Function And Mechanism Of Avirulence Gene PsAvr3c From Phytophthora Sojae

Soybean root rot disease caused by Phytophthora sojae is one of most destructive disease to the soybean production,causing losse of $1-2 billion per year in the world,so use resistant soybean varieties is the most economical and effective way to control the root rot disease.The interaction between soybean and Phytophthora sojae fit for "gene for gene" hypothesis,when Phytophthora sojae carried Avr gene,and soybean carried R gene,these avirulence effector can be recognized by soybean resistance proteins and induce soybean resistance response.To investigate function and mechanism of avirulence gene from Phytophthora sojae not only better understanding molecular machemisms of these avirulence effector in plant-pathogen interaction,but also provide new ideas for control disease.Our study focus on Phytophthora sojae avirulence gene PsAvr3c,which was indentified by our lab before.In this study,we conbined genetics,biochemistry,cell biology and RNA sequencing approaches to find that PsAvr3c can target to GmSKRPs,a novel regulated protein of plant alternative splicing complex to suppress plant immunity.Avr effector genes display different types of allelic variations,including deletion,epigenetic silencing and sequence polymorphism to gain virulence.However,the molecular mechanisms of PsAvr3c genes dodge host immune surveillance remain largely unknown.Site-direct mutagenesis and population studies demonstrated a serine substitution of glycine at position 174 in PsAvr3c,that results in evading Rps3c-mediated soybean immunity.This study show the function and mechanism of PsAvr3c between Phytophthora sojae and soybean interaction.The contents are as follows:PsAvr3c is a virulent effector from Phytophthora sojae.qRT-PCR show that PsAvr3c was high expressed during early stage,this imply that PsAvr3c may play roles during P.sojae infection.We generated three PsAvr3c knockout mutants by using the CRISPR/Cas9 system,the PsAvr3c knockout mutants are less virulent on susceptible soybean hypocotyl compared to control,which demonstrate that PsAvr3c plays an important role during infection.Furthermore,transient expression PsAvr3c in the N.benthamiana or soybean hairy roots can promote pathogen infection,this imply that PsAvr3c is a virulent effector from Phytophthora sojae.In addition,a putative nuclear localization signal(NLS)at the C-terminus of PsAvr3c is predicted by Prosite software,and PsAvr3c accumulated in nucleus dependent on the NLS and localization pattern of PsAvr3c is also required for PsAvr3c enhanced susceptibility.These results provide valuable clues for further study of the virulence mechanism of PsAvr3c.PsAvr3c binds to GmSKRPs protein to reprograms host pre-mRNA splicing and subvert plant immunity.To further elucidate PsAvr3c virulence function,we performed yeast two-hybrid(Y2H)screening to fish for soybean interactors.Here we demonstrate that PsAvr3c physically binds to soybean(Glycine max)serine/lysine/arginine rich proteins GmSKRPs in vivo and in vitro,furthermore,PsAvr3c can stabilize GmSKRPs protein level,it is suggested that GmSKRPs is the host target protein of PsAvr3c.When transient expression of GmSKRP1/2 promotes pathogen infection and transient silencing of GmSKR1/2 in soybean hairy roots enhances resistance to P.sojae,ectopic expression of GFP-GmSKRP1 in N.benthaniiana also promoted P.capsici colonization,these results indicate that SKRP are negative regulators in plant immunity against Phytophthora.To further explore the biological function of GmSKRPs,transient expression and LC-MS/MS analyses found that SKRP associate with spliceosome components SR45 and RBP,immunoprecipitation results also found that GmSKRP1 also associate with U1-70K and U2AF35,these indicated that SKRP are novel proteins that associate with a complex which contains plant spliceosome components.RNA-seq data indicates that alternative splicing of pre-mRNAs from 401 soybean genes,including some defense related genes,are altered in the GmSKRP1 and PsAvr3c over-expressing lines compared to control plants.Representative splicing events mediated by GmSKRPl and PsAvr3c are tested by infection assays or by transient expression in soybean plants.Our results show that a plant pathogen effector can reprogram host pre-mRNA splicing to promote disease,and we propose that pathogens evolved such strategies to defeat host immune systems.GmSKRPs protein phosphorylation and polymer formation may be involved in its negative regulation of plant immunity.GmSKRPs protein are involve in alternative splicing process to negative regulate plant immunity,but how SKRP protein regulate pre-mRNA alternative splicing is unclear.Biochemical assay found that GmSKRP1 is phosphorylated in plant,when mutate all serine,threonine and tyrosine into alanine,GmSKRP1rmut cannot promote infection in N.benthamiana,this indicate that GmSKRPs protein phosphorylation maybe required for its negative regulation of plant immunity.Furthermore,SKRP proteins can interact with themselves in Y2H,and the Monkey tail-like motif in the C-terminal is required for interaction,when deleted the Monkey tail-like motif,SKRP proteins cannot interact with themselves as well as promote infection.The RNA-seq data also show that RNA metabolism,nucleic acid metabolism and gene expression related genes are highly enriched in the GmSKRP1 overexpression samples,furthermore,NBS-LRR genes alternative splicing also altered in the GmSKRP1 over-expressing lines.These studies provide important clues for investigating function of SKRP protein.PsAvr3c evade recognition through 174th amino acids mutation.Avr effector genes display different types of allelic variations,including deletion,epigenetic silencing and sequence polymorphism to dodge host immune surveillance.However,the molecular mechanisms of how PsAvr3c evade Rps3c recognition remains largely unknown.We combined both site direct mutagenesis and population sequence survey to identify a serine substitution of glycine at position 174 in PsAvr3c,that results in evading Rps3c-mediated soybean immunity.Further investigation revealed that this substitution does not affect PsAvr3c nuclear localization,which is required to activate Rps3c,but significantly impairs the binding affinity with a previously identified spliceosome associate protein GmSKRPs.Silencing GmSKRPs specifically impaired the PsAvr3c triggered cell death in Rps3c soybean.Thus the study uncovers that plant pathogen gains adaptation on resistant plant through key amino acid substitution,enabling us to further understand the co-evolution and interaction between P.sojae and soybean.