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ABSTRACTConnections between fungal development and secondary metabolism have been reported previously, but as yet, no comprehensive analysis of a family of secondary metabolites and their possible role in fungal development has been reported. In the present study, mutant strains of the heterothallic ascomycete Cochliobolus heterostrophus, each lacking one of 12 genes (NPS1to NPS12) encoding a nonribosomal peptide synthetase (NRPS), were examined for a role in sexual development. One type of strain (?nps2) was defective in ascus/ascospore development in homozygous ?nps2crosses. Homozygous crosses of the remaining 11 ?npsstrains showed wild-type (WT) fertility. Phylogenetic, expression, and biochemical analyses demonstrated that the NRPS encoded by NPS2is responsible for the biosynthesis of ferricrocin, the intracellular siderophore of C. heterostrophus. Functional conservation of NPS2in both heterothallic C. heterostrophusand the unrelated homothallic ascomycete Gibberella zeaewas demonstrated. G. zeae?nps2strains are concomitantly defective in intracellular siderophore (ferricrocin) biosynthesis and sexual development. Exogenous application of iron partially restored fertility to C. heterostrophusand G. zeae?nps2strains, demonstrating that abnormal sexual development of ?nps2strains is at least partly due to their iron deficiency. Exogenous application of the natural siderophore ferricrocin to C. heterostrophusand G. zeae?nps2strains restored WT fertility. NPS1, a G. zeae NPSgene that groups phylogenetically with NPS2, does not play a role in sexual development. Overall, these data demonstrate that iron and intracellular siderophores are essential for successful sexual development of the heterothallic ascomycete C. heterostrophusand the homothallic ascomycete G. zeae.

ABSTRACTNonribosomal peptides, made by nonribosomal peptide synthetases, have diverse biological activities, including roles as fungal virulence effectors. Inspection of the genome of Cochliobolus heterostrophus, a fungal pathogen of maize and a member of a genus noted for secondary metabolite production, revealed eight multimodular nonribosomal peptide synthase (NPS) genes and three monomodular NPS-like genes, one of which encodes a nonribosomal peptide synthetase/polyketide synthase hybrid enzyme presumed to be involved in synthesis of a peptide/polyketide molecule. Deletion of each NPSgene and phenotypic analyses showed that the product of only one of these genes, NPS6, is required for normal virulence on maize. NPS6 is also required for resistance to hydrogen peroxide, suggesting it may protect the fungus from oxidative stress. This and all other npsmutants had normal growth, mating ability, and appressoria. Real-time PCR analysis showed that expression of all NPSgenes is low (relative to that of actin), that all (except possibly NPS2) are expressed during vegetative growth, and that expression is induced by nitrogen starvation. Only NPS6is unfailingly conserved among euascomycete fungi, including plant and human pathogens and saprobes, suggesting the possibility that NPS6 activity provides oxidative stress protection during both saprobic and parasitic growth.

ABSTRACTPrevious work established that mutations in mitogen-activated protein (MAP) kinase (CHK1) and heterotrimeric G-protein a (Ga) subunit (CGA1) genes affect the development of several stages of the life cycle of the maize pathogen Cochliobolus heterostrophus. The effects of mutating a third signal transduction pathway gene, CGB1, encoding the Gß subunit, are reported here. CGB1is the sole Gß subunit-encoding gene in the genome of this organism. cgb1mutants are nearly wild type in vegetative growth rate; however, Cgb1 is required for appressorium formation, female fertility, conidiation, regulation of hyphal pigmentation, and wild-type virulence on maize. Young hyphae of cgb1mutants grow in a straight path, in contrast to those of the wild type, which grow in a wavy pattern. Some of the phenotypes conferred by mutations in CGA1are found in cgb1mutants, suggesting that Cgb1 functions in a heterotrimeric G protein; however, there are also differences. In contrast to the deletion of CGA1, the loss of CGB1is not lethal for ascospores, evidence that there is a Gß subunit-independent signaling role for Cga1 in mating. Furthermore, not all of the phenotypes conferred by mutations in the MAP kinase CHK1gene are found in cgb1mutants, implying that the Gß heterodimer is not the only conduit for signals to the MAP kinase CHK1module. The additional phenotypes of cgb1mutants, including severe loss of virulence on maize and of the ability to produce conidia, are consistent with CGB1being unique in the genome. Fluorescent DNA staining showed that there is often nuclear degradation in mature hyphae of cgb1mutants, while comparable wild-type cells have intact nuclei. These data may be genetic evidence for a novel cell death-related function of the Gß subunit in filamentous fungi.