Highly efficient gene knockout system in the maize pathogen Colletotrichum graminicola using Agrobacterium tumefaciens-mediated transformation (ATMT)

Colletotrichum graminicola, a hemibiotrophic pathogenic fungus, is the causal agent of anthracnose of maize, which causes significant yield losses worldwide, especially in warm and humid maize production regions. An efficient targeted genes knockout protocol is crucial to explore molecular mechanisms of fungal virulence to the host. In this study, we established a gene knockout transformation system by employing Agrobacterium tumefaciens-mediated transformation to knockout genes in M 1.001 strain of C. graminicola. The conidia germination status, induction medium type, and ratio of Agrobacterium cell and conidia suspension were optimized for the knockout of CgBRN1(OR352905), a gene relating to the fungal melanin biosynthesis pathway. Additionally, CgPKS18 (OR352906) and CgCDC25 (OR352903) were knocked out to test the applicability of the gene knockout transformation system. In this established system, transformation efficiency was 176 transformants per 1 × 105 conidia and the homologous recombination efficiency was 53.3 to 75%. Furthermore, disease index, lesion number and lesion size caused by the three above-mentioned mutant strains were found to be reduced significantly compared to the wild-type strain, which indicated reduction in fungal virulence due to the lack of those genes.