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

Abstract

AbstractIn 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 fungusCochliobolus 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).Tox1is 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 outTox1topology and pinpoint the hypothetical Race O translocation breakpoints corresponding to Race T-specific insertions, we undertook PacBio long-read sequencing which revealedTox1gene arrangement and the breakpoints. SixTox1Agenes are arranged as three small islands in a Race T-specific sea (∼634 kb) of repeats. FourTox1Bgenes 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 facilitatedTox1integration 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 pathogenCochliobolus 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.