Study On Biological Function And Fludioxonil Sensitivity Regulation Mechanism Of Autophagy In Botrytis Cinerea

Gray mold caused by Botrytis cinerea is one of the most serious and harmful diseases in the world.The pathogen has a wide range of hosts and can infect more than 500 plant species,including a variety of fruits,vegetables and ornamentals,causing huge economic losses to agricultural production.At present,due to the lack of effective disease-resistant varieties,the field control of gray mold is mainly based on chemical control.However,with the long-term use of fungicides,and the pathogen has wide-ranging hosts,rapid reproduction and frequent genetic variation,has led to an increasingly prominent problem of resistance to gray mold.Fludioxonil belongs to the class of phenylpyrrole compounds,is a novel,highly effective,low-toxic and broad-spectrum fungicide with special effects on the control of gray mold.The mode of action of fludioxonil is unique and exhibits no cross-resistance with the existing fungicides.The international Fungicide Resistance Action Committee(FRAC)believes that the mechanism of action of fludioxonil is to affect the activity of histidine kinases that are involved in the regulation of osmotic pressure,but the specific mechanism of action is still unclear.To date,there have been reports of low level fludioxonil resistance in the field of other countries.In China,the application of fludioxonil is still in its early stages,however,in 2016,high-level resistant strains of gray mold have been found in the field although the biological fitness of these strains is very low.Autophagy is an evolutionarily conserved process in eukaryotes that allows the turnover of intracellular components.During autophagy process,some of the senescent proteins or damaged organelles are packed by the bilayer membrane phagophores and subsequently transported to lysosomes(animals)or vacuoles(yeast and plants)for degradation and recycling.Recent studies have shown that autophagy is not only a coping strategy when cells acquire energy under starvation,but also participates in many important physiological processes and plays an important role in the process of organisms coping with external stress.Autophagy has been reported in some fungi,and its specific function varies depending on the paratrophy.Therefore,studying the biological function of autophagy in B.cinerea will provide a theoretical basis for scientific control of gray mold and development of new fungicides.In this study,we investigated the mechanism of autophagy to regulate the sensitivity of B.cinerea to fludioxonil.At the same time,the biological roles of autophagy in the development and pathogenesis of B.cinerea was also reported.The main findings are as follows:(1)The role of autophagy in the sensitivity of B.cinerea to fungicides.The key gene deletion mutants of the autophagy pathway in B.cinerea was generated by target gene deletion strategy.Microscopic observation and physiological and biochemical experiments demonstrated that deletion of the key genes in autophagy pathway led to the block of autophagy process in B.cinerea.The sensitivity tests determined the sensitivity of the gene deletion mutants to the commonly used fungicides for controlling gray mold,including phenylpyrrole fludioxonil,dicarboximides iprodione and procymidone,succinate dehydrogenase inhibitor boscalid and pyridylamine fluazinam.The results showed that the block of autophagy process resulted in a significant increase in the sensitivity of B.cinerea to fludioxonil.Similarly,the sensitivity of the mutants to iprodione and procymidone also increased to some extent.However,the sensitivity of the mutants to boscalid and fluazinam remained unchanged.The above results indicate that the autophagy process is involved in the regulation of the sensitivity of B.cinerea to fludioxonil,iprodione and procymidone.(2)Study on the biological function of the autophagy gene BcATG1 in B.cinerea.In Saccharomyces cerevisiae,ATG1 encodes the serine/threonine protein kinase Atgl.When cells are starved,Atgl binds to Atg13 to form a complex to induce autophagy.When the cells are in a nutrient-rich condition,the process of autophagy is inhibited by Atg13 phosphorylation to reduce the binding with Atg1.Homologous alignment identified BcATG1 as a homologous gene of ATG1 in B.cinerea.BcATG1 can functionally recover the tolerance of yeast ATG1 deletion mutant to nitrogen starvation and respond to the induction of carbon and nitrogen starvation.Target gene deletion of BcATG1 results in block of the autophagy process in B.cinerea.The sensitivity tests showed that the BcATGl deletion mutant exhibited increased sensitivity to fludioxonil,iprodione and procymidone.The BcATG1 deletion mutant showed reduced aerial hyphae,abnormal mycelial branching,increased pigmentation,slower growth rate on nutrient-deficient media,decreased conidiation,abnormal conidia morphology,and lost the ability to form sclerotia on PDA plates.Infection tests showed that deletion of BcATG1 affected the formation of infective structures,and weakened the ability of epidermal penetration,which led to a significant reduction in virulence on different host tissues.In addition,the lipid droplet content of the BcATGl deletion mutant was decreased,and glycerol accumulated heavily in mycelia.The phenotypic defects of the BcATG1 deletion mutant were restored by the target gene complementation.The above results indicate that BcATGl plays an important role in the sensitivity regulation to fludioxonil,development and virulence in B.cinerea.(3)Study on the biological functions of the autophagy genes BcATG8 and BcATG4 in B.cinerea.In S.cerevisiae,ATG8 encodes the ubiquitin-like protein Atg8,ATG4 encodes the cysteine protease Atg4.During the formation of autophagosome,Atg8 was first cleaved by Atg4 and then modified by Atg3 and Atg7 to bind phosphatidylethanolamine to form a ubiquitin-like conjugation system.According to the homologous alignment,BcATG4 and BcATG8 were identified in the B.cinerea genome database,and both of them were able to homologously complement the respective yeast deletion mutants,and there was directly physical interaction with each other.The marker of autophagy process in B.cinerea was successfully constructed by GFP fusion protein GFP-BcAtg8.Deletion of BcATG8 or BcATG4 resulted in block of the autophagy process in B.cinerea,and increased sensitivity to fludioxonil,iprodione and procymidone.Both BcATG8 and BcATG4 deletion mutants showed reduced aerial hyphae,slower growth rate,abnormal conidiation,delayed conidial germination under starvation conditions,and lost the ability for sclerotial formation in PDA plates.Infection tests showed that the virulence of both BcATG8 and BcATG4 deletion mutants on different host tissues was decreased significantly.In addition,BcATG8 and BcATG4 affect the lipid droplet metabolism in B.cinerea,and the glycerol content of the deletion mutants increases in a large amount.All the phenotypic defects were recovered by target gene complementation.The above results show that BcATG8 and BcATG4 regulate the sensitivity of B.cinerea to fludioxonil and promote the development and pathogenicity of B.cinerea.(4)Study on the biological functions of the autophagy genes BcATG3 and BcATG7.In S.cerevisiae,ATG3 and ATG7 encode ubiquitin-like activating enzyme E2 and ubiquitin-like activating enzyme E1,respectively,and collaboratively modify ubiquitin-like protein Atg8 to promote the formation of autophagosome.In this study,BcATG3 and BcATG7 were identified from B.cinerea genome database by BLAST search using the respective yeast homolog as query.The physical interaction of BcAtg3 with BcAtg7 was demonstrated by yeast two-hybrid system.Subcellular localization assays showed that BcAtg3 diffused in cytoplasm,and BcAtg7 localized in cytoplasm as pre-autophagosomal structures(PAS).Target gene deletion experiments revealed that both BcATG3 and BcATG7 are essential for autophagy pathway.Notably,the single deletion mutant of BcATG3 and BcATG7 displayed similar biological phenotypes,including the defects in mycelial growth,conidiation and sclerotial formation.Infection tests showed that both BcATG3 and BcATG7 were required for full virulence of B.cinerea.All of these defective phenotypes were rescued by gene complementation.These results indicate that BcATG3 and BcATG7 are necessary for autophagy to regulate vegetative differentiation and pathogenesis in B.cinerea.