Six New Genes Required for Production of T-Toxin, a Polyketide Determinant of High Virulence of Cochliobolus heterostrophus to Maize

Abstract

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.

Cochliobolus heterostrophus Llm1 – A Lae1-like methyltransferase regulates T-toxin production, virulence, and development

“…Double mutant strain 1759-R-12 (MAT1-1; llm1; vel1) is a progeny isolated from a cross between llm1 mutant strain llm1-B1 and vel1 mutant strain 1732-T1-1. Unless mentioned otherwise, all strains were grown on complete medium with xylose (CMX) (Inderbitzin et al, 2010) at 23°C under constant light except for experiments involving OE strains which were grown on CMX supplemented with polygalacturonic acid (PGA) (Wu et al, 2012).…”

“…3B). The expression levels of PKS1 and PKS2, core polyketide synthases required for T-toxin biosynthesis (Baker et al, 2006;Inderbitzin et al, 2010) were upregulated in llm1-deletion strains (Fig. 3C) and decreased in LLM1 OE strains (Fig.…”

“…A LLM1 OE vector, pHPLLM1, carrying the LLM1 coding region driven by the polygalacturonic acid (PGA)-inducible PelA promoter from A. nidulans (Yang et al, 1994) was constructed and transformed into WT strain C4 as described previously Wu et al, 2012). Integrations of all constructs at the intended sites were confirmed by diagnostic PCR reactions, as described previously (Inderbitzin et al, 2010;Wu et al, 2012).…”

“…T-toxin production was assessed using a microbial assay (Ciuffetti et al, 1992;Inderbitzin et al, 2010;Wu et al, 2012), except that mycelial plugs were taken from 8-day-old colonies incubated at 19°C under constant light or constant dark and placed mycelium side up on assay plates. All assays were repeated at least three times.…”

“…Race O, which lacks ability to biosynthesize T-toxin, is only mildly virulent to T-cms corn. To date, nine genes associated with T-toxin production, unique to race T have been characterized (Baker et al, 2006;Inderbitzin et al, 2010;Rose et al, 2002). These genes have complex genome architecture and phylogenetic relationships; they are not clustered like many secondary metabolite genes but distributed in disconnected fragments on two different chromosomes (Kodama et al, 1999); only two share a common evolutionary history (Inderbitzin et al, 2010).…”