Study On The Multilayered Interaction Between Phytophthora Sojae And Soybena In The Apoplast

Oomycetes are eukaryotic organisms that superficially resemble filamentous fungi,but are phylogenetically related to diatoms and brown algae in the stramenopiles.Oomycetes form a deep lineage of eukaryotic organisms that includes a large number of plant pathogens which threaten natural and managed ecosystems.Among these,many destructive pathogens of crops are in the genus Phytophthora.Some species,such as Phytophthora sojae(P.sojae),the agent of soybean(Glycine max)root and stem rot,have caused about 2 billion economic losses each year.P.sojae could deliver a series of extracellular(apoplastic)or intracellular effectors to promote infection by modulating difference plant defense signaling and enable parasitic colonization.While intracellular effectors have been extensively studied,little known about the function of apoplastic effectors of P.sojae.In this paper,we systematically identified and analyzed the extracellular effectors of P.sojae during infection process.Identify the secreted proteome of P.sojae.P.sojae could secrete a range of apoplastic proteins to help its infection during the infection stage.In order to identify those proteins during the infection stage of P.sojae.In this chapter,we extracted the apoplastic proteins of P.sojae from infected soybean leaves at different infection time as well as from synthetic medium cultured pathogen.We identified about 280 apoplastic protein contained signal peptide by (?)KTA and LC-MS/MS.Conserved domain analysis of these protein by HMMER:Forty percent were relation to cell wall degradation,such as glycosyl hydrolase family,pectate lyase family and cutinase family;Ten percent were relation to protein degradation,contained serine protease,aspartic protease and so on.Three major categories of these proteins were classified by RNA-seq data analysis:a)immediately-expressed early during infection,b)expressed later during infection,c)continuous expressed during infection.The result suggested that the apoplast contained many difference kinds of protein and unknown pathogenic mechanisms.Analysis of the virulence function and N-glycosylation of PsXEG1.In our previous work,we identified a novel apoplastic effector-PsXEG1,belonged to GH12 glycosyl hydrolase.Silencing PsXEG1 in the genome of P.sojae reduces the virulence.In this chapter,we found that P.sojae can secrete two different forms of PsXEG1.Further biochemical experiments showed that PsXEGl could undergo N-glycosylation by P.sojae.Mass spectrometry was performed on PsXEG1 protein purified from P.sojae.Two asparagine N174 and N190 were identified as N-glycosylation sites of PsXEG1.In order to verify whether N-glycosylation is involved in the virulence function of PsXEG1,We used the CRISPR/Cas9 to mutate the two PsXEGl glycosylation sites in P.sojae genome.Two independent PsXEG1N174A&190A mutants were identified by PCR,Sanger sequencing traces and single spore purified.Two mutants(T91,T192)shown significant reduction of virulence on etiolated soybean hypocotyl,suggest that N-glycosylation involved in PsXEG1 virulence function.Further studies have shown that N-glycosylation does not affect the secretion of PsXEGl and its enzyme activity.While PsXEG1N174A&N190A was rapidly degraded in the extracellular space but can be inhibited by protease inhibitors,indicated that N-glycosylation was involved in the stability of PsXEG1 affecting its virulence function.Moreover,aspartic acid protease inhibitors could inhibit the degradation of PsXEG1 indicated that aspartic acid protease of soybean was involved in the degradation of PsXEG1.Interestingly,glycosylation also reduced PsXEG1 binding to GmGIP1,according to the results of Co-IP,pull-down,and bio-layer interferometry(BLI)experiments.Together,P.sojae could secrete two different forms of PsXEGl protein by N-glycosylation.N-glycosylation could shield PsXEG1 from aspartic protease degradation and GmGIP1 inhibition.Functional analysis of the soybean aspartic protease GmAP5 in host resistance.To identify the aspartic protease involved in non-glycosylated PsXEG1 degradation,we analyzed the aspartic proteases of soybean.The apoplast-localized PsXEG1 inhibitor,GmGIP 1,is a non-active aspartic protease-like protein,which directly interacts with PsXEG1.This suggested that enzyme-active homologs of GmGIP1 might target PsXEG1 for degradation.Genome-wide analysis identified 60 GmGIP1 homologs,29 of them as putative active aspartic proteases.RNA sequencing showed that two of these homologs exhibited similar timing of transcript accumulation as PsXEG1 during early infection by P.sojae.To test the interaction between PsXEG1 and these two protease candidates,we performed coimmunoprecipitation(Co-IP)assays after transient co-expression of the proteins in N.henthamiana leaves.The results revealed that Glyma.11G215200.1(hereafter named GmAP5),could bind to and degrade PsXEG1;the input western blot confirmed degradation by GmAP5 but not by any of the other co-expressed soybean proteins.Overexpression of GmAP5 in soybean hairy roots enhanced soybean resistance against P.sojae.Together,these results indicated that GmAP5 could contribute positively to soybean resistance against P.sojae,presumably due to PsXEG1 degradation.To assess the N-glycosylation take part in the specificity of the interaction between GmAP5 and PsXEG1 during P.sojae infection,we inoculated soybean hairy roots overexpressing or silencing GmAP5 with two P.sojae strains carrying mutations in their PsXEG1 genes introduced by CRISPR,namely T91(PsXEG1N174A&N190A)and T3(PsXEG1△).The T91 transformant expressing un-glycosylated PsXEG1 regained close to wildtype virulence on GmAP5-silenced transgenic plants.In contrast,T91 infection was drastically reduced compared to wildtype when GmAP5 was over-expressed in the roots.However,when the PsXEG1 knockout line,T3,was tested,GmAP5 silencing or over-expression produced no significant changes in infection levels.These results indicate that the role of GmAP5 in soybean defense depends on the presence of its target PsXEG1 and that N-glycosylation substantially shields PsXEG1 from GmAP5 attack during P.sojae infection.Meanwhile,PsXLP1,the decoy that protects PsXEG1 from GmGIP1,is immune to GmAP5 degradation as a result of the C-terminal deletion it carries.The crucial role of N-glycosylation within the two layers of defense and counter-defense centered on PsXEG1 highlight the critical importance of this conserved apoplastic effector and its post-translational modification in Phytophthora-host co-evolutionary conflict.Functional analysis of soybean secreted protein GmPNGase A.Analysis of P.sojae proteome by N-glycosylation conserved domain(NXS/T)revealed that most secreted proteins contained potential glycosylation sites.Previous studies have found N-glycosylation could protect the effectors from plant proteases degradation and inhibitors inhibition in the apoplast for virulence function.But few known about how the plant repress to the Nglycosylation of pathogens?In this chapter,the extracellular proteins extracted from soybean leaves infected by P.sojae were separated by molecular sieve,and a total of 9 fractions were obtained.Using glycoprotein RNase B as a substrate,the enzyme activity of different fractions was analyzed.A soybean secreted protein of GmPNGase A was identified from apoplastic fluid fractions of soybean leaves infected by P.sojae using LS-MS/MS.It was found that overexpression of GmPNGase A in N.benthamiana could inhibit the infection of P.parasitica,indicating that GmPNGase A is involved in plant resistance to Phytophthora.GmPNGase A purified from Pichia pastoris has de-glycosylase activity,and found that it could remove the N-glycosylation of PsXEG1,an important virulence effector of P.sojae.Furthermore,bioinformatics analysis shown that the homologous gene of GmPNGase A was widely existed in plants,indicating GmPNGase A taken part in host resistance to pathogen.Together,Phytophthora employed N-glycosylation to protect the effectors from plant proteases degradation and inhibitors inhibition in the apoplast for virulence function,while host could secrete GmPNGase A to de-glycosylate of effectors for host resistance.