PNNL

Abstract

Cochliobolus heterostrophus race T, causal agent of Southern Corn Leaf Blight, requires T-toxin (a family of C35 – C49 polyketides) for high virulence on T-cytoplasm maize. Production of T-toxin is controlled by two unlinked loci, Tox1A and Tox1B, carried on 1.2 Mb of DNA not found in race O, a mildly virulent form of the fungus that does not produce T-toxin, or in any other Cochliobolus spp. or closely related fungus. PKS1, a polyketide synthase (PKS)-encoding gene at Tox1A and DEC1, a decarboxylase-encoding gene at Tox1B, are necessary for T-toxin production. Although there is evidence that additional genes are required for T-toxin production, efforts to clone them have been frustrated because the genes are located in highly repeated, A+T-rich DNA. To overcome this difficulty, Ligation specificity-based Expression Analysis Display (LEAD), a comparative AFLP/gel fractionation/capillary sequencing procedure was applied to cDNAs from a near isogenic pair of race T (Tox1+) and race O (Tox1-) strains. This led to discovery of PKS2, a second PKS-encoding gene that maps at Tox1A and is required for both T-toxin biosynthesis and high virulence to maize. Thus, the carbon chain of each T-toxin family member is likely assembled by action of two PKSs, which produce two polyketides, one of which may act as the starter unit for biosynthesis of the mature T-toxin molecule.