Genetic and Physical Interactions between Gα Subunits and Components of the Gβγ Dimer of Heterotrimeric G Proteins in Neurospora crassa

Won, Susan; Michkov, Alexander V.; Kryštofová, Svetlana; Garud, Amruta; Borkovich, Katherine A.

doi:10.1128/ec.00151-12

Cited by 14 publications

(24 citation statements)

References 62 publications

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“…G-proteins and coupled receptors ( gpr )—which are known to be critical regulators of fungal responses to carbon and nitrogen nutrition and major regulators of N. crassa development (38–41)—were differentially expressed. Genes gna-1 and gpr-4 —which regulate the responses to carbon sources (42)—were highly coordinately downregulated in cultures on BM but showed contrasting results in cultures on MSM, upon which gna-1 was upregulated (Fig.…”

Section: Resultsmentioning

confidence: 99%

Metabolism and Development during Conidial Germination in Response to a Carbon-Nitrogen-Rich Synthetic or a Natural Source of Nutrition in Neurospora crassa

Wang

Miguel-Rojas

López-Giráldez

et al. 2019

mBio

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Fungal spores germinate and undergo vegetative growth, leading to either asexual or sexual reproductive dispersal. Previous research has indicated that among developmental regulatory genes, expression is conserved across nutritional environments, whereas pathways for carbon and nitrogen metabolism appear highly responsive—perhaps to accommodate differential nutritive processing. To comprehensively investigate conidial germination and the adaptive life history decision-making underlying these two modes of reproduction, we profiled transcription ofNeurospora crassagerminating on two media: synthetic Bird medium, designed to promote asexual reproduction; and a natural maple sap medium, on which both asexual reproduction and sexual reproduction manifest. A later start to germination but faster development was observed on synthetic medium. Metabolic genes exhibited altered expression in response to nutrients—at least 34% of the genes in the genome were significantly downregulated during the first two stages of conidial germination on synthetic medium. Knockouts of genes exhibiting differential expression across development altered germination and growth rates, as well as in one case causing abnormal germination. A consensus Bayesian network of these genes indicated especially tight integration of environmental sensing, asexual and sexual development, and nitrogen metabolism on a natural medium, suggesting that in natural environments, a more dynamic and tentative balance of asexual and sexual development may be typical ofN. crassacolonies.IMPORTANCEOne of the most remarkable successes of life is its ability to flourish in response to temporally and spatially varying environments. Fungi occupy diverse ecosystems, and their sensitivity to these environmental changes often drives major fungal life history decisions, including the major switch from vegetative growth to asexual or sexual reproduction. Spore germination comprises the first and simplest stage of vegetative growth. We examined the dependence of this early life history on the nutritional environment using genome-wide transcriptomics. We demonstrated that for developmental regulatory genes, expression was generally conserved across nutritional environments, whereas metabolic gene expression was highly labile. The level of activation of developmental genes did depend on current nutrient conditions, as did the modularity of metabolic and developmental response network interactions. This knowledge is critical to the development of future technologies that could manipulate fungal growth for medical, agricultural, or industrial purposes.

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Section: Resultsmentioning

confidence: 99%

Metabolism and Development during Conidial Germination in Response to a Carbon-Nitrogen-Rich Synthetic or a Natural Source of Nutrition in Neurospora crassa

Wang

Miguel-Rojas

López-Giráldez

et al. 2019

mBio

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“…Membrane binding of Ste5 likely concentrates the bound MAP kinases spatially, promoting amplification of the signal [59] , [60] . In N. crassa , how HAM-5 and MAK-2 are recruited to the membrane is still elusive, but is dissimilar from Ste5 since the Gβγ ortholog in N. crassa is not involved in germling fusion [61] , [62] . Other upstream factors shared between S. cerevisiae and N. crassa might regulate the activation of the MAPK pathway.…”

Section: Discussionmentioning

confidence: 99%

HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

et al. 2014

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Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of fungal communication.

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“…Vectors containing predicted GTPase-deficient, constitutively activating mutations gna-1 Q204L (pSVK51), gna-2 Q205L (pSVK52), and gna-3 Q208L (pSVK53) were previously made using site-directed mutagenesis [55]. Electroporation of N .…”

Section: Methodsmentioning

confidence: 99%

Genetic relationships between the RACK1 homolog cpc-2 and heterotrimeric G protein subunit genes in Neurospora crassa

et al. 2019

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Receptor for Activated C Kinase-1 (RACK1) is a multifunctional eukaryotic scaffolding protein with a seven WD repeat structure. Among their many cellular roles, RACK1 homologs have been shown to serve as alternative Gβ subunits during heterotrimeric G protein signaling in many systems. We investigated genetic interactions between the RACK1 homolog cpc-2, the previously characterized Gβ subunit gnb-1 and other G protein signaling components in the multicellular filamentous fungus Neurospora crassa. Results from cell fractionation studies and from fluorescent microscopy of a strain expressing a CPC-2-GFP fusion protein revealed that CPC-2 is a cytoplasmic protein. Genetic epistasis experiments between cpc-2, the three Gα genes (gna-1, gna-2 and gna-3) and gnb-1 demonstrated that cpc-2 is epistatic to gna-2 with regards to basal hyphae growth rate and aerial hyphae height, while deletion of cpc-2 mitigates the increased macroconidiation on solid medium observed in Δgnb-1 mutants. Δcpc-2 mutants inappropriately produce conidiophores during growth in submerged culture and mutational activation of gna-3 alleviates this defect. Δcpc-2 mutants are female-sterile and fertility could not be restored by mutational activation of any of the three Gα genes. With the exception of macroconidiation on solid medium, double mutants lacking cpc-2 and gnb-1 exhibited more severe defects for all phenotypic traits, supporting a largely synergistic relationship between GNB-1 and CPC-2 in N. crassa.

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Genetic and Physical Interactions between Gα Subunits and Components of the Gβγ Dimer of Heterotrimeric G Proteins in Neurospora crassa

Cited by 14 publications

References 62 publications

Metabolism and Development during Conidial Germination in Response to a Carbon-Nitrogen-Rich Synthetic or a Natural Source of Nutrition in Neurospora crassa

Metabolism and Development during Conidial Germination in Response to a Carbon-Nitrogen-Rich Synthetic or a Natural Source of Nutrition in Neurospora crassa

HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

Genetic relationships between the RACK1 homolog cpc-2 and heterotrimeric G protein subunit genes in Neurospora crassa

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