The Virulence Function Of Phytophthora Sojae RXLR Effector Avr1d

Soybean root rot disease caused by Phytophthora sojae Kaufmann&Gerdemann is a devastating disease to soybean（Glycine max）.P.sojae belonging to Stramenopila Oomycota Phytophthora is a hemibiotrophic pathogen which mainly infect soybean.Study the interaction mechanism of soybean and P.sojae will guide the way to breed better resistance cultivar.Effectors as the main tool to manipulate host plant immune system are key factors of the interaction between host plants and biotrophic or hemibiotrophic pathogens.During infection to soybean,P.sojae secrete a large number of RXLR-d EER motif effectors to soybean cells to mediate soybean immune response.Avr1d was a highly expressed effector during early infection stage.But how Avr1d mediate plant immunity and promote P.sojae infection remains unknown.In this study,it was found that effector Avr1d^P6497 localized to the haustoria of the hypha 4-6 hours post infection to soybean.Soybean hairy-roots overexpressing Avr1d were more susceptible to P.sojae,indicating that Avr1d secreted from haustoria possessed a virulence function.The previous work of yeast two-hybrid screening with Avr1d as the bait protein found 4 candidate soybean U-box type E3 ubiquitin ligases including GmPUB13（Glyma12g06860）and its homologous gene GmPUB13L（Glyma11g14910）.The U-box domain of U-box type E3 ubiquitin ligases is required for the association with E2 ubiquitin conjugating enzyme and ubiquitination.The interaction of Avr1d and GmPUB13 was verified by yeast two-hybrid assay and co-immunoprecipitation assay（Co-IP）in this research.And Avr1d co-localized with GmPUB13 at the cytoplasm.Silencing of GmPUB13 and GmPUB13L in soybean hairy-roots increased resistance to P.sojae,which indicated that GmPUB13 and GmPUB13L were associated with soybean immunity to P.sojae.In vitro ubiquitination experiments of this research verified that GmPUB13 possessed E3 ubiquitin ligase activity and showed that GmPUB13 could polyubiquitinate itself.In planta experiment showed that the protein accumulation of GmPUB13 was very low when expressed in N.benthamiana leaves.MG132 improved the protein accumulation of GmPUB13 which indicated that GmPUB13 was degraded by 26S proteasome.This was consistent with the results that GmPUB13 was self-ubiquitinated in the in vitro ubiquitination experiments.To figure out the influence of ligase activity to GmPUB13protein stability,the inactive mutants GmPUB13^C263A and GmPUB13^W290A were expressed in N.benthamiana leaves and the results showed that the protein accumulation of GmPUB13 mutants was significantly higher than that of wild type GmPUB13.Altogether,GmPUB13 could regulate its protein abundance at a low amount through self-ubiquitination.It was observed that the protein accumulation of GmPUB13 was significantly higher when co-expressed with Avr1d in N.benthamiana leaves.To figure out the mechanism of how Avr1d increased the accumulation of GmPUB13 protein,7 soybean E2 ubiquitin conjugating enzymes were found to strongly interact with GmPUB13.But the intensity of the interaction between soybean E2s and GmPUB13 was weaker than the interaction between Avr1d and GmPUB13.And Avr1d interacted with GmPUB13 at the U-box domain,which indicated that Avr1d might compete with E2s for the U-box domain of GmPUB13.Further study showed that Avr1d could block the E3 ubiquitin ligase activity of GmPUB13as well as self-ubiquitination,which was consistent with the results that Avr1d increased the protein accumulation of GmPUB13 in N.benthamiana leaves.Overexpressing GmPUB13in N.benthamiana leaves could not increase P.capsici infection,while overexpressing the inactive but stable mutants GmPUB13^C263A and GmPUB13^W290A increased P.capsici infection.What’s more,the soybean hairy-roots expressing the inactive mutants GmPUB13^C263A and GmPUB13^W290A increased the infection of P.sojae as well as Avr1d knockout P.sojae mutants compared to GmPUB13 and RFP control.All these results indicated that Avr1d blocked the ubiquitination activity of GmPUB13 and utilized the inactivated but stable GmPUB13 to promote P.sojae infection.Mutation of the 90^th residue Phenylalanine（F）to Alanine（A）of Avr1d destructed its interaction with GmPUB13.The mutant Avr1d^F90A could not increase the protein accumulation of GmPUB13,also failed to block the ubiquitin ligase activity of GmPUB13in the ubiquitination experiments in vivo.What’s more,Avr1d^F90A failed to promote P.sojae infection in soybean hairy-roots,which indicated that the interaction with GmPUB13 was required for the function of Avr1d.These results revealed a mechanism that Avr1d targeted GmPUB13 and blocked its enzyme activity to promote P.sojae infection.From the genome and proteome database of soybean,a total of 133 U-box protein encoding genes were predicted and divided into 8 subgroups according to the domains except U-box domain.GmPUB13 and GmPUB13L belonged to the II group（U-box+ARM）.There were 4 U-box proteins among the candidates of Avr1d found in the previous work.In order to study the specificity of U-box proteins that Avr1d interacted with,a total of 27 U-box genes were cloned according to the gene number of each group and the gene transcriptional level post P.sojae infection and 14 of them showed interaction with Avr1d in yeasts.The U-box domain was the key region that Avr1d interacted with these U-box proteins.But no specific amino acid residues in the interacted U-box domains were found.Transcriptome data showed that the gene transcriptional level of the U-box genes interacted with Avr1d were higher than that of the uninteracted genes.These results indicated that many U-box proteins might participated in the immune process of soybean to P.sojae and Avr1d showed a specificity of groups to target a large amount of highly expressed U-box proteins which was consistent with the results that Avr1d was highly expressed at the very early infection stage.Avr1d knockout mutants obtained the pathogenicity in incompatible interaction,and could infect the soybean with resistance gene Rps1d.Though Avr1d targeted to a large amount of highly expressed U-box proteins,knockout of Avr1d had not influence to the pathogenicity of P.sojae P6497 in compatible interactions.Bioinformatics analysis showed that Avh32 was the homologous protein of Avr1d with 35%identities.There were Avr1d in P.sojae strain P7064,but not Avh32 and there were Avh32 in strain P7064 and P7076,but not Avr1d.What’s more,Avh32 and Avr1d^P7064 interacted with GmPUB13 in yeast,which indicated a possibility that Avr1d and Avh32 had similar and redundant function.This provided an explanation that why P.sojae possessed a large amount of effectors.GmPUB13 and AtPUB13 were homologous genes,and Avr1d interacted with AtPUB13 in yeast.To find the substrates of GmPUB13,yeast two-hybrid screening was performed with the mutant GmPUB13^W290A as the bait protein.And there were 85 candidate substrates in the result.According to the annotation,these candidate genes participated in plant growth and immunity,indicating that GmPUB13 regulated plant growth and immunity.A further yeast two-hybrid assay showed that 5 receptor-like kinases domains and 1 intracellular kinase interacted with GmPUB13^W290A in yeast,but not with wild type GmPUB13.These kinases were overexpressed N.benthamiana leaves and plant cell death was found on the N.benthamiana leaves that expressed Gm08g08010 in the condition of low temperature and low relative humidity,indicating that Gm08g08010 might participate in the immunity of plant cell death.But the function of these kinases and how GmPUB13mediate plant immunity by ubiquitinating these substrates remained to be uncovered.