The Studies On The Regulatory Mechanisms Of Three Important Two-component Signal Transduction Systems In Xanthomonas Oryzae Pathovar Oryzae

The genus Xanthomonas comprises an important ubiquitous group of Gram-negative plant pathogenic bacteria belonging to the Gamma subdivision of Proteobacteria.Members of the genus Xanthomonas infect approximately 124 monocotyledonous and 268 dicotyledonous plants.Bacterial blight resulting from Xanthomonas oryzae pv.oryzae(Xoo)infection is one of the most destructive rice diseases.To survive and reproduce in host plant,phytopathogen must sense environmental changes.Therefore,Xanthomonas harbour an array of systems linking environmental stimuli to gene expression changes.The two-component signal transduction system(TCS)is a predominant approach by which bacteria sense and adapt to changing intracellular and extracellular environments.Numerous TCS genes are present in Xanthomonas,but only some genes have been investigated.The present study started with a global investigation of EPS(extracellular polysaccharide),swarming and virulence regulation applied to all TCS histidine kinase(HK)genes in Xoo PX099A.All 41 unique HK genes predicted were knocked out in-frame through homologous double-crossover events using the suicide vector pK18mobsacB.All but two genes,PXO_RS22690 and PXO_RS01335,among the 41 unique HK gene mutants were obtained.StoS(stress tolerance-related oxygen sensor)and SreKRS(salt response kinase,regulator,and sensor)were found to positively regulate EPS production and swarming in Xoo.Surprisingly,the absence of stoS or sreKRS did not attenuate virulence.To better understand the intrinsic functions of StoS and SreKRS,quantitative proteomics isobaric tags for relative and absolute quantitation(iTRAQ)was employed.Consistent with stoS and sreK mutants exhibiting a similar phenotype,the signalling circuits of StoS and SreKRS overlapped.Carbohydrate metabolism proteins and chemotaxis proteins,which could be responsible for EPS and swarming regulation,respectively,were reprogrammed in stoS and sreK mutants.Moreover,StoS and SreKRS were demonstrated to moderate expression of the major virulence factor,hypersensitive response and pathogenicity(Hrp)proteins through the HrpG-HrpX circuit.Most importantly,Xoo equipped with StoS and SreKRS outcompetes strains without StoS or SreKRS in co-infected rice and grows outside the host.Therefore,we propose that StoS and SreKRS adopt a novel strategy involving the moderation of Hrp protein expression and the promotion of EPS and motility to adapt to the environment.The role of all the 39 HK encoding genes on the virulence to susceptible rice breed MH63 was tested.Beside the well functional reported TCS HKs,RpfC and ColS,PhoR was also found to be essential for the full virulence of Xoo PX099A.Unexpectedly,deletion of phoB,the putative cognate RR of phoR,did not affect the virulence of Xoo.The absence of phoR could also result in the growth deficiency of Xoo,which could be one of the reason of the virulence attenuation in phoR mutant.The expression of phoBR was directly transcriptional controlled by PhoB and negatively regulated by PhoR,identified by quantitative reverse transcriptional PCR(qRT-PCR)and confirmed by electrophoretic mobility shift assay(EMSA).The over-expressed PhoB is toxic to Xoo and the expression of PhoB should be strictly controlled for the normal physiological function.The growth deficiency in phoR mutant may be the result of the over-expressed PhoB.To better understand the intrinsic regulation mechanism of PhoB/PhoR,comparative transcriptome was employed between the phoB/phoR mutants and the wild-type strain.PhoB/PhoR regulon was identified by comparative transcriptome and confirmed by qRT-PCR and EMSA.PhoB/PhoR was found to reponse and adapt phosphate limitation,confirmed by qRT-PCR and western-blot.The interesting thing is that PhoB/PhoR was found to regulate the xylose and other nutriments transport via TonB-dependent receptors which are outer membrane proteins mainly known for the transport activity in gram-negative bacteria.Through this regulation Xoo slowdown reproduction speed to adapt phosphate limitation,and that could be one of the reason why the growth of phoR mutant is slower than the wild type strain.