scholarly journals G-protein subunit Gai in mitochondria, MrGPA1, affects conidiation, stress resistance, and virulence of entomopathogenic fungus Metarhizium robertsii

2020 ◽  
Author(s):  
Youmin Tong ◽  
Hao Wu ◽  
Zhenbang Liu ◽  
Zhangxun Wang ◽  
bo huang
Keyword(s):  
Cell Wall ◽  
Thermal Stress ◽  
Antioxidant Capacity ◽  
G Protein ◽  
Uv Irradiation ◽  
Stress Resistance ◽  
Galleria Mellonella ◽  
Cell Wall Integrity ◽  
Protein Subunit ◽  
Metarhizium Robertsii

Abstract G proteins are critical modulators or transducers in various transmembrane signaling systems. They play key roles in numerous biological processes in fungi, including vegetative growth, development of infection-related structures, asexual sporulation, and virulence. However, their function in entomopathogenic fungi remains unclear. Here, we characterized the roles of MrGPA1, a G-protein subunit Gα i , in conidiation, stress resistance, and virulence in Metarhizium robertsii . MrGPA1 was localized in the mitochondria. MrGpa1 deletion resulted in a significant reduction (47%) in the conidiation capacity, and reduced expression of several key conidiation-related genes, including fluG , flbD , brlA , wetA , phiA , and stuA . Further, MrGpa1 disruption resulted in decreased fungal sensitivity to UV irradiation and thermal stress, as determined based on conidial germination of ∆MrGpa1 and wild-type strains. Chemical stress analysis indicated that MrGpa1 contributes to fungal antioxidant capacity and cell wall integrity, but is not involved in antifungal ability and osmotic stress. Importantly, insect bioassays involving (topical inoculation and injection) of Galleria mellonella larvae revealed decreased virulence of ∆MrGpa1 strain after cuticle infection. This was accompanied by decreased rates of appressorium formation and reduced expression of several cuticle penetration-related genes. These observations suggest that MrGpa1 contributes to the regulation of conidiation, UV irradiation, thermal stress response, antioxidant capacity and cell wall integrity in M. robertsii . This gene is also involved in insect cuticle penetration during infection. These findings raise the possibility of designing powerful strategies for genetic improvement of M. robertsii conidiation capacity and virulence for killing pests.

scholarly journals Aspergillus fumigatus G-Protein Coupled Receptors GprM and GprJ Are Important for the Regulation of the Cell Wall Integrity Pathway, Secondary Metabolite Production, and Virulence

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Aílton Pereira da Costa Filho ◽  
Guilherme Thomaz Pereira Brancini ◽  
Patrícia Alves de Castro ◽  
Clara Valero ◽  
Jaire Alves Ferreira Filho ◽  
...  
Keyword(s):  
Cell Wall ◽  
G Protein ◽  
Galleria Mellonella ◽  
G Protein Coupled Receptors ◽  
Cell Wall Integrity ◽  
Fungal Development ◽  
Content Type ◽  
Cell Wall Integrity Pathway ◽  
Mycotoxin Biosynthesis ◽  
G Protein Coupled

ABSTRACT G-protein coupled receptors (GPCRs) are extracellular signaling receptors that sense environmental cues. Fungi sense their environment primarily through GPCR-mediated signaling pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. Aspergillus fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that present a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate compared to the wild-type (WT) strain and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the ΔgprM and ΔgprJ strains and decreased in the overexpression mutants compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. Transcriptome sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the ΔgprM and ΔgprJ strains, at least partially through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signaling. Finally, we show that the ΔgprM and ΔgprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis. IMPORTANCE A. fumigatus is the main etiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immunocompromised humans. Withstanding the host environment is essential for A. fumigatus virulence, and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin, and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella. This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence.

scholarly journals Aspergillus fumigatus G-protein coupled receptors GprM and GprJ are important for the regulation of the cell wall integrity pathway, secondary metabolite production, and virulence

2020 ◽  
Author(s):  
Aílton Pereira da Costa Filho ◽  
Guilherme Thomaz Pereira Brancini ◽  
Patrícia Alves de Castro ◽  
Jaire Alves Ferreira ◽  
Lilian Pereira Silva ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Kinase ◽  
G Protein ◽  
Galleria Mellonella ◽  
G Protein Coupled Receptors ◽  
Cell Wall Integrity ◽  
Fungal Development ◽  
Cell Wall Integrity Pathway ◽  
Mycotoxin Biosynthesis ◽  
G Protein Coupled

AbstractG-protein coupled receptors (GPCRs) are extracellular signalling receptors that sense environmental cues to coordinate a biological response. Fungi sense their environment primarily through GPCR-mediated signalling pathways, which in turn regulate fungal development, metabolism, virulence and mycotoxin biosynthesis. A. fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that presents a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate when compared to the wild-type (WT) strain, and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the ΔgprM and ΔgprJ strains and decreased in the overexpression mutants when compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. RNA-sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the ΔgprM and ΔgprJ strains, and that this regulation occurs, at least partially, through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signalling. Finally, we show that the ΔgprM and ΔgprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis. This work further contributes to unravelling functions of A. fumigatus GPCRs and shows that GprM and GprJ are essential for CWI, secondary metabolite production and virulence.Author summaryA. fumigatus is the main ethiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immuno-compromised humans. Withstanding the host environment is essential for A. fumigatus virulence and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which in turn regulate fungal development, metabolism, virulence and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella. This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence.

scholarly journals AoBck1 and AoMkk1 Are Necessary to Maintain Cell Wall Integrity, Vegetative Growth, Conidiation, Stress Resistance, and Pathogenicity in the Nematode-Trapping Fungus Arthrobotrys oligospora

2021 ◽  
Vol 12 ◽  
Author(s):  
Meihua Xie ◽  
Jiangliu Yang ◽  
Kexin Jiang ◽  
Na Bai ◽  
Meichen Zhu ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Nucleus ◽  
Stress Resistance ◽  
Vegetative Growth ◽  
Cell Wall Integrity ◽  
Cellular Damage ◽  
Cell Wall Synthesis ◽  
Arthrobotrys Oligospora ◽  
Mitogen Activated Protein ◽  
Woronin Body

The cell wall integrity (CWI) pathway is composed of three mitogen-activated protein kinases (MAPKs), Bck1, Mkk1/2, and Slt2, and is one of the main signaling pathways for fungal pathogenesis, cell wall synthesis, and integrity maintenance. In this study, we characterized orthologs of Saccharomyces cerevisiae Bck1 and Mkk1 in the nematode-trapping (NT) fungus Arthrobotrys oligospora by multiple phenotypic comparison, and the regulation of conidiation and cell wall synthesis was analyzed using real-time PCR (RT-PCR). Both ΔAoBck1 and ΔAoMkk1 mutants showed severe defects in vegetative growth, cell nucleus number, and stress resistance. Both the mutants were unable to produce spores, and the transcription of several genes associated with sporulation and cell wall biosynthesis was markedly downregulated during the conidiation stage. Further, cell walls of the ΔAoBck1 and ΔAoMkk1 mutants were severely damaged, and the Woronin body failed to respond to cellular damage. In particular, the mutants lost the ability to produce mycelial traps for nematode predation. Taken together, AoBck1 and AoMkk1 play a conserved role in mycelial growth and development, CWI, conidiation, multi-stress tolerance, trap formation, and pathogenicity. We highlighted the role of AoBck1 and AoMkk1 in regulating the Woronin body response to cellular damage and cell nucleus development in A. oligospora.

scholarly journals Wsc1 and Mid2 Are Cell Surface Sensors for Cell Wall Integrity Signaling That Act through Rom2, a Guanine Nucleotide Exchange Factor for Rho1

2001 ◽  
Vol 21 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Bevin Philip ◽  
David E. Levin
Keyword(s):  
Cell Wall ◽  
Protein Kinase ◽  
Cell Surface ◽  
G Protein ◽  
Guanine Nucleotide Exchange Factor ◽  
Cell Wall Integrity ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

ABSTRACT Wsc1 and Mid2 are highly O-glycosylated cell surface proteins that reside in the plasma membrane of Saccharomyces cerevisiae. They have been proposed to function as mechanosensors of cell wall stress induced by wall remodeling during vegetative growth and pheromone-induced morphogenesis. These proteins are required for activation of the cell wall integrity signaling pathway that consists of the small G-protein Rho1, protein kinase C (Pkc1), and a mitogen-activated protein kinase cascade. We show here by two-hybrid experiments that the C-terminal cytoplasmic domains of Wsc1 and Mid2 interact with Rom2, a guanine nucleotide exchange factor (GEF) for Rho1. At least with regard to Wsc1, this interaction is mediated by the Rom2 N-terminal domain. This domain is distinct from the Rho1-interacting domain, suggesting that the GEF can interact simultaneously with a sensor and with Rho1. We also demonstrate that extracts from wsc1 and mid2 mutants are deficient in the ability to catalyze GTP loading of Rho1 in vitro, providing evidence that the function of the sensor-Rom2 interaction is to stimulate nucleotide exchange toward this G-protein. In a related line of investigation, we identified the PMT2 gene in a genetic screen for mutations that confer an additive cell lysis defect with a wsc1 null allele. Pmt2 is a member of a six-protein family in yeast that catalyzes the first step in O mannosylation of target proteins. We demonstrate that Mid2 is not mannosylated in apmt2 mutant and that this modification is important for signaling by Mid2.

scholarly journals A G protein-coupled receptor-like module regulates Cellulose Synthase secretion from the endomembrane system in Arabidopsis

2020 ◽  
Author(s):  
Heather E. McFarlane ◽  
Daniela Mutwil-Anderwald ◽  
Jana Verbančič ◽  
Kelsey L. Picard ◽  
Timothy E. Gookin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
G Protein ◽  
Protein Complex ◽  
Cellulose Synthase ◽  
Cell Wall Integrity ◽  
Cellulose Synthesis ◽  
Endomembrane System ◽  
G Protein Coupled

AbstractCellulose synthesis is essential for plant morphology, water transport and defense, and provides raw material for biomaterials and fuels. Cellulose is produced at the plasma membrane by Cellulose Synthase (CESA) protein complexes (CSCs). CSCs are assembled in the endomembrane system and then trafficked from the Golgi apparatus and trans-Golgi Network (TGN) to the plasma membrane. Since CESA enzymes are only active in the plasma membrane, control of CSC secretion is a critical step in the regulation of cellulose synthesis. However, the regulatory framework for CSC secretion is not clarified. In this study, we identify members of a family of seven transmembrane domain-containing proteins (7TMs) as important for cellulose production during cell wall integrity stress. 7TM proteins are often associated with guanine nucleotide-binding protein (G) protein signalling and mutants in several of the canonical G protein complex components phenocopied the 7tm mutant plants. Unexpectedly, the 7TM proteins localized to the Golgi apparatus/TGN where they interacted with the G protein complex. Here, the 7TMs and G proteins regulated CESA trafficking, but did not affect general protein secretion. Furthermore, during cell wall stress, 7TMs’ localization was biased towards small CESA-containing vesicles, specifically associated with CSC trafficking. Our results thus outline how a G protein-coupled module regulates CESA trafficking and reveal that defects in this process lead to exacerbated responses upon exposure to cell wall integrity stress.

scholarly journals Schizosaccharomyces pombe Git7p, a Member of the Saccharomyces cerevisiae Sgt1p Family, Is Required for Glucose and Cyclic AMP Signaling, Cell Wall Integrity, and Septation

2002 ◽  
Vol 1 (4) ◽  
pp. 558-567 ◽  
Author(s):  
Kevin Schadick ◽  
H. Matthew Fourcade ◽  
Peter Boumenot ◽  
Jeffrey J. Seitz ◽  
Jennifer L. Morrell ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Schizosaccharomyces Pombe ◽  
G Protein ◽  
Protein Family ◽  
Cell Wall Integrity ◽  
Camp Signaling ◽  
Adenylate Cyclase Activation

ABSTRACT The Schizosaccharomyces pombe fbp1 gene, encoding fructose-1,6-bisphosphatase, is transcriptionally repressed by glucose. Mutations that confer constitutive fbp1 transcription identify git (glucose-insensitive transcription) genes that encode components of a cyclic AMP (cAMP) signaling pathway required for adenylate cyclase activation. Four of these genes encode the three subunits of a heterotrimeric G protein (gpa2, git5, and git11) and a G protein-coupled receptor (git3). Three additional genes, git1, git7, and git10, act in parallel to or downstream from the G protein genes. Here, we describe the cloning and characterization of the git7 gene. The Git7p protein is a member of the Saccharomyces cerevisiae Sgt1p protein family. In budding yeast, Sgt1p associates with Skp1p and plays an essential role in kinetochore assembly, while in Arabidopsis, a pair of SGT1 proteins have been found to be involved in plant disease resistance through an interaction with RAR1. Like S. cerevisiae Sgt1p, Git7p is essential, but this requirement appears to be due to roles in septation and cell wall integrity, which are unrelated to cAMP signaling, as S. pombe cells lacking either adenylate cyclase or protein kinase A are viable. In addition, git7 mutants are sensitive to the microtubule-destabilizing drug benomyl, although they do not display a chromosome stability defect. Two alleles of git7 that are functional for cell growth and septation but defective for glucose-triggered cAMP signaling encode proteins that are altered in the highly conserved carboxy terminus. The S. cerevisiae and human SGT1 genes both suppress git7-93 but not git7-235 for glucose repression of fbp1 transcription and benomyl sensitivity. This allele-specific suppression indicates that the Git7p/Sgt1p proteins may act as multimers, such that Git7-93p but not Git7-235p can deliver the orthologous proteins to species-specific targets. Our studies suggest that members of the Git7p/Sgt1p protein family may play a conserved role in the regulation of adenylate cyclase activation in S. pombe, S. cerevisiae, and humans.

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