SUMMARY: We present an analysis of over 1,100 of the ∼10,000 predicted proteins encoded by the genome sequence of the filamentous fungusNeurospora crassa. Seven major areas ofNeurosporagenomics 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 ofNeurosporagenes do not have homologues in the yeastsSaccharomyces cerevisiaeandSchizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.

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. Fusarium oxysporum is a fungal pathogen that represents a species complex, with members that infect numerous crops. In spite of its importance to agriculture, very little is known about roles of small RNAs in plant immunity against F. oxysporum. In this study, we set up a screen for tomato microRNAs (miRNAs) that correlate with resistance to F. oxysporum f.sp. lycopersici by performing deep sequencing of small RNAs from a resistant and susceptible tomato cultivar. We focused on two miRNAs that are uniquely down-regulated in the resistant cultivar during fungal infection. All predicted targets of these miRNAs encode proteins with NB domains, a motif associated with pathogen resistance in plants. Using a heterologous system, we validated that the miRNAs could regulate expression of four targets. Silencing of the target genes in tomato resulted in decreased immunity to F. oxysporum in the normally resistant cultivar. The finding that none of our 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. Our results suggest that the potential resistance of the susceptible cultivar is insufficiently expressed due to the action of miRNAs.

Summary: 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 Δpre‐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 Gα subunit, GNA‐1, and the Gβ protein, GNB‐1, are essential for female fertility in N. crassa. Trichogynes of Δgna‐1 and Δgnb‐1 mutants displayed severe defects in growth towards and fusion with male cells, similar to that of Δpre‐1 mat A strains. However, the female sterility defect of the Δpre‐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.

Summary: 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.

Neurospora crassa is a self-sterile filamentous fungus with two mating types, mat A and mat a . Its mating involves chemotropic polarized growth of female-specific hyphae (trichogynes) toward male cells of the opposite mating type in a process involving pheromones and receptors. mat A cells express the ccg-4 pheromone and the pre-1 receptor, while mat a strains produce mRNA for the pheromone mfa-1 and the pre-2 receptor; MFA-1 and CCG-4 are the predicted ligands for PRE-1 and PRE-2, respectively. In this study, we generated Δ ccg-4 and Δ mfa-1 mutants and engineered a mat a strain to coexpress ccg-4 and its receptor, pre-2 . As males, Δ ccg-4 mat A and Δ mfa-1 mat a mutants were unable to attract mat a and mat A trichogynes, respectively, and consequently failed to initiate fruiting body (perithecial) development or produce meiotic spores (ascospores). In contrast, Δ ccg-4 mat a and Δ mfa-1 mat A mutants exhibited normal chemotropic attraction and male fertility. Δ ccg-4 Δ mfa-1 double mutants displayed defective chemotropism and male sterility in both mating types. Heterologous expression of ccg-4 enabled mat a males to attract mat a trichogynes, although subsequent perithecial differentiation did not occur. Expression of ccg-4 and pre-2 in the same strain triggered self-stimulation, resulting in formation of barren perithecia with no ascospores. Our results indicate that CCG-4 and MFA-1 are required for mating-type-specific male fertility and that pheromones (and receptors) are initial determinants for sexual identity during mate recognition. Furthermore, a self-attraction signal can be transmitted within a strain that expresses a pheromone and its cognate receptor.

Two-component systems, consisting of proteins with histidine kinase and/or response regulator domains, regulate environmental responses in bacteria, Archaea, fungi, slime molds, and plants. Here, we characterize RRG-1, a response regulator protein from the filamentous fungus Neurospora crassa. The cell lysis phenotype of Δrrg-1 mutants is reminiscent of osmotic-sensitive (os) mutants, including nik-1/os-1 (a histidine kinase) and strains defective in components of a mitogen-activated protein kinase (MAPK) pathway: os-4 (MAPK kinase kinase), os-5 (MAPK kinase), and os-2 (MAPK). Similar to os mutants, Δrrg-1 strains are sensitive to hyperosmotic conditions, and they are resistant to the fungicides fludioxonil and iprodione. Like os-5, os-4, and os-2 mutants, but in contrast to nik-1/os-1 strains, Δrrg-1 mutants do not produce female reproductive structures (protoperithecia) when nitrogen starved. OS-2-phosphate levels are elevated in wild-type cells exposed to NaCl or fludioxonil, but they are nearly undetectable in Δrrg-1 strains. OS-2-phosphate levels are also low in Δrrg-1, os-2, and os-4 mutants under nitrogen starvation. Analysis of the rrg-1^D921Nallele, mutated in the predicted phosphorylation site, provides support for phosphorylation-dependent and -independent functions for RRG-1. The data indicate that RRG-1 controls vegetative cell integrity, hyperosmotic sensitivity, fungicide resistance, and protoperithecial development through regulation of the OS-4/OS-5/OS-2 MAPK pathway.