Abstract
Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia) germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.
Highlights
Since the discovery of the first heterotrimeric G protein in filamentous fungi in the 1990’s [1], G proteins have been found to play key roles in diverse fungal processes ranging from asexual and sexual development to pathogenicity of animal and phytopathogenic fungi
In order to determine whether germination defects contribute to the overall reduction in colony size, we investigated early events during germination of conidia in Dric8 mutants and in strains lacking G protein subunit genes
We report evidence showing that in N. crassa G protein signaling regulates both conidial development and germination, and this is regulated through the non-receptor guanine nucleotide exchange factors (GEFs) RIC8
Summary
Since the discovery of the first heterotrimeric G protein in filamentous fungi in the 1990’s [1], G proteins have been found to play key roles in diverse fungal processes ranging from asexual and sexual development to pathogenicity of animal and phytopathogenic fungi (reviewed in Li et al, 2007). Most fungi possess three Ga subunits and a single Gb and Gc protein, allowing for the assembly of three different heterotrimers. These three Ga subunits can act independently to regulate separate pathways, leading to differing phenotypes for single Ga mutants. All three G proteins are thought to act together to regulate certain processes, as mutants lacking GNA-1 and GNA-3 or all three Ga subunits are severely impaired in growth on solid medium, inappropriately conidiate in submerged liquid culture and do not produce female reproductive structures [6]
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