Functional Analysis Of The Effector Protein PSTG₂3616 In Puccinia Striiformis F.sp.tritici

Wheat stripe rust,cuased by Puccinia striiformis f.sp.tritici?Pst?,pose a great threat to wheat production worldwide.Due to constant virulence variation of Pst,the resistance of wheat cultivar will be overcome and the disease will be prevalent.Because of the lack of efficient and reliable transformation system of Pst,the studies on functional genomics of Pst has lagged behind.Therefore,it is very important to unravell the mechanism of wheat resistance to Pst and the interaction principle between wheat and the rust fugi.The aim of this study is to perform functional analysis of the effector gene PSTG₂3616and to find its roles in virulence and regulating wheat immunity.The full-length cDNA and DNA sequences of PSTG₂3616 were obtained using RT-PCR and PCR technology.Bioinformatic methods were used to analyze molecular properties of PSTG₂3616.The yeast secretion system,agrobacterium-mediated transient expression system,quantitative real-time PCR?qRT-PCR?technique,subcellular localization technique,yeast expression system,virus induced gene silencing?VIGS?technology,the yeast two hybrid screening and prokaryotic expression assays were applied to functional characterization of PSTG₂3616.The full-length of genomic DNA of PSTG₂3616 was 937 bp with three introns.The open reading frame?ORF?of PSTG₂3616 was 699 bp in length,encoding 232 amino acids.Sequence analysis indicated that the signal peptide of PSTG₂3616 was located in the N terminal 1-22 amino acids and the absence of the transmembrane structure and nuclear localization signal?NLS?.The yeast secretion system assays indicated PSTG₂3616 is a typical secreted protein with the signal peptide for protein secretion.The secreted protein PSTG₂3616 can inhibit the PCD triggered by apoptosis protein BAX in mice.qRT-PCR assays revealed that PSTG₂3616 transcriptswas up-regulated during early infection stages of Pst and the maximum expression reached at 24 hpi which were 158-fold as high as that of resting urediospores.However,the expression of PSTG₂3616 was down-regulated gradually after72 hpi till to 264 hpi.Subcellular localization assays revealed that PSTG₂3616 was localized throughout the cells of wheat protoplasts or tobacco cells.The N-terminus of 82 to 183 of PSTG₂3616 plays virulence function.Overexpression of PSTG 23616 affected the cell morphology of fission yeast and decreased the tolerance of yeast cells to H2O2 treatment.Using barely stripe mosaic virus?BSMV?as a transient expression vector in wheat,theexpression of PSTG₂3616 in Pst was suppressed,leading to slower extension of fungal hyphae and reduced production of urediospores.Yeat two-hybrid assay indicated that PSTG₂3616 can interact with TaCAT1,TaCIPK16 and TaCIPK10 in wheat.BiFC assay revealed that PSTG₂3616 can interact with TaCIPK10 in cytoplasm and cell membrane.Furthermore,we obtained the active protein of PSTG₂3616 by using prokaryotic expression assay for its further biochemical characterization.Overall,our data indicate that PSTG₂3616may be involved in the differentiation of the infection structures of Pst and plays a virulence function on wheat via regulating wheat ROS and CBL-CIPK signaling pathways during the interaction between Pst and wheat.