Two Small Non-coding RNAs Target Type ? Secretion System To Confer Virulence In Rice Bacterial Blight Pathogen

Xanthomonas oryzae pathovar oryzae(Xoo)is one of the two variants of Xanthomonas oryzae,causing bacterial blight in rice and leading to severe rice yield reduction.Small non-coding RNA(sRNA)short sequences regulate various biological processes in all organisms,including bacteria that are animal or plant pathogens,as a key post-transcript regulator.Virulent or pathogenicity-associated sRNAs have been increasingly elucidated in animal pathogens but little is known about similar category of sRNAs in plant-pathogenic bacteria.This is particularly true regarding rice bacterial blight pathogen Xoo as studies on the virulent role of Xoo sRNAs is very limited at present.We did three aspects of research work on virulent sRNA in Xoo PX099A strain.Firstly,we identified two virulent sRNAs in the Xoo genome,named trans217 and trans3287.Then we confirmed hrpD1 coding type III secretion system apparatus protein was regulated positively as direct target of sRNA trans217.Finally,the regulating network of sRNA trans217 was revealed by transcriptome sequencing.The purpose of our study is to identify sRNA associated with pathogenicity in Xoo PX099A.The number and genomic distribution of sRNAs in Xoo were determined by bioinformatics analysis based on high throughput sequencing(sRNA-Seq)of the bacterial cultures from virulence-inducing and standard growth media,respectively.A total of 601 sRNAs including 337 cis-sRNAs and 264 trans-sRNAs were identified in the Xoo genome according to its coding region and position information in the genome.Twelve virulent sRNA candidates were screened out based on significant differences of their expression levels between the culture conditions.The differential expression of 12 sRNAs evidenced by the sRNA-Seq data was confirmed by a convincing quantitative method.Based on genetic analysis of Xoo ?sRNA mutants generated by deletion of the 12 single sRNAs,trans217 and trans3287 were characterized as virulent sRNAs.They are essential not only for the formation of bacterial blight in a susceptible rice variety Nipponbare but also for the induction of hypersensitive response(HR)in nonhost plant tobacco.Bacterial T3SS is an important channel for the delivery of effectors to host cells for infecting the host.Similar effects of gene knockout and complementation were found in the expression of hrpG and hrpX genes,which encode regulatory proteins of T3SS.These results suggest that sRNA trans217 and trans3287 are associated with the pathogenicity in rice by regulating T3SS.To confirm this hypothesis,a recombinant vector in which the T3SS effector PthXol fusing with Cya tag was constructed and transferred to PXO99A and PXO99A ?sRNA strains respectively.Consistently,secretion of a type III effector,PthXol,is blocked in ?trans217 or ?trans3287 bacterial cultures but retrieved by genetic complementation to both mutants.These results showed that sRNA trans217 and trans3287 affect pathogenicity in the way of regulating T3SS components and the secretion of PthXo1.sRNA mainly regulates biological functions by interacting with target mRNA,inhibiting or promoting translation,so target identification is an important part of sRNA function research.Firstly,target genes of trans217 was predicted on online platform TargetRNA2.By analyzing the function of trans217 and candidate targets,hrpD1 encoding type III secretion apparatus protein was determined as target gene preliminarily.We used gfp report system to verify that trans217 regulated hrpD1 directly.Trans217 was cloned into a high copy vector pZK001 and hrpD1 was cloned into a low copy vector pXG-1OSF.These two vectors were transformed into E.coli to test whether sRNA trans217 regulates the expression of target gene.The result indicated that trans217 positively regulated the expression of hrpD1 in transcriptional level and protein level,suggesting that hrpD1 is direct target of sRNA trans217.Quantitative PCR and Northern blot detected the expression of hrpD1 with or without the presence of trans217 in vivo.The result showed that the expression of hrpD1 decreased significant because of the absence of trans217.In terms of the subsequent product,no difference was observed in HrpD1 protein fused with HA tag.Hfq protein is one of the bacterial sRNA chaperones.We constructed a deletion mutant of hfq gene and revealed there was no effect on bacterial growth,pathogenicity on rice and the expression of sRNA trans217.We concluded that Hfq protein was not required for sRNA trans217 expression for the first time.Above results show that virulent sRNA trans217 plays an important role in Xoo PXO99A.In order to reveal components regulated by sRNA trans217 based on differential expression of PXO99A and ?trans217 in PSA and XOM2 medium,the transcriptome study was carried out by Huada gene company.The result suggested deletion of sRNA trans217 caused significant differential expression of 619 genes under normal culture conditions,of which 557 genes were up-regulated and 62 genes were down-regulated.In XOM2,729 genes had significant differential expression and 470 genes were up-regulated and 259 genes were down-regulated.Products of these differential genes participate in a multitude of functions,including T3SS,chemotaxis of bacteria,secretion of extracellular enzymes,two-component systems and so on.Five genes were selected for further study out of significant expression and our interest,PXO_RS08490 encoding superoxide dismutase,PXO_RS20075 encoding cell wall hydrolase,PXO_RS23345 encoding avirulence protein,PXO_RS00725 and PXO_RS23350 encoding cellulase-encoding genes.The deletion of PXO_RS08490 and PXO_RS23350 diminished the pathogenicity on rice,respectively.In addition,the lack of PXO_RS08490 gene reduced the tolerance level of PX099A to hydrogen peroxide.Bacterial cellulase activity was not relative with PXO RS23345 and PXO_RS23350.However,bacterial motility was damaged significantly because of deletion of above two genes.These findings suggest that sRNA trans217 regulate many kinds of genes in directly or indirectly manners to regulating bacterial virulence jointly.Taken together,this study proved for the first time that two virulent sRNAs trans217 and trans3287 were key factors related to pathogenicity in host rice and HR in non-host tobacco,and regulated the expression of bacterial T3SS in Xoo PX099A strain.Further,this study firstly utilized green fluorescence reporter system to prove that hrpD1 encoding the T3SS apparatus protein as direct target of sRNA trans217.Regulatory network revealed sRNA trans217 regulated bacterial T3SS,oxidase,extracellular enzymes and many other genes to confer bacterial virulence.The study identified two pathogenic sRNA trans217 and trans3287 in genome-wide,demonstrated that hrpD1 was direct target of sRNA trans217,which encoded the T3SS apparatus protein,and then revealed the regulatory network of sRNA trans217 in transcriptomic level.This study revealed pathogenic mechanism of Xoo PXO99A from virulent sRNA level,enriched bacterial pathogenicity regulatory network,and provided new insights for disease control.