Identification And Functional Analysis Of Genes Involved In DSF-Mediated Quorum Sensing And C-di-GMP Signaling System In Xanthomonas Oryzae Pv.oryzae

Bacterial blight caused by infection of Xanthomonas oryzae pv.oryzae(Xoo) is one of the most important diseases of rice in the world,which becomes an important model system of study of plant-pathogen interactions.Genetic and genomic analysis has greatly improved understanding on the mechanism of its pathogenesis and molecular basis of interactions of Xoo with rice.In particular, availability of whole genome sequencing of rice and three Xoo strains has provided new opportunities and prospective for research on Xoo biology on a genome-wide scale.However,a complex regulatory pathway of cell to cell communication or quorum sensing(QS) was involved in regulation of the pathogenesis of Xoo.However,a little is known about the roles and functions of the genes involved in the global virulence regulation networks(such as QS) in Xoo.The project aimed to identify the novel genes involved in the process and signal pathways and finally develop more innovative,effective and durable strategies and methods for disease controls.To demonstrate if there is QS of Xoo in the infected leaves of rice,real-time quantitative polymerase chain reaction(real-time PCR) assay based on SYBR Green I technology was developed to target lipA and purH for quantification of bacterial population of Xoo in planta."Target gene copies -DNA amount - bacterial population - disease symptoms",a model for molecular quantification of bacterial disease was proposed and verified in bacterial leaf streak of rice caused by X.oryzae pv. oryzicola(Xooc).Bacterial accumulation in rice leaves showing no disease symptoms was observed at 3 d post-inoculation(dpi),then the bacterial density in rice increased significantly 5 dpi with increasing display of bacterial blight,and bacterial numbers reached a peak and maintained a high population at 9-14 dpi when the plants displayed severe disease symptoms.The results showed that 10⁸-10⁹cfu/g leaf tissue,the minimum bacterial density was necessary for the disease development.To identify the regulatory pathway of QS in Xoo,the production of diffusible signaling factor (DSF) was observed in Xoo as assayed in X.campestris pv.campestris(Xcc) strains XC1 using the biosensor strain FE58.Bioinformatic analysis of Xoo genomes showed that rpf gene cluster was highly homologous and conservative in sequence structure between Xoo and Xcc,which was implicated in DSF biosynthesis,signal perception and transduction.The single gene deletion mutantsΔrpfFxoo,ΔrpfCxoo,ΔrpfGxoo and double gene deletion mutantsΔrpfFCxoo andΔrpfFGxoo were generated from wild-type strain PXO99^A by using the marker exchange.Compared to PXO99^A,ΔrpfFxoo,ΔrpfFCxoo andΔrpfFGxoo are deficient in DSF production.Overproduction of DSF was found inΔrpfCxoo,while reduced production of DSF inΔrpfGxoo.DSF production ofΔrpfFxcc,ΔrpfCxcc andΔrpfGxcc mutants can be restored as the wild-type strain XC1 by in trans complementation of rpfFxoo,rpfCxoo and rpfGxoo.All of above mutants exceptΔrpfFxoo were deficient in production of extracellular polysaccharide(EPS).Meanwhile,all the mutants exhibited the reduced bacterial virulence. Results suggested that Xoo virulence depended on cell-to-cell signaling or QS mediated by DSF.The synthesis of DSF depends on RpfFxoo,whereas the RpfCxoo/RpfGxoo two-component system is implicated in DSF perception and signal transduction.RpfCxoo acted to negatively regulate DSF synthesis.RpfGxoo degraded the unusual nucleotide c-di-GMP,leading to activation of a downstream signaling cascade involving other transcriptional regulators.c-di-GMP is a second messenger with the key roles in regulation of a range of biochemical functions in virulence,biofilm formation,motility and environmental adaptation.In the genome of Xoo, 25 proteins with GGDEF,EAL or HD-GYP domains were encoded and involved into the metabolism of c-di-GMP.Mutation of a response regulator RsvRxoo(PXO₀1019),a putative c-di-GMP cyclic diguanylate phosphodiesterase with REC,GGDEF and EAL domains,led to the decreases of EPS production and virulence of Xoo.Either of REC,GGDEF and EAL domains of RsvRxoo was indispensable for its functions in EPS production and virulence expression.Further bioinformatical analysis indicated that RsvR might constitute a two-component regulatory system with RsvSxoo (PXO₀1020),both located in the same operon.Mutation of RsmRxoo(PXO₀4155),a putative c-di-GMP diguanylate cyclase with a GGDEF domain,exhibited reduced motility,but has no effect on virulence or EPS production.E.coli strain DH5αcan be stimulated to produce a high level of DSF by in trans expression of rsvRxoo and rsmRxoo.GGDEF and EAL domains of RsvRxoo are involved in this function.In addition,theΔrpfCxoo andΔrpfGxoo mutant phenotypes can be rescued by in trans expression of rsvRxoo,but rpfCxoo and rpfGxoo can not rescue rsvRxoo mutant phenotypes,suggesting that RsvRxoo and RpfCxoo/RpfGxoo constitute two parallel regulatory systems.Results outlined the existence of a regulatory network that allows Xoo to integrate bacterial population density and environmental cues to modulate virulence and environmental adaptation.In general,the present data indicated that there was a complex virulence regulation networks in Xoo including cell-density-dependent regulation and c-di-GMP signaling system.Xoo coordinated transitions in life histories and infection strategies by collecting signals from the host plant,the environment and their own population density.Two-component signal-transduction systems provided environmental signal inputs to regulate the expression of downstream genes related to virulence factors production.