Validation Of Secretion And Transcriptional Activation Of Phytophthora Sojae And Verticillium Dahliae Selected Effectors, And Identification Of Their Target Proteins

Phytophthora sojae is an important model specie for understanding oomycetes. Phytophthora root rot of soybean caused by P. sojae is a destructive disease in the worldwide. CRN gene family is unique group effectors from oomycetes. P. sojae secretes large number of CRN into the plant cells to suppress host defenses and enhance microbial fitness. Verticillium wilt, caused by Verticillium dahliae, is one of the most damaging diseases of cotton in most cotton-growing countries. This disease is also a major problem on cotton production in China.Effector proteins of biotrophic plant pathogenic fungi and oomycetes are delivered into host cells and play important roles in both disease development and disease resistance response. A mechanism for translocation of oomycete and fungal effectors has recently been uncovered. The PsISO gene of P.sojae and the VdlSO gene of V.dahliae encode isochorismatase protein. PsCRN108and PsCRN78belong to the CRN family gene of P. sojae. It is unknown whether the four genes are secreted. Here, we tested their secretion using yeast signal sequence trap assays, and found all of them could be secreted from yeast cells.Transcription factors (TFs) play crucial roles in transcriptional regulation of gene expression, by mode of activation or repression. Bioinformatics analysis showed that P. sojae cytoplasmic effector Avh138, Avh256, Avh454, Avh62and PsCRN108might have transcriptional activation. The experiment is to test the transcriptional activation of the five effectors using GAL4yeast two-hybrid systems. We showed that the fives effectors could not act as transcriptional activators in yeasts.We also screeded the targets of PsCRN115in the soybean cDNA library using yeast two-hybrid assays. Based on the results of sreening the target in the soybean library using yeast two-hybrid assays and Co-Immunoprecipitation, the genes a-tublin、G protein、 CMPG1、HSP90-2and CAT1a might interact with PsCRN115. By yeast two-hybrid technology to clearify the functional relationships among PsCRN15、PsCRN63and CAT from Nicotiana benthamiana and soybean, we proved that PsCRN115might not directly interact with PsCRN63, but directly interact with CATla, while PsCRN63may directly interact with CAT1a and CAT1b. Accordingly, we deduce that the interaction may play an important role in affecting the defense response of the plant.