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

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