Assessment Of Candidate Avirulence Factors And Effector Functions Of Puccinia Striiformis F.sp.tritici

Stripe rust caused by Puccinia striiformis f.sp.tritici（Pst）is a serious disease that has restricted wheat production worldwide.A large number of research results and production practices at home and abroad have proved that rational use of wheat varieties which are resistant to stripe rust is the safest and most effective way to control stripe rust.The identification of novel pathogenic genes of stripe rust will assist understanding of pathogenesis of Pst and molecular mechanism involving the interaction between Pst and wheat host,which is of not only great scientific importance,but also great significance in agricultural production.However,on the plant side,the research of resistance genes remained at a slow pace due to the large size of the wheat genome,and the interaction mechanism of disease-related genes between wheat and stripe rust is rarely studied.NB-LRR type of wheat resistance genes YrSP and Yr10 were selected as the subject of this research,and Avr1 and Avr2 were chosen to be the candidate pathogenic genes cognate to Yr10.Different biological methods were conducted to explore the biological functions of wheat resistance genes and to identify the interaction between Yr10 and the two candidate avirulence（Avr）genes.Firstly,stripe rust infection experiments confirmed that wheat stripe rust resistance genes YrSP and Yr10 showed different seedling infection types to Pst race CYR32,and the structures of the four proteins were analyzed by bioinformatics.Secondly,different molecular cloning methods were conducted to construct the vectors harboring YrSP,Yr10,Avr1 and Avr2 genes.Transient expression experiments were carried out in the heterologous tobacco system,and it was found that BED-NLR disease-resistant protein YrSP and its“MHD”domain mutant YrSP_D648V did not induce hypersensitive response（HR）reaction.Yr10 and the predicted CC（1-135 aa）domain did not induce cell death.The mutation of“MHD”domain of Yr10 to GAG or VHD resulted in intense cell death.Therefore,it was speculated that the change of the conserved structure of MHD in Yr10 protein would lead to the alternation of the overall conformation,confirming the aspartic acid at position 502was the key residue to induce HR response.During the interaction analysis with two candidate avirulence genes,it was found that Yr10 did not interact with Avr1 and Avr2 in the early stages of expression.But the HR reaction appeared after 7 days of coinfiltration of Avr1-m RFP with Yr10 protein fused with different tags.The interaction between Yr10 and the two candidate Avr genes was further analyzed by yeast two-hybrid and protein immunoprecipitation.The results showed that Yr10 did not interact directly with Avr1 and Avr2 in the yeast system.And oligomeric complex was formed when Yr10 was co-expressed with Avr1 in the western bolt analysis,suggesting Yr10 leads to cell death via puncta formation as in the case of ZAR1.In summary,this study preliminarily explored the resistance mechanism of YrSP and Yr10,and further studied the interaction between Yr10 and candidate Avr genes Avr1 and Avr2.The preliminary analysis of wheat stripe rust resistance protein and the findings of this study lays foundation to conduct further analysis for the verification of Avr Yr10,which is to be the first stripe rust pathogen avirulence gene to date.