Cochliobolus heterostrophus race T, causal agent of southern corn leaf blight, requires T-toxin (a family of C35 to 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 amplified fragment length polymorphism/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 likely is 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.

Southern Corn Leaf Blight, one of the worst plant disease epidemics in modern history, was caused by Cochliobolus heterostrophus race T, which produces T-toxin, a determinant of high virulence to maize carrying Texas male sterile cytoplasm. The genetics of T-toxin production is complex and the evolutionary origin of associated genes is uncertain. It is known that ability to produce T-toxin requires three genes encoded at two unlinked loci, Tox1A and Tox1B, which map to the breakpoints of a reciprocal translocation. DNA associated with Tox1A and Tox1B sums to about 1.2 Mb of A+T rich, repeated DNA that is not found in less virulent race O or other Cochliobolus species. Here, we describe identification and targeted deletion of six additional genes, three mapping to Tox1A and three to Tox1B. Mutant screens indicate that all six genes are involved in T-toxin production and high virulence to maize. The nine known Tox1 genes encode two polyketide synthases (PKS), one decarboxylase, five dehydrogenases, and one unknown protein. Only two have a similar phylogenetic profile. To trace evolutionary history of one of the core PKS, DNA from more than 100 Dothideomycete species were screened for homologs. An ortholog (60% identity) was confirmed in Didymella zeae-maydis, which produces PM-toxin, a polyketide of similar structure and biological specificity as T-toxin. Only one additional Dothideomycete species, the dung ascomycete Delitschia winteri harbored a paralog. The unresolved evolutionary history and distinctive gene signature of the PKS (fast-evolving, discontinuous taxonomic distribution) leaves open the question of lateral or vertical transmission.

No abstract

Genes at two unlinked loci (Tox1A and Tox1B) are required for production of the polyketide T-toxin by Cochliobolus heterostrophus race T, a pathogenic fungus that requires T-toxin for high virulence to maize with T-cytoplasm. Previous work indicated that Tox1A encodes a polyketide synthase (PKS1) required for T-toxin biosynthesis and for high virulence. To identify genes at Tox1B, a wild-type race T cDNA library was screened for genes missing in the genome of a Tox1B deletion mutant. The library was probed, first with a 415-kb NotI restriction fragment from the genome of the Tox1B mutant, then with the corresponding 560-kb fragment from the genome of wild type. Two genes, DEC1 (similar to acetoacetate decarboxylase-encoding genes) and RED1 (similar to genes encoding members of the medium-chain dehydrogenase/reductase superfamily), were recovered. Targeted disruption of DEC1 drastically reduced both T-toxin production and virulence of race T to T-cytoplasm maize, whereas specific inactivation of RED1 had no apparent effect on T-toxin production (as determined by bioassay) or on virulence. DEC1 and RED1 map within 1.5 kb of each other on Tox1B chromosome 6;12 and are unique to the genome of race T, an observation consistent with the hypothesis that these genes were acquired by C. heterostrophus via a horizontal transfer event.

No abstract