The G alpha i homologue gna-1 controls multiple differentiation pathways in Neurospora crassa.

Abstract

Heterotrimeric G proteins are components of principal signaling pathways in eukaryotes. In higher organisms, alpha subunits of G proteins have been divided into four families, Gi, Gs, Gq, and G12. We previously identified a G alpha i homologue gna-1 in the filamentous fungus Neurospora crassa. Now we report that deletion of gna-1 leads to multiple phenotypes during the vegetative and sexual cycles in N. crassa. On solid medium, delta gna-1 strains have a slower rate of hyphal apical extension than wild type, a rate that is more pronounced under hyperosmotic conditions or in the presence of a cellophane overlay. delta gna-1 mutants accumulate less mass than wild-type strains, and their mass accumulation is not affected in the same way by exposure to light. delta gna-1 strains are defective in macroconidiation, possessing aerial hyphae that are shorter, contain abnormal swellings, and differentiate adherent macroconidia. During the sexual cycle, delta gna-1 strains are fertile as males. However, the mutants are female-sterile, producing small, aberrant female reproductive structures. After fertilization, delta gna-1 female structures do not enlarge and develop normally, and no sexual spores are produced. Thus, mutation of gna-1 results in sex-specific loss of fertility.

Roles for Receptors, Pheromones, G Proteins, and Mating Type Genes During Sexual Reproduction in Neurospora crassa

“…Finally, pHK13 (containing the 59 flank) was linearized using BamHI and SpeI and the hph cassette and the 39 flank were inserted, yielding pHK15. pHK15 (2 mg) was linearized with SpeI and used to electroporate wild-type N. crassa strain 74A as described (Ivey et al 1996). Transformants were isolated by plating on FGS plates containing hygromycin.…”

“…For the Dgna-1 strain, a Dpre-1 his-3 mat a female was crossed with a Dgna-1 mat A male, and histidine-requiring hygromycinresistant progeny were selected. The presence of the Dpre-1 and Dgna-1 mutations was verified by Southern analysis as described previously (Ivey et al 1996;Kim and Borkovich 2004). A Dpre-1 Dgna-1 his-3 mat A strain (P1Ga1h3A; Table 1) was then electroporated with pHK46.…”

The Response Regulator RRG-1 Functions Upstream of a Mitogen-activated Protein Kinase Pathway Impacting Asexual Development, Female Fertility, Osmotic Stress, and Fungicide Resistance inNeurospora crassa

“…Homologous recombination events were identified by Southern analysis (Ivey et al, 1996) of genomic DNA from the transformants after digestion with HindIII (Supplemental Figure 1B; data not shown). A 4-kb fragment containing the hph cassette and 3Ј flank from the ⌬rrg-1 cassette (Supplemental Figure 1A) was used as the probe.…”

“…Growth under hyperosmotic conditions was assayed as described previously (Ivey et al, 1996), with details in the figure legend. Photomicroscopy was performed using a SZX9 stereomicroscope or a BX41 compound microscope with a C-4040 digital camera (Olympus, Lake Success, NY).…”

“…To obtain the ⌬rrg-1 mutation in both mating types, the gene replacement construct was transformed into wild-type strains 74A and 74a and histidine auxotrophic strain his3a (Krystofova and Borkovich, 2005). Plasmid pSP1 was linearized by XhoI digestion, and 1 g was used to electroporate 10-d-old macroconidia as described previously (Vann, 1995;Ivey et al, 1996). Transformants were suspended in regeneration agar (Case et al, 1979) and then plated on FIGS medium containing 150 or 200 g/ml hygromycin B.…”