Insertional Mutagenesis and Cloning of the Gene Required for the Biosynthesis of the Non-Host-Specific Toxin in Cochliobolus lunatus that Causes Maize Leaf Spot

Abstract

The maize pathotype Cochliobolus lunatus causes Curvularia leaf spot by producing a non-host-specific toxin known as methyl 5-(hydroxymethyl) furan-2-carboxylate (M5HF2C). However, related research that explores the genes that control the production of this toxin is rare. In the current work, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed to tag the gene required for the biosynthesis of the toxin. Of the 3,000 ATMT transformants recovered, 4 showed a significant decline in pathogenicity on maize leaves; 1 transformant, T806, produced no detectable M5HF2C. Genomic DNA that flanks the integrated plasmid was recovered from one of the mutants. A cosmid clone of the wild-type strain was isolated using the recovered DNA as a probe. The results of the structural and functional analyses of the region corresponding to the tagged site were then used as a basis to successfully clone one gene, called Clt-1. Bioinformatics analysis showed that the gene coded a BTB domain-containing protein that comprises 745 amino acids. Southern analysis revealed that the gene was localized in the genome as a single copy. The essential roles of Clt-1 in both toxin production and pathogenicity were confirmed by gene disruption experiments. In summary, the novel gene Clt-1 is closely associated with toxin production and pathogen virulence in leaves of susceptible varieties.