Analyzing Of Antifungal Activity Mechanism Of Permatin From Naked Oat (Avena Nuda) Seeds

Pathogenesis-related proteins (PR proteins) involved in host defence are the products accumulated by plants when infected by phytopathogenic bacteria,fungi or viruses.PR proteins constitute a large family of proteins and are generally classified into 17 subfamilies based on molecular mass, and biological activity. Proteins that belong to the PR-5 family (PR-5) are also known as thaumatin-like (TL) proteins as they show sequence similarities to the sweet-tasting plant protein thaumatin.PR-5s involve various activities such as antifungal and β-1,3 glucanase.A novel antifungal protein named Permatin isolated from naked oat (Avena nudd) seeds was belong to the PR-5,which was analysised by Matrix-assisted laser desorption ionization-time of flight MS.The gene permatin with an open reading frameof 678bp encoded a protein of 225 amino acids was cloned from Naked Oat Leaves.Permatin contains 8 pairs disulfide bridges formed by 16 conserved cysteine residues and an N-terminal signal peptide targeting mature proteins into the secretory pathway.The protein exhibited antifungal activity against Trametes sp. SQOland Trichoderma spp.The antifungal activity of Permatin was tested against Fusarium oxysporum and Penicillium using filter paper dispersion and morphologic observation.A membrane disruption assay was determined by Delta Vision fluorescence microscope RT.The results indicated that Permatin showed remarkable antifungal activity against Fusarium oxysporum and Penicillium and was capable of causing membrane permeabilization in the fungi Fusarium oxysporum、Penicillium and Saccharomyces cerevisiae.Permatin (22-225aa) was cloned and expressed in E.coli BL21that was still showed a antifungal activity against Trametes sp. SQ01 as before.The result suggests that signal peptide of Permatin doesn’t participate in the antifungal activity. Next,interaction of membrane lipid and Permatin was investigated by fluorescence measurements、Far-UV circular dichroism and PIP StripsTM immunoblotting.The results demonstrate that Permatin could interact with membrane lipid of fungus especially the anionic phospholipids such as phosphoinositide. Fluorescent probe DCFH-DA was used to detect the level of the reactive oxygen species(ROS) of Fusarium oxysporum and Saccharomyces cerevisiae incubated with Nus-permatin or natural permatin. We found that Permatin induce the production of ROS of fungus cells compared with control cells that were not treated with permatin. These results suggest antifungal activity of Permatin involves plasma membrane permeabilization, interaction with the membrane lipid especially the anionic phospholipids,and induction of ROS in fungi Cells.