Functional Study Of Extracellular Proteins Regulated By DSF-Meditated Quorum-Sensing In Xanthomonas Oryzae PV.Oryzicola

Small DSF（Diffusible Signal Factor）-meditated Quorum Sensing （QS） play critical role in the process of infection and pathogenisis of Xanthomonas spp, yet the types and functions of secreted proteins regulated by DSF were still unknown. Bacterial leaf streak （BLS） in rice is an important disease caused by Xanthomonas oryzae pv. oryzicola（Xoc） in China. Xoc was our research target in this study, what we focus on was the types, pathogenic functions and mechanisms of secreted proteins which is regulated by DSF-meditated Quorum Sensing. The research results were as follows:（i） In preliminary study of Zhao Yancun,53proteins which was regulated by DSF were identified in Xoc （Zhao Yancun,2011）.15proteins we were interested in were chosen for futher study, their functions include small-protein mediating QS, cell structure, cell process, oxidative adaptation, intermediary metabolism, protein catabolism and hypothetical functions. Then15proteins encoding genes were in-frame deleted, and four of them, were determined to be required for full virulence in Xoc.The corresponding proteins/genes were XOC₀319（Ax21-like protein, activator of XA21-mediated immunity）, XOC₂128（polygalacturonase）, XOC₃806（protease） and XOC₁601（cysteine protease）, amang them Ax21was also a novel small-protein type QS signal. Phenotypic analysis indicated that the mutation of these four genes differentially affected many important virulence-associated functions, including bacterial colonization, extracellular polysaccharide, cell motility, biofilm formation, protease activity and anti-oxidative ability. The concrete results were as follows:the mutation of these four genes didn’t affect growth rate of Xoc, yet the colonization ability was differentially affected, and the EPS yield decreased when each of these four genes was mutated. Among them, the mutation of XOC₂128and XOC₁601impaired motility of Xoc, the mutation of XOC₀319weakened the formation of biofilms, the mutation of XOC3806or XOC₁601decreased extracellular protease activity, the mutation of XOC₃806and XOC₂128increased anti-oxidative ability. The results of qRT-PCR show that the transcriptional expression level of these genes in Arp/F compared with WT was in some extent depended on nutrient of the medium, and the results were not always consistent with the expression level of these four proteins in ArpfF compared with WT. Meanwhile, these four genes also show differential regulatory roles in transcriptional expression of ten known virulence-associated genes in Xoc. XpsE is a gene of key importance in T2SS, the result of QRT-PCR also show that transcriptional expression level of xpsE decreased significantly in ArpfF, which suggested that the function of T2SS can be regulated by DSF-meditated Quorum Sensing in some extent. In addition, Ax21（activator of XA21-mediated immunity） is a sulfated protein secreted by Xoo that was identified as a trigger of XA21-dependent innate immunity in rice and was considered to be a novel signal molecule of Quorum Sensing; XOC₀319was also an Ax21-like protein, our study show that the functions between these two kind of Quorum Sensing are probably overlapped. In conclusion, our analysis is the first complete list of DSF-regulated extracellular proteins and functions in a Xanthomonas species. Our results show that DSF-type QS played critical roles in regulation of T2SS and Ax21-mediating QS, which sheds light on the role of DSF signaling in Xoc.（ⅱ） Adhesion of bacteria to the host surface is prerequisite for successful invasion, it plays an important role in the early stage of infection, such as leaf attachment and colonization. This process is mediated by specific proteins known as adhesions. So XadA and YapH, which are secreted by T5SS, were chosen for this study. Mutants of xadA and yapH were generated; experiments and analysis about pathogenic mechanism and the relationship with QS were also carried out. We found the homologs of xadA and yapH in the genome sequences of Xoc, they were XOC₃808and XOC₂467, then deletion mutants of them were generated. Here, we provided the evidences to show that both xadA and yapH were involved in leaf attachment, EPS biosynthesis, but not associated with adherence to abiotic surface in Xoc. These two adhesins were required for optimal virulence in Xoc on host rice by spraying inoculation method. We also showed that yapH and xadA displayed differential expression patterns when cultivation in plant-cell mimicking broth and the expression of yapH can be induced by this kind of medium. Interestingly, these two genes were differentially modulated by DSF signal pathway in Xoc, yapH was positively regulated by DSF and Clp while xadA was negatively regulated by them, which suggesting that DSF-meditated QS is probably responsible for the functions of secreted adhensin proteins. Overall, our results provided a first evidence to address the important roles and regulations of two nonfimbrial adhesins in Xoc.