Functional Analysis Of PvRxLR16 And PvRxLR28 Effectors Of Plasmopara Viticola

The RxLR effector family,produced by oomycete pathogens,can manipulate host physiological and biochemical events inside host cells to promote growth and colonization.The oomycete Plasmopara viticola([Berk.et Curt.]Berl.et de Toni)is an obligate biotroph that causes downy mildew,one of the most devastating diseases in grapevine.A total of 31 putative RxLR effectors from a P.viticola isolate 'ZJ-1-1' have been recently identified by RNA-Seq analysis in our lab.However,their roles in pathogenesis are poorly understood.In this study,we attempted to characterize and analyse these RxLR effector candidates identified from P.viticola,with focusing on two of these effectors on their pathogenic virulence,which are of great significance to elucidate the mechanisms underlying the interaction of P.viticola and grapevine.During host infection stages,expression patterns of the effector genes were varied and could be categorized into four different groups.By using transient expression assays in Nicotianal benthamiana,we found that 17 of these effector candidates fully suppressed programmed cell death elicited by a range of cell death-inducing proteins,including BAX,INF1,PsCRN63,PsojNIP,PvRxLR16 and R3a/Avr3a.These results indicate that most PvRxLRs tested in this study can broadly suppress programmed cell death.We also discovered that 6 effector proteins strictly localized to the nucleus,while 16 of them localized to both nucleus and the cytoplasm.The only exception is the effector PvRxLR55 that was only detected in the cell membrane.According to our results,the plant cell nucleus is one of the main targets for pathogen effectors.In order to define the influence of PvRxLRs on plant immunity,we identified a single effector,PvRxLR28,that showed the highest expression level at 6 hpi and have the ability to broadly suppress PCD.Further analysis revealed that its cell death suppression relies on the C-terminus and nuclear localization.Transient and stable expression of PvRxLR28 could impair plant resistance to pathogens and reduces the transcriptional levels of the defense-related genes and impairs the H2O2 accumulation in N.benthamiana,indicating PvRxLR28 may act as broad suppressors of cell death to manipulate immunity in plant.PvRxLR16 is the only effector that can induce cell death in N.benthamiana and it strictly localized to plant nucleus.Deletion mutants,site-specific mutations and localization mutations of PvRxLR 16 were analyzed using agroinfiltration in N.benthamiana.The nuclear localization,W/Y/L motifs,and a putatve N-glycosylation site Asn-240 in C-terminal of PvRxLR16 were essential for cell death-inducing activity.To determine the involvement of SGT1,HSP90,RAR1 and SERK3 in induction of cell death by PvRxLR16,we performed a VIGS assay against these genes in N.benthamiana.Results indicate that PvRxLR16-triggered cell death is dependent on SGT1,Hsp90 and RAR1,but independent of Serk3/Bak1.Some mitogen-activated protein kinases and transcription factors were also involved in the perception of PvRxLR16 by N.benthamiana.Transient expression of PvRxLR16 in N.benthamiana enhanced the expression of defence-associated genes and promoted ROS accumulation to induce plant resistance to Phytophthora capsici.However,the defense responses and disease resistance induced by PvRxLR16 could be suppressed by other PvRxLR effectors,suggesting that it may actually not trigger host cell death or immune responses during physiological infection under natural conditions.Y2H screening the cDNA library of downy mildew early-infected 'Shuanghong' leaves using PvRxLR16 as bait found that it can interact with a CUE motif containing protein.The interaction between PvRxLR16 and VaCUE protein were also verified by bimolecular fluorescence complementation(BiFC)and Co-Immunoprecipitation(Co-IP)assays.The BiFC assay also indicated that the W/Y/L motifs and three putative N-glycosylation sites were essential for their interaction.Although it has been proved that the expression of VaCUE was induced by P.viticola and effector PvRxLR16 interacted with VaCUE,further research is needed to understand specific regulatory mechanism by the effector.