Efficient Agrobacterium tumefaciens-mediated target gene disruption in the maize pathogen Curvularia lunata

Curvularia lunata causes Curvularia leaf spot disease of maize, resulting in periodically significant yield losses around the world. To understand the molecular mechanisms of fungal pathogenicity and virulence factors contributed to the host, here we report a knockout transformation system against target genes. The concentration of conidia, Agrobacterium cell density, and method of co-incubation were optimized for deletion of the gene encoding 1, 3, 8-trihydroxynaphthalene reductase (Brn1), a gene in the melanin biosynthesis pathway, as a test case. Transformants contained a single T-DNA copy. The Brn1 mutant strain was reduced in virulence compared with the wild type strain when inoculated on susceptible maize. Transformation efficiency was 130 +/- 10 transformants per 1 x 10(5) germlings and homologous recombination efficiency was 60.0 to 100.0 %.