The Study Of Virulence Related Regulators HpaR1 And RpfG In The Plant Pathogen Xanthomonas Campestris

With the growing world population,the domestic demand for staple crops is rising.As a consequence plant health is major priority.Understanding how bacterial pathogens regulate and cause disease has become paramount to suggesting strategies for control and improving plant health.Xanthomonas is a large genus of Gram-negative bacteria that cause disease in hundreds of plant hosts.Xanthomonas campestris pv.campestris（Xcc）is an important member of this genus as it causes black rot in many cruciferous plants of major economic importance worldwide.Like other bacterial pathogens,Xcc undergoes various adaptive changes in order to colonize the host and infiltrate the host tissues to cause disease.In order to control such processes Xcc has evolved numerous regulatory systems to sense and respond to environmental stimuli in order to modulate its behavior and the expression of various virulence determinants.The work in this thesis aims to identify novel virulence-related regulators in Xcc and in parallel expand our understanding and function of a previously identified global regulator shown to be involved in pathogensis.The GntR family of regulators is the most abundant and widely distributed group of helix-turn-helix transcriptional regulators in bacteria.Many important GntR family members have been shown to regulate various cellular processes including virulence and anti-microbial resistance.Although the function of the predicted GntR regulators found in the genus Xanthomonas are unknown.Examining the genome of Xcc 8004,we identified six open reading frames that were predicted to encode putative GntR-like regulators.Systematic mutagenesis of all genes encoding predicted GntR regulators and further examination for alterations in plant disease response revealed that one mutant is involved in the hypersensitive response（HR）and virulence.We designated this new identified virulence-related regulator HpaR1.Transcriptional analysis using gusA-reporter fusions and quantitative RT-PCR analyses revealed that HpaRl divergently regulates the expression of genes involved in HR and pathogenicity during host plant colonization or growth in laboratory media.Furthermore,constitutive expression of hrpG,a key regulator of hrp Type Three Secretion System（T3SS）,in the hpaR1 mutant background could bypass the requirement of HpaRl for the induction of wild-type HR,suggesting that HpaRl regulates the expression of hrp genes that encode the T3SS via hrpG.Taken together,this section of work reveals that the transcriptional regulator HpaRl,a member of GntR family,plays an important role in HR and virulence in Xcc.Although,the presence of other regulatory targets remains a possible,we have demonstrated that the first regulator examined HpaRl regulates the expression of hrp genes that encode T3SS via the key hrp regulator HrpG.The second response regulator examined was RpfG.This regulator has been shown to play a major regulatory role in the Diffusible Signal Factor（DSF）cell-to-cell signalling system that is crucial for pathogenesis,and biofilm formation in Xcc.This signalling system is mediated by a cis-unsaturated fatty acid diffusible signal called DSF.The synthesis of DSF is fully dependent on RpfF,whereas DSF perception is believed to involve an unusual two-component regulatory system encoded by the rpfGHC operon,which is adjacent to and convergently transcribed with rpfF.RpfC is a complex sensor kinase and the RpfG encodes a HD-GYP domain that is involved in cyclic di-GMP degradation.Although a number of studies have focused on the regulatory influence of the sensor kinase RpfC,little is known about the full regulatory influence of RpfG in Xcc.To address this issue,we deployed an RNA-Seq approach to compare the transcriptomes of wild-type and rpfG mutant grown in complex media.We identified 112 differentially expressed genes（genes with≥4.0 fold changes and statistically significant are considered dif-ferentially expressed）.Interestedly,a group of novel genes were identified in the wild-type strain and a number of these novel genes were previously unidentified in the reference annotation were differentially expressed in rpfG mutant.Importantly,quantitative RT-PCR confirmed many of transcriptional changes seen by RNA-Seq.14 genes,which were previously unknown to be regulated by RpfG,were chosen from the 112 differentially genes for further study.Disruption of these genes led to significant reduction in virulence in Chinese Radish leaf clipping assay（Virulence grade<level III）,while there was no obvious difference in HR.Additional examination of mutants for changes in virulence traits associated with Rpf/DSF regulation,identified that mutation of XC₂234,XC₃555 and XC₄153 resulted in the decrease in the synthesis of endo-mannanase.While mutations in XC₂234,XC 3555,XC₄153 did not give rise alteration in motility on 0.5%agar.Importantly,in trans expression of full-length genes in corresponding mutant background restored phenotypes towards wild-type suggesting the alternation in phenotype is due in part to disruption of the gene in question.Here the work in this section reveals for the first time the transcriptional scope of regulation by RpfG in Xcc and highlights the complexity of regulation by the Rpf/DSF cell-to-cell signaling system.Furthermore,this global approach identifies a multitude of novel genetic elements regulated by the Rpf/DSF cell-to-cell signaling system that are involved in virulence.Taken together,a new GntR family regulator with a role in virulence was identified in this work and the scope of regulation of previously recognized response regulator RpfG was examined.This work revealed the regulation by which two classes of regulator can play a significant role in virulence and phyto-pathogenesis in Xcc.