Function And Mechanism Of RXLR Effector Avh23 From Phytophthora Sojae

Oomycetes are a group of microbial eukaryotes with a wide range of hosts that can cause disease in plants and animals.The genus Phytophthora contains about 160 species,most of which are the notorious pathogens of crops.For instance,the potato and tomato pathogen Phytophthora infestans triggered the Irish Famine in the 19th century and remains a serious problem worldwide;and remains cause around $6.7 billion in losses per year worldwide;Phytophthora sojae is one of the most destructive pathogens of soybean,causing around $1-2 billion in losses per year worldwide.Due to the lack of understanding of its pathogenesis,which limits the development of disease prevention and control technology.Therefore,study of its pathogenesis will help us find the effective strategy to solve this problem.Bioinformatics analysis found that the genome of the P.sojae encodes about 400 candidate effectors carrying the RXLR-dEER motif,most of which have the function of suppressing the plant immunity.Although several effectors have been shown to suppress plant immunity,the functions of the vast majority of effectors remain unknown.In our previously study,transcriptome analysis showed that a large number of RXLR effector were up-regulated at the stage of infection,and also suppressed the plant immunity.In this study,we further elaborated the mechanism of the key effector Avh23 suppressing plant immunity.Avh23 was induced during infection,and interacted with ADA2,a subunit of histone acetyltransferase(HAT)complex SAGA,to regulate GCN5-mediated histone acetylation levels and thereby suppressed plant immunity and promoted infection.This study elucidates the mechanism by which Avh23 suppresses plant immunity at an epigenetic level.Functional analysis of Avh23.During infection,Avh23 is induced and reaches the maximum at 12-24 hpi.Avh23 has no polymorphism among the four physiological races of P.sojae;and also has no homologous protein in P.parasitica,P.infestans,P.ramorum,P.capsici,Hyaloperonospora arabidopsidi.A domain and motif searches using the SMART database identified two internal repeats(IRs),namely,IR1,and IR2,in the C-terminus of Avh23.Avh23 can suppress Bax,INF1,Avh241,Avh238 and XEG1 induced cell death.Furthermore,the P.sojae mutants exhibited the decreasion of pathogenicity.Taken together,These results demonstrated that Avh23 plays an important role during P.sojae infection of soybean.Molecular target identification of Avh23 suppressing plant immunity.Expressing of Avh23 in soybean hairy roots and Nicotiana benthamiana leaves promotes the Phytophthora infection,indicating that Avh23 can suppress the plant immunity.To better understand the virulence mechanism of Avh23,we performed yeast two-hybrid(Y2H)screens using a P.sojae-infected soybean cDNA library and investigated proteins potentially associating with Avh23 in host cells.One gene that was repeatedly identified in four independent screens encodes a protein that is similar to the ADA2 subunit of the prototypical nucleosome-acetylating modification complex SAGA.ADA2 is a subunit of HAT complex SAGA,associating with the catalytic subunit GCN5 to regulate the function of SAGA complex.We found that Avh23 interacted with plant ADA2 proteins,and the FVxR(Phe-Val-any-Arg)sequences in the C-terminal of Avh23 are required for the interaction.Avh23 binds to the ADA2 subunit and interferes the association of ADA2 with the catalytic subunit GCN5,thereby disrupts ADA2-GCN5 subcomplex formation.As such,Avh23 suppresses H3K9 acetylation levels mediated by the ADA2/GCN5 module and increases plant susceptibility.Moreover,Avh23 re-localizes GmADA2-1 from nuclear speckles to nucleoplasm.Silencing of ADA2/GCN5 in soybean hairy roots and N.benthamiana resultes in promoting infection,and the levels of H3K9ac were decreased in GmADA2 or GmGCN5-silenced hairy roots.These results indicate that ADA2 and GCN5 positively regulate plant resistance to Phytophthora.The reduced virulence of Avh23 mutant T94 can be partially restored in GmADA2 or GmGCN5-silenced soybean roots,indicating that Avh23 promotes Phytophthora infection by affecting the function of ADA2-GCN5 subcomplex.Taken together,these results demonstrate that Avh23 reduces GCN5-mediated H3K9ac levels in soybean in an AD A2-dependent manner to promote infection.Avh23 regulates plant immunity in a GCN5-dependent manner.ADA2 and GCN5 play an important role in responding to abiotic stress,but their function in response to biotic factors is unclear.Both ADA2 and GCN5 positively regulate plant resistance to Phytophthora,but the specific mechanism is unclear.In this study,we use RNA-seq to explore how ADA2 and GCN5 regulate plant immunity.RNA-seq data showed that 484 genes were up-regulated and 1712 genes were down-regulated in the Avh23-expressing(35S-Avh23)hairy roots compared with the control hairy roots(EV);1206 genes were up-regulated and 3202 genes were down-regulated in the GmGCN5-silenced(GmGCN5-RNAi)hairy roots.These results suggesting that a large number of genes are down-regulated in 35S-Avh23 and GmGCN5-RNAi samples.The DEG(Differentially expressed genes)profiles of PsAvh23-expressing and GmGCN5-silenced roots exhibited a positive Pearson correlation coefficient(R2=0.73).Moreover,there are 1528 genes overlapped in PsAvh23-expressing roots and GmGCN5-silenced roots,which are 190 genes up-regulation and 1338 genes down-regulation,including 141 genes involved plant defense.We further determined the impact of PsAvh23,GmGCN5 and GmADA2 on the H3K9ac levels at the promoter regions of the ten defense-related genes using chromatin immunoprecipitation(ChIP)followed by qPCR.Taken together,these results demonstrate that Avh23 reduces the levels of GCN5-mediated H3K9ac to suppress plant defense gene expression,and enhances plant susceptibility.