Genomic And Molecular Genetic Analyses Of Secondary Metabolism, Toxin Production, And Iron Homeostasis In Cochliobolus Heterostrophus

Cochliobolus heterostrophus is a model necrotrophic maize pathogen. In 1970, a new and highly virulent race, race T, swept the US east coast, armed with a novel secondary metabolite Host Selective Toxin (HST), T-toxin. The genetic and molecular toolkit developed to characterize the genetics of T-toxin production is combined with genomic resources for C. heterostrophus and related species herein. Comparative genomics of lab and field strains (Tox+ race T and Tox- race O), and other Cochliobolus species, revealed that suites of secondary metabolism nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes are astoundingly diverse among species but remarkably conserved among isolates of the same species. Strain unique NRPSs and PKSs may produce HSTs, such as PKS1 and PKS2 that biosynthesize T-toxin. These race T specific genes map to the genetically complex Tox1 locus, which is associated with a reciprocal translocation of race O chromosomes. Two candidate reciprocal translocation breakpoint locations were found by whole-genome alignment of race T and O assemblies. Phylogenetic analyses of all ten C. heterostrophus Tox1-affiliated proteins revealed that Didymella zeae-maydis and Leptosphaeria maculans each possess complete but slightly divergent Tox1 loci, while Talaromyces stipitatus possesses a distant ortholog of C. heterostrophus PKS1. Genes at the L. maculans Tox1 locus are clustered, in contrast to the disconnected and scattered nature of C. heterostrophus Tox1. In vitro T-toxin activity assays demonstrated that C. heterostrophus, D. zeae-maydis, and T. stipitatus all display T-toxin-like activity. Broadly conserved secondary metabolite genes produce metabolites with core biological functions. NPS2 and NPS6, encoding NRPSs producing iron-chelating siderophores, are key examples. In C. heterostrophus, deletion of these results in loss of extracellular siderophore biosynthesis, attenuated virulence, hypersensitivity to oxidative and iron-depletion stress, and defective sexual spore development. In addition to siderophores, fungi can also utilize high affinity Reductive Iron Assimilation (RIA) mechanisms to acquire iron. Characterization of genetic mutants found that RIA is dispensable for C. heterostrophus. When RIA/siderophore polymutants were created, however, basic and severe morphological defects occurred. The role of HapX, an iron-responsive transcription factor, was also investigated. HapX is required for full virulence, iron-depletion tolerance, and sexual development