Functional Analysis Of The Effector Protein PSTG＿15539 In Puccinia Striiformis F.sp.Tritici

Stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),is one of the most prevalent and damaging diseases in wheat worldwide.New strains with significantly increased aggressiveness overcome those resistant varieties and have recently caused devastating epidemics at continental scales.One important limitation of research progress for obligate biotrophic fungi is the inherent difficulty in generating transgenic strains.Due to stripe rust disease gene function research is lagging behind,disease prevention and control is always in a blind state.Comprehending the pathogenesis of wheat stripe rust and the mechanism of wheat-Pst play important roles in disease prevention and control.The aim of this study was to identify the function of PSTG＿15539 gene and its interaction with host plant,and the results will provided a theoretical basis for analyzing the growth and development of the pathogen and its function in pathogenicity of Pst.The full-length c DNA of PSTG-15539 were obtained by RT-PCR.The characteristics of the amino acid sequence encoded by the effector protein were analyzed by bioinformatics software.The yeast secretion system,Agrobacterium mediated transient expression,real-time fluorescence quantitative RT-PCR(q RT-PCR)technology,subcellular localization,type III secretion system(T3SS),virus induced gene silencing(VIGS),and yeast two hybrid library screening were used to study the gene function.Sequence analysis showed that the open reading frame(ORF)of PSTG＿15539 was 624 bp in length and encoded 208 amino acids.The bioinformatics software predicted that the signal peptide was located at the N-terminal 1-20 amino acids without the transmembrane structure and the nuclear localization signal.Recombinant Agrobacterium tumefaciens containing PSTG＿15539 could inhibit programmed cell death(PCD)induced by BAX.q RT-PCR assay showed that PSTG＿15539 was up-regulated at the early infection stage of Pst,and reached the peak at 6 h and 5d.Subcellular localization assays indicated that PSTG＿15539 was distributed throught the cells of wheat protoplasts.PSTG＿15539 was secreted into wheat leaves by bacterial T3 SS and found to inhibit the PTI and ETI of wheat.Transient silencing of PSTG＿15539 resulted in inhibition of the growth and development of Pst and decreased urediospore production.The yeast two hybrid assay indicated that Ta SPOP can interact with PSTG＿15539,and BIFC assay further verified that PSTG＿15539 and Ta SPOP interacts on the cell membrane.These results indicate that PSTG＿15539 plays virulence roles in the process of stripe rust fungus and wheat interaction.