Functional Analysis Of Disulfide Isomerase Gene Pst16567 In Puccinia Striiformis F.sp.tritici

Wheat stripe rust is an important fungal disease in wheat production.At present,there is no perfect genetic transformation system for Puccinia striiformis f.sp.tritici(Pst),and the pathogenic mechanism of Pst is not systematically studied.The variation of Pst is frequent,leading to the passive control of wheat stripe rust.Studying the gene function of Pst at molecular level is of great significance for systematically understanding the pathogenic mechanism of Pst and controlling wheat stripe rust.We screened a highly up-regulated gene Pst16567 during the interaction between wheat and Pst from the transcriptome database,and its function was preliminarily analyzed.It was found that Pst16567 was an important pathogenic gene in Pst,which provided a theoretical basis for further research on the pathogenic mechanism mediated by this gene.In this paper,Pst16567 was cloned from the c DNA of Pst by PCR.The full length of Pst16567 was 792 bp,encoding 263 amino acids.Sequence analysis indicated that Pst16567 encodes a disulfide isomerase with a signal peptide harboring 21 amino acids at N-terminal.We found that the signal peptide has secretory function verified by the validation system of secretory function of signal peptides.Transient overexpression of Pst16567 in tobacco could not inhibit programmed cell death induced by BAX.Quantitative real-time PCR(q RT-PCR)analysis showed that Pst16567 was up-regulated at the early stage of the infection,with the highest expression level at 24 hours post inoculation(hpi).Subcellular localization results showed that Pst16567 was located in the nucleus,cytoplasm and plasma membrane of wheat protoplasts.Overexpression of Pst16567 in yeast resulted in significant changes in cell morphology and shorter cell length.BSMV-mediated host-induced gene silencing(HIGS)was used to silence Pst16567.The transcriptional level of Pst16567 was significantly inhibited at 24 and 48 hpi.Compared with the controls,the sporulation of Pst in the Pst16567-silenced plants decreased significantly.At the same time,histological observation showed that the length of hypha decreased significantly at 48 hpi,and the infection area decreased significantly at 120 hpi.The results showed that the pathogenicity of Pst decreased significantly by silencing Pst16567.The transcription levels of defense-related genes in Pst16567-silenced plants were analyzed by q PT-PCR.Compared with the control plants,the expression of Ta SOD(superoxide dismutase)increased significantly at 24 hpi,while the expression of Ta NOX(plasma-membrane NADPH oxidase)decreased significantly at 24 hpi.The c DNA library in association with the compatible interaction between wheat and Pst was screened by yeast two-hybrid technology using Pst16567 as the bait protein.Candidate targets related to cell signal transduction,such as calcium-dependent protein kinase,were obtained.Therefore,we infer that Pst16567 has an important role in the growth and pathogenicity of Pst.