The Nuclear Dbf2-Related Kinase COT1 and the Mitogen-Activated Protein Kinases MAK1 and MAK2 Genetically Interact to Regulate Filamentous Growth, Hyphal Fusion and Sexual Development in Neurospora crassa

Abstract

Ndr kinases, such as Neurospora crassa COT1, are important for cell differentiation and polar morphogenesis, yet their input signals as well as their integration into a cellular signaling context are still elusive. Here, we identify the cot-1 suppressor gul-4 as mak-2 and show that mutants of the gul-4/mak-2 mitogen-activated protein (MAP) kinase pathway suppress cot-1 phenotypes along with a concomitant reduction in protein kinase A (PKA) activity. Furthermore, mak-2 pathway defects are partially overcome in a cot-1 background and are associated with increased MAK1 MAPK signaling. A comparative characterization of N. crassa MAPKs revealed that they act as three distinct modules during vegetative growth and asexual development. In addition, common functions of MAK1 and MAK2 signaling during maintenance of cell-wall integrity distinguished the two ERK-type pathways from the p38-type OS2 osmosensing pathway. In contrast to separate functions during vegetative growth, the concerted activity of the three MAPK pathways is essential for cell fusion and for the subsequent formation of multicellular structures that are required for sexual development. Taken together, our data indicate a functional link between COT1 and MAPK signaling in regulating filamentous growth, hyphal fusion, and sexual development.

TheSaccharomyces cerevisiae PRM1Homolog inNeurospora crassaIs Involved in Vegetative and Sexual Cell Fusion Events but Also Has Postfertilization Functions

“…DPrm1 mutants show a comparable reduction in trichogyne-conidium fusion during fertilization as during germling fusion: Many fusion mutants in N. crassa are defective in the production of prefruiting bodies (protoperithecia) that function as female reproductive structures (Pandey et al 2004;Maerz et al 2008), suggesting that hyphal fusion contributes to the formation of these structures. We therefore evaluated whether the DPrm1 mutants form normal protoperithecia.…”

“…The promoter of PRM1 has multiple sites for binding of the transcription factor Ste12p, which directly regulates genes involved in shmoo formation and mating cell fusion (Zeitlinger et al 2003). Interestingly, mutations in the ortholog of STE12 in N. crassa, called pp-1, also show defects in germling/hyphal fusion (Li et al 2005) (A. Fleißner and N. L. Glass, unpublished data), as do strains containing mutations in orthologs of the pheromone response MAPK pathway (mak-2, nrc-1, and mek-2) (Pandey et al 2004;Maerz et al 2008). However, N. crassa pheromone mutants, although infertile as a male, undergo normal germling and hyphal fusion (Kim and Borkovich 2006).…”

“…Analysis of the conservation of genes involved in mating cell fusion in S. cerevisiae and N. crassa reveal that most of the components of the signal transduction/polarization pathway are highly conserved . In fact, mutations in N. crassa genes encoding the orthologs of the S. cerevisiae pheromone response MAP kinase pathway (STE11, STE7, and FUS3) result in strains that are defective in germling and hyphal fusion (Pandey et al 2004;Maerz et al 2008). So far, homologs of S. cerevisiae genes involved in membrane merger, including FUS1, FUS2, and FIG1 have not been identified in the N. crassa genome ).…”

Mutations in the Saccharomyces cerevisiae Kinase Cbk1p Lead to a Fertility Defect That Can Be Suppressed by the Absence of Brr1p or Mpt5p (Puf5p), Proteins Involved in RNA Metabolism

“…These different interactions are summarized in the model in Figure 8. Given the links between Cbk1p and Mpt5p, and Mpt5p and the m-MAPK and f-MAPK pathways, it is interesting to note that the Neurospora crassa homolog of Cbk1p, COT1, has recently been shown to interact genetically with two MAP kinases, MAK1 and MAK2, which participate in the regulation of filamentous growth, hyphal fusion, and sexual development (Maerz et al 2008). It is clear that the RAM network interacts with Ssd1p, Brr1p, and Mpt5p to regulate a series of parallel, and probably at least partially redundant, pathways controlling aspects of cell separation, cell integrity, mating, and polarized growth (Figure 8).…”

A versatile set of Lifeact-RFP expression plasmids for live-cell imaging of F-actin in filamentous fungi

“…Therefore, pALxLifeact transformant selection with nourseothricin was the preferred option, as it is much more stringent than ignite per se and has no reported problems in conferring cross-resistance to other selection markers, such as hygromycin B (Kück and Hoff, 2006). Nourseothricin has been successfully used for the transformation of several yeast species including Saccharomyces cerevisiae (Goldstein and McCusker, 1999), Schizosaccharomyces pombe (Hentges et al, 2005), Candida albicans (Shen et al, 2005) and Cryptococcus neoformans (McDade and Cox, 2001), and more recently in an increasing number of filamentous fungal species, including Acremonium chrysogenum and Sordaria macrospora (Kück and Hoff, 2006), as well as Neurospora crassa (Maerz et al, 2009;Maerz et al, 2008).…”