title: The guanine nucleotide exchange factor RIC8 regulates conidial germination through Gα proteins in Neurospora crassa.
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.
authors.affiliation: Department of Plant Pathology and Microbiology and Institute for Integrative Genome Biology, University of California Riverside, Riverside, California, USA.
authors.forename: Carla J
authors.initials: CJ
authors.lastname: Eaton
authors.forename: Ilva E
authors.initials: IE
authors.lastname: Cabrera
authors.forename: Jacqueline A
authors.initials: JA
authors.lastname: Servin
authors.forename: Sara J
authors.initials: SJ
authors.lastname: Wright
authors.forename: Murray P
authors.initials: MP
authors.lastname: Cox
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Borkovich
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D022081:Cell Surface Extensions; D017124:Conserved Sequence; D005656:Fungal Proteins; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000096:biosynthesis; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015398:Signal Transduction; D013172:Spores, Fungal / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D059008:Time-Lapse Imaging; D014617:Vacuoles / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D005656:Fungal Proteins; D044385:GTP-Binding Protein alpha Subunits; D020662:Guanine Nucleotide Exchange Factors; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein