Functional Analysis Of Two Phytophthora Sojae RxLR Effectors PsAvh181 And PsAvh320

Soybean root rot caused by Phytophthora sojae is a devastating disease in the soybean production process,and the economic loss caused by P.sojae is as high as several billion dollars every year.P.sojae secreted effectors to help itself infecting host plants.Among these effectors,one kind of effectors located in plant cells intracellular space were named Rx LR effectors because of its special conserved N-terminal Rx LR(Arg-x-Lys-Arg)domain.Rx LR effectors were secreted into plant cells by P.sojae and localized in different parts of plant cells to attack and inhibit the components of plant immune system.Therefore,studying the function of Rx LR effectors is helpful to reveal the pathogenic mechanism of P.sojae.At the same time,do research on the interaction between effectors and target proteins of effectors can help us understand the process of host plant immune defense,and provide new guidance for the prevention of soybean root rot in practical application.In the previously work of our laboratory,we analysis the function of two early stage up-regulated Rx LR effectors Ps Avh181 and Ps Avh320.This research revealed the toxic mechanism of these two effectors by studying their target proteins in plants.The mechanism of Rx LR effector molecules Ps Avh181 and Ps Avh320 in the process of infection was preliminarily elucidated.Functional analysis of P.sojae Rx LR effector Ps Avh181: Previous studies in our laboratory showed that Ps Avh181,an effector of P.sojae,was up-regulated at the early stage of infection,indicating that Ps Avh181 might be an effector related to P.sojae infection.In this study,CRISPR-Cas9 was used to knock out Ps Avh181 in P.sojae.It was found that the pathogenicity of the mutant to soybean hypocotyls was decreased.Meanwhile,overexpression of Ps Avh181 in soybean hairy roots showed that overexpression of Ps Avh181 in soybean hairy roots could effectively promote the infection of P.sojae.The results showed that Ps Avh181 is important for soybean infection.In order to further analyze the function of Ps Avh181,we overexpressed Ps Avh181 in Nicotiana benthamiana and observed the subcellular localization of Ps Avh181.The results showed that Ps Avh181 was localized in the plasma membrane.In order to further determine the key domain of Ps Avh181 plasma membrane localization,we predicted the secondary structure of Ps Avh181 and constructed mutants based on the predicted secondary structure.By observing the subcellular localization of Ps Avh181,it was determined that the key region for the plasma membrane localization of Ps Avh181 was its N-terminal 70-95 amino acids domain,and the virulence function of Ps Avh181 depended on its plasma membrane localization.To sum up,we found that Ps Avh181 is a plasma membrane localized effector that promotes P.sojae infection,and the virulence function of Ps Avh181 depends on its plasma membrane localization.Target proteins screening and mechanism analysis of P.sojae Rx LR effector Ps Avh181: In order to further analyze the mechanism of the effector Ps Avh181 that promote infection,we used co-immunoprecipitation and protein mass spectrometry to screen the target proteins of Ps Avh181 in plants.After analyzing the sequencing results of protein mass spectrometry,according to the characteristics of Ps Avh181 located in the plasma membrane,we cloned the candidate proteins that Ps Avh181 may target in plants.After in vivo immunoprecipitation and in vitro interaction verification,N-ethylmaleimide sensitive factor attachment protein(Gm SNAP-1)was identified as the target protein of Ps Avh181.The subcellular localization of Ps Avh181 and Gm SNAP-1 was observed by laser confocal microscope.It was found that Ps Avh181 and Gm SNAP-1 were co-localized in the plasma membrane.SNAP is an important component of SNARE(SNAP receptor)complex.Overexpression of Gm SNAP-1 in soybean hairy roots showed that Gm SNAP-1 could positively regulate plant immunity,and the resistance of soybean hairy roots was weakened when silencing Gm SNAPs and its homologous genes.SNAP carries NSF(N-ethylmaleimide sensitive factor)to provide energy for the disassembly and recycling steps in the process of SNARE complex reassembly.Further studies showed that Ps Avh181 could interfere with the interaction between Gm SNAP-1 and Gm NSF both in vivo and in vitro.In order to explore the mechanism of Ps Avh181 regulating plant immunity by affecting the interaction between Gm SNAP-1 and Gm NSF,combined with previous research results: Gm SNAP-1,as an important component of SNARE,plays an important role in the process of vesicle transport.In order to verify whether Ps Avh181 affects the secretion of plant intercellular disease resistance protein by affecting the normal function of SNARE complex,we overexpressed Ps Avh181 in N.benthamiana leaves and soaked N.benthamiana leaves with zoospores released by P.capsici to induce the expression of disease resistance related proteins in N.benthamiana,and then extract N.benthamiana apoplastic fluid for protein mass spectrometry identification.Based on the results of mass spectrometry,the experiment was designed to clone the apoplastic defense proteins Gm GIP1,P69 B and Gm AP1.After these proteins were co-expressed with Ps Avh181 in N.benthamiana leaves,the secretion of Gm GIP1 and P69 B co-expressed with Ps Avh181 was significantly less than that of the control GFP protein However,Gm AP1 did not change.The above results showed that Ps Avh181 could inhibit the secretion of Gm GIP1 and P69 B,but could not inhibit the secretion of Gm AP1.In conclusion,Ps Avh181 manipulates plant immunity by interacting with Gm SNAP-1 and interfering with the interaction between Gm SNAP-1 and Gm NSF and inhibiting the secretion of Gm GIP1 and P69 B to apoplast space.Virulence function analysis of P.sojae Rx LR effector Ps Avh320: The preliminary analysis and screening results in our laboratory showed that there were more than 300 Rx LR effectors in P.sojae.In order to study more systematically that effectors attack plant target proteins with different subcellular localization,we selected a nuclear specific effector molecule Ps Avh320 to study its virulence function.After Ps Avh320 was knocked out in P.sojae by CRISPR-Cas9,it was found that the virulence of Ps Avh320 knocked-out mutant to soybean hypocotyls was weakened,and overexpression of Ps Avh320 in soybean hairy roots could promote the infection of Phytophthora to soybean hairy roots.The results showed that Ps Avh320 has virulence function when P.sojae infect hosts.In order to further study the virulence mechanism of Ps Avh320,we analyzed the sequence characteristics of Ps Avh320 and found that it contained nuclear localization signal.The localization of Ps Avh320 was observed by laser confocal microscopy.It was found that Ps Avh320 was localized in the nucleus and cytoplasm of plants.Mutation of the key site of nuclear localization signal can change the localization of Ps Avh320 and make it lose nuclear localization and locate in plant cytoplasm.Interestingly,even if Ps Avh320 lost its nuclear localization,its virulence function was not affected by its different localization.It was found that Ps Avh320 could interact with Gm SAMS by immunoprecipitation and protein mass spectrometry.Gm SAMS plays an important role in plant ethylene synthesis and methylation pathways,and participates in the regulation of plant developmental immunity.This study shows that Ps Avh320 is a virulence effector and plays a virulence role by targeting plant protein GmSAMS.