INVESTIGATIONS INTO THE ORIGIN AND EVOLUTION OF THE TOX1 LOCUS OF COCHLIOBOLUS HETEROSTROPHUS RACE T

Abstract

In 1970, a highly virulent race of a fungal plant pathogen emerged, resulting in the most severe plant disease epidemic in US history at that time, the Southern Corn Leaf Blight epidemic. Cochliobolus heterostrophus race T, differed from the original race by production of a toxin (T-toxin). T-toxin biosynthesis is governed by the Tox1 locus, composed of nine genes intimately associated with the breakpoints of a reciprocal translocation. Genomically, the locus is fragmented onto four small scaffolds; the precise location and organization of the genes unknown. Here, Tox1 orthologs were found in other fungi in a compact, collinear organization; and revealed the presence of a 10th Tox1 gene. Comparative analyses indicated that these share an ancient evolutionary origin with Tox1, with evidence for vertical and horizontal transmission, and may be foreign to C. heterostrophus race T. Most Tox1-like clusters share two additional genes, encoding an ATP-binding cassette transporter (ABC), and an oxidoreductase (Oxr1), loss of which may have represented a critical turning point in the evolution of Tox1. Deletion of the ABC and OXR1 genes in Corynespora cassiicola, did not result in T-toxin production; however, gene expression data indicated that the ABC and OXR1 genes were not co-expressed with Tox1, therefore the ABC and Oxr1 may have roles outside of Tox1. Heterologous expression of the genes in C. heterostrophus race T resulted in reduced growth and T-toxin relative to wild-type in the dark; however, in the light, the Ch[CcABC] mutant recovered growth, but not T-toxin production; whereas the Ch[CcOXR1] mutant recovered T-toxin production, but not growth, indicating that the proteins may play a role in light-dependent biology of C. heterostrophus race T. A major caveat is that the genes integrated at ectopic locations. Most importantly, long-read sequencing has facilitated fine-scale mapping of the Tox1 locus. Finally, the topological organization of the Tox1 genes has been resolved, as has the breakpoints of the reciprocal translocation. While the origin of Tox1 remains a mystery, this work represents a landmark with a new vista in the search to understand the origin and evolution of Tox1, and C. heterostrophus race T.