Early Colony Establishment in Neurospora crassa Requires a MAP Kinase Regulatory Network

Abstract

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 Dpp-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 (Dham-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 Dham-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. CELL fusion between genetically identical cells is important in development (for example, myoblast fusion during muscle formation) and occurs in many multicellular organisms from simple ascomycete fungi to mammals (Chen et al. 2007;Aguilar et al. 2013). Cell fusion between genetically identical cells can be mediated by cells that have differentiated, but in some cases, also between cells in an identical developmental state, for example, cell fusion between germinating asexual spores (conidia) of filamentous fungi (Pandey et al. 2004;Roca et al. 2005a;Read et al. 2010). In filamentous fungi, these fusions are integral to the formation of an interconnected hyphal network, which mediates genetic mixing and the sharing of resources (Simonin et al. 2012;Roper et al. 2013). How this process is initiated and maintained and what proteins are involved are still mostly unknown.In filamentous ascomycete fungi, a conserved MAP kinase pathway that is involved in pheromone response and mating in Saccharomyces cerevisiae (Ste11, Ste7, and Fus3) (Bardwell 2005) is required for cell fusion and heterokaryon formation during vegetative growth (Hou et al. 2002;Wei et al. 2003;Pandey et al. 2004;Fu et al. 2011;Jun et al. 2011;Dettmann et al. 2012). This conserved pathway also plays a role in sexual development and secondary metabolism and is required for the virulence of both plant and animal fungal pathogens (Roman et al. 2007;Rispail and Di Piet...

Chemotropism and Cell Fusion in Neurospora crassa Relies on the Formation of Distinct Protein Complexes by HAM-5 and a Novel Protein HAM-14

“…Genes important for germling/hyphal fusion include genes of the MAK-2 MAP kinase pathway and upstream factors (Fu et al 2011;Dettmann et al 2014), members of the cell wall integrity (CWI) MAP kinase pathway (Fu et al 2011(Fu et al , 2014, the STRIPAK complex (Simonin et al 2010;Dettmann et al 2013), the NADPH oxidation complex (Cano-Dominguez et al 2008;Fu et al 2011Fu et al , 2014, a number of transcription factors (Aldabbous et al 2010;Fu et al 2011;Leeder et al 2013), and genes encoding uncharacterized proteins (Fu et al 2011(Fu et al , 2014. Previously, we showed that HAM-5-GFP localizes to puncta in Dmak-2, Dham-7, and Dham-11 fusion mutants (Jonkers et al 2014).…”

“…In fungi, vegetative fusion enables a fungal colony to share resources and has implications for fitness and virulence (Craven et al 2008;Fricker et al 2009;Eaton et al 2011;Richard et al 2012;Simonin et al 2012;Roper et al 2013;Chagnon 2014). In the filamentous ascomycete fungus, Neurospora crassa, genetically identical germinated asexual spores (germlings) undergo fusion to form a syncytium that is an interconnected network of fused cells Leeder et al 2013;Herzog et al 2015). An important pathway required for both germling and hyphal fusion is a conserved mitogen-activated protein kinase (MAPK) cascade that includes the MAPK kinase kinase NRC-1, the MAPK kinase MEK-2, the MAPK MAK-2, and the scaffold protein, HAM-5 (Pandey et al 2004;Dettmann et al 2012Dettmann et al , 2014Jonkers et al 2014).…”

“…Future work will continue to dissect the molecular mechanism of how a germling switches from one state to another and how the germlings send and receive signals. Importantly, to fully understand the process of germling communication, the elusive receptors/ ligands for initiating this process require identification; so far numerous screens of the N. crassa deletion collection have failed to reveal candidates (Fu et al 2011;Read et al 2012;Leeder et al 2013).…”

Identification and Characterization of LFD-2, a Predicted Fringe Protein Required for Membrane Integrity during Cell Fusion in Neurospora crassa

“…PRM1 is the ortholog of S. cerevisiae Prm1p, and N. crassa ⌬Prm1 mutants have a defect in membrane fusion similar to that observed for S. cerevisiae prm1⌬ mutants (21). LFD-1 was recently identified as playing a role in membrane merger by screening for genes regulated by PP-1, a transcription factor that regulates genes associated with cell fusion in N. crassa (22,23). In addition to defects in membrane merger, the absence of PRM1 or LFD-1 also led to significant cell lysis of adhered germlings.…”

Mating-type genes and hyphal fusions in filamentous basidiomycetes

“…Importantly, MAK-2 kinase was shown to phosphorylate the N-terminus of MOB-3, which could lead, with the help of HAM-2 and HAM-3 associations to the transport and accumulation of MAK-1 kinase into the nucleus to activate the CWI pathway regulating transcription factors. N. crassa hyphal fusion mutants also include the genes encoding transcription factors ADV-1, ADA-3, RCO-1, SNF5 and PP1 (Fu et al, 2011a(Fu et al, , 2014Leeder et al, 2013). These transcription factors are possibly needed to activate genes involved in cell communication and hyphal growth in CATs and hyphal fusions .…”