HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa.

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of fungal communication.

Identification and characterization of LFD1, a novel protein involved in membrane merger during cell fusion in Neurospora crassa.

Despite its essential role in development, molecular mechanisms of membrane merger during cell-cell fusion in most eukaryotic organisms remain elusive. In the filamentous fungus Neurospora crassa, cell fusion occurs during asexual spore germination, where genetically identical germlings show chemotropic interactions and cell-cell fusion. Fusion of germlings and hyphae is required for the formation of the interconnected mycelial network characteristic of filamentous fungi. Previously, a multipass membrane protein, PRM1, was characterized and acts at the step of bilayer fusion in N. crassa. Here we describe the identification and characterization of lfd-1, encoding a single pass transmembrane protein, which is also involved in membrane merger. lfd-1 was identified by a targeted analysis of a transcriptional profile of a transcription factor mutant (Δpp-1) defective in germling fusion. The Δlfd-1 mutant shows a similar, but less severe, membrane merger defect as a ΔPrm1 mutant. By genetic analyses, we show that LFD1 and PRM1 act independently, but share a redundant function. The cell fusion frequency of both Δlfd-1 and ΔPrm1 mutants was sensitive to extracellular calcium concentration and was associated with an increase in cell lysis, which was suppressed by a calcium-dependent mechanism involving a homologue to synaptotagmin.

Oscillatory recruitment of signaling proteins to cell tips promotes coordinated behavior during cell fusion.

Cell-cell communication is essential for coordinating physiological responses in multicellular organisms and is required for various developmental processes, including cell migration, differentiation, and fusion. To facilitate communication, functional differences are usually required between interacting cells, which can be established either genetically or developmentally. However, genetically identical cells in the same developmental state are also capable of communicating, but must avoid self-stimulation. We hypothesized that such cells must alternate their physiological state between signal sending and receiving to allow recognition and behavioral changes. To test this hypothesis, we studied cell communication in the filamentous fungus Neurospora crassa, a simple and experimentally amenable model system. In N. crassa, germinating asexual spores (germlings) of identical genotype chemotropically sense others in close proximity, show attraction-mediated directed growth, and ultimately undergo cell fusion. Here, we report that two proteins required for cell fusion, a MAP kinase (MAK-2) and a protein of unknown molecular function (SO), exhibit rapid oscillatory recruitment to the plasma membranes of interacting germlings undergoing chemotropic interactions via directed growth. Using an inhibitable MAK-2 variant, we show that MAK-2 kinase activity is required both for chemotropic interactions and for oscillation of MAK-2 and SO to opposing cell tips. Thus, N. crassa germlings undergoing chemotropic interactions rapidly alternate between two different physiological states, associated with signal delivery and response. Such spatiotemporal coordination of signaling allows genetically identical and developmentally equivalent cells to avoid self-stimulation and to coordinate their behavior to achieve the beneficial physiological outcome of cell fusion.

Characterisation of Aspergillus nidulans polarisome component BemA.

BemA, the orthologue of Saccharomyces cerevisiae Bem1p, was identified through genome sequence comparison. We have shown that it plays a similar role to Bem1p in yeast, acting as a cell growth protein. Deletion of the gene produced a moderately abnormal hyphal tip morphology, and had an extremely detrimental effect on conidiospore production, with development stalling after conidiophore vesicle production. It was also shown that BemA is required for vacuole fusion, similar to Bem1p. This role is dependent on the first SH3 domain of the protein, whose deletion has no detectable effect on cell growth. Localisation studies showed that BemA formed a clear cap at hyphal tips, analogous to the S. cerevisiae polarisome. The relationship between BemA and SepA, a spitzenkörper protein, was investigated. It was found that localisation of the proteins were interdependent, and a conditional double mutant was inviable.

Early colony establishment in Neurospora crassa requires a MAP kinase regulatory network.

Vegetative fusion is essential for the development of an interconnected colony in many filamentous fungi. In the ascomycete fungus Neurospora crassa, vegetative fusion occurs between germinated conidia (germlings) via specialized structures termed "conidial anastomosis tubes" (CATs) and between hyphae within a mature colony. In N. crassa, both CAT and hyphal fusion are under the regulation of a conserved MAP kinase cascade (NRC1, MEK2, and MAK2). Here we show that the predicted downstream target of the MAK2 kinase pathway, a Ste12-like transcription factor known as PP1, regulates elements required for CAT and hyphal fusion. The PP1 regulatory network was revealed by expression profiling of wild type and the Δpp-1 mutant during conidial germination and colony establishment. To identify targets required for cell fusion more specifically, expression-profiling differences were assessed via inhibition of MAK2 kinase activity during chemotropic interactions and cell fusion. These approaches led to the identification of new targets of the cell fusion pathway that, when mutated, showed alterations in chemotropic signaling and cell fusion. In particular, conidial germlings carrying a deletion of NCU04732 (Δham-11) failed to show chemotropic interactions and cell fusion. However, signaling (as shown by oscillation of MAK2 and SO to CAT tips), chemotropism, and cell fusion were restored in Δham-11 germlings when matched with wild-type partner germlings. These data reveal novel insights into the complex process of self-signaling, germling fusion, and colony establishment in filamentous fungi.

The social network: deciphering fungal language.

It has been estimated that up to one quarter of the world's biomass is of fungal origin, comprising approximately 1.5 million species. In order to interact with one another and respond to environmental cues, fungi communicate with their own chemical languages using a sophisticated series of extracellular signals and cellular responses. A new appreciation for the linkage between these chemical languages and developmental processes in fungi has renewed interest in these signalling molecules, which can now be studied using post-genomic resources. In this Review, we focus on the molecules that are secreted by the largest phylum of fungi, the Ascomycota, and the quest to understand their biological function.

Use of laccase as a novel, versatile reporter system in filamentous fungi.

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (Km = 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.

A rapid method for promoter exchange in Aspergillus nidulans using recombinant PCR.

Recombinant PCR has been used to generate linear fragments for promoter replacement by transformation in Aspergillus nidulans. A cassette vector carrying the pyr-4 non-homologous selectable marker and conditional promoter Pr-alcA was constructed for use as a template for PCR, and is suitable for testing the function of essential genes. Two genes involved in polar growth, cotA and bemA, were used to assess the system. Efficient targeting was possible with both genes using approximately 500bp of flanking homologous sequence. Depending on yield, the linear PCR product could be used directly for transformation, or after first cloning into a suitable vector. bemA, a putative homologue of the Saccharomyces cerevisiae BEM1 gene was identified through sequence comparison. In A. nidulans, this protein appears to have a similar role to the yeast Bem1p, which acts as a scaffold protein involved in the establishment of cell polarity.