The NDR Kinase Scaffold HYM1/MO25 Is Essential for MAK2 MAP Kinase Signaling in Neurospora crassa

Abstract

Cell communication is essential for eukaryotic development, but our knowledge of molecules and mechanisms required for intercellular communication is fragmentary. In particular, the connection between signal sensing and regulation of cell polarity is poorly understood. In the filamentous ascomycete Neurospora crassa, germinating spores mutually attract each other and subsequently fuse. During these tropic interactions, the two communicating cells rapidly alternate between two different physiological states, probably associated with signal delivery and response. The MAK2 MAP kinase cascade mediates cell–cell signaling. Here, we show that the conserved scaffolding protein HYM1/MO25 controls the cell shape-regulating NDR kinase module as well as the signal-receiving MAP kinase cascade. HYM1 functions as an integral part of the COT1 NDR kinase complex to regulate the interaction with its upstream kinase POD6 and thereby COT1 activity. In addition, HYM1 interacts with NRC1, MEK2, and MAK2, the three kinases of the MAK2 MAP kinase cascade, and co-localizes with MAK2 at the apex of growing cells. During cell fusion, the three kinases of the MAP kinase module as well as HYM1 are recruited to the point of cell–cell contact. hym-1 mutants phenocopy all defects observed for MAK2 pathway mutants by abolishing MAK2 activity. An NRC1-MEK2 fusion protein reconstitutes MAK2 signaling in hym-1, while constitutive activation of NRC1 and MEK2 does not. These data identify HYM1 as a novel regulator of the NRC1-MEK2-MAK2 pathway, which may coordinate NDR and MAP kinase signaling during cell polarity and intercellular communication.

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

“…Western blots were probed with antip44/42 antibodies, which specifically recognize phosphorylated MAK-2 (Pandey et al 2004). As seen with other fusion mutants (Dettmann et al 2012Fu et al 2014), phosphorylated MAK-2 was present in Dham-14 strains, but to a lower level (61.2 612.8%, P , 0.05, Student's t-test) than in WT germlings (Figure 7,F and G;Figure S7,D and E).…”

“…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). During germling fusion, chemotrophic growth is associated with oscillation of the MAK-2/MEK-2/NRC-1/HAM-5 complex and the regulatory adaptor subunit STE-50 to the fusion tips of germlings, conidial anastomosis tubes (CATs), and to the tips of hyphae undergoing chemotropic interactions prior to cell fusion (fusion hyphae) (Fleissner et al 2009;Dettmann et al 2012Dettmann et al , 2014Jonkers et al 2014 signaling complex is perfectly out of phase with a second protein, called SOFT, which is also essential for chemotropic interactions and cell fusion (Fleissner et al 2005).…”

“…Since cellular localization of HAM-14 in the Pham14-ham-14-gfp strain could not be determined, it is possible that puncta observed in the Ptef-1-ham-14-gfp strains are not associated with or required for HAM-14 function. In heterokaryotic strains expressing both the nuclear marker histone H1-dsRed and ham-14-gfp, nonoverlapping fluorescence in germlings or hyphae was observed ( Figure S6A), indicating that HAM-14 is excluded from the nucleus, similar to HAM-5, MEK-2, NRC-1, and SOFT (Fleissner et al 2009;Dettmann et al 2012Dettmann et al , 2014Jonkers et al 2014).…”

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

“…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 Pietro 2010;Bayram et al 2012).…”

“…During chemotropic interactions, NRC1, MEK2, and MAK2 oscillate together from CAT tip to cytoplasm. The oscillation of NRC1/MEK2/MAK2 occurs every 4 min and is perfectly out of phase with a second protein, SO, which also oscillates from CAT tip to cytoplasm in communicating germling pairs (Fleissner et al 2009b;Dettmann et al 2012). A STE12 homolog has also been identified in N. crassa (pp-1).…”