Mitogen-Activated Protein Kinase Cascade Required for Regulation of Development and Secondary Metabolism in Neurospora crassa

Abstract

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 ⌬mik-1 mutants and almost abolished in ⌬mek-1 and ⌬mak-1 strains. In contrast, the normally rare asexual spores, arthroconidia, were abundant in cultures of the three mutants. ⌬mik-1, ⌬mek-1, and ⌬mak-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 ⌬mik-1 and ⌬mek-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.

Circadian Activation of the Mitogen-Activated Protein Kinase MAK-1 Facilitates Rhythms in Clock-Controlled Genes in Neurospora crassa

“…Therefore, we focused further efforts on defining the MAK-1 pathway. MAK-1 was previously shown to be phosphorylated after treatment with a ␤-1,3 glucan inhibitor, aculeacin A (29), suggesting that the MAK-1 pathway functions in the cell wall stress response pathway in Neurospora, similar to Slt2 in S. cerevisiae (43). Slt2 is also activated in response to increased temperatures, which can destabilize the integrity of the cell wall (44).…”

“…In previous studies of Neurospora, the phosphorylation state of MAK-1 and MAK-2 has been detected using an available phospho-specific antibody against the mammalian homologs p44 and p42 (29,42). As expected, using this antibody in Western blots, bands were observed corresponding to the predicted sizes for MAK-1 (46.8 kDa) and MAK-2 (40.8 kDa) that were absent in the ⌬mak-1 and ⌬mak-2 strains, respectively (Fig.…”

“…In addition to OS-2, there are two other MAPKs present in Neurospora; MAK-1, a homologue of Slt2 in S. cerevisiae and extracellular signal-regulated kinase 1/2 (ERK1/2) in mammals, is predicted to be involved in cell wall integrity and maintenance, while MAK-2, a homologue of Fus3 in S. cerevisiae, and also ERK1/2 in mammals, has been implicated in the sexual cycle, conidiation, hyphal fusion, and vegetative growth (26)(27)(28)(29)(30). In this study, we show that the phosphorylation state of MAK-1 and MAK-2 is rhythmically controlled by the circadian clock; however, we focus on characterizing the circadian regulation of the MAK-1 pathway.…”

“…Although predictions can be made based on homology, there have been no extensive studies to identify the genetic targets of the MAK-1 pathway in Neurospora. Phenotypic analyses of deletion mutants of the MAK-1 pathway have shown various levels of perturbation in the cell wall, increased melanin biosynthesis, and decreased growth rate, polarity, hyphal fusion, conidiation, and sexual reproduction (28,29).…”

Mitogen-activated protein kinase signaling pathways of the tangerine pathotype of Alternaria alternata

“…12,53,60 We also found that the A. alternata Slt2 is involved in regulating biosynthesis of secondary metabolites, because Dslt2 mutant accumulated lower levels of ACT toxin and melanin. 45 The levels of ACT toxin and melanin were restored in a strain that expresses a wild-type copy of AaSlt2.…”