scholarly journals Molecular Mechanisms Controlling the Disease Cycle in the Vascular Pathogen Verticillium dahliae Characterized Through Forward Genetics and Transcriptomics

2020 ◽  
Vol 33 (6) ◽  
pp. 825-841
Author(s):  
Jorge L. Sarmiento-Villamil ◽  
Nicolás E. García-Pedrajas ◽  
M. Carmen Cañizares ◽  
María D. García-Pedrajas
Keyword(s):  
G Protein ◽  
Verticillium Dahliae ◽  
Forward Genetics ◽  
Phenotypic Characterization ◽  
Insertion Mutant ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Wild Type ◽  
Disease Cycle ◽  
Dna Insertion

The soil-borne pathogen Verticillium dahliae has a worldwide distribution and a plethora of hosts of agronomic value. Molecular analysis of virulence processes can identify targets for disease control. In this work, we compared the global gene transcription profile of random T-DNA insertion mutant strain D-10-8F, which exhibits reduced virulence and alterations in microsclerotium formation and polar growth, with that of the wild-type strain. Three genes identified as differentially expressed were selected for functional characterization. To produce deletion mutants, we developed an updated version of one-step construction of Agrobacterium-recombination-ready plasmids (OSCAR) that included the negative selection marker HSVtk (herpes simplex virus thymidine kinase gene) to prevent ectopic integration of the deletion constructs. Deletion of VdRGS1 (VDAG_00683), encoding a regulator of G protein signaling (RGS) protein and highly upregulated in the wild type versus D-10-8F, resulted in phenotypic alterations in development and virulence that were indistinguishable from those of the random T-DNA insertion mutant. In contrast, deletion of the other two genes selected, vrg1 (VDAG_07039) and vvs1 (VDAG_01858), showed that they do not play major roles in morphogenesis or virulence in V. dahliae. Taken together the results presented here on the transcriptomic analysis and phenotypic characterization of D-10-8F and ∆VdRGS1 strains provide evidence that variations in G protein signaling control the progression of the disease cycle in V. dahliae. We propose that G protein–mediated signals induce the expression of multiple virulence factors during biotrophic growth, whereas massive production of microsclerotia at late stages of infection requires repression of G protein signaling via upregulation of VdRGS1 activity.

scholarly journals A Regulator of G Protein Signaling-containing Kinase Is Important for Chemotaxis and Multicellular Development inDictyostelium

2003 ◽  
Vol 14 (4) ◽  
pp. 1727-1743 ◽  
Author(s):  
Binggang Sun ◽  
Richard A. Firtel
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Kinase Activity ◽  
Site Directed Mutagenesis ◽  
Membrane Localization ◽  
Pleckstrin Homology Domain ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Wild Type ◽  
Gene Encoding

We have identified a gene encoding RGS domain-containing protein kinase (RCK1), a novel regulator of G protein signaling domain-containing protein kinase. RCK1 mutant strains exhibit strong aggregation and chemotaxis defects. rck1 null cells chemotax ∼50% faster than wild-type cells, suggesting RCK1 plays a negative regulatory role in chemotaxis. Consistent with this finding, overexpression of wild-type RCK1 reduces chemotaxis speed by ∼40%. On cAMP stimulation, RCK1 transiently translocates to the membrane/cortex region with membrane localization peaking at ∼10 s, similar to the kinetics of membrane localization of the pleckstrin homology domain-containing proteins CRAC, Akt/PKB, and PhdA. RCK1 kinase activity also increases dramatically. The RCK1 kinase activity does not rapidly adapt, but decreases after the cAMP stimulus is removed. This is particularly novel considering that most other chemoattractant-activated kinases (e.g., Akt/PKB, ERK1, ERK2, and PAKa) rapidly adapt after activation. Using site-directed mutagenesis, we further show that both the RGS and kinase domains are required for RCK1 function and that RCK1 kinase activity is required for the delocalization of RCK1 from the plasma membrane. Genetic evidence suggests RCK1 function lies downstream from Gα2, the heterotrimeric G protein that couples to the cAMP chemoattractant receptors. We suggest that RCK1 might be part of an adaptation pathway that regulates aspects of chemotaxis in Dictyostelium.

scholarly journals Heterotrimeric G-Protein Signaling Is Required for Cellulose Degradation in Neurospora crassa

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Logan A. Collier ◽  
Arit Ghosh ◽  
Katherine A. Borkovich
Keyword(s):  
G Protein ◽  
Cellulose Degradation ◽  
Cellulase Activity ◽  
Cellulase Production ◽  
Camp Signaling ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Wild Type ◽  
Posttranscriptional Control ◽  
Heterotrimeric G Protein Signaling

ABSTRACT The filamentous fungus Neurospora crassa decomposes lignocellulosic biomass to generate soluble sugars as carbon sources. In this study, we investigated a role for heterotrimeric G-protein signaling in cellulose degradation. Loss of the Gα subunit genes gna-1 and gna-3, the Gβ subunit genes gnb-1 and cpc-2, the Gγ gene gng-1, or the gene for downstream effector adenylyl cyclase (cr-1) resulted in loss of detectable cellulase activity. This defect was also observed in strains expressing a constitutively active version of gna-3 (gna-3Q208L). We found that GNA-1 levels are greatly reduced in Δgna-3, Δgnb-1, and Δgng-1 strains, likely contributing to cellulase defects in these genetic backgrounds. The observation that gna-3Q208L Δgnb-1 strains exhibit cellulase activity, despite greatly reduced levels of GNA-1 protein, is consistent with positive control of cellulase production by GNA-3 that is manifested in the absence of gnb-1. Expression patterns for five cellulase genes showed that Δgna-1, Δgnb-1, and Δgna-3 mutants produce less cellulase mRNA than the wild type, consistent with transcriptional regulation. Δcpc-2 mutants had wild-type levels of cellulase transcripts, suggesting posttranscriptional control. In contrast, results for Δcr-1 mutants support both transcriptional and posttranscriptional control of cellulase activity by cAMP signaling. Cellulase activity defects in Δgna-3 mutants were fully remediated by cAMP supplementation, consistent with GNA-3 operating upstream of cAMP signaling. In contrast, cAMP addition only partially corrected cellulase activity defects in Δgna-1 and Δgnb-1 mutants, suggesting participation of GNA-1 and GNB-1 in additional cAMP-independent pathways that control cellulase activity. IMPORTANCE Filamentous fungi are critical for the recycling of plant litter in the biosphere by degrading lignocellulosic biomass into simpler compounds for metabolism. Both saprophytic and pathogenic fungi utilize plant cell wall-degrading enzymes to liberate carbon for metabolism. Several studies have demonstrated a role for cellulase enzymes during infection of economically relevant crops by fungal pathogens. Especially in developing countries, severe plant disease means loss of entire crops, sometimes leading to starvation. In this study, we demonstrate that G-protein signaling is a key component of cellulase production. Therefore, understanding the role of G-protein signaling in the regulation of the unique metabolism of cellulose by these organisms can inform innovations in strain engineering of industrially relevant species for biofuel production and in combatting food shortages caused by plant pathogens.

scholarly journals Identification of Critical Residues Involved in Ligand Binding and G Protein Signaling in Human Somatostatin Receptor Subtype 2

Endocrinology ◽  
2012 ◽  
Vol 153 (6) ◽  
pp. 2747-2755 ◽  
Author(s):  
Jesse J. Parry ◽  
Ronald Chen ◽  
Rebecca Andrews ◽  
Kimberly A. Lears ◽  
Buck E. Rogers
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Calcium Release ◽  
Expression Profiles ◽  
Somatostatin Receptor ◽  
Receptor Subtype ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Wild Type ◽  
Somatostatin Receptor Subtype 2

G protein signaling through human somatostatin receptor subtype 2 (SSTR2) is well known, but the amino acids involved in stimulation of intracellular responses upon ligand binding have not been characterized. We constructed a series of point mutants in SSTR2 at amino acid positions 89, 139, and 140 in attempts to disrupt G protein signaling upon ligand binding. The aspartic acid changes at position 89 to either Ala, Leu, or Arg generated mutant receptors with varying expression profiles and a complete inability to bind somatostatin-14 (SST). Mutations to Asp 139 and Arg 140 also led to varying expression profiles with some mutants maintaining their affinity for SST. Mutation of Arg 140 to Ala resulted in a mutated receptor that had a Bmax and dissociation constant (Kd) similar to wild-type receptor but was still coupled to the G protein as determined in both a cAMP assay and a calcium-release assay. In contrast, mutation of Asp 139 to Asn resulted in a mutated receptor with Bmax and Kd values that were similar to wild type but was uncoupled from G protein-mediated cAMP signaling, but not calcium release. Thus, we identified mutations in SSTR2 that result in either receptor expression levels that are similar to wild type but is completely ablated for ligand binding or a receptor that maintains affinity for SST and is uncoupled from G protein-mediated cAMP signaling.

scholarly journals Up-regulation of Endogenous RGS2 Mediates Cross-desensitization between Gs and Gq Signaling in Osteoblasts

2006 ◽  
Vol 281 (43) ◽  
pp. 32684-32693 ◽  
Author(s):  
Anju Anne Roy ◽  
Caroline Nunn ◽  
Hong Ming ◽  
Min-Xu Zou ◽  
Josef Penninger ◽  
...  
Keyword(s):  
G Protein ◽  
Calcium Release ◽  
Inositol Phosphates ◽  
Primary Cultures ◽  
G Protein Signaling ◽  
Rgs Proteins ◽  
Camp Accumulation ◽  
Protein Signaling ◽  
Wild Type

Regulator of G protein signaling (RGS) proteins limit G protein signals. In this study, we investigated the role of RGS2 in the control of G protein signaling cascades in osteoblasts, the cells responsible for bone formation. Expression of RGS2 was up-regulated in primary cultures of mouse calvarial osteoblasts by parathyroid hormone-related peptide (PTHrP)-(1-34), which stimulates Gs signaling. RGS2 was also up-regulated by extracellular ATP, which selectively activates Gq, as well as by forskolin and phorbol myristate acetate, which activate targets downstream of Gs and Gq, respectively. To assess the role of endogenous RGS2, we characterized Gs and Gq signaling in osteoblasts derived from wild type and rgs2-/- mice. Under control conditions, nucleotide-stimulated calcium release, endothelin-stimulated accumulation of inositol phosphates, and PTHrP-stimulated cAMP accumulation were equivalent in osteoblasts isolated from wild type and rgs2-/- mice. Thus, basal levels of endogenous RGS2 do not appear to regulate Gs or Gq signaling in osteoblasts. Interestingly, forskolin treatment of wild type but not rgs2-/- osteoblasts suppressed both endothelin-stimulated accumulation of inositol phosphates and nucleotide-stimulated calcium release, indicating that up-regulation of RGS2 by Gs signaling desensitizes Gq signals. Furthermore, pretreatment with ATP suppressed PTHrP-dependent cAMP accumulation in wild type but not rgs2-/- osteoblasts, implying that up-regulation of RGS2 by Gq signaling desensitizes Gs signals. Our findings demonstrate that endogenously expressed RGS2 can limit Gs signaling. Moreover, up-regulation of RGS2 contributes to cross-desensitization of Gs- and Gq-coupled signals.

scholarly journals Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins

2013 ◽  
Vol 289 (3) ◽  
pp. 1580-1591 ◽  
Author(s):  
Buxin Chen ◽  
David P. Siderovski ◽  
Richard R. Neubig ◽  
Mark A. Lawson ◽  
JoAnn Trejo
Keyword(s):  
G Protein ◽  
Protein Complex ◽  
Adaptor Protein ◽  
Surface Expression ◽  
G Protein Signaling ◽  
Rgs Proteins ◽  
Protein Signaling ◽  
Wild Type ◽  
Protease Activated Receptor 1 ◽  
G Protein Coupled

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of “regulator of G protein signaling” (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 420AKKAA424 mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

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