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

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.

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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 ◽

10.1128/mbio.02458-20 ◽

2020 ◽

Vol 11 (5) ◽

Cited By ~ 1

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.

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Biased Signaling: Distinct Ligand-directed Plasma Membrane Signalosomes Using a Common RGS/G protein Core

10.1101/666578 ◽

2019 ◽

Author(s):

Timothy J. Ross-Elliott ◽

Justin Watkins ◽

Xiaoyi Shan ◽

Fei Lou ◽

Bernd Dreyer ◽

...

Keyword(s):

Plasma Membrane ◽

G Protein ◽

Lipid Rafts ◽

Protein Complex ◽

Heterotrimeric G Protein ◽

Unknown Mechanism ◽

Protein Core ◽

Protein System ◽

Biased Signaling ◽

G Protein Coupled

Biased signaling occurs when different ligands that are directed at the same receptor launch different cellular outcomes. Because of their pharmacological importance, we know the most about biased ligands and little is known about other mechanisms to achieve signaling bias. In the canonical animal G protein system, endocytosis of a 7-transmembrane GPCR is mediated by arrestins to propagate or arrest cytoplasmic signaling depending on the bias. In Arabidopsis, GPCRs are not required for G protein coupled signaling because the heterotrimeric G protein complex spontaneously exchanges nucleotide. Instead, the prototype 7-transmembrane Regulator of G Signaling 1 protein AtRGS1 modulates G signaling and through ligand-dependent endocytosis, de-repression of signaling is initiated but canonical arrestins are not involved. Endocytosis initiates from two separate pools of plasma membrane: sterol-dependent domains, possibly lipid rafts, and a clathrin-accessible neighborhood, each with a select set of discriminators, activators, and newly-discovered arrestin-like adaptors. Different trafficking origins and trajectories lead to different cellular outcomes. Thus, compartmentation with its attendant signalosome architecture is a previously unknown mechanism to drive biased signaling.

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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

10.1101/2020.08.03.235119 ◽

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.

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Cellulose synthase complexes display distinct dynamic behaviors during xylem transdifferentiation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1802113115 ◽

2018 ◽

Vol 115 (27) ◽

pp. E6366-E6374 ◽

Cited By ~ 21

Author(s):

Yoichiro Watanabe ◽

Rene Schneider ◽

Sarah Barkwill ◽

Eliana Gonzales-Vigil ◽

Joseph L. Hill ◽

...

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Cell Expansion ◽

Secondary Wall ◽

Transition Period ◽

Cellulose Synthase ◽

Wall Thickening ◽

Cellulose Synthesis ◽

Primary Wall ◽

Dynamic Behaviors

In plants, plasma membrane-embedded CELLULOSE SYNTHASE (CESA) enzyme complexes deposit cellulose polymers into the developing cell wall. Cellulose synthesis requires two different sets of CESA complexes that are active during cell expansion and secondary cell wall thickening, respectively. Hence, developing xylem cells, which first undergo cell expansion and subsequently deposit thick secondary walls, need to completely reorganize their CESA complexes from primary wall- to secondary wall-specific CESAs. Using live-cell imaging, we analyzed the principles underlying this remodeling. At the onset of secondary wall synthesis, the primary wall CESAs ceased to be delivered to the plasma membrane and were gradually removed from both the plasma membrane and the Golgi. For a brief transition period, both primary wall- and secondary wall-specific CESAs coexisted in banded domains of the plasma membrane where secondary wall synthesis is concentrated. During this transition, primary and secondary wall CESAs displayed discrete dynamic behaviors and sensitivities to the inhibitor isoxaben. As secondary wall-specific CESAs were delivered and inserted into the plasma membrane, the primary wall CESAs became concentrated in prevacuolar compartments and lytic vacuoles. This adjustment in localization between the two CESAs was accompanied by concurrent decreased primary wall CESA and increased secondary wall CESA protein abundance. Our data reveal distinct and dynamic subcellular trafficking patterns that underpin the remodeling of the cellulose biosynthetic machinery, resulting in the removal and degradation of the primary wall CESA complex with concurrent production and recycling of the secondary wall CESAs.

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A G protein-coupled receptor-like module regulates cellulose synthase secretion from the endomembrane system in Arabidopsis

Developmental Cell ◽

10.1016/j.devcel.2021.03.031 ◽

2021 ◽

Author(s):

Heather E. McFarlane ◽

Daniela Mutwil-Anderwald ◽

Jana Verbančič ◽

Kelsey L. Picard ◽

Timothy E. Gookin ◽

...

Keyword(s):

G Protein ◽

Cellulose Synthase ◽

Endomembrane System ◽

G Protein Coupled Receptor ◽

G Protein Coupled

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Faculty Opinions recommendation of A G protein-coupled receptor is a plasma membrane receptor for the plant hormone abscisic acid.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1068817.523909 ◽

2007 ◽

Author(s):

Martin Robert McAinsh

Keyword(s):

Plasma Membrane ◽

Abscisic Acid ◽

G Protein ◽

Plant Hormone ◽

Membrane Receptor ◽

G Protein Coupled Receptor ◽

Plasma Membrane Receptor ◽

G Protein Coupled

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Gβγ translocation to the Golgi apparatus activates ARF1 to spatiotemporally regulate G protein-coupled receptor signaling to MAPK

Journal of Biological Chemistry ◽

10.1016/j.jbc.2021.100805 ◽

2021 ◽

pp. 100805

Author(s):

Mostafa Khater ◽

Christian N. Bryant ◽

Guangyu Wu

Keyword(s):

Golgi Apparatus ◽

G Protein ◽

Receptor Signaling ◽

G Protein Coupled Receptor ◽

G Protein Coupled

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Thyrotropin-releasing hormone activates Ca2+ efflux. Evidence suggesting that a plasma membrane Ca2+ pump is an effector for a G-protein-coupled Ca(2+)-mobilizing receptor.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)47360-7 ◽

1994 ◽

Vol 269 (49) ◽

pp. 30854-30860

Author(s):

E J Nelson ◽

P M Hinkle

Keyword(s):

Plasma Membrane ◽

G Protein ◽

Thyrotropin Releasing Hormone ◽

Releasing Hormone ◽

G Protein Coupled

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Putative PmrA and PmcA are important for normal growth, morphogenesis and cell wall integrity, but not for viability in Aspergillus nidulans

Microbiology ◽

10.1099/mic.0.080119-0 ◽

2014 ◽

Vol 160 (11) ◽

pp. 2387-2395 ◽

Cited By ~ 7

Author(s):

Hechun Jiang ◽

Feifei Liu ◽

Shizhu Zhang ◽

Ling Lu

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Aspergillus Nidulans ◽

Normal Growth ◽

Cell Wall Integrity ◽

Plasma Membrane Atpase ◽

Growth Defects ◽

Double Deletion ◽

Intracellular Ca ◽

P Type

P-type Ca2+-transporting ATPases are Ca2+ pumps, extruding cytosolic Ca2+ to the extracellular environment or the intracellular Ca2+ store lumens. In budding yeast, Pmr1 (plasma membrane ATPase related), and Pmc1 (plasma membrane calcium-ATPase) cannot be deleted simultaneously for it to survive in standard medium. Here, we deleted two putative Ca2+ pumps, designated AnPmrA and AnPmcA, from Aspergillus nidulans, and obtained the mutants ΔanpmrA and ΔanpmcA, respectively. Then, using ΔanpmrA as the starting strain, the promoter of its anpmcA was replaced with the alcA promoter to secure the mutant ΔanpmrAalcApmcA or its anpmcA was deleted completely to produce the mutant ΔanpmrAΔpmcA. Different from the case in Saccharomyces cerevisiae, double deletion of anpmrA and anpmcA was not lethal in A. nidulans. In addition, deletion of anpmrA and/or anpmcA had produced growth defects, although overexpression of AnPmc1 in ΔanpmrAalcApmcA could not restore the growth defects that resulted from the loss of AnPmrA. Moreover, we found AnPmrA was indispensable for maintenance of normal morphogenesis, especially in low-Ca2+/Mn2+ environments. Thus, our findings suggest AnPmrA and AnPmcA might play important roles in growth, morphogenesis and cell wall integrity in A. nidulans in a different way from that in yeasts.

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