Multiple calcium signaling genes play a role in the circadian period of Neurospora crassa.

The Ca2+ signaling genes cpe-1, plc-1, ncs-1, splA2, camk-1, camk-2, camk-3, camk-4, cmd, and cnb-1 are necessary for a normal circadian period length in Neurospora crassa. In addition, the Q10 values ranged between 0.8 and 1.2 for the single mutants lacking cpe-1, splA2, camk-1, camk-2, camk-3, camk-4, and cnb-1, suggesting that the circadian clock exhibits standard temperature compensation. However, the Q10 value for the ∆plc-1 mutant was 1.41 at 25 and 30 °C, 1.53 and 1.40 for the ∆ncs-1 mutant at 20 and 25 °C, and at 20 and 30 °C, respectively, suggesting a partial loss of temperature compensation in these two mutants. Moreover, expression of frq, a regulator of the circadian period, and the blue light receptor wc-1, were increased >2-fold in the Δplc-1, ∆plc-1; ∆cpe-1, and the ∆plc-1; ∆splA2 mutants at 20 °C. The frq mRNA level was increased >2-fold in the Δncs-1 mutant compared to the ras-1bd strain at 20 °C. Therefore, multiple Ca2+ signaling genes regulate the circadian period, by influencing expression of the frq and wc-1 genes that are critical for maintaining the normal circadian period length in N. crassa.

Regulator of G Protein Signaling Proteins Control Growth, Development and Cellulase Production in Neurospora crassa.

Heterotrimeric (αßγ) G protein signaling pathways are critical environmental sensing systems found in eukaryotic cells. Exchange of GDP for GTP on the Gα subunit leads to its activation. In contrast, GTP hydrolysis on the Gα is accelerated by Regulator of G protein Signaling (RGS) proteins, resulting in a return to the GDP-bound, inactive state. Here, we analyzed growth, development and extracellular cellulase production in strains with knockout mutations in the seven identified RGS genes (rgs-1 to rgs-7) in the filamentous fungus, Neurospora crassa. We compared phenotypes to those of strains with either knockout mutations or expressing predicted constitutively activated, GTPase-deficient alleles for each of the three Gα subunit genes (gna-1Q204L, gna-2Q205L or gna-3Q208L). Our data revealed that six RGS mutants have taller aerial hyphae than wild type and all seven mutants exhibit reduced asexual sporulation, phenotypes shared with strains expressing the gna-1Q204L or gna-3Q208L allele. In contrast, Δrgs-1 and Δrgs-3 were the only RGS mutants with a slower growth rate phenotype, a defect in common with gna-1Q204L strains. With respect to female sexual development, Δrgs-1 possessed defects most similar to gna-3Q208L strains, while those of Δrgs-2 mutants resembled strains expressing the gna-1Q204L allele. Finally, we observed that four of the seven RGS mutants had significantly different extracellular cellulase levels relative to wild type. Of interest, the Δrgs-2 mutant had no detectable activity, similar to the gna-3Q208L strain. In contrast, the Δrgs-1 and Δrgs-4 mutants and gna-1Q204L and gna-2Q205L strains exhibited significantly higher cellulase activity than wild type. With the exception of sexual development, our results demonstrate the greatest number of genetic interactions between rgs-1 and gna-1 and rgs-2 and gna-3 in N. crassa.

Targeted Metabolomics Using LC-MS in Neurospora crassa.

The filamentous fungus Neurospora crassa has historically been a model for understanding the relationship between genes and metabolism-auxotrophic mutants of N. crassa were used by Beadle and Tatum to develop the one-gene-one-enzyme hypothesis for which they earned the Nobel Prize in 1958. In the ensuing decades, several techniques have been developed for the systematic analysis of metabolites in N. crassa and other fungi. Untargeted and targeted approaches have been used, with a focus on secondary metabolites over primary metabolism. Here, we describe a pipeline for sample preparation, metabolite extraction, Liquid Chromatography-Mass Spectrometry (LC-MS), and data analysis that can be used for targeted metabolomics of primary metabolites in N. crassa. Liquid cultures are grown with shaking in a defined minimal medium and then collected using filtration. Samples are lyophilized for 2 days at -80°C, pulverized, and mixed with a solution to extract polar metabolites. The metabolites are separated and identified using LC-MS, with downstream analysis using Skyline interpretive software. Relative levels of hundreds of metabolites can be detected and compared across strains. © 2022 Wiley Periodicals LLC. Basic Protocol: Metabolite extraction and detection from Neurospora crassa cell cultures using Liquid Chromatography-Mass Spectrometry.

Fol-milR1, a pathogenicity factor of Fusarium oxysporum, confers tomato wilt disease resistance by impairing host immune responses.

Although it is well known that miRNAs play crucial roles in multiple biological processes, there is currently no evidence indicating that milRNAs from Fusarium oxysporum f. sp. lycopersici (Fol) interfere with tomato resistance during infection. Here, using sRNA-seq, we demonstrate that Fol-milR1, a trans-kingdom small RNA, is exported into tomato cells after infection. The knockout strain ∆Fol-milR1 displays attenuated pathogenicity to the susceptible tomato cultivar 'Moneymaker'. On the other hand, Fol-milR1 overexpression strains exhibit enhanced virulence against the resistant cultivar 'Motelle'. Several tomato mRNAs are predicted targets of Fol-milR1. Among these genes, Solyc06g007430 (encoding the CBL-interacting protein kinase, SlyFRG4) is regulated at the posttranscriptional level by Fol-milR1. Furthermore, SlyFRG4 loss-of-function alleles created using CRISPR/Cas9 in tomato ('Motelle') exhibit enhanced disease susceptibility to Fol, further supporting the idea that SlyFRG4 is essential for tomato wilt disease resistance. Notably, our results using immunoprecipitation with specific antiserum suggest that Fol-milR1 interferes with the host immunity machinery by binding to tomato ARGONAUTE 4a (SlyAGO4a). Furthermore, virus-induced gene silenced (VIGS) knock-down SlyAGO4a plants exhibit reduced susceptibility to Fol. Together, our findings support a model in which Fol-milR1 is an sRNA fungal effector that suppresses host immunity by silencing a disease resistance gene, thus providing a novel virulence strategy to achieve infection.

Small RNA Isolation and Library Construction for Expression Profiling of Small RNAs from Neurospora crassa and Fusarium oxysporum and Analysis of Small RNAs in Fusarium oxysporum-Infected Plant Root Tissue.

Due to crucial roles in gene regulation, noncoding small RNAs (sRNAs) of 20-30 nucleotides (nt) have been intensively studied in mammals and plants and are implicated in significant diseases and metabolic disorders. Elucidation of biogenesis mechanisms and functional characterization of sRNAs is often achieved using tools such as separation of small-sized RNA and deep sequencing. Although RNA interference pathways, such as quelling and meiotic silencing, have been well-described in Neurospora crassa, knowledge of sRNAs in other filamentous fungi is still limited compared to other eukaryotes. As a prerequisite for study, isolation and sequence analysis of sRNAs is necessary. We developed a protocol for isolation and library construction of sRNAs of 20-30 nt for deep sequencing in two filamentous fungi, N. crassa and Fusarium oxysporum f.sp. lycopersici. Using 200-300 µg total RNA, sRNA was isolated by size-fractionation and ligated with adapters and amplified by RT-PCR for deep sequencing. Sequence analysis of several cDNA clones showed that the cloned sRNAs were not tRNAs and rRNAs and were fungal genome-specific. In order to validate fungal miRNAs that were imported into the host cell, we developed a straightforward method to isolate protoplasts from tomato roots infected by Fusarium oxysporum f.sp. lycopersici using enzymatic digestion.

Heterotrimeric G-Protein Signaling Is Required for Cellulose Degradation in Neurospora crassa.

The filamentous fungus Neurospora crassa decomposes lignocellulosic biomass to generate soluble sugars as carbon sources. In this study, we investigated a role for heterotrimeric G-protein signaling in cellulose degradation. Loss of the Gα subunit genes gna-1 and gna-3, the Gß subunit genes gnb-1 and cpc-2, the Gγ gene gng-1, or the gene for downstream effector adenylyl cyclase (cr-1) resulted in loss of detectable cellulase activity. This defect was also observed in strains expressing a constitutively active version of gna-3 (gna-3Q208L ). We found that GNA-1 levels are greatly reduced in Δgna-3, Δgnb-1, and Δgng-1 strains, likely contributing to cellulase defects in these genetic backgrounds. The observation that gna-3Q208L Δgnb-1 strains exhibit cellulase activity, despite greatly reduced levels of GNA-1 protein, is consistent with positive control of cellulase production by GNA-3 that is manifested in the absence of gnb-1 Expression patterns for five cellulase genes showed that Δgna-1, Δgnb-1, and Δgna-3 mutants produce less cellulase mRNA than the wild type, consistent with transcriptional regulation. Δcpc-2 mutants had wild-type levels of cellulase transcripts, suggesting posttranscriptional control. In contrast, results for Δcr-1 mutants support both transcriptional and posttranscriptional control of cellulase activity by cAMP signaling. Cellulase activity defects in Δgna-3 mutants were fully remediated by cAMP supplementation, consistent with GNA-3 operating upstream of cAMP signaling. In contrast, cAMP addition only partially corrected cellulase activity defects in Δgna-1 and Δgnb-1 mutants, suggesting participation of GNA-1 and GNB-1 in additional cAMP-independent pathways that control cellulase activity.IMPORTANCE Filamentous fungi are critical for the recycling of plant litter in the biosphere by degrading lignocellulosic biomass into simpler compounds for metabolism. Both saprophytic and pathogenic fungi utilize plant cell wall-degrading enzymes to liberate carbon for metabolism. Several studies have demonstrated a role for cellulase enzymes during infection of economically relevant crops by fungal pathogens. Especially in developing countries, severe plant disease means loss of entire crops, sometimes leading to starvation. In this study, we demonstrate that G-protein signaling is a key component of cellulase production. Therefore, understanding the role of G-protein signaling in the regulation of the unique metabolism of cellulose by these organisms can inform innovations in strain engineering of industrially relevant species for biofuel production and in combatting food shortages caused by plant pathogens.

Clustering analysis of large-scale phenotypic data in the model filamentous fungus Neurospora crassa.

BACKGROUND: With 9730 protein-coding genes and a nearly complete gene knockout strain collection, Neurospora crassa is a major model organism for filamentous fungi. Despite this abundance of information, the phenotypes of these gene knockout mutants have not been categorized to determine whether there are broad correlations between phenotype and any genetic features. RESULTS: Here, we analyze data for 10 different growth or developmental phenotypes that have been obtained for 1168 N. crassa knockout mutants. Of these mutants, 265 (23%) are in the normal range, while 903 (77%) possess at least one mutant phenotype. With the exception of unclassified functions, the distribution of functional categories for genes in the mutant dataset mirrors that of the N. crassa genome. In contrast, most genes do not possess a yeast ortholog, suggesting that our analysis will reveal functions that are not conserved in Saccharomyces cerevisiae. To leverage the phenotypic data to identify pathways, we used weighted Partitioning Around Medoids (PAM) approach with 40 clusters. We found that genes encoding metabolic, transmembrane and protein phosphorylation-related genes are concentrated in subsets of clusters. Results from K-Means clustering of transcriptomic datasets showed that most phenotypic clusters contain multiple expression profiles, suggesting that co-expression is not generally observed for genes with shared phenotypes. Analysis of yeast orthologs of genes that co-clustered in MAPK signaling cascades revealed potential networks of interacting proteins in N. crassa. CONCLUSIONS: Our results demonstrate that clustering analysis of phenotypes is a promising tool for generating new hypotheses regarding involvement of genes in cellular pathways in N. crassa. Furthermore, information about gene clusters identified in N. crassa should be applicable to other filamentous fungi, including saprobes and pathogens.

Broad Substrate-Specific Phosphorylation Events Are Associated With the Initial Stage of Plant Cell Wall Recognition in Neurospora crassa.

Fungal plant cell wall degradation processes are governed by complex regulatory mechanisms, allowing the organisms to adapt their metabolic program with high specificity to the available substrates. While the uptake of representative plant cell wall mono- and disaccharides is known to induce specific transcriptional and translational responses, the processes related to early signal reception and transduction remain largely unknown. A fast and reversible way of signal transmission are post-translational protein modifications, such as phosphorylations, which could initiate rapid adaptations of the fungal metabolism to a new condition. To elucidate how changes in the initial substrate recognition phase of Neurospora crassa affect the global phosphorylation pattern, phospho-proteomics was performed after a short (2 min) induction period with several plant cell wall-related mono- and disaccharides. The MS/MS-based peptide analysis revealed large-scale substrate-specific protein phosphorylation and de-phosphorylations. Using the proteins identified by MS/MS, a protein-protein-interaction (PPI) network was constructed. The variance in phosphorylation of a large number of kinases, phosphatases and transcription factors indicate the participation of many known signaling pathways, including circadian responses, two-component regulatory systems, MAP kinases as well as the cAMP-dependent and heterotrimeric G-protein pathways. Adenylate cyclase, a key component of the cAMP pathway, was identified as a potential hub for carbon source-specific differential protein interactions. In addition, four phosphorylated F-Box proteins were identified, two of which, Fbx-19 and Fbx-22, were found to be involved in carbon catabolite repression responses. Overall, these results provide unprecedented and detailed insights into a so far less well known stage of the fungal response to environmental cues and allow to better elucidate the molecular mechanisms of sensory perception and signal transduction during plant cell wall degradation.

Genetic relationships between the RACK1 homolog cpc-2 and heterotrimeric G protein subunit genes in Neurospora crassa.

Receptor for Activated C Kinase-1 (RACK1) is a multifunctional eukaryotic scaffolding protein with a seven WD repeat structure. Among their many cellular roles, RACK1 homologs have been shown to serve as alternative Gß subunits during heterotrimeric G protein signaling in many systems. We investigated genetic interactions between the RACK1 homolog cpc-2, the previously characterized Gß subunit gnb-1 and other G protein signaling components in the multicellular filamentous fungus Neurospora crassa. Results from cell fractionation studies and from fluorescent microscopy of a strain expressing a CPC-2-GFP fusion protein revealed that CPC-2 is a cytoplasmic protein. Genetic epistasis experiments between cpc-2, the three Gα genes (gna-1, gna-2 and gna-3) and gnb-1 demonstrated that cpc-2 is epistatic to gna-2 with regards to basal hyphae growth rate and aerial hyphae height, while deletion of cpc-2 mitigates the increased macroconidiation on solid medium observed in Δgnb-1 mutants. Δcpc-2 mutants inappropriately produce conidiophores during growth in submerged culture and mutational activation of gna-3 alleviates this defect. Δcpc-2 mutants are female-sterile and fertility could not be restored by mutational activation of any of the three Gα genes. With the exception of macroconidiation on solid medium, double mutants lacking cpc-2 and gnb-1 exhibited more severe defects for all phenotypic traits, supporting a largely synergistic relationship between GNB-1 and CPC-2 in N. crassa.

The SNARE protein FolVam7 mediates intracellular trafficking to regulate conidiogenesis and pathogenicity in Fusarium oxysporum f. sp. lycopersici.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are conserved in fungi, plants and animals. The Vam7 gene encodes a v-SNARE protein that involved in vesicle trafficking in fungi. Here, we identified and characterized the function of FolVam7, a homologue of the yeast SNARE protein Vam7p in Fusarium oxysporum f. sp. lycopersici (Fol), a fungal pathogen of tomato. FolVam7 contains SNARE and PX (Phox homology) domains that are indispensable for normal localization and function of FolVam7. Targeted gene deletion showed that FolVam7-mediated vesicle trafficking is important for vegetative growth, asexual development, conidial morphology and plant infection. Further cytological examinations revealed that FolVam7 is localized to vesicles and vacuole membranes in the hyphae stage. Moreover, the ΔFolvam7 mutant is insensitive to salt and osmotic stresses and hypersensitive to cell wall stressors. Taken together, our results suggested that FolVam7-mediated vesicle trafficking promotes vegetative growth, conidiogenesis and pathogenicity of Fol.

FRG3, a Target of slmiR482e-3p, Provides Resistance against the Fungal Pathogen Fusarium oxysporum in Tomato.

The vast majority of plant disease resistance (R) genes encode nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins, which specifically determine the plant immune response and have been demonstrated to be targets of several microRNA (miRNA) families. The fungus Fusarium oxysporum f. sp. lycopersici (FOL) causes vascular wilt disease in tomato worldwide. Here, we explored a possible role for FGR3 in tomato defense against FOL. FRG3 is a predicted NBS-LRR like gene that is targeted by slmiR482e-3p, a member of slmiR482 miRNA family. Northern blot data demonstrated that all seven members of the slmiR482 family were regulated in diverse ways after infection by FOL. The ability of FRG3 to be regulated by slmiR482e-3p was confirmed at the transcript level by co-expression studies in Nicotiana benthamiana. A virus-induced gene silencing (VIGS) approach revealed that FRG3 confers resistance to the Motelle tomato cultivar. Taken together, our study has identified a novel R gene, FRG3, which is targeted by slmiR482e-3p at the transcript level, and is necessary for resistance to tomato wilt disease in planta.

Functional Profiling of Transcription Factor Genes in Neurospora crassa.

Regulation of gene expression by DNA-binding transcription factors is essential for proper control of growth and development in all organisms. In this study, we annotate and characterize growth and developmental phenotypes for transcription factor genes in the model filamentous fungus Neurospora crassa We identified 312 transcription factor genes, corresponding to 3.2% of the protein coding genes in the genome. The largest class was the fungal-specific Zn2Cys6 (C6) binuclear cluster, with 135 members, followed by the highly conserved C2H2 zinc finger group, with 61 genes. Viable knockout mutants were produced for 273 genes, and complete growth and developmental phenotypic data are available for 242 strains, with 64% possessing at least one defect. The most prominent defect observed was in growth of basal hyphae (43% of mutants analyzed), followed by asexual sporulation (38%), and the various stages of sexual development (19%). Two growth or developmental defects were observed for 21% of the mutants, while 8% were defective in all three major phenotypes tested. Analysis of available mRNA expression data for a time course of sexual development revealed mutants with sexual phenotypes that correlate with transcription factor transcript abundance in wild type. Inspection of this data also implicated cryptic roles in sexual development for several cotranscribed transcription factor genes that do not produce a phenotype when mutated.

Calcineurin Subunits A and B Interact to Regulate Growth and Asexual and Sexual Development in Neurospora crassa.

Calcineurin is a calcium/calmodulin dependent protein phosphatase in eukaryotes that consists of a catalytic subunit A and a regulatory subunit B. Previous studies in the filamentous fungus Neurospora crassa had suggested that the catalytic subunit of calcineurin might be an essential protein. We generated N. crassa strains expressing the A (cna-1) and B (cnb-1) subunit genes under the regulation of Ptcu-1, a copper-responsive promoter. In these strains, addition of bathocuproinedisulfonic acid (BCS), a copper chelator, results in induction of cna-1 and cnb-1, while excess Cu2+ represses gene expression. Through analysis of these strains under repressing and inducing conditions, we found that the calcineurin is required for normal growth, asexual development and female fertility in N. crassa. Moreover, we isolated and analyzed cnb-1 mutant alleles generated by repeat-induced point mutation (RIP), with the results further supporting roles for calcineurin in growth and fertility in N. crassa. We demonstrated a direct interaction between the CNA-1 and CNB-1 proteins using an assay system developed to study protein-protein interactions in N. crassa.

Global Analysis of Predicted G Protein-Coupled Receptor Genes in the Filamentous Fungus, Neurospora crassa.

G protein-coupled receptors (GPCRs) regulate facets of growth, development, and environmental sensing in eukaryotes, including filamentous fungi. The largest predicted GPCR class in these organisms is the Pth11-related, with members similar to a protein required for disease in the plant pathogen Magnaporthe oryzae. However, the Pth11-related class has not been functionally studied in any filamentous fungal species. Here, we analyze phenotypes in available mutants for 36 GPCR genes, including 20 Pth11-related, in the model filamentous fungus Neurospora crassa. We also investigate patterns of gene expression for all 43 predicted GPCR genes in available datasets. A total of 17 mutants (47%) possessed at least one growth or developmental phenotype. We identified 18 mutants (56%) with chemical sensitivity or nutritional phenotypes (11 uniquely), bringing the total number of mutants with at least one defect to 28 (78%), including 15 mutants (75%) in the Pth11-related class. Gene expression trends for GPCR genes correlated with the phenotypes observed for many mutants and also suggested overlapping functions for several groups of co-transcribed genes. Several members of the Pth11-related class have phenotypes and/or are differentially expressed on cellulose, suggesting a possible role for this gene family in plant cell wall sensing or utilization.

Metabolic Impacts of Using Nitrogen and Copper-Regulated Promoters to Regulate Gene Expression in Neurospora crassa.

The filamentous fungus Neurospora crassa is a long-studied eukaryotic microbial system amenable to heterologous expression of native and foreign proteins. However, relatively few highly tunable promoters have been developed for this species. In this study, we compare the tcu-1 and nit-6 promoters for controlled expression of a GFP reporter gene in N. crassa. Although the copper-regulated tcu-1 has been previously characterized, this is the first investigation exploring nitrogen-controlled nit-6 for expression of heterologous genes in N. crassa. We determined that fragments corresponding to 1.5-kb fragments upstream of the tcu-1 and nit-6 open reading frames are needed for optimal repression and expression of GFP mRNA and protein. nit-6 was repressed using concentrations of glutamine from 2 to 20 mM and induced in medium containing 0.5-20 mM nitrate as the nitrogen source. Highest levels of expression were achieved within 3 hr of induction for each promoter and GFP mRNA could not be detected within 1 hr after transfer to repressing conditions using the nit-6 promoter. We also performed metabolic profiling experiments using proton NMR to identify changes in metabolite levels under inducing and repressing conditions for each promoter. The results demonstrate that conditions used to regulate tcu-1 do not significantly change the primary metabolome and that the differences between inducing and repressing conditions for nit-6 can be accounted for by growth under nitrate or glutamine as a nitrogen source. Our findings demonstrate that nit-6 is a tunable promoter that joins tcu-1 as a choice for regulation of gene expression in N. crassa.

MicroRNAs suppress NB domain genes in tomato that confer resistance to Fusarium oxysporum.

MicroRNAs (miRNAs) suppress the transcriptional and post-transcriptional expression of genes in plants. Several miRNA families target genes encoding nucleotide-binding site-leucine-rich repeat (NB-LRR) plant innate immune receptors. The fungus Fusarium oxysporum f. sp. lycopersici causes vascular wilt disease in tomato. We explored a role for miRNAs in tomato defense against F. oxysporum using comparative miRNA profiling of susceptible (Moneymaker) and resistant (Motelle) tomato cultivars. slmiR482f and slmiR5300 were repressed during infection of Motelle with F. oxysporum. Two predicted mRNA targets each of slmiR482f and slmiR5300 exhibited increased expression in Motelle and the ability of these four targets to be regulated by the miRNAs was confirmed by co-expression in Nicotiana benthamiana. Silencing of the targets in the resistant Motelle cultivar revealed a role in fungal resistance for all four genes. All four targets encode proteins with full or partial nucleotide-binding (NB) domains. One slmiR5300 target corresponds to tm-2, a susceptible allele of the Tomato Mosaic Virus resistance gene, supporting functions in immunity to a fungal pathogen. The observation that none of the targets correspond to I-2, the only known resistance (R) gene for F. oxysporum in tomato, supports roles for additional R genes in the immune response. Taken together, our findings suggest that Moneymaker is highly susceptible because its potential resistance is insufficiently expressed due to the action of miRNAs.

Global analysis of serine/threonine and tyrosine protein phosphatase catalytic subunit genes in Neurospora crassa reveals interplay between phosphatases and the p38 mitogen-activated protein kinase.

Protein phosphatases are integral components of the cellular signaling machinery in eukaryotes, regulating diverse aspects of growth and development. The genome of the filamentous fungus and model organism Neurospora crassa encodes catalytic subunits for 30 protein phosphatase genes. In this study, we have characterized 24 viable N. crassa phosphatase catalytic subunit knockout mutants for phenotypes during growth, asexual development, and sexual development. We found that 91% of the mutants had defects in at least one of these traits, whereas 29% possessed phenotypes in all three. Chemical sensitivity screens were conducted to reveal additional phenotypes for the mutants. This resulted in the identification of at least one chemical sensitivity phenotype for 17 phosphatase knockout mutants, including novel chemical sensitivities for two phosphatase mutants lacking a growth or developmental phenotype. Hence, chemical sensitivity or growth/developmental phenotype was observed for all 24 viable mutants. We investigated p38 mitogen-activated protein kinase (MAPK) phosphorylation profiles in the phosphatase mutants and identified nine potential candidates for regulators of the p38 MAPK. We demonstrated that the PP2C class phosphatase pph-8 (NCU04600) is an important regulator of female sexual development in N. crassa. In addition, we showed that the Δcsp-6 (ΔNCU08380) mutant exhibits a phenotype similar to the previously identified conidial separation mutants, Δcsp-1 and Δcsp-2, that lack transcription factors important for regulation of conidiation and the circadian clock.

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.

Genetic and physical interactions between Gα subunits and components of the Gßγ 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 Gß subunit (GNB-1), and one Gγ subunit (GNG-1) have been functionally characterized, but genetic epistasis relationships between Gß 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 Gßγ 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 Gßγ 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 Gßγ dimer. The finding that the Gßγ 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.