Functional analysis of effector PvCRN91 from Plasmopara viticola

【Objective】 To clone PvCRN91 gene from Plasmopara viticola and verify its functions,which could pro-vide theoretical reference for revealing the pathogenic mechanism of P. viticola. 【Method】The full-length of PvCRN91gene was amplified using molecular cloning techniques,and the bioinformatics characteristics of encoded protein wereanalyzed. The effector protein PvCRN91 was transiently expressed on Nicotiana benthamiana. The performance of effec-tor protein PvCRN91 on tobacco leaves and effects on cell necrosis induced by Phytophthora infesfans elicitor and mousepro-apoptotic protein BAX were observed. The pBWA（V）HS-PvCRN91-GFP fusion expression vector was constructedand transiently expressed in grape leaves and tobacco leaves. The leaves were inoculated with P. viticola and Phytophthoranicotianae to analyze its ability to promote pathogen infection. The pBI221-PvCRN91-GFP expression vector was con-structed and transiently expressed in tobacco leaves, and the subcellular localization of PvCRN91 protein was analyzed.The pBWA（V）HS-PvCRN91-3×Flag fusion expression vector was constructed, and the target proteins interacting withPvCRN91 were screened by immunoprecipitation-mass spectrometry（IP-MS）. 【Result】 The coding region（CDS） ofgene PvCRN91 was 351 bp,encoding 116 amino acids residues with about 13.03 kD molecular weight. It did not containsignal peptides, nuclear localization sequences（ NLS） and transmembrane domains, but contained 1 Crinkler domain andconsensus disorder prediction domain. The secondary structure of PvCRN91 consisted of α-helix（31.90%）, extendedchain（22.41%） and random coil（45.69%）. Sequence comparison showed that there were sequences with high consis-tency（56.60%-74.77%） with PvCRN91 protein in various phytophthora, water mold, peronospora and aphanomyces,suggesting that PvCRN91 was a conserved effector. PvCRN91 did not have the ability to trigger cell necrosis in tobacco,but completely inhibited INF1-and BAX-induced programmed cell death（ PCD） reaction. PvCRN91 effector could pro-mote the infection of P. viticola and P. nicotianae. PvCRN91 protein subcellular location was in cytoplasm and nucleus,and it was speculated that it interacted with host target protein in cytoplasm or nucleus to inhibit host immune response.IP-MS method initially screened 252 target proteins that might interact with PvCRN91 and affect the physiological, bio-chemical and defense responses of tobacco through multiple metabolic pathways. 【Conclusion】 PvCRN91 is a virulencefactor that may manipulate host target proteins in the cytoplasm or nucleus to reduce plant defense responses and promotepathogen infection.