A pheromone receptor gene, pre-1, is essential for mating type-specific directional growth and fusion of trichogynes and female fertility in Neurospora crassa

Neurospora crassa is a heterothallic filamentous fungus with two mating types, mat a and mat A. Its mating involves differentiation of female reproductive structures (protoperithecia) and chemotropic growth of female-specific hyphae (trichogynes) towards a cell of the opposite mating type in a pheromone-mediated process. In this study, we characterize the pre-1 gene, encoding a predicted G-protein-coupled receptor with sequence similarity to fungal pheromone receptors. pre-1 is most highly expressed in mat A strains under mating conditions, but low levels can also be detected in mat a strains. Analysis of pre-1 deletion mutants showed that loss of pre-1 does not greatly affect vegetative growth, heterokaryon formation or male fertility in either mating type. Protoperithecia from Deltapre-1 mat A mutants do not undergo fertilization; this defect largely stems from an inability of their trichogynes to recognize and fuse with mat a cells. Previous work has demonstrated that the Galpha subunit, GNA-1, and the Gbeta protein, GNB-1, are essential for female fertility in N. crassa. Trichogynes of Deltagna-1 and Deltagnb-1 mutants displayed severe defects in growth towards and fusion with male cells, similar to that of Deltapre-1 mat A strains. However, the female sterility defect of the Deltapre-1 mat A mutant could not be complemented by constitutive activation of gna-1, suggesting additional layers of regulation. We propose that PRE-1 is a pheromone receptor coupled to GNA-1 that is essential for the mating of mat A strains as females, consistent with a role in launching the pheromone response pathway in N. crassa.     

Positive regulation of adenylyl cyclase activity by a galphai homolog in Neurospora crassa

GNA-1 and GNA-2 are two G protein alpha subunits from the filamentous fungus Neurospora crassa. Loss of gna-1 leads to multiple phenotypes, while Deltagna-2 strains do not exhibit visible defects. However, Deltagna-1Deltagna-2 mutants are more affected in Deltagna-1 phenotypes. Here we report a biochemical investigation of the roles of GNA-1 and GNA-2 in cAMP metabolism. Assays of Mg2+ ATP-dependent adenylyl cyclase activity (+/-GppNHp) in extracts from submerged cultures indicated that Deltagna-2 strains were normal, whereas Deltagna-1 and Deltagna-1Deltagna-2 strains had only 10-15% the activity of the wild-type control. Levels of the Gbeta protein, GNB-1, were normal in Deltagna-1 strains, excluding altered GNB-1 production as a factor in loss of adenylyl cyclase activity. Steady-state cAMP levels in Deltagna-1 and Deltagna-1Deltagna-2 mutants were reduced relative to wild-type under conditions that result in morphological abnormalities (solid medium), while levels in submerged culture were normal. cAMP phosphodiesterase activities in submerged cultures of Deltagna-1 and/or Deltagna-2 strains were lower than in wild-type; the individual deletions were additive in decreasing activity. These results suggest that in submerged culture, N. crassa, like mammalian systems, possesses compensatory mechanisms that maintain cAMP at relatively constant levels. Furthermore, the finding that Mg2+ATP-dependent adenylyl cyclase activity in wild-type cell extracts could be inhibited using anti-GNA-1 IgG suggests that GNA-1 directly interacts with adenylyl cyclase in N. crassa.     

Genetic and Physical Interactions between Gα Subunits and Components of the Gβγ Dimer of Heterotrimeric G Proteins in Neurospora crassa

Heterotrimeric G proteins are critical regulators of growth and asexual and sexual development in the filamentous fungus Neurospora crassa. Three Gα subunits (GNA-1, GNA-2, and GNA-3), one Gβ subunit (GNB-1), and one Gγ subunit (GNG-1) have been functionally characterized, but genetic epistasis relationships between Gβ and Gα subunit genes have not been determined. Physical association between GNB-1 and FLAG-tagged GNG-1 has been previously demonstrated by coimmunoprecipitation, but knowledge of the Gα binding partners for the Gβγ dimer is currently lacking. In this study, the three N. crassa Gα subunits are analyzed for genetic epistasis with gnb-1 and for physical interaction with the Gβγ dimer. We created double mutants lacking one Gα gene and gnb-1 and introduced constitutively active, GTPase-deficient alleles for each Gα gene into the Δgnb-1 background. Genetic analysis revealed that gna-3 is epistatic to gnb-1 with regard to negative control of submerged conidiation. gnb-1 is epistatic to gna-2 and gna-3 for aerial hyphal height, while gnb-1 appears to act upstream of gna-1 and gna-2 during aerial conidiation. None of the activated Gα alleles restored female fertility to Δgnb-1 mutants, and the gna-3^Q208L allele inhibited formation of female reproductive structures, consistent with a need for Gα proteins to cycle through the inactive GDP-bound form for these processes. Coimmunoprecipitation experiments using extracts from the gng-1-FLAG strain demonstrated that the three Gα proteins interact with the Gβγ dimer. The finding that the Gβγ dimer interacts with all three Gα proteins is supported by epistasis between gnb-1 and gna-1, gna-2, and gna-3 for at least one function.     

GPR-4 is a predicted G-protein-coupled receptor required for carbon source-dependent asexual growth and development in Neurospora crassa

The filamentous fungus Neurospora crassa is able to utilize a wide variety of carbon sources. Here, we examine the involvement of a predicted G-protein-coupled receptor (GPCR), GPR-4, during growth and development in the presence of different carbon sources in N. crassa. Deltagpr-4 mutants have reduced mass accumulation compared to the wild type when cultured on high levels of glycerol, mannitol, or arabinose. The defect is most severe on glycerol and is cell density dependent. The genetic and physical relationship between GPR-4 and the three N. crassa Galpha subunits (GNA-1, GNA-2, and GNA-3) was explored. All three Galpha mutants are defective in mass accumulation when cultured on glycerol. However, the phenotypes of Deltagna-1 and Deltagpr-4 Deltagna-1 mutants are identical, introduction of a constitutively activated gna-1 allele suppresses the defects of the Deltagpr-4 mutation, and the carboxy terminus of GPR-4 interacts most strongly with GNA-1 in the yeast two-hybrid assay. Although steady-state cyclic AMP (cAMP) levels are normal in Deltagpr-4 strains, exogenous cAMP partially remediates the dry mass defects of Deltagpr-4 mutants on glycerol medium and Deltagpr-4 strains lack the transient increase in cAMP levels observed in the wild type after addition of glucose to glycerol-grown liquid cultures. Our results support the hypothesis that GPR-4 is coupled to GNA-1 in a cAMP signaling pathway that regulates the response to carbon source in N. crassa. GPR-4-related GPCRs are present in the genomes of several filamentous ascomycete fungal pathogens, raising the possibility that a similar pathway regulates carbon sensing in these organisms.     

RIC8 is a guanine-nucleotide exchange factor for Galpha subunits that regulates growth and development in Neurospora crassa

Heterotrimeric (αβγ) G proteins are crucial components of eukaryotic signal transduction pathways. G-protein-coupled receptors (GPCRs) act as guanine nucleotide exchange factors (GEFs) for Gα subunits. Recently, facilitated GDP/GTP exchange by non-GPCR GEFs, such as RIC8, has emerged as an important mechanism for Gα regulation in animals. RIC8 is present in animals and filamentous fungi, such as the model eukaryote Neurospora crassa, but is absent from the genomes of baker's yeast and plants. In Neurospora, deletion of ric8 leads to profound defects in growth and asexual and sexual development, similar to those observed for a mutant lacking the Gα genes gna-1 and gna-3. In addition, constitutively activated alleles of gna-1 and gna-3 rescue many defects of Δric8 mutants. Similar to reports in Drosophila, Neurospora Δric8 strains have greatly reduced levels of G-protein subunits. Effects on cAMP signaling are suggested by low levels of adenylyl cyclase protein in Δric8 mutants and suppression of Δric8 by a mutation in the protein kinase A regulatory subunit. RIC8 acts as a GEF for GNA-1 and GNA-3 in vitro, with the strongest effect on GNA-3. Our results support a role for RIC8 in regulating GNA-1 and GNA-3 in Neurospora.     

Severe impairment of growth and differentiation in a Neurospora crassa mutant lacking all heterotrimeric G alpha proteins

Heterotrimeric G alpha proteins play a critical role in regulating growth and differentiation in filamentous fungi. No systematic analysis of functional relationships between subunits has been investigated. This study explores the relative contributions of Neurospora crassa G alpha subunits, gna-1, gna-2, and gna-3, in directing development by analyzing strains deleted for various combinations of these genes. Although viable, mutants lacking all G alpha subunits or gna-1 and gna-3 are severely restricted in apical growth, forming small colonies. These strains form little aerial hyphae during asexual development on solid medium and exhibit inappropriate sporulation in submerged cultures. Similar to all strains carrying the Delta gna-1 mutation, these mutants are female sterile. Defects attributed to gna-2 are observed only in conjunction with the loss of gna-1 or gna-3, suggesting a minor role for this G alpha in N. crassa biology. Results from analysis of adenylyl cyclase and epistatic studies with the cAMP-dependent protein kinase regulatory subunit (mcb) indicate separate functions for GNA-1 and GNA-3 in cAMP metabolism and additional cAMP-independent roles for GNA-1. These studies indicate that although G alpha subunits are not essential for viability in filamentous fungi, their loss results in an organism that cannot effectively forage for nutrients or undergo asexual or sexual reproduction.     

Regulation of conidiation and adenylyl cyclase levels by the Galpha protein GNA-3 in Neurospora crassa

We have identified a new gene encoding the G protein alpha subunit, gna-3, from the filamentous fungus Neurospora crassa. The predicted amino acid sequence of GNA-3 is most similar to the Galpha 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 Deltagna-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 Deltagna-3 strains in standing liquid cultures. Solid medium augmented with exogenous cAMP suppresses the premature conidiation defect, but aerial hypha formation is still reduced. Submerged-culture conidiation is refractory to cAMP but is suppressed by peptone. In addition, Deltagna-3 submerged cultures express the glucose-repressible gene, qa-2, to levels greatly exceeding those observed in the wild type under carbon-starved conditions. Deltagna-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 Deltagna-3 strains are decreased 90% relative to the wild type under a variety of growth conditions. Reduced intracellular cAMP levels in the Deltagna-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.     

The Guanine Nucleotide Exchange Factor RIC8 Regulates Conidial Germination through Gα Proteins in Neurospora crassa

Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia) germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.     

Overlapping Functions for Two G Protein α Subunits in Neurospora Crassa

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.     

The G alpha i homologue gna-1 controls multiple differentiation pathways in Neurospora crassa.

Heterotrimeric G proteins are components of principal signaling pathways in eukaryotes. In higher organisms, alpha subunits of G proteins have been divided into four families, Gi, Gs, Gq, and G12. We previously identified a G alpha i homologue gna-1 in the filamentous fungus Neurospora crassa. Now we report that deletion of gna-1 leads to multiple phenotypes during the vegetative and sexual cycles in N. crassa. On solid medium, delta gna-1 strains have a slower rate of hyphal apical extension than wild type, a rate that is more pronounced under hyperosmotic conditions or in the presence of a cellophane overlay. delta gna-1 mutants accumulate less mass than wild-type strains, and their mass accumulation is not affected in the same way by exposure to light. delta gna-1 strains are defective in macroconidiation, possessing aerial hyphae that are shorter, contain abnormal swellings, and differentiate adherent macroconidia. During the sexual cycle, delta gna-1 strains are fertile as males. However, the mutants are female-sterile, producing small, aberrant female reproductive structures. After fertilization, delta gna-1 female structures do not enlarge and develop normally, and no sexual spores are produced. Thus, mutation of gna-1 results in sex-specific loss of fertility.     

A catalytic subunit of cyclic AMP-dependent protein kinase, PKAC-1, regulates asexual differentiation in Neurospora crassa

A cyclic AMP (cAMP)-dependent protein kinase pathway has been shown to regulate growth, morphogenesis and virulence in filamentous fungi. However, the precise mechanisms of regulation through the pathway remain poorly understood. In Neurospora crassa, the cr-1 adenylate cyclase mutant exhibits colonial growth with short aerial hyphae bearing conidia, and the mcb mutant, a mutant of the regulatory subunit of cAMP-dependent protein kinase (PKA), shows the loss of growth polarity at the restrictive temperature. In the present study, we isolated mutants of the catalytic subunit of the PKA gene pkac-1 through the process of repeat-induced point mutation (RIP). PKA activity of the mutants obtained through RIP was undetectable. The genome sequence predicts two distinct catalytic subunit genes of PKA, named pkac-1 (NCU06240.1, AAF75276) and pkac-2 (NCU00682.1), as is the case in most filamentous fungi. The results suggest that PKAC-1 works as the major PKA in N. crassa. The phenotype of the pkac-1 mutants included colonial growth, short aerial hyphae, premature conidiation on solid medium, inappropriate conidiation in submerged culture, and increased thermotolerance. This phenotype of pkac-1 mutants resembled to that of cr-1 mutants, except that the addition of cAMP did not rescue the abnormal morphology of pkac-1 mutants. The loss of growth polarity at the restrictive temperature in the mcb mutant was suppressed by pkac-1 mutation. These results suggest that the signal transduction pathway mediated by PKAC-1 plays an important role in regulation of aerial hyphae formation, conidiation, and hyphal growth with polarity.     

Ggamma1 + Ggamma2 not equal to Gbeta: heterotrimeric G protein Ggamma-deficient mutants do not recapitulate all phenotypes of Gbeta-deficient mutants

Heterotrimeric G proteins are signaling molecules ubiquitous among all eukaryotes. The Arabidopsis (Arabidopsis thaliana) genome contains one Galpha (GPA1), one Gbeta (AGB1), and two Ggamma subunit (AGG1 and AGG2) genes. The Gbeta requirement of a functional Ggamma subunit for active signaling predicts that a mutant lacking both AGG1 and AGG2 proteins should phenotypically resemble mutants lacking AGB1 in all respects. We previously reported that Gbeta- and Ggamma-deficient mutants coincide during plant pathogen interaction, lateral root development, gravitropic response, and some aspects of seed germination. Here, we report a number of phenotypic discrepancies between Gbeta- and Ggamma-deficient mutants, including the double mutant lacking both Ggamma subunits. While Gbeta-deficient mutants are hypersensitive to abscisic acid inhibition of seed germination and are hyposensitive to abscisic acid inhibition of stomatal opening and guard cell inward K+ currents, none of the available Ggamma-deficient mutants shows any deviation from the wild type in these responses, nor do they show the hypocotyl elongation and hook development defects that are characteristic of Gbeta-deficient mutants. In addition, striking discrepancies were observed in the aerial organs of Gbeta- versus Ggamma-deficient mutants. In fact, none of the distinctive traits observed in Gbeta-deficient mutants (such as reduced size of cotyledons, leaves, flowers, and siliques) is present in any of the Ggamma single and double mutants. Despite the considerable amount of phenotypic overlap between Gbeta- and Ggamma-deficient mutants, confirming the tight relationship between Gbeta and Ggamma subunits in plants, considering the significant differences reported here, we hypothesize the existence of new and as yet unknown elements in the heterotrimeric G protein signaling complex.     

Analysis of the pdx-1 (snz-1/sno-1) region of the Neurospora crassa genome: correlation of pyridoxine-requiring phenotypes with mutations in two structural genes

We report the analysis of a 36-kbp region of the Neurospora crassa genome, which contains homologs of two closely linked stationary phase genes, SNZ1 and SNO1, from Saccharomyces cerevisiae. Homologs of SNZ1 encode extremely highly conserved proteins that have been implicated in pyridoxine (vitamin B6) metabolism in the filamentous fungi Cercospora nicotianae and in Aspergillus nidulans. In N. crassa, SNZ and SNO homologs map to the region occupied by pdx-1 (pyridoxine requiring), a gene that has been known for several decades, but which was not sequenced previously. In this study, pyridoxine-requiring mutants of N. crassa were found to possess mutations that disrupt conserved regions in either the SNZ or SNO homolog. Previously, nearly all of these mutants were classified as pdx-1. However, one mutant with a disrupted SNO homolog was at one time designated pdx-2. It now appears appropriate to reserve the pdx-1 designation for the N. crassa SNZ homolog and pdx-2 for the SNO homolog. We further report annotation of the entire 36,030-bp region, which contains at least 12 protein coding genes, supporting a previous conclusion of high gene densities (12,000-13,000 total genes) for N. crassa. Among genes in this region other than SNZ and SNO homologs, there was no evidence of shared function. Four of the genes in this region appear to have been lost from the S. cerevisiae lineage.