Academic journal article Genetics

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

Academic journal article Genetics

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

Article excerpt

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 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.

CELL fusion between genetically identical cells is important in development (for example, myoblast fusion during muscle formation) and occurs in many multicellular organ- isms 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 differenti- ated, but in some cases, also between cells in an identical developmental state, for example, cell fusion between germi- nating asexual spores (conidia) of filamentous fungi (Pandey et al. 2004; Roca et al. 2005a; Read et al. 2010). In filamen- tous fungi, these fusions are integral to the formation of an interconnected hyphal network, which mediates genetic mix- ing 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 (Stell, 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 re- quired for the virulence of both plant and animal fungal pathogens (Roman et al. 2007; Rispad and Di Pietro 2010; Bayram et al. 2012). In S. cerevisiae, reception of a mating- type-specific pheromone signal results in the activation of Stell (MEKK), Ste7 (MEK), and Fus3 (MAPK) (Bardwell 2005). After Fus3 is activated, it enters the nucleus and phosphorylates Stel2, a mating-type-specific transcription factor, as well as two regulators of Stel2, Digl and Dig2 (Blackwell et al. 2007). Activated Stel2 regulates the ex- pression of many genes involved in the mating process, both indirectly and directly by binding pheromone re- sponse elements in target promoters (Zeitlinger et al. 2003).

Stel2-like proteins have also been studied in filamentous fungi, where they play essential roles in development and pathogenicity (Alspaugh et al. 1998; Vallim et al. 2000; Borneman et al. 2001; Park et al. 2002; Tsuji et al. 2003; Li et al. 2005; Nolting and Poggeler 2006; Ren et al. 2006; Tollot et al. …

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