Anitifungal Mechanisms Of Chitosan Against Penicillium Expansum:A Study Of Membrane Trafficking Pathways Of Chitosan And Its Molecular Targets

Blue mold decay,caused by species of Penicillium expansum,is the main postharvest diseases of apples.Bseides causing the rot of apple and huge economic losses,P.expansum has also serious threaten to public health since it produces patulin.Chitosan is a natural polysaccharide,which has the effect of stimulating plant resistance and inhibiting pathogenic microorganisms,so it is widely used in the prevention and control of post-harvest diseases of fruits and vegetables.The results of previous studies in our laboratory indicated that chitosan could significantly inhibit P.expansum and was up-take into the cell via an energy-dependent manner,suggesting that there may be an intracellular antifungal pattern.In this study,we aimed to explore the transmembrane transport pathway of chitosan and the role of intracellular chitosan in its antifungal process with reverse genetic and biochemical analysis,to explore the antifungal activity and the possible molecular mechanisms involved.The main research results obtained in this paper are as follows:(1)Three kinds of specific blockers of the endocytosis pathway(chlorpromazine hydrochloride,genistein,and amiloride hydrochloride)were used in vitro and in vivo experiments,and it was found that they can inhibit the up-take of chitosan into the cell.The inhibition rate of chitosan on blue mold decay has decreased respectively by19.3%,11.4%,3.3% and 15.1%,after treatment with chlorpromazine hydrochloride and genistein,chlorpromazine hydrochloride and amiloride hydrochloride,genistein and amiloride hydrochloride,chlorpromazine hydrochloride and genistein and amiloride hydrochloride,respectively.The results indicated that chitosan could enter the cell of P.expansum possibly through the clathrin-mediated and caveolae-mediated endocysis.(2)Three genes PeCAM,PePLD and PeEps involved in the chitosan endocytosis pathway were selected for gene knockout,and mutant strains of ?PeCAM,?PePLD and ?PeEps were successfully obtained.Compared with the wild type,the spores of the mutant strains became larger;the color of colonies were greener and the growth rate of colonies increased significantly,but the mycelium growth decreased;thepathogenicity of mutants weakened and the production capacity of patulin did not change significantly.Compared with the type,the inhibition rates of chitosan on the growth of the mutant strains ?PeCAM,?PePLD and ?PeEps were respectively decreased by 49.1%,62.6% and 52.0%,and the sensitivity of the mutant strain to chitosan was significantly reduced,which further clarified the transmembrane transport pathway of chitosan and the role of intracellular chitosan in the antifungal action.(3)Gel shift assay showed that chitosan can interact with DNA of P.expansum,and block the migration of DNA in the gel;the increasing of ultraviolet absorption of DNA with the presence of chitosan further showed that this macromolecule can bind to DNA thereby destroy the duplex structure of DNA;the results of in vitro DNA amplification showed that chitosan can interfere with DNA replication,which indicated that DNA may be the intracellular target for antifungal action of chitosan against P.expansum.