A ToxA-like protein from Cochliobolus heterostrophus induces light-dependent leaf necrosis and acts as a virulence factor with host selectivity on maize

ToxA, the first discovered fungal proteinaceous host-selective toxin (HST), was originally identified in 1989 from the tan spot fungus Pyrenophora tritici-repentis (Ptr). About 25 years later, a homolog was identified in the leaf/glume blotch fungus Stagonospora nodorum (Parastagonospora nodorum), also a pathogen of wheat. Here we report the identification and function of a ToxA-like protein from the maize pathogen Cochliobolus heterostrophus (Ch) that possesses necrosis-inducing activity specifically against maize. ChToxA is encoded by a 535-bp open reading frame featuring a ToxA-specific intron with unusual splicing sites (5′-ATAAGT…TAC-3′) at conserved positions relative to PtrToxA. The protein shows 64% similarity to PtrToxA and is predicted to adopt a similar three-dimensional structure, although lacking the arginyl-glycyl-aspartic acid (RGD) motif reported to be required for internalization into sensitive wheat mesophyll cells. Reverse-transcriptase PCR revealed that the ChTOXA gene expression is up-regulated in planta, relative to axenic culture. Plant assays indicated that the recombinant ChToxA protein induces light-dependent leaf necrosis in a host-selective manner on maize inbred lines. Gene deletion experiments confirmed that ChtoxA mutants are reduced in virulence on specific ChToxA-sensitive maize lines, relative to virulence caused by wild-type strains. Database searches identified potential ChToxA homologues in other plant-pathogenic ascomycetes. Sequence and phylogenetic analyses revealed that the corresponding ToxA-like proteins include one member recently shown to be associated with formation of penetration hypha. These results provide the first evidence that C. heterostrophus is capable of producing proteinaceous HSTs as virulence factors in addition to well-known secondary metabolite-type toxins produced biosynthetically by polyketide synthase megaenzymes. Further studies on ChToxA may provide new insights into effector evolution in host-pathogen interactions.