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.

Figure 1.—

gul-4/Δmak-2 strains suppress the cot-1(ts) growth defects. (A) The indicated strains were germinated and grown on minimal media plates for 3 days at 37°. Note the increased colony diameters of cot-1(ts);gul-4 and cot-1(ts);Δmak-2 compared to cot-1(ts). Bar, 1 cm. (B) Results of temperature-shift experiments, in which strains grown at 25° and shifted to 37° for 8 hr illustrate pointed growth-arrested tips of cot-1(ts), cot-1(ts);Δos-2, and cot-1(ts);Δmak-1, while dome-shaped slow-growing apices are visible in cot-1(ts),gul-4 and cot-1(ts);Δmak-2. Bar, 20 μm. (C) The indicated strains were transformed with mak-2 expression vector or the empty vector as control and grown on minimal media plates supplemented with 30 μg/ml nourseothricin for 3 days at 37°. Bar, 1 cm. (D) Western blot analysis of cell extracts probed with anti-COT1 antibodies indicate that deleting any of the three MAPKs does not affect COT1 expression (top) and that the gulliver-like suppression of the cot-1(1) phenotype by Δmak-2 at restrictive temperature is independent of the presence of the COT1 67-kDa band (arrow on bottom).

Figure 2.—

PKA activity is reduced in Δmak-2. (A) Morphology of Δmak-2; mcb(14-4) and mcb(14-4);Δmak-2 germinated for 12 hr at 37°. Bar, 20 μm. (B) Growth of cot-1(ts) and cot-1(ts);Δmak-2 on minimal media and media supplemented with 500 μm 8-Br-cAMP at restrictive temerature. (C) PKA activity in extracts of germinating conidia of wild type, cot-1(ts), mcb(14-4), Δmak-2, and cot-1(ts);Δmak-2, 11 hr post-inoculation, relative to wild type. Cultures were incubated in prewarmed liquid Vogel's minimal medium at 36° and were assayed for PKA activity. Data presented in the graph are means of at least four independent experiments with two replicates each. Standard errors are shown. (Bottom) A selected experiment demonstrating the nonphosphorylated and phosphorylated (indicating PKA activity) fluorescent Kemptide substrates, migrated to the anode and cathode of the agarose gel, respectively. The PepTag assays utilize fluorescent peptide substrates specific for PKA. Phosphorylation of the substrate by PKA alters the peptide's net charge from +1 to −1, allowing separation of the phosphorylated substrate from the nonphosphorylated on the agarose gel.

Figure 3.—

Comparative characterization of the N. crassa MAP kinase mutants. (A) Colony morphology, asexual development, and hyphal morphology (top and middle, respectively; bar, 20 μm) of the indicated strains grown on minimal media plates. Sexual development (bottom) was induced by growth for 5 days on cornmeal agar. The insets illustrate the terminal morphology of the female reproductive structures (protoperithecia). Bar, 10 μm. (B) Growth of the three MAPK mutants on gradient plates supplemented with the indicated additives. Conidia (5 × 10³) were inoculated for each spot. Wedges denote the compound gradient. To restrict the radial growth rates of the strains, all plates were supplemented with 1% sorbose in addition to the indicated additives.

Figure 4.—

mak-2 pathway defects are suppressed when COT1 activity is reduced. (A) cot-1(ts);Δmak-2 grown at 25° in race tubes has an intermediate tip-extension rate (A) and generates intermediate amounts of aerial hyphae and conidia (B) when compared to the parental strains. (C) Time course of protoperithecia formation by cot-1(ts);Δmak-2. Bars, 100 μm (5 and 10 days overview), 10 μm (5-day inset), and 25 μm (10-day inset).

Figure 5.—

Hyphal fusion is dependent on the three MAP kinase modules. Microscopic analysis of the indicated strains grown for 2 days on minimal media plates at 25°. Note that the three MAPK mutants show extended cell–cell contacts, but no distinct fusion bridges, which are clearly visible in wild type, cot-1(ts), and cot-1(ts);Δmak-2. Bar, 5 μm.

Figure 6.—

MAK1 activity is increased in cot-1(ts). (A) Total soluble protein (100 μg/lane) was extracted from the indicated strains grown in the presence or absence of stress inducers (1 m NaCl, 7 mm H₂O₂). The blot was probed with anti-phospho-ERK (α-P-ERK) and anti-phospho-p38 (α-P-p38) antibodies to detect activated MAK1, MAK2, and OS2 kinase. (B) For the temperature-shift experiments, total soluble protein (50 μg/lane) of the indicated strains grown at 25° and shifted to 37° for 12 hr was extracted and the blot was probed with anti-phospho-ERK (α-P-ERK) antibody (top). To confirm equal loading, the blot was stripped and reprobed with α-tubulin antibody (bottom). lrg-1 is an unrelated temperature-sensitive hyperbranching mutant used as a control.

Figure 7.—

Model summarizing the components and functions of the three N. crassa MAPK modules and cross-communication between COT1, MAP kinase, and PKA signaling pathways. Details are discussed in the text.