Screening And Functional Analysis Of Plant Immune-Inducing Proteins From Fusarium Graminearum

Fusarium graminearum is one of the most important pathogens of crops,causing serious losses in agricultural production.It can infect cereal crops such as wheat,barley and rice,and causes plant diseases such as ear rot and scab.It can also co-infect soybeans with Phytophthora sojae,causing soybean root rot.Chemical pesticides and resistant varieties are effective to protect plant against pathogens.However,the long-term irrational use of chemical pesticides will cause the pathogens to develop resistance and threaten the environment and the safety of agricultural products.The high frequency of evolution on avirulence genes in pathogens makes resistance breeding using resistance genes not always durable.Therefore,it is essential to develop improved biological pesticides for sustainable agricultural development.Plant innate immunity has the advantages of preventiveness,systemicity,safety,and stability.We hope to identify extracellular effectors of pathogens that induce immunity,and use effectors as probes to study plant immune mechanisms and provide a theoretical basis for creating new plant immune inducers.In this study,we first identified plant immune-inducing proteins in the culture filtrate of F.graminearum.Through bioinformatics analysis,six putative proteins that induce plant immunity were discovered.On this basis,we analyzed the function of two candidate proteins Fg5 and Fg12 to investigate whether they can induce plant immune response.1.Screening and identification of plant immune-inducing proteins in F.graminearum.In this study,we found that the culture filtrate of F.graminearum can induce Nicotiana benthamiana leaves cell death,and its ability to induce cell death is lost after boiling.It is speculated that the culture filtrate contains some substances with plant immune-inducing activity.Then we collected the culture filtrate and conducted mass spectrometry analysis to identify the potential proteins that might induce plant immune activity.In total,72 genes were identified.61 of these genes were successfully cloned and screened on N.benthamiana leaves.Six effectors were finally selected,which may have the activity to induce plant immunity.2.Functional analysis of plant-immunity inducing activity of Fg5.We investigated the function of Fg5 in inducing plant immunity.The result of bioinformatics analysis revealed that Fg5 is a member of the GH12 family and has glycosyl hydrolase domain.Fg5 has xyloglucan hydrolase activity and its ability to induce plant cell death depends on its signal peptide but not on its enzyme activity.Purified Fg5 protein was obtained through prokaryotic expression in Escherichia coli to further investigate its plant immune inducing activity.Both Fg5 and its mutant protein without enzyme activity could induce cell death and up-regulate the expression of defense-related genes in N.benthamiana.In addition,the purified protein was used to treat N.benthamiana leaves and soybean hypocotyls,and then inoculated with tobacco mosaic virus,F.graminearum,P.sojae and Pythium ultimum,respectively.The results showed that Fg5 induces disease resistance in tobacco to TMV,and also induces disease resistance in soybean to different pathogens,which indicating that Fg5 has the activity to induce plant immunity.3.Functional analysis of plant-immunity inducing activity of Fg12.Bioinformatics analysis found that Fg12 is a secreted ribonuclease that is widely present in different fungi,and the homologous genes of Fg12 in different fungi can induce N.benthamiana leaves cell death.The ability of Fg12 to induce plant cell death depends on its enzyme activity and signal peptide,as well as on light conditions.By prokaryotic expressing Fg12 in E.coli,we obtained the protein of Fg12 and then purified it.We found that only in high concentration can Fg12 induce cell death in N.benthamiana leaves.Fg12 is a fully functional RNases active against plant total RNA.Treating N.benthamiana with Fg12 found that it induces up-regulated expression of defense-related genes.By silencing BAK1 and SOBIR1 in N.benthamiana,we found that the ability of Fg12 to induce cell death is independent of BAK1 and SOBIR1.Besides,Fg12 induces disease resistance in soybean to F graminearum.The results demonstrated that Fg12 has the activity to induce plant immunity.