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

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.     

Mitogen-activated protein kinase cascade required for regulation of development and secondary metabolism in Neurospora crassa

Mitogen-activated protein kinase (MAPK) signaling cascades are composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In this study, we characterize components of a MAPK cascade in Neurospora crassa (mik-1, MAPKKK; mek-1, MAPKK; and mak-1, MAPK) homologous to that controlling cell wall integrity in Saccharomyces cerevisiae. Growth of basal hyphae is significantly reduced in mik-1, mek-1, and mak-1 deletion mutants on solid medium. All three mutants formed short aerial hyphae and the formation of asexual macroconidia was reduced in Deltamik-1 mutants and almost abolished in Deltamek-1 and Deltamak-1 strains. In contrast, the normally rare asexual spores, arthroconidia, were abundant in cultures of the three mutants. Deltamik-1, Deltamek-1, and Deltamak-1 mutants were unable to form protoperithecia or perithecia when used as females in a sexual cross. The MAK-1 MAPK was not phosphorylated in Deltamik-1 and Deltamek-1 mutants, consistent with the involvement of MIK-1, MEK-1, and MAK-1 in the same signaling cascade. Interestingly, we observed increased levels of mRNA and protein for tyrosinase in the mutants under nitrogen starvation, a condition favoring sexual differentiation. Tyrosinase is an enzyme that catalyzes production of the secondary metabolite l-DOPA melanin. These results implicate the MAK-1 pathway in regulation of development and secondary metabolism in filamentous fungi.     

The osmoregulatory pathway represses mating pathway activity in Saccharomyces cerevisiae: isolation of a FUS3 mutant that is insensitive to the repression mechanism.

Mitogen-activated protein (MAP) kinase cascades are conserved signal transduction pathways that are required for eukaryotic cells to respond to a variety of stimuli. Multiple MAP kinase pathways can function within a single cell type; therefore, mechanisms that insulate one MAP kinase pathway from adventitious activations by parallel pathways may exist. We have studied interactions between the mating pheromone response and the osmoregulatory (high-osmolarity glycerol response [HOG]) pathways in Saccharomyces cerevisiae which utilize the MAP kinases Fus3p and Hog1p, respectively. Inactivating mutations in HOG pathway kinases cause an increase in the phosphotyrosine content of Fus3p, greater expression of pheromone-responsive genes, and increased sensitivity to growth arrest by pheromone. Therefore, the HOG pathway represses mating pathway activity. In a HOG1+ strain, Fus3p phosphotyrosine increases modestly and transiently following an increase in the extracellular osmolarity; however, it increases to a greater extent and for a sustained duration in a hog1-delta strain. Thus, the HOG-mediated repression of mating pathway activity may insulate the mating pathway from activation by osmotic stress. A FUS3 allele whose gene product is resistant to the HOG-mediated repression of its phosphotyrosine content has been isolated. This mutant encodes an amino acid substitution in the highly conserved DPXDEP motif in subdomain XI. Other investigators have shown that the corresponding amino acid is also mutated in a gain-of-function allele of the MAP kinase encoded by the rolled locus in Drosophila melanogaster. These data suggest that the DPXDEP motif plays a role in the negative regulation of MAP kinases.     

Importance of MAP Kinases during Protoperithecial Morphogenesis in Neurospora crassa

In order to produce multicellular structures filamentous fungi combine various morphogenetic programs that are fundamentally different from those used by plants and animals. The perithecium, the female sexual fruitbody of Neurospora crassa, differentiates from the vegetative mycelium in distinct morphological stages, and represents one of the more complex multicellular structures produced by fungi. In this study we defined the stages of protoperithecial morphogenesis in the N. crassa wild type in greater detail than has previously been described; compared protoperithecial morphogenesis in gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; confirmed that all three MAP kinase cascades are required for sexual development; and showed that the three different cascades each have distinctly different functions during this process. However, only MAP kinases equivalent to the budding yeast pheromone response and cell wall integrity pathways, but not the osmoregulatory pathway, were essential for vegetative cell fusion. Evidence was obtained for MAP kinase signaling cascades performing roles in extracellular matrix deposition, hyphal adhesion, and envelopment during the construction of fertilizable protoperithecia.     

Specificity of MAP kinase signaling in yeast differentiation involves transient versus sustained MAPK activation

Signals transmitted by common components often elicit distinct (yet appropriate) outcomes. In yeast, two developmental options-mating and invasive growth-are both regulated by the same MAP kinase cascade. Specificity has been thought to result from specialized roles for the two MAP kinases, Kss1 and Fus3, and because Fus3 prevents Kss1 from gaining access to the mating pathway. Kss1 has been thought to participate in mating only when Fus3 is absent. Instead, we show that Kss1 is rapidly phosphorylated and potently activated by mating pheromone in wild-type cells, and that this is required for normal pheromone-induced gene expression. Signal identity is apparently maintained because active Fus3 limits the extent of Kss1 activation, thereby preventing inappropriate signal crossover.     

A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C.

Mitogen-activated protein (MAP) kinases are activated in response to a variety of stimuli through a protein kinase cascade that results in their phosphorylation on tyrosine and threonine residues. The molecular nature of this cascade is just beginning to emerge. Here we report the isolation of a Saccharomyces cerevisiae gene encoding a functional analog of mammalian MAP kinases, designated MPK1 (for MAP kinase). The MPK1 gene was isolated as a dosage-dependent suppressor of the cell lysis defect associated with deletion of the BCK1 gene. The BCK1 gene is also predicted to encode a protein kinase which has been proposed to function downstream of the protein kinase C isozyme encoded by PKC1. The MPK1 gene possesses a 1.5-kb uninterrupted open reading frame predicted to encode a 53-kDa protein. The predicted Mpk1 protein (Mpk1p) shares 48 to 50% sequence identity with Xenopus MAP kinase and with the yeast mating pheromone response pathway components, Fus3p and Kss1p. Deletion of MPK1 resulted in a temperature-dependent cell lysis defect that was virtually indistinguishable from that resulting from deletion of BCK1, suggesting that the protein kinases encoded by these genes function in a common pathway. Expression of Xenopus MAP kinase suppressed the defect associated with loss of MPK1 but not the mating-related defects associated with loss of FUS3 or KSS1, indicating functional conservation between the former two protein kinases. Mutation of the presumptive phosphorylated tyrosine and threonine residues of Mpk1p individually to phenylalanine and alanine, respectively, severely impaired Mpk1p function. Additional epistasis experiments, and the overall architectural similarity between the PKC1-mediated pathway and the pheromone response pathway, suggest that Pkc1p regulates a protein kinase cascade in which Bck1p activates a pair of protein kinases, designated Mkk1p and Mkk2p (for MAP kinase-kinase), which in turn activate Mpk1p.     

Pheromone-induced polarization is dependent on the Fus3p MAPK acting through the formin Bni1p

During mating, budding yeast cells reorient growth toward the highest concentration of pheromone. Bni1p, a formin homologue, is required for this polarized growth by facilitating cortical actin cable assembly. Fus3p, a pheromone-activated MAP kinase, is required for pheromone signaling and cell fusion. We show that Fus3p phosphorylates Bni1p in vitro, and phosphorylation of Bni1p in vivo during the pheromone response is dependent on Fus3p. fus3 mutants exhibited multiple phenotypes similar to bni1 mutants, including defects in actin and cell polarization, as well as Kar9p and cytoplasmic microtubule localization. Disruption of the interaction between Fus3p and the receptor-associated Galpha subunit caused similar mutant phenotypes. After pheromone treatment, Bni1p-GFP and Spa2p failed to localize to the cortex of fus3 mutants, and cell wall growth became completely unpolarized. Bni1p overexpression suppressed the actin assembly, cell polarization, and cell fusion defects. These data suggest a model wherein activated Fus3p is recruited back to the cortex, where it activates Bni1p to promote polarization and cell fusion.     

Effect of 5-azacytidine on the light-sensitive formation of sexual and asexual reproductive structures in wc-1 and wc-2 mutants of Neurospora crassa

Under the conditions of nitrogen starvation, illumination by blue light of wc-1 and wc-2 mutants of the ascomycete Neurospora crassa failed to stimulate the formation of protoperithecia and inhibit conidiation (contrary to what was observed in the mycelium of the wild-type fungus). The data obtained indicate that wc-1 and wc-2 genes of N. crassa are involved in light-dependent formation of protoperithecia and conidia. The effects of 5-azacytidine (an inhibitor of DNA methylation) under the same experimental conditions suggest that the balance between the formation of sexual and asexual reproductive structures, maintained in N. crassa, depends on genome methylation processes sensitive to the action of light, which is mediated by the photoreceptor complex of WC proteins.     

Isolation of new white collar mutants of Neurospora crassa and studies on their behavior in the blue light-induced formation of protoperithecia

White collar (wc) mutants of Neurospora crassa are thought to be regulatory mutants blocked in the photoinduction of carotenogenesis. Eight new wc mutants have been isolated after UV mutagenesis; their morphology and linear growth rate are not altered, although blue light-induced carotenogenesis is completely blocked. All of the wc mutations fall into two complementation groups corresponding to the already-known wc-1 and wc-2 loci. It is shown that the wc mutations impair another blue light effect, the photoinduction of protoperithecia formation, as well as the low constitutive production of protoperithecia in the dark. These effects are not due to the lack of carotenoids since the albino mutants show a normal sexual development. The pleiotropic effects of the mutations in the wc genes indicate that they play a key role in the mechanisms of regulation of the blue light-induced responses of N. crassa.     

WSC-1 and HAM-7 Are MAK-1 MAP Kinase Pathway Sensors Required for Cell Wall Integrity and Hyphal Fusion in Neurospora crassa

A large number of cell wall proteins are encoded in the Neurospora crassa genome. Strains carrying gene deletions of 65 predicted cell wall proteins were characterized. Deletion mutations in two of these genes (wsc-1 and ham-7) have easily identified morphological and inhibitor-based defects. Their phenotypic characterization indicates that HAM-7 and WSC-1 function during cell-to-cell hyphal fusion and in cell wall integrity maintenance, respectively. wsc-1 encodes a transmembrane protein with extensive homology to the yeast Wsc family of sensor proteins. In N. crassa, WSC-1 (and its homolog WSC-2) activates the cell wall integrity MAK-1 MAP kinase pathway. The GPI-anchored cell wall protein HAM-7 is required for cell-to-cell fusion and the sexual stages of the N. crassa life cycle. Like WSC-1, HAM-7 is required for activating MAK-1. A Δwsc-1;Δham-7 double mutant fully phenocopies mutants lacking components of the MAK-1 MAP kinase cascade. The data identify WSC-1 and HAM-7 as the major cell wall sensors that regulate two distinct MAK-1-dependent cellular activities, cell wall integrity and hyphal anastomosis, respectively.     

The Cryptococcus neoformans STE11alpha gene is similar to other fungal mitogen-activated protein kinase kinase kinase (MAPKKK) genes but is mating type specific

Partial sequence analysis of the Cryptococcus neoformans MATalpha mating type locus revealed the presence of a gene with substantial sequence similarity to other fungal mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK) genes. The C. neoformans gene, designated STE11alpha, showed the highest degree of similarity to the Neurospora crassa nrc-1, Schizosaccharomyces pombe byr2 and Saccharomyces cerevisiae STE11 genes. A polymerase chain reaction-mediated sib-selection technique was successfully adapted for the purpose of disrupting STE11alpha. C. neoformans ste11alphaDelta mutants were found to be sterile, consistent with the phenotypes of ste11 and byr2 mutants in S. cerevisiae and S. pombe respectively. Haploid ste11alphaDelta mutants were also found to be unable to produce hyphae, suggesting that the C. neoformans gene is functionally conserved when compared with its S. cerevisiae MAPKKK counterpart. Comparison of the wild-type STE11alpha strain with a ste11alphaDelta disruptant for virulence using the mouse model showed that the ste11alphaDelta strain was less virulent, but the difference was only minor. In spite of some of the conserved functions of STE11alpha, linkage analysis showed that STE11alpha is only found in mating type alpha strains. These results demonstrate that, although functionally conserved, the mating pathway in C. neoformans has a unique organization.     

The Fus3/Kss1 MAP kinase homolog Amk1 regulates the expression of genes encoding hydrolytic enzymes in Alternaria brassicicola

Mitogen-activated protein (MAP) kinases have been shown to be required for virulence in diverse phytopathogenic fungi. To study its role in pathogenicity, we disrupted the Amk1 MAP kinase gene, a homolog of the Fus3/Kss1 MAP kinases in Saccharomyces cerevisiae, in the necrotrophic Brassica pathogen, Alternaria brassicicola. The amk1 disruption mutants showed null pathogenicity on intact host plants. However, amk1 mutants were able to colonize host plants when they were inoculated on a physically damaged host surface, or when they were inoculated along with nutrient supplements. On intact plants, mutants expressed extremely low amounts of several hydrolytic enzyme genes that were induced over 10-fold in the wild-type during infection. These genes were also dramatically induced in the mutants on wounded plants. These results imply a correlation between virulence and the expression level of specific hydrolytic enzyme genes plus the presence of an unidentified pathway controlling these genes in addition to or in conjunction with the Amk1 pathway.     

Twitchin kinase interacts with MAPKAP kinase 2 in Caenorhabditis elegans striated muscle

In Caenorhabditis elegans, twitchin is a giant polypeptide located in muscle A-bands. The protein kinase of twitchin is autoinhibited by 45 residues upstream (NL) and 60 residues downstream (CRD) of the kinase catalytic core. Molecular dynamics simulation on a twitchin fragment revealed that the NL is released by pulling force. However, it is unclear how the CRD is removed. To identify proteins that may remove the CRD, we performed a yeast two-hybrid screen using twitchin kinase as bait. One interactor is MAK-1, C. elegans orthologue of MAPKAP kinase 2. MAPKAP kinase 2 is phosphorylated and activated by p38 MAP kinase. We demonstrate that the CRD of twitchin is important for binding to MAK-1. mak-1 is expressed in nematode body wall muscle, and antibodies to MAK-1 localize between and around Z-disk analogues and to the edge of A-bands. Whereas unc-22 mutants are completely resistant, mak-1 mutants are partially resistant to nicotine. MAK-1 can phosphorylate twitchin NL-Kin-CRD in vitro. Genetic data suggest the involvement of two other mak-1 paralogues and two orthologues of p38 MAP kinase. These results suggest that MAK-1 is an activator of twitchin kinase and that the p38 MAP kinase pathway may be involved in the regulation of twitchin.     

Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue

Neurospora crassa osmosensitive (os) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl. We found that os-2 and os-5 mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. To understand the relationship between osmoregulation and fungicide resistance, we cloned the os-2 gene by using sib selection. os-2 encodes a putative mitogen-activated protein (MAP) kinase homologous to HOG1 and can complement the osmosensitive phenotype of a Saccharomyces cerevisiae hog1 mutant. We sequenced three os-2 alleles and found that all of them were null with either frameshift or nonsense point mutations. An os-2 gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides. Conversely, os-2 mutants transformed with the wild-type os-2 gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides. Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in os-2 mutants. Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst. These results suggest that the hyperosmotic stress response pathway of N. crassa is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive os-2 MAP kinase pathway.     

A Highlights from MBoC Selection: Combined computational and experimental analysis reveals mitogen-activated protein kinase–mediated feedback phosphorylation as a mechanism for signaling specificity

Different environmental stimuli often use the same set of signaling proteins to achieve very different physiological outcomes. The mating and invasive growth pathways in yeast each employ a mitogen-activated protein (MAP) kinase cascade that includes Ste20, Ste11, and Ste7. Whereas proper mating requires Ste7 activation of the MAP kinase Fus3, invasive growth requires activation of the alternate MAP kinase Kss1. To determine how MAP kinase specificity is achieved, we used a series of mathematical models to quantitatively characterize pheromone-stimulated kinase activation. In accordance with the computational analysis, MAP kinase feedback phosphorylation of Ste7 results in diminished activation of Kss1, but not Fus3. These findings reveal how feedback phosphorylation of a common pathway component can limit the activity of a competing MAP kinase through feedback phosphorylation of a common activator, and thereby promote signal fidelity.     

A pheromone receptor gene, pre-1, is essential for mating type-specific directional growth and fusion of trichogynes and female fertility in Neurospora crassa

Neurospora crassa is a heterothallic filamentous fungus with two mating types, mat a and mat A. Its mating involves differentiation of female reproductive structures (protoperithecia) and chemotropic growth of female-specific hyphae (trichogynes) towards a cell of the opposite mating type in a pheromone-mediated process. In this study, we characterize the pre-1 gene, encoding a predicted G-protein-coupled receptor with sequence similarity to fungal pheromone receptors. pre-1 is most highly expressed in mat A strains under mating conditions, but low levels can also be detected in mat a strains. Analysis of pre-1 deletion mutants showed that loss of pre-1 does not greatly affect vegetative growth, heterokaryon formation or male fertility in either mating type. Protoperithecia from Deltapre-1 mat A mutants do not undergo fertilization; this defect largely stems from an inability of their trichogynes to recognize and fuse with mat a cells. Previous work has demonstrated that the Galpha subunit, GNA-1, and the Gbeta protein, GNB-1, are essential for female fertility in N. crassa. Trichogynes of Deltagna-1 and Deltagnb-1 mutants displayed severe defects in growth towards and fusion with male cells, similar to that of Deltapre-1 mat A strains. However, the female sterility defect of the Deltapre-1 mat A mutant could not be complemented by constitutive activation of gna-1, suggesting additional layers of regulation. We propose that PRE-1 is a pheromone receptor coupled to GNA-1 that is essential for the mating of mat A strains as females, consistent with a role in launching the pheromone response pathway in N. crassa.