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Efficient Gene Knockout in the Maize Pathogen Setosphaeria turcica Using Agrobacterium tumefaciens-Mediated Transformation

First author: Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY 14853 and College of Plant Protection, Shenyang Agricultural University, Shenyang 110161, China; second, third, fourth, fifth, and sixth authors: Department of Plant Pathology and Plant-Microbe Biology, Cornell University; fourth author: State Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China; and fifth author: College of Plant Science, Jilin University, Changchun 130062, China.

Setosphaeria turcica, a hemibiotrophic pathogenic dothideomycete, is the causal agent of Northern Leaf Blight of maize, which periodically causes significant yield losses worldwide. To explore molecular mechanisms of fungal pathogenicity and virulence to the host, an efficient targeted gene knockout transformation system using Agrobacterium tumefaciens was established with field collected strains. The starting materials, incubation time, induction medium type, Agrobacterium cell density, and method of co-incubation were optimized for deletion of 1,3,8-trihydroxynaphthalene reductase, a gene in the melanin biosynthesis pathway, as a test case. Four additional genes were deleted in two different S. turcica field isolates to confirm robustness of the method. One of these mutant strains was reduced in virulence compared with the wild-type strain when inoculated on susceptible maize. Transformation efficiency was ≈20 ± 3 transformants per 1× 10⁶ germlings and homologous recombination efficiency was 33.3 to 100%.

Additional keyword: ECP6.