Functional Characterization And Mechanism Analysis Of Phytophthora Sojae RXLR Effector Avh238 And Avh241

Phytophthora sojae was identified as a fast-spreading and devastating root and stem rot pathogen of soybean.The disease has threatened the yield of soybean in almost all soybean production regions around the globe.Resistant varieties are effective to protect soybean against P.sojae,but the high fruquency of evolution on avirulence genes in P.sojae makes resistance breeding using Rps genes not always keep for long a time.In order to develop improved methods for controlling P.sojae infection,it is essential to understand the mechanisms by which these pathogens break down plant defences.Bioinformatics analysis shows that the genome of the soybean pathogen P.sojae encodes nearly 400 candidate effectors carrying the host cell entry motif RxLR-dEER.Previously,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.Here we described the function of two P.sojae RXLR effector Avh238 and Avh241 during infection,and investigated the molecular pathogenesis involved,respectively.The main results are showed as follows:Natural variation and cell death induction of P.sojae RXLR effector Avh238.We mapped the regions responsible for Avh238 function in plant cell death induction using a strategy that combines investigation of natural variation and large-scale mutagenesis assays.The correlation between cellular localization and Avh238 functions was also evaluated.We found that Avh238 induced cell death in several different plants,the 79th residue(histidine or leucine)of Avh238 determined its cell death-inducing activity.Silencing of NbMEK2 in Nicotiana benthamiana attenuated the cell death.Transient expression of Avh238 in Nicotiana benthamiana revealed that nuclear localization is essential for triggering cell death.Our results demonstrate a representative example of an essential Phytophthora RXLR effector that can evolve to escape recognition by the host by mutating one nucleotide site in planta.Analysis of the virulence function of RXLR effector Avh238.By using CRISPR/Cas9 mediated gene disruption,we found that Avh238 is essential for the full virulence of P.sojae in soybean.To investigate whether the virulence function of Avh238 is associated with its cytoplasmic localization,we used the nuclear-and cytoplasmic-targeted Avh238 to see which one could suppress INF 1-triggered cell death N.benthmiana leaves,and the results showed that the cytoplasmic localization of Avh238 is required for the suppression of INF 1-induced cell death.The deletion mutants further revealed that the 53 amino acids in the C terminal of Avh238 is sufficient to suppress INF 1-induced cell death.Transiently overexpression of Avh238 and its mutants or overexpression of Avh238P7076-GFP in P.capsici significantly increased the virulence of P.capsici in N.benthamiana leaves also confirmed its virulence function.Replace of Avh238P6497 by Avh238P7076,which can not induce cell death in N.benthamiana leaves,did not influence its pathogenicity,indicating that the cell death-inducing activity did not contribute to its virulence function.Target identification and virulent mechanism of Avh238.By using coimmunoprecipitation(co-IP)and liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis,we identified the 1-aminocyclopropane-1-carboxylate synthase(ACS)isoforms,key enzymes in ethylene(ET)biosynthesis,as a host target of Avh238.We show that Avh238 interacts with soybean ACSs(GmACSs)in vivo and in vitro.By destabilizing GmACSs,Avh238 suppresses ACS-participated ethylene(ET)biosynthesis and facilitates Phytophthora infection.Silencing of GmACSs or inhibition of ET signaling increases susceptibility to P.sojae infection,supporting a role for GmACSs and ET in anti-P.sojae immunity.Moreover,wild-type P.sojae but not the Avh238-disrupted mutants,inhibits ET induction and promotes P.sojae multiplication in soybean.This work highlights the ET biosynthesis pathway as an anti-P.sojae defense mechanism and a direct effector target.Target identification and related mechanism of Avh241.By using coimmunoprecipitation(co-IP)and liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis in Nicotiana benthamiana,we identified the NDR1(non-race-specific disease resistance 1)isoforms as a host target of Avh241.We show that Avh241 interacts with N.benthamiana and soybean NDR1(NbNDRl,GmNDR1-1,GmNDR1-2)in vivo.NDR1 was reported as a resistance signaling component for the recognition of effectors by matching R proteins.Avh241 induces cell death in virous non-host plants,we investigate whether NDR1 involved in the cell death that triggered by Avh241.The results showed that silencing of NbNDR1 does not affect the cell death triggered by Avh241,indicating that NDR1 may involved in the virulence function of Avh241.NDR1 play an important role in plant ETI by interacting with RIN4,then we speculate whether Avh241 disrupt the interaction between NDR1 and RIN4.However,we found that Avh241 can not disrupt the interaction between NDR1 and RIN4.On the other hand,overexpression of NDR1 in N.benthamiana followed by inoculation with P.capsici experiment demonstrated that NDR1 induce resistance to P.capsici in N.benthamiana,indicating the important role of NDR1 in plant immunity.Further study found that the dimerization of NDR1 is important for its role in anti-Phytophthora.Importantly,Avh241 disrupted the dimerization of NDR1 and promoted the infection of P.sojae.