, Y.P., Zhang, Y., Li, J.L., Yan, Q. Kogenaru, S., Hu, X.F. TARGET AUDIENCES: Scientific community PROJECT MODIFICATIONS: Nothing significant to report during this reporting period
Impacts
The quorum sensing pathway of Xanthomonas spp. consists of three major QS components, RpfF, RpfC and RpfG. In order to further characterize the QS regulatory network in Xanthomonas citri subsp. citri, we investigated the RpfF, RpfC, and RpfG regulons by using transcriptome analyses. Comparison of the transcriptomes of the QS mutants (rpfF, rpfC, and rpfG) with that of the wild-type strain revealed a core group of genes controlled by all the three QS components, suggesting that the RpfC-RpfG two-component system is a major and conserved signal perception and transduction system for DSF family signal-mediated QS in XCC. The unique genes controlled by RpfF alone indicate the complexity of the QS pathway and the involvement of additional sensory mechanisms in XCC. The unique genes controlled by RpfC and RpfG, respectively, support the possibility that RpfC and RpfG play broader roles in gene regulation other than transduction of DSF signals. To investigate the virulence mechanism of the citrus canker pathogen, eighty-two genes were identified in our study that contribute to citrus canker symptoms development caused by X. citri subsp. citri including genes that are involved in the type II secretion system, type III secretion system (T3SS), and T3SS effectors, the quorum sensing system, extracellular polysaccharide (EPS) and lipopolysaccharide (LPS) synthesis, and general metabolic pathways. In conclusion, we have significantly advanced our understanding of the putative genetic determinants of the virulence mechanism of X. citri subsp. citri by identifying 59 putative virulence-related genes, including 27 novel genes. In our study, we further investigated a novel gene designated as nlxA (Novel LPS cluster gene of X. citri subsp. citri) in the LPS cluster of X. citri subsp. citri 306. Our results indicate that nlxA is required for O-polysaccharide biosynthesis by encoding a putative rhamnosyltransferase. We further characterized the bdp24 (XAC3110) gene (designated as gpsX) using genetic complementation assays and expanded the knowledge about the function of the gpsX gene in Xac pathogenesis by investigating the roles of gpsX in EPS and LPS production, cell motility, biofilm formation on host leaves, stress tolerance, growth in planta, and host virulence of the citrus canker bacterium. Additionally, we compared RNA-seq and microarray in gene expression study by profiling the transcriptome from the wild-type and the hrpX mutant strains of γ-Proteobacterium X. citri subsp. citri. Our comparative analysis indicated that gene expression levels quantified by RNA-seq and microarray well-correlated both at absolute as well as relative levels (Spearman correlation-coefficient, rs > 0.76). Further, 72% of the already known HrpX target genes were detected by both RNA-seq and microarray, while, the remaining 28% could only be detected by either one of the methods. This study has significantly advanced our understanding of the regulome of the critical transcriptional factor HrpX. Our study demonstrated that RNA-seq and microarray complement each other in transcriptome profiling.
Publications
- 1. Kogenaru, S.,Yan, Q., Guo, Y., and Wang, N. 2012 RNA-seq and microarray complement each other in transcriptome profiling. BMC Genomics 13:629.
- 2. Guo, Y., Zhang, Y., Li, J.L., Wang, N. (2012). Diffusible signal factor-mediated quorum sensing plays a central role in coordinating gene expression of Xanthomonas citri subsp. citri. Molecular Plant-Microbe Interactions 25, 165 to 179.
- 3. Yan, Q. , and Wang, N. (2012). High throughput screening and analysis of genes of Xanthomonas citri subsp. citri involved in citrus canker symptom development. Molecular Plant-Microbe Interactions 25, 69 to 84.
- 4. Li J., and Wang N. (2012). The gpsX gene encoding glycosyltransferase is important for polysaccharide production and required for full virulence in Xanthomonas citri subsp. citri. BMC Microbiology 2012, 12:31 (doi:10.1186/1471-2180-12-31).
- 5. Yan, Q., Hu, X.F., Wang, N. (2012). The novel virulence related gene nlxA is involved in the production of lipopolysaccharide and extracellular polysaccharide, motility, biofilm formation and stress resistance of Xanthomonas citri subsp. citri. Molecular Plant Pathology 13:923 to 34.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: These findings have been shared to communities through presentations at the APS meeting and citrus canker workshop, and four publications. PARTICIPANTS: Wang, N, Guo, Y.P., Jones, J., and Yan, Q. Aritua, V., Kumar, D., Yu, F.H, Graham, J.H., Setubal, J.C., Zhang, Y., Li, J.L. TARGET AUDIENCES: Scientific community PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The quorum sensing pathway of Xanthomonas spp. consists of three major QS components, RpfF, RpfC and RpfG. In order to further characterize the QS regulatory network in Xanthomonas citri subsp. citri, we investigated the RpfF, RpfC, and RpfG regulons by using transcriptome analyses. Comparison of the transcriptomes of the QS mutants (rpfF, rpfC, and rpfG) with that of the wild-type strain revealed a core group of genes controlled by all the three QS components, suggesting that the RpfC-RpfG two-component system is a major and conserved signal perception and transduction system for DSF family signal-mediated QS in XCC. The unique genes controlled by RpfF alone indicate the complexity of the QS pathway and the involvement of additional sensory mechanisms in XCC. The unique genes controlled by RpfC and RpfG, respectively, support the possibility that RpfC and RpfG play broader roles in gene regulation other than transduction of DSF signals. To investigate the virulence mechanism of the citrus canker pathogen, eighty-two genes were identified in our study that contribute to citrus canker symptoms development caused by X. citri subsp. citri including genes that are involved in the type II secretion system, type III secretion system (T3SS), and T3SS effectors, the quorum sensing system, extracellular polysaccharide (EPS) and lipopolysaccharide (LPS) synthesis, and general metabolic pathways. In conclusion, we have significantly advanced our understanding of the putative genetic determinants of the virulence mechanism of X. citri subsp. citri by identifying 59 putative virulence-related genes, including 27 novel genes. Accumulating evidence suggests that the formation of biofilms on citrus leaves plays an important role in the epiphytic survival of this pathogen prior to the development of canker disease. Here, we report a genome-scale study of Xcc biofilm formation in which we identified 92 genes, including 33 novel genes involved in biofilm formation. This work is the first report on a genome-wide scale of the genetic processes of biofilm formation in plant pathogenic bacteria. The report provides significant new information about the genetic determinants and regulatory mechanism of biofilm formation. Xanthomonas axonopodis pv. citrumelo (Xacm) is a citrus pathogen causing citrus bacterial spot disease that is geographically restricted within the state of Florida. Illumina, 454 sequencing and optical mapping were used to obtain a complete genome sequence of Xacm strain F1, 4.9Mb in size. Comparative genomic analysis showed differences in several gene clusters like Type 3 effectors, Type 4 secretion system, lipopolysaccharide synthesis and others. In addition to pthA, effectors such as xopE3, xopAI and hrpW were absent in Xacm while present in Xcc. These effectors might be responsible for survival and reduced virulence of this pathogen on citrus compared to Xac. We also identified unique effectors in Xacm that may be related to the different host range as compared to Xcc. Comparison of the complete genome sequence of Xacm to Xcc and Xcv provides valuable insights into the mechanism of bacterial virulence and host-specificity.
Publications
- Jalan, N., Aritua, V., Kumar, D., Yu, F.H., Jones, J.B., Graham, J.H., Setubal, J.C., and Wang, N. 2011 Comparative genomic analysis of Xanthomonas axonopodis pv. citrumelo F1 causing citrus bacterial spot and related strains provides insights into virulence and host-specificity. Journal of Bacteriology 2011 193:6342-57.
- Li, J. and Wang, N. 2011b Genome-wide mutagenesis of Xanthomonas axonopodis pv. citri reveals novel genetic determinants and regulation mechanisms of biofilm formation. PLoS ONE 6(7): e21804. doi:10.1371/journal.pone.0021804.
- Guo, Y., Zhang, Y., Li, J.L., Wang, N. 2011b DSF-mediated quorum sensing plays a central role in coordinating gene expression of Xanthomonas citri subsp. citri. Molecular Plant-Microbe Interactions (advance online publication 2011b Oct 13.).
- Yan, Q., and Wang, N. 2012 High-throughput screening and analysis of genes of Xanthomonas citri subsp. citri involved in citrus canker symptom development Molecular Plant-Microbe Interactions 2012 25:69-84.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: These findings have been shared to communities through the APS meeting and four publications. PARTICIPANTS: Wang, N, Guo, Y.P., Sagaram, U.S., Kim, J.S., Li, J., Wang, N., Guo, Y., Figueiredo, F., Jones, J., and Yan, Q. TARGET AUDIENCES: Scientific community PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
To understand the virulence mechanisms of X. citri subsp. citri (XCC), we designed and conducted genome-wide microarray analyses to characterize the HrpG and HrpX regulons, which are critical for the pathogenicity of XCC. Our analyses revealed that 232 genes and 181 genes belonged to the HrpG and HrpX regulons, respectively. Our results showed that HrpG and HrpX regulated all 24 type III secretion system genes, 23 type III secretion system effector genes, and 29 type II secretion system substrate genes. Our data revealed that XCC regulates multiple cellular activities responding to the host environment, such as amino acid biosynthesis, oxidative phosphorylation, pentose-phosphate pathway, transport of sugar, iron and potassium, and the phenolic catabolism through HrpX and HrpG. Our results suggest that HrpG and HrpX interplay with global signaling network and co-ordinate the expression of multiple virulence factors for modification and adaption of host environment during XCC infection. We characterized the galU gene of Xcc. The galU mutants (F6 and D12) were identified in an Xcc EZ-Tn5 Tnp transposon library. Further study showed that galU was required for biosynthesis of extracellular polysaccharides (EPS), capsular polysaccharide (CPS), and biofilm formation. Mutation of galU caused loss of pathogenicity on grapefruit. Loss of pathogenicity of the galU mutant results from its inability to grow in planta rather than from its effect on virulence genes. These data indicate that the galU gene contributes to Xcc growth in the intercellular spaces and is involved in EPS and CPS synthesis and biofilm formation. We characterized the role of the two-component regulatory system ColR/ColS in the pathogenicity of XCC. The colS mutants (256A10 and 421E7), colR mutants (386C6 and 417E10), and the colR-colS double mutant (306DSR) all lost pathogenicity and showed no symptoms on the grapefruit leaves using either pressure infiltration or spray method for inoculation. Mutation of colS or colR significantly reduced X. citri subsp. citri growth in planta. The ColR/ColS system also played important roles in bacterial biofilm formation in glass tubes and on leaf surfaces, lipopolysaccharide (LPS) production, catalase activity and tolerance against environmental stresses including phenol, copper and hydrogen peroxide. Two EZ-Tn5 transposon mutants of Xcc strain 306 affected in biofilm formation were isolated; and subsequent analyses led to the identification of a novel gene locus XAC3596 (designated as wxacO) encoding a putative transmembrane protein and the rfbC gene encoding a truncated O-antigen biosynthesis protein. These mutants also showed reduced virulence and decreased growth on host leaves when inoculated by spray. Affected phenotypes of the wxacO and rfbC mutants were complemented to wild-type levels by the intact wxacO and rfbC genes, respectively. This report identified a new gene influencing LPS production by Xcc. In addition, our results suggested that a structurally intact LPS is critical for survival in the phyllosphere and virulence of Xcc.
Publications
- Guo, Y.P., Sagaram, U.S., Kim, J.S., and Wang, N. 2010. Requirement of the galU gene for polysaccharide production by and pathogenicity and growth in planta of Xanthomonas citri subsp. citri. Applied and Environmental Microbiology 76:2234 2242.
- Li, J. and Wang, N. 2010 Characterization of a hypothetical gene wxacO as well as rfbC involved in lipopolysaccharide biosynthesis, biofilm formation, stress tolerance and virulence of Xanthomonas citri subsp. citri strain 306. Molecular Plant Pathology (Article first published online: 6 DEC 2010 DOI: 10.1111/j.1364-3703.2010.00681.x).
- Guo, Y., Figueiredo, F., Jones, J., and Wang, N. 2010 HrpG, together with HrpX, play global roles in coordinating different virulence traits of Xanthomonas axonopodis pv. citri Molecular Plant-Microbe Interactions (Accepted).
- Qing Yan and Nian Wang 2010 The ColR/ColS two-component system plays multiple roles in the pathogenicity of the citrus canker pathogen Xanthomonas citri subsp. citri J. Bacteriol. (published online ahead of print, doi:10.1128/JB.01415-10).
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Research activities on Functional genomics study of citrus and Xanthomonas axonopodis pv. citri (Xac) interaction are described below: Microarray study. In our current study, mutants of key regulators including hrpX, hrpG, rpfF, rpfC, and rpfG have been generated for Xac 306. All the mutants have shown major effects on virulence indicating their importance in controlling the complex virulence gene network. An Agilent microarray is being used to study the genes controlled by those key regulators both in vitro and in planta. Genome sequencing. Identification of critical virulence factors is a crucial step toward a comprehensive understanding of bacterial pathogenesis, host-species specificity, and invasion of different tissues thus to design new management strategies for long term control. Treatment of citrus with copper-based bactericides is one of the most common practices used for control. However, there is potential for horizontal gene transfer of copper resistance genes from other closely and distantly related bacterial strains, which will drastically reduce the efficacy of copper bactericides. Currently, copper resistant strains of other xanthomonads, including X. a. pv. citrumelo, the citrus bacterial spot pathogen, have been isolated from fields in Florida. Understanding the potential mechanisms of copper resistance in Xac and potential horizontal gene transfer of this resistance to Xac is also important for the long-term management of citrus canker. 454, solexa, and sanger sequencing has been used to sequence several Xac related strains including: X. a. pv. aurantifolii B and C strains, Xac Aw and A* strains which have restricted host range compared to the A type strain, X. axonopodis pv. citrumelo strains (copper resistant and non-copper resistant), and Argentinian strain (copper resistant). Comparative genomics analysis is being performed to identify novel genes which may contribute to host-species specificity and copper resistance. Novel and critical virulence genes and copper resistance genes will be characterized using genetic and functional analyses. Critical genes involved in virulence and biofilm. In order to facilitate the systematic identification of critical genes involved in Xac and citrus interaction, we constructed a library containing 20,000 Xac mutants using transposon insertion mutagenesis. Mutants affected in virulence and biofilm formation are being screened. The initial screening of virulence mutants was conducted using pressure infiltration method and confirmed again with pressure infiltration and/or spray inoculation. The initial screening of biofilm mutants were performed using 96-well plates/pin - crystal violet staining. PARTICIPANTS: Jeong-soon Kim; Yinping Guo; Neha Jalan; Pankaj Trivedi; Jinyun Li, Yan Qing; Lin Yang; Vladimir Kolbasov; James Graham; Jeffery Jones TARGET AUDIENCES: Citrus growers and other stakeholders, citrus research community, plant pathology research community PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Microarray study. Microarray analysis is being conducted. Genome sequencing. 454, solexa, and sanger sequencing has been completed to sequence several Xac related strains including: X. a. pv. aurantifolii B and C strains, Xac Aw and A* strains which have restricted host range compared to the A type strain, X. axonopodis pv. citrumelo strains (copper resistant and non-copper resistant), and Argentinian strain (copper resistant). Critical genes involved in virulence and biofilm. Currently, total 112 mutants with reduced virulence and 69 mutants with abnormal biofilm formation of Xac have been identified by screening the mutant library. Among the screened mutants, mutated genes of more than 30 mutants have been identified including pthA and hrpM mutants. Importantly, mutation of six hypothetical genes showed dramatic virulence reduction and characterization is under way. Most mutated genes in our study have not been characterized previously in Xac or closely related strains, such as the genes encoding LPS biosynthesis, and the type II secretion system. Two mutants with disrupted galU genes encoding UTP-glucose-1-phosphate uridylyltransferase have been studied extensively. This is our initial use of the transposon approach and the result has been submitted for publication. In this study, we characterized the galU gene of Xac. The galU mutants (F6 and D12) were identified in an Xac EZ-Tn5 <R6Kγori/KAN-2>Tnp transposon library. Rescue cloning, sequence analysis and Southern blot analysis indicated that both of the two mutants had single copy of EZ-Tn5 transposon insertion in galU on Xac chromosome. Further study showed that galU was required for biosynthesis of extracellular polysaccharides (EPS), capsular polysaccharide (CPS), and biofilm formation. Mutation of galU caused loss of pathogenicity on grapefruit. Loss of pathogenicity of the galU mutant results from its inability to grow in planta rather than from its effect on virulence genes. Quantitative reverse transcription PCR assays indicate that mutation of galU did not impair the gene expression of key virulence genes such as pthA of Xac. Although D12 showed similar growth rate as the wild type strain in nutrient broth medium, its population failed to establish in the intercellular spaces of citrus leaves. Co-inoculation of a galU mutant with wild type strain did not promote the growth of the galU mutant in planta. Defects of EPS and CPS production, pathogenicity, and growth of the galU mutant in planta were complemented to wild type level using plasmid pCGU2.1 containing an intact galU gene. These data indicate that the galU gene contributes to Xac growth in the intercellular spaces and is involved in EPS and CPS synthesis and biofilm formation. This study will help identify putative targets for antimicrobial development and design new management strategies.
Publications
- Guo, Y.P., Sagaram, U.S., Kim J.S., and Wang N. 2009 The galU gene is essential for growth of Xanthomonas axonopodis pv. citri in planta. Applied and Environmental Microbiology. (Submitted).
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