Shared and Independent Roles for a Gα _i Protein and Adenylyl Cyclase in Regulating Development and Stress Responses in Neurospora crassa

Abstract

Growth and development are regulated using cyclic AMP (cAMP)-dependent and -independent pathways in Neurospora crassa. The cr-1 adenylyl cyclase mutant lacks detectable cAMP and exhibits numerous defects, including colonial growth habit, short aerial hyphae, premature conidiation on plates, inappropriate conidiation in submerged culture, and increased thermotolerance. Evidence suggests that the heterotrimeric G␣ protein GNA-1 is a direct positive regulator of adenylyl cyclase. ⌬gna-1 strains are female-sterile, and ⌬gna-1 strains have reduced apical extension rates on normal and hyperosmotic medium, greater resistance to oxidative and heat stress, and stunted aerial hyphae compared to the wild-type strain. In this study, a ⌬gna-1 cr-1 double mutant was analyzed to differentiate cAMP-dependent and -independent signaling pathways regulated by GNA-1. ⌬gna-1 cr-1 mutants have severely restricted colonial growth and do not produce aerial hyphae on plates or in standing liquid cultures. Addition of cAMP to plates or standing liquid cultures rescues cr-1, but not ⌬gna-1 cr-1, defects, which is consistent with previous results demonstrating that ⌬gna-1 mutants do not respond to exogenous cAMP. The females of all strains carrying the ⌬gna-1 mutation are sterile; however, unlike cr-1 and ⌬gna-1 strains, the ⌬gna-1 cr-1 mutant does not produce protoperithecia. The ⌬gna-1 and cr-1 mutations were synergistic with respect to inappropriate conidiation during growth in submerged culture. Thermotolerance followed the order wild type < ⌬gna-1 < cr-1 ‫؍‬ ⌬gna-1 cr-1, consistent with a cAMP-dependent process. Taken together, the results suggest that in general, GNA-1 and CR-1 regulate N. crassa growth and development using parallel pathways, while thermotolerance is largely dependent on cAMP.Development in fungal systems frequently occurs in response to specific environmental cues and stressors. In the presence of abundant nutrients, the filamentous fungus Neurospora crassa extends hyphae that elongate and fuse to form the multicellular mycelium (for a review, see reference 51). Desiccation or nutrient deprivation causes the mycelium to differentiate aerial hyphae that give rise to conidiophores and multinucleate asexual spores, macroconidia (referred to here as conidia). Elaboration of conidiophores and production of conidia require an air-water interface; however, submerged cultures can be induced to undergo conidiation by carbon or nitrogen starvation or exposure to high temperatures (7,18,43,55). Nitrogen limitation initiates the sexual cycle by stimulating production of female reproductive structures, or protoperithecia (reviewed in reference 44). Fertilization by a conidium or hypha of the opposite mating type results in formation of the fertilized structure (perithecium), within which sexual spores (ascospores) develop. Mature ascospores are subsequently ejected from the perithecium in the direction of blue light.Heterotrimeric GTP-binding proteins, consisting of ␣, ␤, and ␥ subunits, transduce various environmental signals ...

The genome sequence of the filamentous fungus Neurospora crassa

“…Components of the macroconidiation pathway have been identified in both Neurospora and the filamentous fungus A. nidulans, and known upstream signalling proteins seem to be conserved in both species 58 . In contrast, there is little conservation of downstream components between the two fungi.…”

The GanB Gα-Protein Negatively Regulates Asexual Sporulation and Plays a Positive Role in Conidial Germination in Aspergillus nidulansSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession no. AF198116.

“…GanB has conserved domains that are considered mutants from VER7 and six from RMS011 were isolated to be directly involved in interaction with the guanine and the deletion of ganB was checked by genomic Southnucleotide found in all G␣-proteins (Simon et al 1991; ern analysis. The fact that the transcripts of the ganB Skiba et al 1996;Bohm et al 1997), as well as the myrisgene were not detected by Northern analysis of these toylation site in the N terminus (Buss et al 1987). The mutants and sequencing the flanking region of inserted amino acid sequence of GanB shows 75-90% identity argB showed that the deletion was successful (data not to the amino acids of GpaB (Liebmann et al 2003) and shown).…”

“…The cAMP/PKA system is also involved downstream of group III G␣-protein signaling GTP bound and the free ␤␥-complex may interact with many types of downstream effectors such as enzymes in some processes such as cell proliferation, development, stress response, mating, and virulence (Kü bler (adenylate cyclases, phosphodiesterases, phospholipases, etc.) and ion channels, which in turn initiate signals via et al 1997;Kruger et al 1998;Alspaugh et al 2002;Ivey et al 2002;Liebmann et al 2003). second messengers such as cyclic AMP, diacylglycerol, inositol triphosphate, and/or critical ions in the signal A dominant activating mutation in the fadA gene, encoding a G␣-subunit of the filamentous fungus Aspertransduction pathway (Neer 1995;Bohm et al 1997;Neves et al 2002).…”

“…( Figure 1) play important roles in the GTPase activity that hydrolyzes GTP bound to G␣ protein to GDP (Simon Northern blot analysis showed that the ganB transcript et al 1991;Skiba et al 1996;Bohm et al 1997). Mutations in the conformational change that accompanies guaat either of these sites largely abolish the hydrolytic nine nucleotide exchange (Kurjan 1992; Bohm et al activity of intrinsic GTPase and thus stabilize the active, 1997).…”

A Mitogen-Activated Protein Kinase Pathway Essential for Mating and Contributing to Vegetative Growth in Neurospora crassa

“…To examine the effect of downstream flanking region was ligated into the BamHI site cAMP on production of aerial hyphae, 2 mm cAMP (Sigma, St. of pBP27. The 2-kb XhoI-XhoI upstream flanking region of Louis) was added to standing VM liquid cultures as described mak-2 was PCR amplified from plasmid pBP30 with T7 and previously Ivey et al 2002). To test for MAK1 (containing an XhoI site) primers and ligated into the conidiation in submerged liquid cultures, conidia were inocuXhoI site to produce pBP-KOMAK2.…”