A polyketide synthase is required for fungal virulence and production of the polyketide T-toxin.

Abstract

Race T of the fungal pathogen Cochliobolus heterostrophus is highly virulent toward Texas male sterile (T) maize and differs from its relative, race O, at a locus (Tox1) that is responsible for the production of T-toxin, a family of linear long-chain (C35 to E41) polyketides. In a previous study, the restriction enzyme-mediated integration procedure was used to mutagenize and tag Tox1. Here, we report that the DNA recovered from the insertion site of one mutant encodes a 7.6-kb open reading frame (2530 amino acids) that identifies a multifunctional polyketide synthase (PKS)-encoding gene (PKS1) with six catalytic domains arranged in the following order, starting at the N terminus: beta-ketoacyl synthase, acyltransferase, dehydratase, enoyl reductase, beta-ketoacyl reductase, and acyl carrier protein. PKS1 is interrupted by four apparent introns (74, 57, 49, and 41 bp) and exists in the genome as a single copy surrounded by highly repetitive, A + T-rich DNA. When PKS1 in race T was inactivated by targeted gene disruption, T-toxin production and high virulence were eliminated, indicating that this PKS is required for fungal virulence. Race O strains, which do not produce T-toxin, lack a detectable homolog of PKS1, suggesting that race T may have acquired PKS1 by horizontal transfer of DNA rather than by vertical inheritance from an ancestral strain.

Cochliobolus heterostrophus T-toxin gene expression modulation via G protein and MAPK pathways

“…Tox1B includes two genes, DEC1 (similar to acetoacetate decarboxylaseencoding genes) and RED1 (similar to genes encoding members of the medium-chain dehydrogenase/ reductase superfamily) (Rose et al 2002). DEC1 and RED1 map within 1.5 kb of each other on the Tox1B chromosome 6;12 ( Figure 2B) 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 (Yang et al 1996). 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 or virulence (Rose et al 2002).…”

A PKS gene, pks-1, is involved in chaetoglobosin biosynthesis, pigmentation and sporulation in Chaetomium globosum

“…To further confirm transformants, genomic DNA was extracted as previously described by Raeder and Broda [37], and Southern blot analysis was performed according to the method of Yang et al [18]. DNA samples, digested with enzymes as indicated in the figure, were resolved on 0.8% agarose gels, and transferred onto Magmaprobe Nylon Transfer Membrane-N + (Osmonics, Minnetonka, MN, USA).…”

“…Molecular studies have also been performed to determine the biosynthetic principle of important bioactive polyketides, such as aflatoxin [17][18][19][20][21][22]. Regulatory networks governing secondary metabolism have been gradually uncovered in several fungal species, for example, the central roles of the G protein-cAMP-PKA pathway and the LaeA and VeA protein complex have been determined [19,[23][24][25].…”

Functional replacement of the ketosynthase domain of FUM1 for the biosynthesis of fumonisins, a group of fungal reduced polyketides

“…1) [21]. This organization is almost identical to several other PKSs for the biosynthesis of reduced polyketides, such as LDKS and LNKS for lovastatin in A. terreus [15] and PKS1 for T-toxins in Cochliobolus heterostrophus [32], although the polyketide chain-length of fumonisins, lovastatin, and T-toxins is substantially different from each other. The objectives of this study were to test whether a heterologous PKS is functionally interchangeable with FUM1 in F. verticillioides and whether such a swapping would result in polyketide products with an altered chain-length.…”

“…In experiments using PCR to quickly screen putative mutants, genomic DNA was mini-prepared from the mutants by using UltraClean TM Microbial DNA Isolation Kit (Mo Bio, Fig. 3d, f) was amplifi ed by PCR using primers tKS-F/tKS-R (Table 1) with C. heterostrophus clone pF5P1 [32] as template. The probe t1 (703 bp) and t2 (681 bp) in Fig.…”