Functional Analysis Of Autophagy-related Genes BcATG26,BcATG17 And BcATG14 In Botrytis Cinerea

Botrytis cinerea can cause gray mould disease on numerous economically important crops and lead serious economic losses.But few of researches about autophagy in B.Cinerea,cellular autophagy plays an important role in eukaryotic organisms,which can maintain physiological balance in cell and help cell to survive adversity.Many researches show the great importance of macroautophagy in fungal development and pathogenicity.we download nucleotide sequences of BC1G-12424,BC1G₁1270 and BC1G₀1868 from B.cinerea database,analyse genetic structure and homology,use the Split-marker method for gene knockout.Finally,the hypha growth,sclerotia forming,nutritional requirements,pathogenicity and so on are determined.Following results have been achieved:?1?We use BLAST search on NCBI and reconstruct phylogenetic tree,we found BC1G-12424,BC1G₁1270 and BC1G₀1868 are highly conserved with ATG26 orthologs,ATG17 orthologs and ATG14 orthologs of other fungal and mammalian species,respectively,so named BcATG26,BcATG17 and BcATG14.?2?To generate disruptants of BcATG26,BcATG17 and BcATG14,we use the Split-marker method for gene knockout,transformants are verified by PCR and Soutnern blot,get the gene delection mutants ?BcATG26,?BcATG17 and ?BcATG14.?3?The results show BcATG26,BcATG17 and BcATG14 play important role in autophagy process.The hyphae of the wild type strain B05.10,?BcATG26-13,?BcATG17-6 and ?BcATG14-107 are stained with MDC,The appearance of vacuoles corresponds to the accumulation of autophagic bodies and autophagosomes in the hyphae of the wild type strain B05.10,while as no appearance of autophagic bodies and autophagosomes in the hyphae of gene delection mutants ?BcATG26-13,?BcATG17-6 and ?BcATG14-107,autophagy in gene delection mutants are blocked.?4?The results show autophagy affect hypha growth and sclerotia forming.The delection mutant ?BcATG26-13 cultures on PDA medium,forms sclerotia but sclerotia black delay compared with B05.10 and are still yellow for 15 days.The delection mutant ?BcATG14-107 on PDA medium forms so small sclerotia.The radial growths of delection mutant ?BcATG26-13,?BcATG17-6 and ?BcATG14-107 culture on 1/8 PDA medium,DM medium,DM-C medium as well as DM-N medium are markedly reduced compared with B05.10.?5?The results show autophagy affect pathogenicity.Pathogenicity of delection mutant ?BcATG26-13,?BcATG17-6 and ?BcATG14-107 weaken greatly on isolated or living tobacco leaves as well as on strawberry leaves compared with B05.10?P < 0.01?.?6?The results show autophagy has connection with B.cinerea resistance to reactive oxygen species?ROS?and phytoalexin?camalexin?.The delection mutant ?BcATG26-13,?BcATG17-6 and ?BcATG14-107 are more sensitive to hydrogen peroxide and phytoalexin?camalexin?compared with the wild type strain B05.10.All the results show that autophagy in B.cinerea is not only involved in cellular processes such as cell differentiation,sclerotia forming and pathogenicity,but aslo in pathogen resistance to reactive oxygen species?ROS?and phytoalexin?camalexin?.Those rusults are important for prevention and treatment of grey mould disease.