Project description:Xanthomonas axonopodis pv. citri (XAC) is the causal agent of citrus canker which is one of the most serious diseases of citrus. To understand the virulence mechanism of XAC, we designed and conducted genome-wide microarray analyses to characterize the regulons of HrpG and HrpX, which are critical for the pathogenicity of XAC. Our analyses revealed that the expression of 232 genes and 181 genes belonged to the HrpG and HrpX regulons, respectively. Totally, 123 genes were overlapped in the two regulons at any of the three time-points. Our results showed that HrpG and HrpX regulated all 25 T3SS genes, 22 T3SS effector genes, and 29 T2SS substrate genes. Our data showed that XAC 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, the phenolic compounds catabolism pathway (pcaQ) through HrpX and HrpG. We also identified 124 and 90 unknown genes controlled by HrpG and HrpX, respectively. Our data suggest that a cross-talk exists between HrpG and quorum sensing system in XAC. We also identified genes involved in chemotaxis and flagellar biosynthesis, production of extracellular enzymes, metabolic pathways, as well as signal transduction controlled by HrpG/HrpX. 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 XAC infection. Two mutants and three time-points experiment. Four biological and dye-swap replicates of each strain per each time-point were used. Totally, there were three datasets of hrpG mutant: 1) hrpG mutant vs. wild type strain at time-point A; 2) hrpG mutant vs. wild type strain at time-point B; 3) hrpG mutant vs. wild type strain at time-point C. There are also three datasets of hrpX mutant: 1) hrpX mutant vs. wild type strain at time-point A; 2) hrpX mutant vs. wild type strain at time-point B; 3) hrpX mutant vs. wild type strain at time-point C.

Project description:We have used the citrus GeneChip array (GPL5731) to survey the transcription profiles of sweet orange in response to the bacterial pathogens Xanthomonas axonopodis pv. citri (Xac) and Xanthomonas axonopodis pv. aurantifolii (Xaa). Xac is the causal agent of the citrus canker disease on a wide range of citrus species, including sweet oranges (Citrus sinensis). On the other hand, Xaa is pathogenic to Mexican lime (Citrus aurantifolia) only, and in sweet orange it triggers a defense response. In order to identify the genes induced during the defense response (Xaa-responsive genes) or citrus canker development (Xac-responsive genes), we conducted microarrays hybridization experiments at 6 and 48 hours after bacterial infiltration (habi). The analysis revealed that genes commonly modulated by Xac and Xaa are associated with basal defenses normally triggered by pathogen-associated molecular patterns, including those involved in reactive oxygen species production and lignification. Significantly, Xac-infected leaves showed considerable changes in the transcriptional profiles of defense-, cell wall-, vesicle trafficking- and cell growth-related genes between 6 and 48 habi. This is consistent with the notion that Xac suppresses host defenses near the beginning of the infection and simultaneously changes the physiological status of the host to promote cell enlargement and division. Finally, Xaa triggered a MAP kinase signaling pathway involving WRKY and ethylene-responsive transcriptional factors known to activate downstream defense genes. Keywords: Comprehensive transcriptional analysis of the Citrus-Xanthomonas interaction Adult leaves of sweet orange were infiltrated with the bacterial suspensions or water (mock control). Two stages were selected after bacterial infiltration for RNA extraction and hybridization on Affymetrix microarrays. In total, these experiments consist of two biological replicates of six samples: water-infiltrated leaves, Xaa-infiltrated leaves and Xac-infiltrated leaves, at both 6 and 48 (habi).

Project description:Xanthomonas is one important model microbe to study the molecular determinants of virulence and host range of pathogens since Xanthomonas is capable of infecting numerous monocotyledonous and dicotyledonous plants. Among the plant diseases caused by Xanthomonas, X. citri subsp. citri (Xcc) causes citrus canker, which has significant impact on citrus production. Xcc is classified into different strains primarily by host range including A and Aw. The A (Asiatic) strain (XccA) has a wide host range and is most virulent, whereas Aw (Wellington) strain has restricted host range including Key or Mexican lime and alemow. We hypothesized that not only gene content but also gene expression contributes to the difference in virulence and host range of closely related strains. To test our hypothesis, comparative genomic and transcriptome analyses were conducted to study the two closely related Xcc A and Aw strains. The genome of X. citri subsp. citri strain Aw12879 (Xcaw) was completely sequenced using 454 Pyrosequencing, Illumina sequencing and Optical mapping. The finished genome (5.3 Mb chromosome and two plasmids pXcaw19 and pXcaw58) of Xcaw was annotated, curated and compared with XccA genome. Protein blast revealed multiple genes including type III secretion system (TIIISS) effectors xopAF and xopAG are present in Xcaw but absent in XccA. Comparative genomic analysis showed various changes in genes encoding LPS and type IV secretion system. Furthermore, RNA-Seq was used to compare expression profile of Xcaw and XccA in nutrient rich (NB) medium and XVM2 medium which is known to mimic the intercellular space of plant cells using Illumina sequencing. Multiple avirulence/effector genes were over-expressed in Xcaw compared to XccA which might contribute to the limited host range of Xcaw compared to XccA. The overexpression of genes involved in cell wall degradation, attachment, ROS (reactive oxygen species) scavenging, nutrient transportation in XccA might contribute to its expanding of host range. Our data suggest that both gene content and gene expression contribute to difference in virulence and host range of bacterial pathogens. This study lays the foundation to further characterize the mechanisms for virulence and host range of strains of X. citri subsp. citri and other bacterial pathogens. mRNA expression profiles of Xcc strain A and Aw were generated in 2 media: NB and XVM2 by deep sequencing, in triplicate, using Illumina GAII.

Project description:Xanthomonas is one important model microbe to study the molecular determinants of virulence and host range of pathogens since Xanthomonas is capable of infecting numerous monocotyledonous and dicotyledonous plants. Among the plant diseases caused by Xanthomonas, X. citri subsp. citri (Xcc) causes citrus canker, which has significant impact on citrus production. Xcc is classified into different strains primarily by host range including A and Aw. The A (Asiatic) strain (XccA) has a wide host range and is most virulent, whereas Aw (Wellington) strain has restricted host range including Key or Mexican lime and alemow. We hypothesized that not only gene content but also gene expression contributes to the difference in virulence and host range of closely related strains. To test our hypothesis, comparative genomic and transcriptome analyses were conducted to study the two closely related Xcc A and Aw strains. The genome of X. citri subsp. citri strain Aw12879 (Xcaw) was completely sequenced using 454 Pyrosequencing, Illumina sequencing and Optical mapping. The finished genome (5.3 Mb chromosome and two plasmids pXcaw19 and pXcaw58) of Xcaw was annotated, curated and compared with XccA genome. Protein blast revealed multiple genes including type III secretion system (TIIISS) effectors xopAF and xopAG are present in Xcaw but absent in XccA. Comparative genomic analysis showed various changes in genes encoding LPS and type IV secretion system. Furthermore, RNA-Seq was used to compare expression profile of Xcaw and XccA in nutrient rich (NB) medium and XVM2 medium which is known to mimic the intercellular space of plant cells using Illumina sequencing. Multiple avirulence/effector genes were over-expressed in Xcaw compared to XccA which might contribute to the limited host range of Xcaw compared to XccA. The overexpression of genes involved in cell wall degradation, attachment, ROS (reactive oxygen species) scavenging, nutrient transportation in XccA might contribute to its expanding of host range. Our data suggest that both gene content and gene expression contribute to difference in virulence and host range of bacterial pathogens. This study lays the foundation to further characterize the mechanisms for virulence and host range of strains of X. citri subsp. citri and other bacterial pathogens.

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A time-course transcriptome study of hrpG and hrpX regulons in Xanthomonas axonopodis pv. citri

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