Tobacco (Nicotiana tabacum L. cv. Xanthi) XD cells containing nitrate and nitrate reductase stopped producing nitrite after approximately 1 hour when incubated under anaerobic conditions. The cessation of nitrite production was not due to an inactivation of the nitrate reducing system. This was shown by the ability of the cells to resume anaerobic nitrite production at a rate similar to the initial rate of nitrite production upon exposure to nitrate, monohydroxy alcohols or pyrazole. Cessation of nitrite production also could not be attributed to leakage of nitrate from the cells. Although some nitrate did leak from the cells, most of the nitrate was still in the cells by the time anaerobic nitrite production ceased. We infer the existence of a small metabolic pool and a large storage pool of nitrate, such that nitrite production ceases when the metabolic pool is depleted of nitrate. The metabolic pool of nitrate in tobacco cells decreased 170-fold as the culture aged from 3 to 5 days. However, total cellular nitrate during this period remained relatively constant. Anaerobic nitrite production by barley (Hordeum vulgare) aleurone layers and corn (Zea mays) leaf sections also ceased after only a small fraction of endogenous nitrate was reduced and resumed again upon addition of exogenous nitrate. In contrast to that found with tobacco cells, the metabolic pool of nitrate in corn leaf sections remained constant with age, while total endogenous nitrate increased. These results were interpreted to mean that higher plants in general contain metabolic and storage pools of nitrate, the properties of which vary with species and physiological variables.

Homothallic Didymella zeae-maydis undergoes sexual reproduction by selfing. Sequence analysis of the mating type (MAT) locus from this fungus revealed that MAT carries both MAT1-1-1 and MAT1-2-1 genes found in heterothallic Dothideomycetes, separated by ∼1.0 kb of noncoding DNA. To understand the mechanistic basis of homothallism in D. zeae-maydis, each of the MAT genes was deleted and the effects on selfing and on ability to cross in a heterothallic manner were determined. The strain carrying an intact MAT1-1-1 but defective MAT1-2-1 gene (MAT1-1-1;ΔMAT1-2-1) was self-sterile, however strains carrying an intact MAT1-2-1 but defective MAT1-1-1 gene (ΔMAT1-1-1;MAT1-2-1), when selfed, showed delayed production of a few ascospores. Attempts to cross the two MAT deletion strains yielded fewer ΔMAT1-1-1;MAT1-2-1 than MAT1-1-1;ΔMAT1-2-1 progeny and very few ascospores overall compared to WT selfs. This study demonstrates that, as in the other homothallic Dothideomycetes, both MAT genes are required for full fertility, but that, in contrast to other cases, the presence of a single MAT1-2-1 gene can induce homothallism, albeit inefficiently, in D. zeae-maydis.

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

Cochliobolus heterostrophus race T, causal agent of southern corn leaf blight, requires T-toxin (a family of C35 to C49 polyketides) for high virulence on T-cytoplasm maize. Production of T-toxin is controlled by two unlinked loci, Tox1A and Tox1B, carried on 1.2 Mb of DNA not found in race O, a mildly virulent form of the fungus that does not produce T-toxin, or in any other Cochliobolus spp. or closely related fungus. PKS1, a polyketide synthase (PKS)-encoding gene at Tox1A, and DEC1, a decarboxylase-encoding gene at Tox1B, are necessary for T-toxin production. Although there is evidence that additional genes are required for T-toxin production, efforts to clone them have been frustrated because the genes are located in highly repeated, A+T-rich DNA. To overcome this difficulty, ligation specificity-based expression analysis display (LEAD), a comparative amplified fragment length polymorphism/gel fractionation/capillary sequencing procedure, was applied to cDNAs from a near-isogenic pair of race T (Tox1+) and race O (Tox1-) strains. This led to discovery of PKS2, a second PKS-encoding gene that maps at Tox1A and is required for both T-toxin biosynthesis and high virulence to maize. Thus, the carbon chain of each T-toxin family member likely is assembled by action of two PKSs, which produce two polyketides, one of which may act as the starter unit for biosynthesis of the mature T-toxin molecule.

Redox sensing is a ubiquitous mechanism regulating cellular activity. Fungal pathogens face reactive oxygen species produced by the host plant's oxidative burst in addition to endogenous reactive oxygen species produced during aerobic metabolism. An array of preformed and induced detoxifying enzymes, including superoxide dismutase, catalases, and peroxidases, could allow fungi to infect plants despite the oxidative burst. We isolated a gene (CHAP1) encoding a redox-regulated transcription factor in Cochliobolus heterostrophus, a fungal pathogen of maize. CHAP1 is a bZIP protein that possesses two cysteine-rich domains structurally and functionally related to Saccharomyces cerevisiae YAP1. Deletion of CHAP1 in C. heterostrophus resulted in decreased resistance to oxidative stress caused by hydrogen peroxide and menadione, but the virulence of chap1 mutants was unaffected. Upon activation by oxidizing agents or plant signals, a green fluorescent protein (GFP)-CHAP1 fusion protein became localized in the nucleus. Expression of genes encoding antioxidant proteins was induced in the wild type but not in chap1 mutants. Activation of CHAP1 occurred from the earliest stage of plant infection, in conidial germ tubes on the leaf surface, and persisted during infection. Late in the course of infection, after extensive necrotic lesions were formed, GFP-CHAP1 redistributed to the cytosol in hyphae growing on the leaf surface. Localization of CHAP1 to the nucleus may, through changes in the redox state of the cell, provide a mechanism linking extracellular cues to transcriptional regulation during the plant-pathogen interaction.

ABSTRACT Nonribosomal 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 NPS gene 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 nps mutants had normal growth, mating ability, and appressoria. Real-time PCR analysis showed that expression of all NPS genes 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 NPS6 is 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.

Insertional mutants of the fungal maize pathogen Cochliobolus heterostrophus were screened for altered virulence. One mutant had 60% reduction in lesion size relative to WT but no other detectable change in phenotype. Analysis of sequence at the insertion site revealed a gene ( CPS1 ) encoding a protein with two AMP-binding domains. CPS1 orthologs were detected in all Cochliobolus spp. examined, in several other classes of ascomycete fungi, and in animals but not in basidiomycete fungi, bacteria, or plants. Phylogenetic analysis suggested that CPS1 represents a previously undescribed subset of adenylate-forming enzymes that have diverged from certain acyl-CoA ligases, which in bacteria are involved in biosynthesis of nonribosomal peptides or polyketide/peptide hybrids. Disruption of CPS1 caused reduced virulence of both race T and race O of C. heterostrophus on maize, of Cochliobolus victoriae on oats, and of Gibberella zeae on wheat. These results suggest that CPS1 functions as a general fungal virulence factor in plant pathogenic ascomycetes.

A commonly used method for fungal gene deletion is introduction of linear DNA consisting of a selectable marker gene flanked on both sides by short stretches of DNA that target a gene of interest (Wirsel et al 1996 Curr. Genet 29:241-249). Gene deletion in Cochliobolus heterostrophus and Gibberella zeae occurs efficiently with this approach. To facilitate deletion construct synthesis, we have applied the "split-marker” deletion strategy previously developed for Saccharomyces cerevisiae (Fairhead et al. 1996 Yeast 12:1439-57; Fairhead et al. 1998 Gene 223:33-46). Here, we describe both fusion PCR-based and plasmid-based deletion methods using this strategy with PEG-mediated protoplast transformation (Turgeon et al, 1985 Mol. Gen. Genet. 201:450-453). These methods are predicted to work well with any transformable fungus that undergoes homologous recombination between chromosomal and introduced DNA sequences.

Complete inventories of kinesins from three pathogenic filamentous ascomycetes, Botryotinia fuckeliana, Cochliobolus heterostrophus, and Gibberella moniliformis, are described. These protein sequences were compared with those of the filamentous saprophyte, Neurospora crassa and the two yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Data mining and phylogenetic analysis of the motor domain yielded a constant set of 10 kinesins in the filamentous fungal species, compared with a smaller set in S. cerevisiae and S. pombe. The filamentous fungal kinesins fell into nine subfamilies when compared with well-characterized kinesins from other eukaryotes. A few putative kinesins (one in B. fuckeliana and two in C. heterostrophus) could not be defined as functional, due to unorthodox organization and lack of experimental data. The broad representation of filamentous fungal kinesins across most of the known subfamilies and the ease of gene manipulation make fungi ideal models for functional and evolutionary investigation of these proteins.

Fungal type I polyketides (PKs) are synthesized by PK synthases (PKSs) and include well known secondary metabolites such as the anticholesterol drug lovastatin and the potent natural carcinogen aflatoxin. Other type I PKs are known to be virulence factors for some plant pathogens and pigments such as melanin. in this study, a phylogenomic approach was used to investigate the origin and diversity of fungal genes encoding putative PKSs that are predicted to synthesize type I PKs. The resulting genealogy, constructed by using the highly conserved PKS ketosynthase (KS) domain, indicated that: (i) Species within subphylum Pezizomycotina (phylum Ascomycota) but not early diverging ascomycetes, like Saccharomyces cerevisiae (Saccharomycotina) or Schizosaccharomyces pombe (Taphrinomycotina), had large numbers (7-25) of PKS genes. (ii) Bacteria and fungi had separate groups of PKS genes; the few exceptions are the likely result of horizontal gene transfer from bacteria to various sublineages of fungi. (iii) The bulk of genes encoding fungal PKSs fell into eight groups. Four groups were predicted to synthesize variously reduced PKs, and four groups were predicted to make unreduced PKs. (iv) Species within different classes of Pezizomycotina shared the same groups of PKS genes. (v) Different fungal genomes shared few putative orthologous PKS genes, even between closely related genomes in the same class or genus. (vi) The discontinuous distributions of orthologous PKSs among fungal species can be explained by gene duplication, divergence, and gene loss; horizontal gene transfer among fungi does not need to be invoked.

ABSTRACT Two-component phosphorelay systems are minimally comprised of a histidine kinase (HK) component, which autophosphorylates in response to an environmental stimulus, and a response regulator (RR) component, which transmits the signal, resulting in an output such as activation of transcription, or of a mitogen-activated protein kinase cascade. The genomes of the yeasts Saccharomyces cerevisiae , Schizosaccharomyces pombe , and Candida albicans encode one, three, and three HKs, respectively. In contrast, the genome sequences of the filamentous ascomycetes Neurospora crassa , Cochliobolus heterostrophus ( Bipolaris maydis ), Gibberella moniliformis ( Fusarium verticillioides ), and Botryotinia fuckeliana ( Botrytis cinerea ) encode an extensive family of two-component signaling proteins. The putative HKs fall into 11 classes. Most of these classes are represented in each filamentous ascomycete species examined. A few of these classes are significantly more prevalent in the fungal pathogens than in the saprobe N. crassa , suggesting that these groups contain paralogs required for virulence. Despite the larger numbers of HKs in filamentous ascomycetes than in yeasts, all of the ascomycetes contain virtually the same downstream histidine phosphotransfer proteins and RR proteins, suggesting extensive cross talk or redundancy among HKs.

The genome of the maize pathogen Cochliobolus heterostrophus encodes three unlinked monofunctional catalase-encoding (CAT) genes that singly or in combination could offer protection against the harmful effects of oxidative stress. Phylogenetic analysis placed the CAT2 and CAT3 proteins in a cluster with large subunit catalases (CAT3 has a secretory signal sequence and was grouped with known secreted catalases), whereas CAT1 clustered with small subunit catalases. Single, double, and triple cat mutants were created and screened for sensitivity to hydrogen peroxide and altered virulence on maize. All mutants deficient in CAT3 had enhanced sensitivity to hydrogen peroxide, as compared with wild type or with mutants deficient in CAT1, CAT2, or both. All catalase-deficient mutants had normal virulence to maize. Thus, the secreted CAT3 protein protects the fungus from oxidative stress during vegetative growth, but members of this enzyme family, alone or in combination, are not essential for virulence.

Genes at two unlinked loci (Tox1A and Tox1B) are required for production of the polyketide T-toxin by Cochliobolus heterostrophus race T, a pathogenic fungus that requires T-toxin for high virulence to maize with T-cytoplasm. Previous work indicated that Tox1A encodes a polyketide synthase (PKS1) required for T-toxin biosynthesis and for high virulence. To identify genes at Tox1B, a wild-type race T cDNA library was screened for genes missing in the genome of a Tox1B deletion mutant. The library was probed, first with a 415-kb NotI restriction fragment from the genome of the Tox1B¯ mutant, then with the corresponding 560-kb fragment from the genome of wild type. Two genes, DEC1 (similar to aceto-acetate decarboxylase-encoding genes) and RED1 (similar to genes encoding members of the medium-chain dehydro-genase/reductase superfamily), were recovered. Targeted disruption of DEC1 drastically reduced both T-toxin production and virulence of race T to T-cytoplasm maize, whereas specific inactivation of RED1 had no apparent effect on T-toxin production (as determined by bioassay) or on virulence. DEC1 and RED1 map within 1.5 kb of each other on Tox1B chromosome 6;12 and are unique to the genome of race T, an observation consistent with the hypothesis that these genes were acquired by C. heterostrophus via a horizontal transfer event.

The filamentous fungal genetics community has enthusiastically embraced the utilization of genomics technologies to resolve long-standing issues in fungal biology. For example, such technologies have been proposed to study the mechanics of tip growth, photoreception, gene silencing, the molecular basis of conidiation, the pathway leading to sexual reproduction, and mechanisms of pathogenesis. These studies have provided a refreshing change of pace in research on filamentous fungi, which has lagged behind that on other eukaryotes in the exploitation of genome-wide methodologies. Despite the late start, several fungal genome sequencing projects are underway. The resulting databases will allow the comprehensive analysis of developmental processes that are characteristic of fungi, including the molecular nature of pathogenicity. DNA databases underpin analyses of the fungal transcriptome, proteome, and metabolome. This combined information will contribute to our basic understanding of not only the mechanics of infection but also the evolution of pathogenicity.

Mating type (MAT) genes were cloned from three members of the Gibberella/Fusarium complex that differ in reproductive mode: heterothallic G. fujikuroi, homothallic G. zeae, and asexual F. oxysporum. The G. fujikuroi MAT locus organization is typical of other heterothallic pyrenomycetes characterized to date; i.e., there are three genes at MAT1-1 and one at MAT1-2. G. zeae has homologues of all four genes encoded by the two G. fujikuroi MAT idiomorphs, tightly linked on the same chromosome, interspersed with sequences unique to G. zeae. Field isolates of F. oxysporum, although asexual, have either the MAT1-1 or the MAT1-2 genes found in sexual species and these genes are highly similar to those of heterothallic G. fujikuroi. RT-PCR analysis proved that the F. oxysporum MAT genes are expressed and that all putative introns found in each of the four MAT genes in G. fujikuroi and F. oxysporum are removed. Apparent failure of F. oxysporum to reproduce sexually could not be attributed to mutations in the MAT genes themselves.

The new sesterterpenoid 6-epi-3-anhydroophiobolin B (1) and six known ophiobolins were isolated from the extracts of the fungus Cochliobolus heterostrophus race O. The structure of 6-epi-3-anhydroophiobolin B was deduced from analysis of spectral data and the structural characterization of dehydration and dimerization products. Ophiobolin A (2) showed potent activity in cytotoxicity assays and marginal activity in antimalarial assays.