Functional Characterization Of Stripe Rust Fungus Effector Pst03495 And Generation Of Its RNAi Transgenic Wheat Plants

Wheat（Triticum aestivum）is an important crop in China with a long planting history.Stripe rust,caused by Puccinia striiformis f.sp.tritici（Pst）is one of the major threat to wheat production wordwide.Cultivation of resistant varieties is the one of the most economical and effective way to control wheat stripe rust.However,the rapid mutation of Pst and the emergence of new virulent races gradually overcome the race-specific resistance.Being a biotrophic fungus,Pst uptakes nutrients from living cells through haustoria and releases effector proteins to regulate host immune responses.Therefore,it is urgent to strengthen the functional research on effector of wheat stripe rust and further using Host-Induced Gene Silencing（HIGS）technique to create durable wheat resistant materials.A candidate effector Pst03495 was cloned and indentified.We characterized Pst03495using different methods and used HIGS technique to create durable wheat resistant materials,and the main results were as follows.1.Sequence analysis indicated that Pst03495 contains a N-terminal signal peptide and an Inhibitor-I9 domain at C-terminal,encoding a serine protease inhibitor.q RT-PCR analysis showed that Pst03495 was highly expressed during the early stage of Pst infection.Phylogenetic analysis indicated that homologs of Pst03495 were mainly found in rust fungi.2.Yeast signal sequence trap system confirmed that the signal peptide of Pst03495 was functional.Transcient expression of Pst03495 in Nicotiana benthamiana showed that Pst03495 was malinly distributed in apoplast.Protease activity analysis of apoplastic fluid collected from tobacco leaves showed that Pst03495 inhibited the activity of plant protease in apoplast.3.Pst03495 inhibited the cell death in N.benthamiana induced by Pst elicitor-like protein Pst322,and its inhibitory function was depended on the domain of Inhibitor-I9.In addition,protease inhibitor activity analysis confirmed that Pst03495 inhibited the S8 family serine protease Subtilisin A.In conclusion,Inhibitor-I9 domain is essential for both cell death suppression and protease inhibitor activity of Pst03495.4.Using host induced gene silencing technique,.After inoculating the Pst virulent race CYR31,transient silencing of Pst03495 significantly suppressed the length of hyphae and the mycelial growth area.These results indicate that the Pst03495 is involved in the pathogenicity of Pst.5.Pst03495-RNAi transgenic wheat plants were generated by Agrobacterium-mediated genetic transformation technology.Evaluation of disease resistance was conducted by inoculating the Pst virulent race CYR32.Compared with wild-type plants,Two T₂ transgenic lines,L5 and L8,showed significantly less sporulation,suppressed length of hyphae and suppressed mycelial growth area.In addition,after inoculation of avirulent race CYR23,L5and L8 showed significantly expanded the reactive oxygen species（ROS）area and enhanced apoplastic protease activity compared with wild-type plants.In conclusion,the resistance of Pst03495-RNAi transgenic plants to stripe rust is significantly enhanced.Functional characterization of the effector Pst03495 in Puccinia striiformis f.sp.tritici and the resistant materials of Pst03495-RNAi was preliminarily created,which will provide genetic resources and theoretical basis for the long-term control of stripe rust.