Search

Histidine kinase (HK) phosphorelay signaling is a major mechanism by which fungi sense their environment. The maize pathogen Cochliobolus heterostrophus has 21 HK genes, 4 candidate response regulator (RR) genes (SSK1, SKN7, RIM15, REC1), and 1 gene (HPT1) encoding a histidine phosphotransfer domain protein. Because most HKs are expected to signal through RRs, these were chosen for deletion. Except for pigment and slight growth alterations for rim15 mutants, no measurable altered phenotypes were detected in rim15 or rec1 mutants. Ssk1p is required for virulence and affects fertility and proper timing of sexual development of heterothallic C. heterostrophus. Pseudothecia from crosses involving ssk1 mutants ooze masses of single ascospores, and tetrads cannot be found. Wild-type pseudothecia do not ooze. Ssk1p represses asexual spore proliferation during the sexual phase, and lack of it dampens asexual spore proliferation during vegetative growth, compared to that of the wild type. ssk1 mutants are heavily pigmented. Mutants lacking Skn7p do not display any of the above phenotypes; however, both ssk1 and skn7 mutants are hypersensitive to oxidative and osmotic stresses and ssk1 skn7 mutants are more exaggerated in their spore-type balance phenotype and more sensitive to stress than single mutants. ssk1 mutant phenotypes largely overlap hog1 mutant phenotypes, and in both types of mutant, the Hog1 target gene, MST1, is not induced. ssk1 and hog1 mutants were examined in the homothallic cereal pathogen Gibberella zeae, and pathogenic and reproductive phases of development regulated by Ssk1 and Hog1 were found to mirror, but also vary from, those of C. heterostrophus.

Connections 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 (NPS1 to 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 nps2 crosses. Homozygous crosses of the remaining 11 nps strains showed wild-type (WT) fertility. Phylogenetic, expression, and biochemical analyses demonstrated that the NRPS encoded by NPS2 is responsible for the biosynthesis of ferricrocin, the intracellular siderophore of C. heterostrophus. Functional conservation of NPS2 in both heterothallic C. heterostrophus and the unrelated homothallic ascomycete Gibberella zeae was demonstrated. G. zeae nps2 strains are concomitantly defective in intracellular siderophore (ferricrocin) biosynthesis and sexual development. Exogenous application of iron partially restored fertility to C. heterostrophus and G. zeae nps2 strains, demonstrating that abnormal sexual development of nps2 strains is at least partly due to their iron deficiency. Exogenous application of the natural siderophore ferricrocin to C. heterostrophus and G. zeae nps2 strains restored WT fertility. NPS1, a G. zeae NPS gene 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. heterostrophus and the homothallic ascomycete G. zeae.

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.

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.

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.

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.

ABSTRACTTwo-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 albicansencode 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.

About 15000 species of ascomycete fungi lack a known sexual state. For fungi with asexual states in the anamorph genera Embellisia, Ulocladium, and Alternaria, six species have known sexual states but more than 50 species do not. In sexual filamentous ascomycetes, opposite mating type information at the MAT1 locus regulates mating and the opposite mating type genes each have a clonal, non-recombining phylogenetic history. We used PCR to amplify and sequence fragments of the opposite mating type genes from three supposedly asexual species, A. brassicae, A. brassicicola and A. tenuissima. Each haploid fungal isolate had just one mating type, but both mating types were present in all the three species. We sequenced the ribosomal ITS regions for isolates of opposite mating type, for the three asexual species and four known related sexual species. In a phylogenetic analysis including other ITS sequences from GenBank®, the three asexual species were not closely related to any of the known sexual species. Isolates of opposite mating type but the same species had identical ITS sequences. During any period of asexual evolutionary history, lineages of each mating type would have had a separate evolutionary history and any ITS substitutions shared between isolates of opposite mating type would have had to accumulate by convergence. Allowing for varying substitution rates and assuming a Poisson distribution of substitutions, the probability that isolates of opposite mating type shared an ITS substitution through convergence was low. This suggests that isolates of opposite mating type of A. brassicae, A. brassicicola and A. tenuissima were exchanging substitutions through sexual or parasexual reproduction while the ITS was evolving. If sexuality was lost, it was lost after the period of evolutionary history represented by the shared substitutions.

A gene (NhKIN1) encoding a kinesin was cloned fromNectria haematococcagenomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed thatNhKIN1belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkörper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkörper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkörper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology.

Twenty-three strains of Cochliobolus heterostrophus were examined for the presence of plasmid DNA. One isolate, T40, contained a 1.9 kb sequence which occurred as a series of circular head-to-tail multimers with from 1 to 17 or more monomers per plasmid molecule. The plasmid was cloned in pBR322 to facilitate analysis. It was homologous to the mitochondrial chromosome of isolate T40 as well as to the mitochondrial DNAs of C. heterostrophus isolates that did not contain the plasmid; each isolate, including T40, had only one copy of the plasmid sequence integrated into the mitochondrial chromosome and the sequence mapped at the same location in all isolates tested. In the T40 isolate there were about 30 excised copies per chromosome in addition to the single integrated sequence. Presence of the plasmid had no apparent effect on the structural integrity of the mitochondrial chromosome. There was no detectable homology between the plasmid and either C. heterostrophus nuclear DNA or plasmids that have been isolated from mitochondria of Neurospora or Podospora. A circular map was constructed which has 6 sites for hexan-ucleotide-recognizing enzymes and the region of the splice site; no sites were detected in the plasmid for an additional 17 restriction enzymes. The plasmid functioned as an ARS (autonomously replicating sequence) in yeast, although it was highly unstable compared to other ARSs.

Cochliobolus heterostrophus protoplasts transformed with a plasmid carrying the Aspergillus nidulans amdS gene (Hynes et al. 1983) gave rise to colonies on a selective medium that did not support significant growth of wild type cells. The plasmid integrated at a single chromosomal locus in each transformant analyzed and the site of integration differed among transformants. Some transformants had one copy of the plasmid, others had multiple copies tandemly arranged and oriented head-to-tail. Both single and multiple copies segregated meiotically as single genes and were mitotically stable on either selective or nonselective medium. The andS gene is advantageous for transformation of genetically undeveloped fungi because it is selectable in wild type cells in organisms that lack a functional amdS gene, thus eliminating the need for induced mutations in recipient strains. Moreover, there is no background due to reversion of a counter-selected mutant allele.

Fungus-plant interactions involve complex developmental processes in which a variety of fungal and plant molecules are required to determine whether the outcome is a susceptible reaction (successful fungal colonization of plant tissues) or a resistant reaction (the plant mounts a defence that aborts fungal invasion). To understand the molecular basis of fungal disease, it is necessary to identify the fungal molecules that areessentialfor pathogenic processes, and to distinguish them from molecules that may be present during infection but not critical to its outcome. Molecular-genetic technology has been developed for fungal pathogens and used to evaluate the roles of fungal molecules in fungal infection processes. Although the field is in its infancy, several molecules have already been proven as essential components of fungal pathogenesis. Some are clearly involved in the adhesion and penetration phases of infection, i.e. hydrophobins, melanin, glycerol, cutinase, and components of signal transduction pathways (which mediate colonization as well), whereas others are required for colonization of plant tissues after penetration, i.e. toxins that induce susceptibility, toxins that induce resistance, and enzymes that inactivate plant defence mechanisms. Molecular-genetic manipulation has also been used to show that certain candidates for roles in pathogenesis are in fact not involved in any detectable way.

An astral pulling force helps to elongate the mitotic spindle in the filamentous ascomycete, Nectria haematococca. Evidence is mounting that dynein is required for the formation of mitotic spindles and asters. Obviously, this would be an important mitotic function of dynein, since it would be a prerequisite for astral force to be applied to a spindle pole. Missing from the evidence for such a role of dynein in aster formation, however, has been a dynein mutant lacking mitotic asters. To determine whether or not cytoplasmic dynein is involved in mitotic aster formation in N. haematococca, a dynein-deficient mutant was made. Immunocytochemistry visualized few or no mitotic astral microtubules in the mutant cells, and studies of living cells confirmed the veracity of this result by revealing the absence of mitotic aster functions in vivo: intra-astral motility of membranous organelles was not apparent; the rate and extent of spindle elongation during anaphase B were reduced; and spindle pole body separation almost stopped when the anaphase B spindle in the mutant was cut by a laser microbeam, demonstrating unequivocally that no astral pulling force was present. These unique results not only provide a demonstration that cytoplasmic dynein is required for the formation of mitotic asters in N. haematococca; they also represent the first report of mitotic phenotypes in a dynein mutant of any filamentous fungus and the first cytoplasmic dynein mutant of any organism whose mitotic phenotypes demonstrate the requirement of cytoplasmic dynein for aster formation in vivo.

We document that the absence of a single gene encoding a DNase in a fungal plant pathogen results in significantly reduced virulence to a plant host. We compared a wild-type strain of the maize pathogen Cochliobolus heterostrophusand an isogenic mutant lacking a candidate secreted DNase-encoding gene and demonstrated that the mutant is reduced in virulence on leaves and on roots. There are no previous reports of deletion of such a gene from either an animal or plant fungal pathogen accompanied by comparative assays of mutants and wild type for alterations in virulence. We observed DNase activity, in fungal culture filtrates, that is Mg2+dependent and induced when plant host leaf material is present. Our findings demonstrate not only that fungi use extracellular DNases (exDNases) for virulence, but also that the relevant molecules are deployed in above-ground leaves as well as below-ground plant tissues. Overall, these data provide support for a common defense/counter defense virulence mechanism used by animals, plants, and their fungal and bacterial pathogens and suggest that components of the mechanism might be novel targets for the control of plant disease.