Functional Analysis Of RxLR Effector Avh262 And Avh172 From Phytophthora Sojae

The oomycete genus Phytophthora contains over 120 species,most of which are destructive plant pathogens.Phytophthora sojae which causes Phytophthora root and stem rot of soybean is a soil-borne and hemibiotrophic oomycete pathogen that has become endemic in almost all of the world’s soybean production regions.This pathogen results in average annual losses of approximately one to two billion US dollars worldwide.To control this disease,there is an urgent need to understand the mechanisms underlying the interaction between P.sojae and soybean.Phytophthora pathogens secrete an array of specific effector proteins,such as RxLR effectors,to disarm host defenses and promote colonization in plants.Up to date,only several RxLR effectors were found to play an essential role in Phytophthora infection,mainly by targeting host immunity,and the function of the vast majority of the Phytophthora effectors remains unknown.The genome of the soybean pathogen P.sojae encodes nearly 400 candidate effectors carrying the host cell entry motif RxLR-dEER.In previous studies we found transcriptional programming and functional interactions within such RxLR effector repertoire,but the detail molecular mechanisms to suppress plant immunity are still largely unknown.The subcellular localizations of 38 effectors were observed when expressed in N.benthamiana,and we chose vh262 and Avhl72 for the detail functional analysis.Meanwhile we identified the differential regulation of defense-related proteins in soybean during compatible and incompatible interactions between Phytophthora sojae and soybean by comparative proteomic analysis.Firstly,we analyzed the subcellular localizations of effectors,which are the virulence essential effectors,cell death-inducer or PTI/ETI suppressors.We found there is 26effectors contain the NLS(Nuclear localization signal)sequences,which the 8 effectors of them we identified localize to the nucleus,such as Avh52.The localization of Avh52 is irregular in the nucleus,which the NLS is essential for the localization and enhancing the Phytophthora infection.Meanwhile,subcellular localizations of 38 effectors were observed when expressed in N.benthamiana,including the cytoplasm,plasma membrane,and/or cell nucleus.Most of the effectors localize to the nucleus and/or cytoplasm,effectors localize around the haustoria in infected plant cells were also observed,including Avh172,Avh262and Avrld.Interestingly,we found that Avh262 localized to the microtubule in plant cell,a novel subcellular localization pattern that has never been reported.When expressed in N.benthamiana,Avh262 could transfer to microtubule.In this study,we also performed detail functional analysis on the RxLR effector PsAvh262.We showed that the RxLR effector PsAvh262 from Phytophthora sojae,which is highly expressed during early stages of infection,is essential for the virulence of P.sojae.In addition,transient expression of PsAvh262 enhances the susceptibility of soybean(Glycine max)hairy roots and Nicotiana benthamiana leaves to Phytophthora pathogens.Using in planta co-immunoprecipitation(Co-IP)followed by liquid chromatography-tandem mass spectrometry(LC-MS/MS)and pull-down assays,plant redundant Endoplasmic Reticulum(ER)-luminal Binding immunoglobulin Proteins(BiPs)from N.benthamiana and soybean were identified as host targets of Avh262.Avh262 and its targets could localize to the ER and around the haustoria during the infection.Avh262could stabilize BiPs such that a high level of BiPs is maintained.Meanwhile,the Bips-binding domain is essential for the function in promoting the infection and stabilizing the BiPs.We also found that BiPs act as a negative regulator of plant resistance to Phytophthora since transient expression of BiPs in soybean hairy roots or N.benthamiana leaves enhances susceptibility and the NbBiP5-silencing can enhance plant resistance.BiPs and Avh262 attenuate BCL-2-associated protein X(BAX)-triggered cell death.In contrast,upon infection of Avh262-silenced transformants the plant BiPs could not be stabilized and cell death occurred.Taken together,our results indicate that during early infection stage,the RxLR effector Avh262 stabilizes host BiPs to attenuate ER stress-triggered cell death,enhances the tolerance to the infection and thereby promotes Phytophthora colonization.As an IE(Immediately-Early)and essential effector,Avh172 was strongly expressed during infection.During the infection of P.sojae-Avh172mRFP,the Avhl72 localized around/to the penetration site and haustoria in the infected soybean cells.We had the similar result that the subcellular localization of eGFP-Avh172 associated with extra-haustorial membrane in host cells during the early infection periods of P.capsici:mRFP.When expressing GFP-Avh172 in N.benthamiana,we found Avh172from different races showed different localization to the nuclear or cytoplasm,but all the Avh172 variants could suppress the effector-triggered cell death.We found the C terminus of Avh172 plays a crucial role for the functions in suppressing effector-triggered cell death and localization to the nucleolus.In addition,we also expressed the Avh172 in the soybean hairy roots to identify four candidate targets by Co-IP and LC-MS/MS.Our findings suggest that during infection Avh172 could be secreted to the host cells and localized around haustoria to suppress the ETI and contribute to the virulence of P.sojae.To better understand the response of soybean seedlings to the stress of infection by virulent and avirulent pathogens at the proteomic level,proteins extracted from the hypocotyls of soybean reference cultivar Williams 82 infected by P.sojae P6497 and P7076,respectively,were analyzed by two-dimensional gel electrophoresis.83 protein spots were differently expressed and successfully identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.A defense-related network for soybean infected by virulent and avirulent pathogens was proposed based on the majority of the 83 defense-responsive proteins,and defense-related pathway genes supplemented by a quantitative reverse transcription PCR assay.We found reactive oxygen species(ROS)burst,the expression levels of genes related to salicylic acid(SA)signal pathway and biosynthesis of isoflavones were significantly up-regulated in the resistant soybean.Our results imply that following the P.sojae infection,ROS,SA signal pathway and the biosynthesis of isoflavones in soybean play major roles in defense against P.sojae.This research will facilitate further investigation of the molecular regulatory mechanism of the defense response in soybean following infection by P.sojae.