Functional Studies Of The Type ? Effectors In Xanthomonas Oryzae Pv.Oryzicola

Xanthomonas oryzae pv.oryzicola(Xoc),the causal agent of bacterial leaf streak,is one of the most important bacterial pathogens in rice.Little has been known about molecular mechanisms underlying Xoc pathogenicity and the function(s)of non transcription activator-like(non-TAL)type III effectors in Xoc virulence and pathogenicity so far.In this study,we examined virulence functions of the non-TAL effectors in Xoc and molecular mechanisms underlying virulence functions of the effectors AvrBs2 and XopC2 through gene knock-out,transgenic and biochemical approaches.First,virulence to rice of the Xoc mutants with the deletion of 14 individual non-transcription activator-like effector genes was investigated.The results revealed that the avrBs2 knock-out mutant was significantly attenuated in virulence under the tested experimental conditions.By contrast,the xopAA deletion caused enhanced virulence to a certain rice cultivar,Jingang 30.Second,virulence function of AvrBs2 was further explored using the avrBs2 transgenic rice plants and cell suspensions.Pathogen-associated molecular pattern(PAMP)-triggered immune responses including generation of reactive oxygen species and induced expression of pathogenesis-related genes were strongly suppressed in the AvrBs2-expressing transgenic rice cell suspensions.Meanwhile,induced expression of AvrBs2 in the transgenic rice plants also inhibited PAMP-triggered expression of defense-related genes.Although not inhibiting flg22-induced activation of mitogen-activated protein kinases,heterologous expression of AvrBs2 greatly promotes disease progression in rice caused by two important bacterial pathogens X.oryzae pvs.oryzae and oryzicola.Collectively,these results indicate that AvrBs2 is an essential virulence factor that contributes to Xoc virulence through inhibiting defense responses and promoting bacterial multiplication in monocot rice.Furthermore,target proteins of the Xoc effector XopC2 in host rice were confirmed through bimolecular fluorescence complementation,pull-down and coimmuniprecipitation assays.XopC2 physically interacted with OsSLRl,a key protein in gibberellic acid(GA)signaling transduction and OsJAZ5,OsJAZ7,OsJAZ8 and OsJAZ9,key proteins in jasmonate(JA)signaling.OsSLRl and OsJAZ3,OsJAZ9 could also interact in vivo and in vitro.In addition,we demonstrated that OsSLRl interacted with XopC2 and OsJAZs through its GRAS domain,and that OsJAZ9 interacted with XopC2 and OsSLRl through its ZIM and Jas domain,respectively.Finally,it was demonstrated that XopC2 could physically interact with AtJAZ 1,AtJAZ4,AtJAZ6,AtJAZ9 and AtJAZ11 of Arabidopsis.Virulence function of XopC2 was further confirmed using the xopC2 transgenic Arabidopsis plants.Heterologous expression of XopC2 in Arabidopsis greatly promoted JA-induced expression of AtPDF1.2 and AtLOX2.By contrast,XopC2 inhibited salicylic acid-induced AtPRl expression and pathogen-triggered stomata closure.Moreover,XopC2 could promote disease progression in Arabidopsis caused by the coronatine-deficient strain Pseudomonas syringae pv.tomato(Pst)DC3118.The results suggest that XopC2 functions similarly to coronatine and suppresses stomata-mediated defenses and thus disarms plant immunity.In summary,our results indicate that AvrBs2 is a key virulence factor in X.oryzae pv.oryzicola and contributes to bacterial virulence through suppressing plant immunity.Meanwhile,we revealed that the Xoc effector XopC2 disrupted plant immunity through interacting with OsSLR1 and OsJAZ proteins.The findings in this study lay a solid foundation to elucidate molecular mechanisms underlying Xoc pathogenicity.