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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.

BackgroundWith 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.ResultsHere, 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.ConclusionsOur 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.

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.

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.

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 Gβ subunits during heterotrimeric G protein signaling in many systems. We investigated genetic interactions between the RACK1 homolog cpc-2, the previously characterized Gβ 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.

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.

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.

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 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.

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.

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.

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.

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 (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.

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.

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.

We present an analysis of over 1,100 of the approximately 10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.

The article analyzes heterotrimeric G protein in filamentous fungi by identifying G protein-coupled receptor genes. The sensing of nutrients and host signals of G proteins is useful for pathogenesis.

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.

Author(s): James E. Galagan (corresponding author) [1]; Sarah E. Calvo [1]; Katherine A. Borkovich [2]; Eric U. Selker [3]; Nick D. Read [4]; David Jaffe [1]; William FitzHugh [5]; Li-Jun [...]