Life Cycle
The ascomycete N. crassa has defined asexual and sexual cycles (Fig. 20). Under nutrient-rich conditions, N. crassa pro-liferates by the extension and branching of multinucleate veg-etative hyphal cells to form a multicellular mycelium. During the vegetative cycle, N. crassa produces two types of vegetative spores, macroconidia and microconidia (reviewed in refer-ences 65 and 264). Macroconidia are multinucleate and differ-entiate from specific organs called conidiophores that arise from aerial hyphae. Microconidia differentiate from vegetative mycelia, are smaller and uninucleate, and germinate poorly.
N. crassa is heterothallic and has two mating types, A and a.
The sexual cycle initiates in response to nitrogen starvation (191). Either A or a cells of vegetative hyphae produce
fruiting-body precursors called protoperithecia. Specialized hyphae called trichogynes grow directionally from the protoperithe-cium toward an opposite mating type cell such as a conidium or a hyphal fragment, which functions as a male gamete. Once a nucleus from a male gamete enters a protoperithecium, the protoperithecium becomes a perithecium. The male and fe-male nuclei divide as a dikaryon in ascogenous hyphae. Dikary-otic cells at the crest of hook-shaped structures, croziers, un-dergo premeiotic DNA synthesis and nuclear fusion (karyogamy) to produce the only diploid cells in the life cycle of N. crassa.
Meiosis and one round of mitosis immediately follow to pro-duce eight-spored asci. The mature ascospores are ejected and can be induced to germinate by heat shock or exposure to ethers, producing cells that reenter the vegetative phase of the life cycle.
In response to nutrient deprivation, desiccation, and light, N.
crassa initiates asexual conidiation (65, 264). The major deter-minants regulating the onset of conidiation are the availability of carbon source (181, 233) and light (153). In response to light, N. crassa cultures produce conidia faster and in greater numbers compared to cultures grown in complete darkness. In addition to these environmental factors, the endogenous clock of the organism affects the conidiation process: conidiation occurs during the subjective morning. Clock-controlled genes expressed during the subjective morning have been found to encode proteins expressed during conidiation (25). Therefore, to complete fungal development in response to various extra-cellular and intraextra-cellular stimuli, signal transduction pathways sensing these stimuli must function coordinately.
cAMP Signaling Pathway
In N. crassa, two G␣ subunit genes, gna-1 and gna-2, have been identified (285) (Fig. 14 and 21). The deduced amino acid sequence of GNA1 has homology with members of the G␣i family; for example, it is 55.2% identical to rat Gi2. GNA1 also possesses consensus sequences for addition of myristic acid
FIG. 21. Model for signal transduction pathways in N. crassa. A cAMP signaling pathway consisting of G protein␣ subunits (GNA1 and GNA2), adenylyl cyclase (CR1), and a PKA regulatory subunit (MCB) is involved in conidiation, morphogenesis, mating, and stress tolerance. Ras-, MAP kinase (MAPK)-, and PKA-related kinase-mediated signaling pathways also regulate conidiation. How signaling is coordinated between these pathways remains to be elucidated.
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and labeling by pertussis toxin, which are diagnostic of the Gi family (285). GNA2 is most similar to S. pombe Gpa1 (47.7%
identity), which is involved in pheromone sensing.
Disruption of the gna-1 gene causes pleiotropic defects in asexual and sexual development (119, 120, 316). During vege-tative growth,⌬gna-1 strains exhibit slower apical extension of hyphae and slower growth, and hyperbranching of the myce-lium occurs on solid minimal medium (119). Addition of NaCl, KCl, or sorbitol exacerbates the growth defect of⌬gna-1 mu-tants on solid medium. Furthermore,⌬gna-1 strains have de-fects in forming aerial hyphae and macroconidia. In addition to defects in morphogenesis,⌬gna-1 mutants show a higher ca-rotenoid content than wild-type strains (316). Caca-rotenoids have been shown to provide resistance to oxidative stress through quenching of free radicals (197, 248). Furthermore, resistance to oxidative stress is often linked to heat tolerance.
Consistent with this, the⌬gna-1 strain is more resistant to heat and oxidative stress by hydrogen peroxide (316). During sexual development,⌬gna-1 mutants differentiate small and infertile protoperithecia and produce fewer protoperithecia than wild-type strains (119).
Strains expressing activated alleles of the gna-1 gene (R178C and Q204L) exhibit opposite phenotypes with respect to de-fects observed in⌬gna-1 strains (316). In contrast to ⌬gna-1 strains, gna-1R178C and gna-1Q204L mutant strains produce longer, more abundant aerial hyphae than the wild type. In spite of the presence of more aerial hyphae, the activated-allele-containing strains produce almost the same amount of conidia as the wild type. Although ⌬gna-1 strains produce fewer aerial hyphae, they also make the same amount of conidia as the wild type. Both activated gna1 alleles comple-ment the sensitivity of⌬gna-1 mutant strains to NaCl, KCl, and sorbitol (316). gna-1R178Cand gna-1Q204Lmutant strains pro-duce less carotenoids than the wild type and are more sensitive to heat and hydrogen peroxide. In sexual development, the Q204L and R178C strains are female fertile but produce fewer perithecia than the wild type (316).
Some of the phenotypes observed in the⌬gna-1, gna-1R178C, and gna-1Q204L strains are consistent with their effects on cAMP production. As described below, adenylyl cyclase-defec-tive cr-1 mutants exhibit morphological aberrations, defeccyclase-defec-tive conidium formation, and thermotolerance. Moreover, previ-ous studies suggest that aerial hyphal development is positively regulated by cAMP (205, 273, 316). Carotenoid accumulation has been shown to be negatively correlated with cAMP levels (138). In accord with these findings, cAMP levels in the⌬gna-1 mutants are lower than those in the wild type, and levels of cAMP in the gna-1R178C and gna-1Q204L mutant strains are higher (120, 316). In vitro experiments have demonstrated that GNA1 positively regulates adenylyl cyclase activity, possibly via direct interactions with adenylyl cyclase (120). Interestingly, a mutation in the gna-1 locus affects both phosphodiesterase and adenylyl cyclase activities (120).
Deletion of gna-2 or introduction of an activated allele (gna-2R179C or gna-2Q205L) does not result in major abnormalities during vegetative growth or sexual development (16). How-ever, combination of the⌬gna-2 and ⌬gna-1 mutations exac-erbates ⌬gna-1 mutant phenotypes (16, 120). ⌬gna-1 ⌬gna-2 double mutants show a slower rate of hyphal apical extension than⌬gna-1 strains on hyperosmotic solid medium containing sorbitol, KCl, or NaCl (16). Adenylyl cyclase and phosphodi-esterase activities in the⌬gna-1 ⌬gna-2 strains are lower than those in the ⌬gna-1 single mutant strains (120). During the sexual cycle,⌬gna-1 ⌬gna-2 mutant strains produce fewer pro-toperithecia, which fail to differentiate ascospores (16). These
data indicate that GNA1 and GNA2 have overlapping roles in both the asexual and sexual cycles.
Adenylyl Cyclase
Two mutations of N. crassa that cause defects in cAMP metabolism have been isolated (241): one is frost (fr) (250, 251), and the other is crisp-1 (cr-1) (274). Originally, these mutations were characterized as morphological, causing a co-lonial morphology due to hyperbranching of hyphae (89, 228).
Both mutants exhibit reduced adenylyl cyclase activity and low endogenous levels of cAMP (274). The abnormal morphology of fr mutants is not corrected by exogenous cAMP or phos-phodiesterase inhibitors, indicating that the morphology of fr mutants is not caused by low endogenous cAMP levels (241).
Very recently, the fr gene was isolated and found to encode a homolog of the S. cerevisiae Cdc1 protein (258), which regu-lates Mn2⫹homeostasis in S. cerevisiae (221, 222). The fr gene product may also be involved in Mn2⫹homeostasis and regu-lation of hyphal morphology (258).
cr-1 mutant strains exhibit colonial growth with few or no aerial hyphae and undergo a characteristic uniform, dense, and premature conidiogenesis. Even in liquid culture, germ tubes emerging from conidia differentiate early into branched con-idiophores (123). Another characteristic of the cr-1 mutant is an inability to use glycerol and other carbon sources (275).
Exogenous cAMP or dibutyryl-cAMP stimulates the rate of mycelial elongation and formation of aerial hyphae in cr-1 mutants (273, 274). These observations suggest that a defect in cAMP production is the primary cause of the cr-1 phenotype.
Consistent with this, the cr-1 gene was found to encode aden-ylyl cyclase (134, 135). As observed in S. cerevisiae, adenaden-ylyl cyclase-deficient mutant strains exhibit increased thermotoler-ance (58, 254). In summary, in N. crassa adenylyl cyclase is involved in hyphal tip growth, formation of aerial hyphae, conidiation, and thermotolerance.
Regulatory Subunit of cAMP-Dependent Protein Kinase N. crassa mcb mutant strains lacking the PKA regulatory subunit exhibit temperature-sensitive morphological abnor-malities (35). mcb mutant strains also show a complete loss of growth polarity, which is restricted to the apical cell of hyphal filaments when strains are cultured at 37°C (35). The isotropic mutant hyphae are distended at the restrictive temperature, resulting in an increase in hyphal length as well as hyphal diameter. This phenotype is not suppressed by osmotic stabi-lization, indicating that cell wall integrity is not compromised in the mcb mutant at 37°C.
Cytological studies on the mcb mutants revealed that these strains have defects in cytoskeleton organization (35). In wild-type hyphae, actin patches are concentrated at the hyphal tip and associated with the cell cortex. When mcb mutant hyphae are incubated at 37°C, actin patches are uniformly distributed over the surface of hyphae but are still concentrated at sites of septation. Calcofluor white staining revealed septal abnormal-ities in mcb mutants at 37°C. Some septa were immediately adjacent to each other, and most septa were asymmetric. Cy-toplasmic microtubule tracks in wild-type cells are located in regions of hyphae and extend from distal to apical regions of hyphae. In the mcb mutant, the hyphae themselves are shorter and organized in overlapping and concentrated networks at 37°C.
The predicted amino acid sequence of the MCB protein has similarity to regulatory subunits of cAMP-dependent protein kinase (PKA), including S. cerevisiae Bcy1 (62%) and S. pombe Cgs1 (57%) (35). Thus, it is most likely that N. crassa PKA
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regulates hyphal growth polarity, possibly through organization of the cytoskeleton. As mentioned above, components of the cAMP-mediated signaling pathway, including GNA1, GNA2, and adenylyl cyclase (CR1), are involved in morphology and conidiation. Morphogenesis depends on polarized hyphal growth, which is governed by hyphal extension. Conidiation requires polarized growth of hyphae. Therefore, growth polar-ity and cytoskeletal organization regulated by PKA must be important for conidiation as well as morphology.
Surprisingly, the cytological defects observed in mcb mutant strains with constitutive PKA activity were suppressed by ad-enylyl cyclase (cr-1) mutations, and it was concluded that the cr-1 mutation is epistatic to the mcb mutation (35). This is surprising because a mutant lacking the regulatory subunit of PKA should have constitutive PKA activity that is no longer regulated by cAMP. Possible explanations are that there is a second PKA regulatory subunit in N. crassa, that there is a second target of cAMP in N. crassa, or that the mcb mutation is a hypomorphic allele that could retain residual regulation by cAMP. Further studies will be required to resolve this issue.
Ras Homologs NC-ras and NC-ras2
Two ras homologs, NC-ras and smco7/NC-ras2, have been identified in N. crassa (11, 125). NC-ras was isolated from an N.
crassa cDNA library using the S. cerevisiae RAS2 gene as the probe (11). NC-ras2 was isolated from an N. crassa genomic library using the mammalian v-H-ras gene as the probe (125).
Although the roles of the N. crassa ras genes are largely un-known, the NC-ras2 gene has been deleted in N. crassa (125).
The NC-ras2 gene is not essential for growth, but mutation confers slow growth and an abnormal morphology during veg-etative culture on solid medium. The growth rate of the mu-tants is about one-tenth that of wild-type cells. The NC-ras2 mutants produce more branching hyphae at peripheral regions of the colony. In addition, the apical compartment in these mutants is shorter and thinner than in the wild type. Because growth rate and morphology depend on hyphal growth, which is limited to the apex of the hyphae in filamentous fungi, the NC-RAS2 protein appears to regulate apical growth of hyphae.
Moreover, the NC-ras2 mutants produce fewer aerial hyphae and conidia than the wild type, suggesting that NC-RAS2 pro-tein also controls formation of aerial hyphae and conidia (125).
The morphological characteristics of the NC-ras2 mutant are very similar to those of a morphological mutation, smco7 (semicolonial-7), which had been mapped on the same linkage group as NC-ras2 (89, 199). In fact, the smco7 mutation is an allele of NC-ras2 (125).
MAPKKK homolog and cAMP-Dependent Protein Kinase-Related Kinase
To isolate N. crassa mutations affecting the regulation of the clock-controlled gene 1 (ccg-1) (173), the nrc-1 and nrc-2 mu-tants were isolated by insertional mutagenesis (136). The ccg-1 gene is one of the earliest genes expressed during conidiation, and its expression is regulated by extracellular glucose levels, circadian rhythm, and light (14, 173, 190). ccg-1 is induced greater than 500-fold in response to glucose limitation (190, 295) and regulated 5- to 10-fold by the circadian clock, with the highest level of expression in the morning, when conidiation occurs (173). ccg-1 expression is also induced by blue light (14).
Because expression of the ccg-1 gene is regulated by three distinct stimuli which induce conidiation, it was anticipated that mutants defective in conidiation could be isolated with ccg-1 as a reporter gene. In fact, in both the nrc-1 and nrc-2
mutants, ccg-1 is constitutively expressed and conidiation is defective (136).
Although exposure to air and glucose limitation are neces-sary for conidiation, the nrc-1 mutant can produce conidio-phores in glucose-rich solid and liquid medium. Even in con-stant darkness, the nrc-1 mutant can differentiate and produce conidia more efficiently than the wild-type strain. Therefore, in the nrc-1 mutant, conidiation occurs constitutively and the asexual developmental program is no longer regulated by glu-cose and light levels, suggesting that the nrc-1 gene is necessary to repress asexual development. In the sexual cycle, the nrc-1 mutant exhibits a female sterile phenotype and fails to produce protoperithecia because of constitutive entry into the conidia-tion program and failure to produce vegetative hyphae from which protoperithecia arise. The nrc-1 mutant also shows an ascospore-lethal phenotype when crossed with a wild-type strain as the male partner. Thus, the nrc-1 gene has roles in ascospore development and asexual development.
The deduced amino acid sequence of the nrc-1 gene reveals homology to the S. cerevisiae STE11 and S. pombe byr2 gene products, both of which are MAPKKKs (136, 232, 294). The carboxy-terminal region containing the protein kinase catalytic domain is the most highly conserved. The Ste11 and byr2 kinases are necessary for pheromone-induced sexual differen-tiation in budding and fission yeasts. Moreover, the S. cerevi-siae MAP kinase signaling pathways in which Ste11 functions are also involved in haploid invasive growth and diploid pseudohyphal growth (see section on S. cerevisiae). Interest-ingly, inactivation of the nrc-1 gene results in enhanced inva-sive growth in N. crassa (136).
On the other hand, a mutation in the nrc-2 gene results in a complete loss of vegetative hyphae (136). The nrc-2 mutant strain produces thin and meandering hyphae that closely re-semble wild-type aerial hyphae. Although some of the mutant hyphae generate chains of proconidia, these conidia do not mature fully and the conidia remain attached together (136).
During sexual development, the nrc-2 mutant fails to produce protoperithecia, resulting in female sterility. This may be at-tributable to derepression of conidial differentiation (136).
The predicted amino acid sequence of NRC2 has the highest level of amino acid sequence identity with putative serine/
threonine protein kinases encoded by the KIN82 and YNR047w genes in S. cerevisiae and the kad5⫹gene in S. pombe (136).
These kinases are closely related to cAMP-dependent protein kinases, but their cellular functions are unknown. Although both nrc-2 and nrc-1 mutant strains were isolated as mutants that fail to repress ccg-1 expression and are unable to regulate conidiation, it is unclear whether the NRC2 kinase acts in the same signaling cascade as the NRC1 MAPKKK.
Conclusions
In summary, a cAMP signaling pathway regulates morphol-ogy, conidiation, mating, and responses to heat shock and oxidative stress in N. crassa. The mcb mutant phenotypes sug-gest that regulation of morphology and conidiation may in-volve integrity of cellular growth polarity and cytoskeletal or-ganization. The similarity in phenotypes between mutations affecting adenylyl cyclase (cr-1), the MAPKKK (nrc-1), and the PKA homolog (nrc-2) suggests that they coordinately regulate development, possibly in two parallel signaling pathways (MAP kinase and cAMP-PKA), as is the case in S. cerevisiae, S.
pombe, C. albicans, U. maydis, and C. neoformans. Further studies are required to establish the relationships among these signaling pathways. In addition to the mutants discussed here, more than 100 mutants of N. crassa exhibiting abnormal
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phology have been isolated (229). N. crassa is genetically trac-table, and the genome sequencing project for this organism is in progress. Thus, N. crassa is an excellent model organism for the study of signaling cascades regulating morphogenesis and differentiation in filamentous fungi.