Function Research Of Methylation Related Genes In Botrytis Cinerea

Botrytis cinerea is an important plant-pathogenic fungus with world-wide distribution. This pathogen attacks more than 200 plant species, and is responsible for significant annual yield loss of many economically important crops. Epigenetic modification of DNA and DNA-associated histone proteins have been demonstrated to play important roles in the growth and development of plants and animals. To illuminate the function of this modification in B. cinerea, genes encoding the DNA methyltransferase（DIM-2）, heterochromatin protein 1（HP1）, and histone 3 lysine 9 methyltransferase（DIM-5） were identified by Blastp. These genes were named as Bcdim-2（BC1G₁2419）, Bchp1（BC1G₀6432）, and Bcdim-5（BC1G₁1188） respectively. The function of DIM-2, HP1, DIM-5 and their homologous proteins have been well studied in animals, plants and some filamentous fungus, while their roles in the plant-pathogenic fungus B. cinereaare still not clear. Here in, the function of Bcdim-2, Bchp1 and Bcdim-5 were studied in B. cinerea, and the primary results are shown: 1. The Bcdim-2 gene contains 4 exons and 3 introns, encoding 1025 amino acids. Bcdim-2 was predicted containing a DNA methylase domain; The Bchp1 gene contains 6 exons and 6 introns, encoding 244 amino acids and containing two typical conserved domains of HP1: CD（Chromo Domain） and CSD（Chrome Shadow Domain）; The Bcdim-5 gene contains 4 exons and 3 introns, encoding 357 amino acids and containing three conserved domains: Pre-SET, Post-SET and SET domain. 2. QRT-PCR results showed that Bcdim-2, Bchp1 and Bcdim-5 express during vegetative growth, sclerotia formation and infection stages. Bcdim-2 is upregulated during infection,Bchp1 is downregulated in the stage of sclerotia formation and infection. However, the expression of Bcdim-5 hasno significant difference in the three stages. 3. The gene knockout mutants of Bcdim-2, Bchp1 and Bcdim-5 were obtained according tothe principle of homologous recombination. The results showed that the deletion of Bcdim-2 and Bchp1 have no effect on the growth, hyphal tip morphology, sclerotia formation, conidia productionand virulence of B. cinerea. But theΔBcdim-5strains showed obvious abnormal phyneotype, including lower growth rate, later sclerotia formation, lesser and smaller sclerotia, less conidia production. The virulence ofΔBcdim-5strains was also reduced on the leaves of Arabidopsis thaliana, soybean and tomato. 4. QRT-PCR was used to detect the expression level of some pathogenicity-related genes in the wild type strain B05.10 and ΔBcdim-5 strain. The results showed that most of these genes were downregulated in the ΔBcdim-5 strain, indicating that these genes maybe regulated by the modification of DNA-associated histone proteins and contributed to the virulence reduction of in B. cinerea. Taken together, our results demonstrated that Bcdim-5 plays an important role in the growth, sporulation, sclerotia formation and virulence in B.cinerea,but the deletion of Bcdim-2 and Bchp1 has no obvious effect on the phyneotype of B.cinerea. These results suggested that H3K9 methylation is indispensable and involved in the regulation of key biological processes in B. cinerea, but DNA methylation is not associated with the growth and development in B. cinerea.