A catalytic subunit of cyclic AMP-dependent protein kinase, PKAC-1, regulates asexual differentiation in Neurospora crassa

Abstract

A cyclic AMP (cAMP)-dependent protein kinase pathway has been shown to regulate growth, morphogenesis and virulence in filamentous fungi. However, the precise mechanisms of regulation through the pathway remain poorly understood. In Neurospora crassa , the cr-1 adenylate cyclase mutant exhibits colonial growth with short aerial hyphae bearing conidia, and the mcb mutant, a mutant of the regulatory subunit of cAMP-dependent protein kinase (PKA), shows the loss of growth polarity at the restrictive temperature. In the present study, we isolated mutants of the catalytic subunit of the PKA gene pkac-1 through the process of repeat-induced point mutation (RIP). PKA activity of the mutants obtained through RIP was undetectable. The genome sequence predicts two distinct catalytic subunit genes of PKA, named pkac-1 (NCU06240.1, AAF75276) and pkac-2 (NCU00682.1), as is the case in most filamentous fungi. The results suggest that PKAC-1 works as the major PKA in N. crassa. The phenotype of the pkac-1 mutants included colonial growth, short aerial hyphae, premature conidiation on solid medium, inappropriate conidiation in submerged culture, and increased thermotolerance. This phenotype of pkac-1 mutants resembled to that of cr-1 mutants, except that the addition of cAMP did not rescue the abnormal morphology of pkac-1 mutants. The loss of growth polarity at the restrictive temperature in the mcb mutant was suppressed by pkac-1 mutation. These results suggest that the signal transduction pathway mediated by PKAC-1 plays an important role in regulation of aerial hyphae formation, conidiation, and hyphal growth with polarity.

Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

“…However, the different mutants are different from the wt in both microscopic and macroscopic phenotypic features. A characteristic that seems to be a common feature of R mutants of filamentous fungi is a slower radial growth, since besides our own results, this has also been reported for N. crassa, C. lagenarium, A. fumigatus, and A. niger (2,46,51,54). Germination rates of the fungal R mutant strains are lower than those of the wt strains.…”

“…Thus, zygomycetes seem to be the only class of fungi that have more than one isoform for the R subunit (1,2,8,9,11,14,26,46,50,51,52,54). The corresponding mutants with mutations in the genes encoding the PKA regulatory subunit have been obtained from several of these fungi, and there does not seem to be a unique way in how fungi respond to the deletion of the R subunit, since some of them, such as A. niger (46), have the same level of PKA activity as in the wild-type cell, whereas in others, such as C. neoformans or A. fumigatus (11,54), the PKA activity is increased.…”

“…Sporulation or conidiation defects also appear to be common to these mutants in filamentous fungi, such as A. niger, A. fumigatus, or C. lagenarium (46,51,54). Some R subunit mutants also show a loss of growth polarity, as in A. niger, C. albicans, and N. crassa (2,8,46). In plant-and human-pathogenic fungi such as U. maydis, C. albicans, and C. neoformans, R subunits are also required for filamentous growth (8,11,12,14).…”

Role for Protein Kinase A in the Neurospora Circadian Clock by Regulating White Collar-Independent frequency Transcription through Phosphorylation of RCM-1

“…In Neurospora, pkac-1 (NCU00682) and pkac-2 (NCU06240) encode the PKA catalytic subunits, and PKAC-1 is the major catalytic subunit (32,42). To determine whether the WC-independent frq transcription in mcb mutant is due to the increase of PKA activity but not due to a role of MCB in another process, we introduced a QA-inducible Myc-His-tagged PKAC-1 construct into the wild type or a pkac-1 KO , wc-1 KO , or wc-2 KO single mutant strain.…”

A High-Density Single Nucleotide Polymorphism Map for Neurospora crassa

“…Asexual development in Neurospora and other filamentous fungi is a complex and incompletely understood process, integrating signals from the circadian clock, metabolic state, and various types of external and internal stress (Turian and Bianchi 1971). This developmental process involves multiple intracellular signaling events including MAPK (Pandey et al 2004), cAMP (Banno et al 2005), and Ras-dependent pathways (Belden et al 2007). We identified a binuclear zincfinger transcription factor as a downstream regulator of conidiation (similar to another conidiation gene, fluffy; Bailey and Ebbole 1998), which is regulated at the transcriptional level in a time-of-day and Ras-dependent manner.…”