| Xanthomonas oryzae pv.oryzicola of X.oryzae pathogenic variant which belongs to the kingdom Prokaryotae in the division Gracilicutes,is the causal agent of the bacterial leaf streak disease in rice.The disease mainly causes rice leaf damage and is a serious threat in tropical and subtropical regions of rice cultivation.X.oryzae consists of two pathogenic variants,X.oryzae pv.oryzae(Xoo)and X.oryzae pv.oryzicola(Xoc).Although Xoo and Xoc belong to X.oryzae,their infection mechanism of rice plantis is different.Little is known about the pathogenic molecular mechanisms of Xoc compared with Xoo.Quorum sensing(QS)is a chemical signal-dependent and widely conserved cell-cell communication mechanism,that coordinates various bacterial behaviour and gene expression patterns in a cell density-dependent manner,by which the bacteria can rely on population density or growth stage to regulate gene expression.Quorum sensing of Xanthomonas spp.bacteria is mainly dependent on the diffusible signal molecules(DSF),and this system is highly conserved in Xanthomonas spp.In a previous study,we confirmed Xoc has DSF-dependent quorum sensing system,and rpfF gene is involved in the synthesis of DSF signal molecules.The rpfF mutant completely lost the ability to synthesize DSF signaling molecules and also lost pathogenicity ability in rice.In order to better understand quorum sensing system,and how to regulate pathogenicity of Xoc,we took a two-dimensional protein electrophoresis approach;comparative analysis of the intracellular protein expression profiles of the wild-type strain Rs105 and the rpfF mutant was carried out,and ultimately 18 proteins were identified using bioinformatics technology.The predicted functions of these 18 proteins were related to flagellar formation,anti-oxidative stress and nitrogen conversion and so on,in which a protein encoded by the gene hshB were involved.The gene hshB is necessary for pathogenicity of Xoc,and this gene is absent in Xoo.The gene hshB is regulated by the quorum sensing signal DSF and global regulatory factor clp.Deletion of the gene hshB,severely weakened pathogenic of Xoc.This reduced the ability of Xoc to secrete extracellular proteases and extracellular polysaccharides.The ability of hydrogen peroxide tolerance in Xoc was also reduced.Transcription of the gene hshB is induced by nutrient poor conditions,but specific mechanism is not clear.In this study,we first analyzed biological function domain of gene hshB by bioinformatics and further determined the biological characteristics of gene hshB.We found that cell motility of the hshB mutant was significantly lower compared to the wild-type strain.Further,RNA-seq revealed that after gene hshB deletion in Xoc,305 genes were significantly differentially expressed,and functions of these differentially expressed genes related to pathogenicity of bacteria,protease secretion,carbon metabolism,nitrogen metabolism,anti-oxidative stress,cell motility,chemotaxis and biofilm formation.Futher enrichment analysis found out that these differentially expressed genes focused on two aspects:cell motility and cell signal transduction.These results not only prove the accuracy of the determined biological characteristics of cell motility,but also provide direction for the next study.Futher analysis of RNA-seq data,indicated that genes regulated by hshB are also involved in bacterial biofilm formation.Through a variety of methods,we confirmed that mutation of the hshB gene indeed enhanced bacterial biofilm formation in comparison with the wild-type.The phenotypic mutation of hshB enhancing bacterial biofilm formation,its ability to impair cell motility alongside existing literature reveals that there is a relationship between gene hshB with c-di-GMP receptor YajQ.And we speculate that the gene hshB plays an important role in Xoc by regulating c-di-GMP concentration.This study not only reveals a new function in cell motility of gene hshB,but initially reveals regulating mechanisms of gene hshB in pathogenic bacteria,extracellular proteases,anti-oxidative stress and cell motility,etc.,as well as identify and validate the gene hshB also play an important role in the regulation of biofilm formation in Xoc by explored in depth RNA-seq data.The mechanism of gene hshB is closely related to c-di-GMP.These findings provide research directions for further specific mechanisms of gene hshB studies.After thorough research and analysis of gene hshB,we established that gene hshB in Xoc is located in the nodB-rghB locus,which is absent in Xoo.In the present study,we identified two additional genes within this locus(hshA and hshC)that were unique to Xoc compared to Xoo,and we found that the transcription of these genes was regulated by DSF signaling.The mutation of these genes impaired the virulence of the wild-type strain when using a low inoculation density of Xoc.In contrast to hshB,the mutation of these genes did not have any visible effect on characterised virulence-related functions,including;in vitro growth,extracellular polysaccharide production,extracellular protease activity and anti-oxidative ability.However,we found that mutation of hshA or hshC impaired the plant growth and epiphytic survival ability of Xoc cells,which was one of the probable mechanisms involving these genes in virulence.Collectively,our studies of Xoc have identified two novel DSF-controlled virulence-associated genes(hshA and hshC),which will add to our understanding of the regulatory mechanisms of conserved DSF virulence signalling in Xanthomonas species.In summary,this research not only reveals new functions of gene hshB in cell motility and biofilm formation,but also reveals through RNA-seq data possible mechanisms in regulation of many cell traits of gene hshB.We also established that gene hshB is located at the nodB-rghB gene cluster.Further,we identified two novel DSF-controlled virulence-associated genes(hshA and hshC).All these findings provide research directions for further studies of specific mechanisms of gene hshB.Knowledge from these studies will help us further understand Xoc,for better prevention and management of rice bacterial leaf streak disease. |
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