We have identified a new gene encoding the G protein ␣ subunit, gna-3, from the filamentous fungus Neurospora crassa. The predicted amino acid sequence of GNA-3 is most similar to the G␣ proteins MOD-D, MAGA, and CPG-2 from the saprophytic fungus Podospora anserina and the pathogenic fungi Magnaporthe grisea and Cryphonectria parasitica, respectively. Deletion of gna-3 leads to shorter aerial hyphae and premature, dense conidiation during growth on solid medium or in standing liquid cultures and to inappropriate conidiation in submerged culture. The conidiation and aerial hypha defects of the ⌬gna-3 strain are similar to those of a previously characterized adenylyl cyclase mutant, cr-1. Supplementation with cyclic AMP (cAMP) restores wild-type morphology to ⌬gna-3 strains in standing liquid cultures. Solid medium augmented with exogenous cAMP suppresses the premature conidiation defect, but aerial hypha formation is still reduced. Submergedculture conidiation is refractory to cAMP but is suppressed by peptone. In addition, ⌬gna-3 submerged cultures express the glucose-repressible gene, qa-2, to levels greatly exceeding those observed in the wild type under carbon-starved conditions. ⌬gna-3 strains exhibit reduced fertility in homozygous crosses during the sexual cycle; exogenous cAMP has no effect on this phenotype. Intracellular steady-state cAMP levels of ⌬gna-3 strains are decreased 90% relative to the wild type under a variety of growth conditions. Reduced intracellular cAMP levels in the ⌬gna-3 strain correlate with lower adenylyl cyclase activity and protein levels. These results demonstrate that GNA-3 modulates conidiation and adenylyl cyclase levels in N. crassa.G-protein-coupled receptors (GPCRs) are a family of seven transmembrane helix receptors that bind ligands such as neurotransmitters, pheromones, and odorants. GPCRs are associated with heterotrimeric G proteins, consisting of ␣, ␤, and ␥ subunits (for a review, see reference 82). In the inactive state, the heterotrimer is docked at the receptor and GDP is bound to the G␣ subunit. Binding of a ligand activates the receptor, leading to the exchange of GDP for GTP on G␣ and the subsequent dissociation of G␣-GTP from the G␤␥ moiety. G␣-GTP and the G␤␥ dimer can activate downstream effectors, such as enzymes and ion channels, to produce a response to the signal (for a review, see reference 7). The response is terminated and the cycle is completed with hydrolysis of GTP by G␣. The GDP-bound G␣ protein reassociates with G␤␥, and the heterotrimer is then able to bind to the receptor to await the next cycle of activation (for reviews, see references 10 and 20).Neurospora crassa is a filamentous fungus that has a complex life cycle due to its ability to produce both asexual and sexual spores (for a review, see reference 71). Grown with adequate nitrogen, N. crassa remains in the asexual cycle and extends basal hyphae to form a complex, intertwined network called a mycelium. Asexual spores, or conidia, are produced by two pathways: macroconidiation and micr...

Biological control of chestnut blight caused by the filamentous ascomycete Cryphonectria parasitica can be achieved with a virus that infects this fungus. This hypovirus causes a perturbation of fungal development that results in low virulence (hypovirulence), poor asexual sporulation, and female infertility without affecting fungal growth in culture. At the molecular level, the virus is known to affect the transcription of a number of fungal genes. Two of these genes, Vir1 and Vir2, produce abundant transcripts in noninfected strains of the fungus, but the transcripts are not detectable in virus-infected strains. We report here that these two genes encode the pheromone precursors of the Mat-2 mating type of the fungus; consequently, these genes have been renamed Mf2/1 and Mf2/2. To determine if the virus affects the mating systems of both mating types of this fungus, the pheromone precursor gene, Mf1/1, of a Mat-1 strain was cloned and likewise was found to be repressed in virus-infected strains. The suppression of transcription of the pheromone precursor genes of this fungus could be the cause of the mating defect of infected strains of the fungus. Although published reports suggest that a G␣ i subunit may be involved in this regulation, our results do not support this hypothesis. The prepropheromone encoded by Mf1/1 is structurally similar to that of the prepro-p-factor of Schizosaccharomyces pombe. This is the first description of the complete set of pheromone precursor genes encoded by a filamentous ascomycete.Chestnut blight, caused by the ascomycete Cryphonectria parasitica, is one of the most devastating plant diseases in recorded history. Because of this disease, only occasional root sprouts grow where the American chestnut (Castanea dentata) was once dominant in eastern North America. A similar fate for the European chestnut (C. sativa) in European forests and orchards was prevented by a naturally occurring biological control of the fungus. The basis of this biological control is infection of C. parasitica by double-stranded RNA viruses which reduce the virulence of the fungus, causing a condition termed hypovirulence. The effect of the virus on reducing the virulence of this plant pathogen is the basis of an effective biological control of chestnut blight (1,7,13,16,24,25).One of these hypoviruses, CHV1-713, causes no detectable effects on the growth rate of its host in culture but perturbs normal developmental processes such as sporulation and virulence. The visible symptoms of CHV1-infected colonies growing on agar are a white rather than orange colony color and poor asexual sporulation, providing a useful phenotype for the study of this virus. The sexual cycle of the fungus is also perturbed by the virus, with the female parent being sterile. At the molecular level, the virus causes transcriptional down-regulation of a number of host genes (10). The cloning and identification of the function of some of these genes have provided us with a group of molecular markers of virus infection. We have previo...

In an effort to understand the role of hydroxyproline-rich glycoproteins (HRGPs) in plant cell wall structure, we studied the distribution and physical properties of PC-1-like proteins (PC-1 being the major pericarp HRGP) throughout sporophytic tissues of two maize (Zea mays L.) varieties. We determined total amounts of hydroxyproline, an indicator of HRGPs, and did tissue print and Western blot analysis. We found hydroxyproline in cell walls of stems, leaves, roots, tassels, and silks. We also observed reactivity of anti-PC-1 monoclonal antibodies with anatomical prints of these tissues on nitrocellulose paper. Stem nodes and silks contained the most hydroxyproline and exhibited the strongest reaction with the antibody. PC-1 was localized in vascular bundles and the epidermis of stem tissue.

Heterotrimeric G proteins, consisting of α, β and γ subunits, mediate a variety of signaling pathways in eukaryotes. We have previously identified two genes, gna-1 and gna-2, that encode G protein α subunits in the filamentous fungus Neurospora crassa. Mutation of gna-1 results in female infertility and sensitivity to hyperosmotic media. In this study, we investigate the expression and functions of gna-2. Results from Western analysis and measurements of gna-2 promoter-lacZ fusion activity indicate that gna-2 is expressed during the vegetative and sexual cycle of N. crassa in both A and a mating types. Activating mutations predicted to abolish the GTPase activity of GNA-2 cause subtle defects in aerial hyphae formation and conidial germination. Extensive phenotypic analysis of Δgna-2 strains did not reveal abnormalities during vegetative or sexual development. In contrast, deletion of gna-2 in a Δgna-1 strain accentuates the Δgna-1 phenotypes. Δgna-1 Δgna-2 strains have a slower rate of hyphal apical extension than Δgna-1 strains on hyperosmotic media. Moreover, Δgna-1 Δgna-2 mutants have more pronounced defects in female fertility than Δgna-1 strains. We propose that gna-1 and gna-2 have overlapping functions and may constitute a gene family. This is the first report of G protein α subunits with overlapping functions in eukaryotic microbes.