Shared and independent roles for a Galpha(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 Galpha protein GNA-1 is a direct positive regulator of adenylyl cyclase. deltagna-1 strains are female-sterile, and deltagna-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 deltagna-1 cr-1 double mutant was analyzed to differentiate cAMP-dependent and -independent signaling pathways regulated by GNA-1. deltagna-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 deltagna-1 cr-1, defects, which is consistent with previous results demonstrating that deltagna-1 mutants do not respond to exogenous cAMP. The females of all strains carrying the deltagna-1 mutation are sterile; however, unlike cr-1 and deltagna-1 strains, the deltagna-1 cr-1 mutant does not produce protoperithecia. The deltagna-1 and cr-1 mutations were synergistic with respect to inappropriate conidiation during growth in submerged culture. Thermotolerance followed the order wild type < deltaga-1 < cr-1 = deltagna-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.

FIG. 1.

Levels of Gα, Gβ, and adenylyl cyclase proteins. (A) Levels of GNA-1, GNA-2, and GNB-1. Samples containing 30 μg of total membrane protein from strains with the indicated genotypes were subjected to Western blot analysis using specific antisera. WT, wild type. (B) Levels of CR-1. Samples containing 30 μg of total membrane protein were analyzed by Western blot analysis using a CR-1 antiserum. The position of full-length CR-1 in the wild-type (WT) and Δgna-1 mutant preparations is indicated to the right of the blot. The migration position of the amino-terminal 56-kDa CR-1 protein fragment (IB) used as an antigen is indicated. The asterisk indicates the 150-kDa species found in the cr-1 strain preparation.

FIG. 2.

Female fertility. Strains with the indicated genotypes were cultured on SCM for 7 days prior to fertilization using wild-type (WT) strain 74-OR23-1A or Sta73a conidia. Perithecia were photographed 6 days after fertilization. Perithecia (in cr-1 and wild-type strains) and aberrant perithecia (in a Δgna-1 strain) are indicated by arrows.

FIG. 3.

Vegetative phenotypes in the presence of an air-water interface. (A) Growth of colonies on solid medium. Strains with the indicated genotypes were grown on VM plates at room temperature under constant light and photographed when the Δgna-1 colony had reached the edge of the plate (approximately 36 h). Wild-type colonies are approximately two times larger than Δgna-1 colonies under these conditions (data not shown). (B) Standing liquid cultures. Strains were inoculated into 2-ml portions of liquid VM and incubated in a stationary position first for 3 days at 30°C in the dark and then for 4 days at room temperature in constant light. The presence (+) or absence (−) of 2.0 mM cAMP is indicated below the test tubes.

FIG. 4.

Thermotolerance assays. The viability of 3-h-old germlings was determined after exposure to a lethal temperature (52°C) with (induced thermotolerance) and without (uninduced thermotolerance) prior exposure to a sublethal heat shock temperature (45°C). The error bars show standard errors of the means.

FIG. 5.

Conidiophore development and conidiation-specific gene expression in submerged cultures. (A) Conidiophore development. Conidia were inoculated into liquid VM at either 1 × 10⁶ or 3 × 10⁶ cells/ml, incubated with shaking in the dark at 30°C for 16 h, and then photographed. Arrows indicate conidiophores. (B) Correlation between conidiophore development and conidiation-specific gene expression. Total RNA (20 μg) isolated from strains with the indicated genotypes was analyzed by Northern blot analysis using a ³²P-labeled con-10 cDNA as a conidiation-specific probe; the cox-5 gene was used as a probe to control for RNA loading and transfer.