Identification And Functional Analysis Of Quorum-sensing Regulated Genes In Xanthomonas Oryzae Pv.Oryzicola

Xanthomonas oiyzae pv.oryzicola?Xoc?is a Xanthomonas oryzae pathovar,which caused bacterial leaf streak?BLS?in rice?Oryza sativa?.BLS constrains production of this staple crop in much of Asia and parts of Africa.Xoc penetrates the leaf mainly through leaf stomata,multiplies in the substomatal cavity and colonizes the parenchyma apoplast,and causes interveinal lesions.Diffusible signal factor?DSF?-dependent quorum sensing?QS?system is an important component part of the global regulation networks in the Xanthomonas genus,which regulates multifaceted biological functions at the community level,such as biosynthesis of virulence factors and biofilm structure.Recently,information about genome and proteome analysis has been increasing,especially for organisms that have a known genome,as a high number of sequences are available for direct comparing.Therefore,high-throughput identification of QS-mediated genes is feasible by microarray,transcriptome sequencing or two-dimensional gel electrophoresis?2-DE?at the whole-genome/proteome level,respectively.Thus,the objective of this study is to analyze the function of the DSF-dependent QS system in Xoc,identify the DSF-regulated pathogenic genes by comparative proteomic analysis,and analyze functions and transcriptional regulation of these genes.First,in this study,BLASTN analysis showed that rpf gene cluster was high conserved in Xanthomonas genus.The rpf gene cluster was cloned from Xoc strain Rs105,the core genes of which included rpfF,rpfC and rpfG.It was observed that the rpfF mutant lost the ability to produce DSF molecular compared to the wild-type strain Rs105,the mutation of rpfG impaired biosynthesis level of DSF,and rpfC and rpfC/rpfG?double genes?mutants all excessively produced DSF.Compared to the wild-type strain,mutations of rpfF,rpfC and rpfG resulted in virulence loss of Xoc,and decreased biosynthesis level of extracellular polysaccharide?EPS?at 26.9-48.5%.In L medium,Xoc wild-type strain was growing at planktonic pattern,but mutations of rpfF,rpfC and rpfG led to Xoc cell aggregation at the wall of the flaks at the air-liquid interfaces,rpfG mutant generated reticulation biofilm at the bottom of the flaks,but rpfC/rpfG mutant only generated reticulation biofilm at the bottom of the flaks.In addition,mutations of rpfF,rpfC and rpfG did not influence extracellular protease activity of Xoc,and could still excitated hypersensitive response?HR?at no-host tobacco.Then,comparative analysis of the intracellular proteomic was carried out between Xoc wild-type strain and rpfF in-frame deletion mutant[did not synthesize diffusible signal factor?DSF?]by 2-DE.The results clearly revealed that 48 protein spots were differentially expressed above the threshold ratio of 1.5 fold.Among them,18 proteins were identified by MS/MS,which were involved in nitrogen transfer,protein folding,elimination of superoxide radicals and flagellar formation.Among them,RS27 is a metallo-dependent hydrolase involved in secondary metabolite biosynthesis,transport and catabolism,which is encoded by Xoryp₀10100018570?named hshB?.These results indicated that DSF might play an important role in virulence and growth of Xoc by mediating expression of proteins.Based on deletion mutantion of genes,a novel virulence gene hshB of Xoc was identified,which encoded a putative secreted protein with a signal peptide.Sequencing and Southern blot analysis demonstrated that hshB is relatively conserved in the genus Xanthomonas,but the homologous gene of hshB was not found in X.oryzae pv.oryzae.Reverse transcription polymerase chain reaction?RT-PCR?analysis showed that hshB and its upstream gene,Xoryp₀10100018565?named hshA?,are co-transcribed in Xoc.Subsequent experimental results indicated that mutation of hshB remarkably impaired the virulence,extracellular protease activity,EPS production,growth in minimal medium and resistance to oxidative stress and bismerthiazol of Xoc.Mutation of clp,which encodes a global regulator,also gave similar phenotypes.Real-time PCR assays showed that hshB transcription was positively regulated by clp and DSF,and greatly induced by poor nutrition.This study not only found a new pathogenic gene regulated by a DSF-dependent QS system but also demonstrated why mutation of this gene leads to complete loss of virulence in terms of virulence factors,overcoming the oxidative burst of the host and growth of Xoc.The expression and transport of extracellular virulence factors were orchestrated by DSF-dependent QS system in Xanthomonas genus.In this study,the extraction method of total extracellular proteins from Xanthomonas genus was improved,by which the total extracellular protein was extracted from the culture supernate of each Xoc strain,respectively.Then,comparative analysis of the extracellular proteomic was carried out between Xoc wild-type strain Rs105 and rpfF in-frame deletion mutant?did not biosynthesize DSF molecular?'by 2-DE.The results showed that the expressions of 53 protein spots were regulated by DSF?>1.5?.Among them,50 protein spots were identified by MS/MS,47 of which were encoded by 32 genes in Xoc BLS256 genome.These genes involved in many biological functions,such as host cell wall degrading,flagellar formation,elimination of superoxide radicals,protein transport,signal transduction and nucleic acid metabolism.Nine genes of them were mutated by in-frame deletion.The results showed that the mutation of Xoryp₁0330?encoding polygalacturonase?and Xoryp₁8750?encoding serine proteases?impaired virulence of Xoc,respectively.Amino acid sequences of 35 proteins were analyzed by SignalP 3.0 Server respectively,which showed that 18 of them possessed a signal peptide at N terminal.This result indicated that extracellular proteins were mainly secreted by type ? secretion system.