Turgeon, B. G. | Search bioRxiv

bioRxiv2025

Cochliobolus miyabeanus species-specific identification using nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS)-encoding genes associated with virulence to rice

Cochliobolus miyabeanus causes brown spot disease of rice. For necrotrophic Cochliobolus spp., some small molecule products of nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) are known pathogenicity and/or virulence factors, often acting in a host-specific manner. Previous whole genome analyses across Cochliobolus species identified 11 NRPS- and 21 PKS-encoding genes in C. miyabeanus. First purpose of the current study was to examine the effect of deletion of ten of the discontinuously distributed genes (corresponding to JGI protein IDs 4446, 5802, 6546, 7015, 9064, 41693, 41753, 83551, 98843 and 107726) on virulence of C. miyabeanus to rice. A second purpose, based on results of the first, was to develop a PCR method to specifically identify C. miyabeanus and to distinguish it from other closely related Cochliobolus spp. We show that deletion of seven of the genes resulted in mutants that were reduced in virulence compared to the wild-type strain WK1C. Wild-type developed large dark brown necrotic lesions surrounded by chlorotic halos, while the mutants produced smaller, light brown spots with chlorosis. These results suggest that the products of these genes contribute to brown spot disease. In addition, we identified PKS and NPS SNPs that discriminate between C. miyabeanus and other species of Cochliobolus.

genetics↗

bioRxiv2023

T-toxin virulence genes: unconnected dots in a sea of repeats

In 1970, the Southern Corn Leaf Blight epidemic ravaged US fields to great economic loss. The outbreak was caused by never-before-seen, super-virulent, Race T of the fungus Cochliobolus heterostrophus. The functional difference between Race T and O, the previously known, far less aggressive strain, is production of T-toxin, a host-selective polyketide. Super-virulence is associated with [~]1 Mb of Race T- specific DNA; only a fraction encodes T-toxin biosynthetic genes (Tox1). Tox1 is genetically and physically complex, with unlinked loci (Tox1A, Tox1B) genetically inseparable from breakpoints of a Race O reciprocal translocation that generated hybrid Race T chromosomes. Previously, we identified ten genes for T-toxin biosynthesis. Unfortunately, high depth, short-read sequencing placed these genes on four small, unconnected scaffolds surrounded by repeated A+T rich sequence, concealing context. To sort out Tox1 topology and pinpoint the hypothetical Race O translocation breakpoints corresponding to Race T-specific insertions, we undertook PacBio long-read sequencing which revealed Tox1 gene arrangement and the breakpoints. Six Tox1A genes are arranged as three small islands in a Race T-specific sea ([~]634 kb) of repeats. Four Tox1B genes are linked, on a large loop of Race T-specific DNA ([~]210 kb). The race O breakpoints are short sequences of race O-specific DNA; corresponding positions in race T are large insertions of race T-specific, A+T rich DNA, often with similarity to transposable (predominantly Gypsy) elements. Nearby, are Voyager Starship elements and DUF proteins. These elements may have facilitated Tox1 integration into progenitor Race O and promoted large scale recombination resulting in race T. ImportanceIn 1970 a corn disease epidemic ravaged fields in the US to great economic loss. The outbreak was caused by a never-before seen, super-virulent strain of the fungal pathogen Cochliobolus heterostrophus. This was a plant disease epidemic, however, the current COVID-19 pandemic of humans is a stark reminder that novel, highly virulent, pathogens evolve with devastating consequences, no matter what the host-animal, plant, or other organism. Long read DNA sequencing technology allowed in depth structural comparisons between the sole, previously known, much less aggressive, version of the pathogen and the super-virulent version and revealed, in meticulous detail, the structure of the unique virulence-causing DNA. These data are foundational for future analysis of mechanisms of DNA acquisition from a foreign source.

genomics↗