Functional Analysis Of Flid,Flik And Ssb_Xoc In Pathogenesis Of Xanthomonas Oryzae On Rice

Rice bacterial blight,caused by Xanthomonas oryzae pv.oryzae（Xoo）,is one of the most destructive diseases in the rice ecosystem,and quite often outbreaks intermittently and regionally in rice-growing regions of China.When the disease occurs,it severely reduces rice yield and quality,leading to huge economic loss in agriculture.Xoo produces a variety of pathogenic factors,including extracellular polysaccharides（EPS）,extracellular enzymes,type-III secreted effectors（T3SE）,biofilms,and quorum-sensing signals,which interfere with or destroy plant normal physiological processes and immunity,thereby causing bacterial blight in rice.Xoo also produces harpin-like proteins,including single-stranded DNA binding protein（Ssb）,to elicit hypersensitive response（HR）,a programmed cell death as a defense,on nonhost plants.It has been found that flagellar proteins of plant pathogenic bacteria not only induce basic defense in plants,but also participate in the pathogenesis of pathogenic bacteria.However,the roles of Xoo flagellar proteins and harpin-like proteins in invading host plants are still not fully elucidated.In this study,the two flagellar proteins,FliD and FliK of Xoo strain PXO99^A,were selected to investigate whether or not their secretion is through the T3SS by using an working system in which HR is generated by an AvrXa10-Xa10 manner in rice.The first 50 amino acid fragments of FliD and FliK were separately fused to the N-terminus truncated AvrXa10 which had a 28 amino acid deletion at the N-terminus.The fusion genes were then transferred into PXO99^A and a T3SS-deficient mutant PΔhrcU,respectively,and then the transformants were inoculated in a rice cultivar IRBB10 which contain an R gene Xa10.Interestingly,the fusion of N-terminal truncated AvrXa10 with FliK triggered HR on Xa10-harboring rice plants,implying the coevolutionary origins between flagellar type-III secretion system（fTTSS）and the type-III secretion system（T3SS）.To investigate the functions of FliD and FliK in bacterial virulence,the encoding genes of the two flagellar proteins were mutated using a SacB knockout vector,and finally the mutants ofΔfliD and ΔfliK were obtained.Compared with the wild-type,the two mutants of ΔfliD and ΔfliK did not change bacterial growth,but showed abnormal flagellar structures and reduction in bacterial motility,chemotaxis,biofilm formation and EPS production.All of the deficient phenotypes mentioned above could be recovered to the wild-type levels by the complemented strains of the two mutants.In addition,the expression levels of two disease resistance genes,OsPAL1 and OsPBZ1 in rice plants were significantly up-regulated and the lesion lengths in rice plants were both decreased byΔfliD andΔfliK,indicating that FliD and FliK were required for full virulence of Xoo.On the other hand,ΔfliD and ΔfliK were still able to stimulate HR in nonhost tobacco,while the expression levels of the defense-related genes PTI,NPR1 and APX and HR marker genes HIN1,HSR203J and rbohA in tobaccos were all up-regulated.Western blot assay indicated that mutations in fliD and fliK had no effect on the secretion of FlgD protein,which is secreted through the fTTSS.Taking together,these results demonstrated that the two flagellar proteins FliD and FliK played an essential role in the bacterial flagellum formation and virulence in plants.The previous study of our lab found that Ssb_Xoc is an harpin-like protein that triggers HR in tobacco.The protein is rich in glycine,thermostable,and lack cysteine residues.Ssb-treated tobacco and Arabidopsis plants showed improved growth and enhanced resistance to fungal pathogen infection.In this study,we used the Agrobacterium-mediated method to transfer the Ssb_Xoc gene into Nicotiana benthamiana,confirmed by（q）RT-PCR and Southern blot.Compared to the wild-type,the Ssb_Xoc transgenic lines exhibited increased growth both at the seedling and adult stages.Two growth-related genes EXPA1 and EIN2 were significantly increased in transgenic plants.When inoculated with Hpa1 protein and the nonpathogenic Pseudomonas syringae pv.tomato DC3000（PstDC3000）,Ssb _Xococ transgenic tobacco produced HR earlier than the wild-type.Correspondingly,the expression levels of the pathogenesis-related（PR）genes,PR1aand SGT1,HR marker genes,HIN1 and HSR203J,and a MAPK signaling-dependent gene MPK3,were significantly increased in Ssb_Xoc transgenic tobacco than those in the wild-type.Ssb_Xoc transgenic plants were also more resistant to the pathogen of P.syringae pv.tabaci（P.s.tabaci）,and the expressions of PR genes,including PR1a,PR2,PR4 and SGT1,were much more up-regulated than those in the wild-type.Under salt and drought stresses,the Ssb_Xoc transgenic tobacco showed seed germination rates enhanced,leaf chlorophyll retention,and proline and malondialdehyde（MDA）contents decreased.Correspondingly,the transcription levels of three tress-related genes,APX,GPX and CAT1,were significantly increased in Ssb_Xoc transgenic tobaccos.All the results suggested that Ssb_Xoc transgenic tobacco showed plant growth enhanced,HR production increased and abiotic and biotic stress tolerance improved by up-regulating the expression levels of plant growth-related,HR marker,PR and stress-related genes.