The Candida albicans ELMO homologue functions together with Rac1 and Dck1, upstream of the MAP Kinase Cek1, in invasive filamentous growth

Regulation of Rho G‐proteins is critical for cytoskeletal organization and cell morphology in all eukaryotes. In the human opportunistic pathogen Candida albicans, Rac1 and its activator Dck1, a member of the CED5, Dock180, myoblast city family of guanine nucleotide exchange factors, are required for the budding to filamentous transition during invasive growth. We show that Lmo1, a protein with similarity to human ELMO1, is necessary for invasive filamentous growth, similar to Rac1 and Dck1. Furthermore, Rac1, Dck1 and Lmo1 are required for cell wall integrity, as the deletion mutants are sensitive to cell wall perturbing agents, but not to oxidative or osmotic stresses. The region of Lmo1 encompassing the ELMO and PH‐like domains is sufficient for its function. Both Rac1 and Dck1 can bind Lmo1. Overexpression of a number of protein kinases in the rac1, dck1 and lmo1 deletion mutants indicates that Rac1, Dck1 and Lmo1 function upstream of the mitogen‐activated protein kinases Cek1 and Mkc1, linking invasive filamentous growth to cell wall integrity. We conclude that the requirement of ELMO/CED12 family members for Rac1 function is conserved from fungi to humans.     

Mutations in SNF1 complex genes affect yeast cell wall strength

The trimeric SNF1 complex from Saccharomyces cerevisiae, a homolog of mammalian AMP-activated kinase, has been primarily implicated in signaling for the utilization of alternative carbon sources to glucose. We here find that snf1 deletion mutants are hypersensitive to different cell wall stresses, such as the presence of Calcofluor white, Congo red, Zymolyase or the glucan synthase inhibitor Caspofungin in the growth medium. They also have a thinner cell wall. Caspofungin treatment triggers the phosphorylation of the catalytic Snf1 kinase subunit at Thr210 and removal of this phosphorylation site by mutagenesis (Snf1-T210A) abolishes the function of Snf1 in cell wall integrity. Deletion of the PFK1 gene encoding the α-subunit of the heterooctameric yeast phosphofructokinase suppresses the cell wall phenotypes of a snf1 deletion, which suggests a compensatory effect of central carbohydrate metabolism. Epistasis analyses with mutants in cell wall integrity (CWI) signaling confirm that the SNF1 complex and the CWI pathway independently affect yeast cell integrity.     

Histone acetyltransferase GCN5 contributes to cell wall integrity and salt stress tolerance by altering the expression of cellulose synthesis genes

Excess soluble salts in soil are harmful to the growth and development of most plants. Evidence is emerging that the plant cell wall is involved in sensing and responding to salt stress, but the underlying mechanisms are not well understood. We reveal that the histone acetyltransferase General control non‐repressed protein 5 (GCN5) is required for the maintenance of cell wall integrity and salt stress tolerance. The levels of GCN5 mRNA are increased in response to salt stress. The gcn5 mutants exhibited severe growth inhibition and defects in cell wall integrity under salt stress conditions. Combining RNA sequencing and chromatin immunoprecipitation assays, we identified the chitinase‐like gene CTL1, polygalacturonase involved in expansion‐3 (PGX3) and MYB domain protein‐54 (MYB54) as direct targets of GCN5. Acetylation of H3K9 and H3K14 mediated by GCN5 is associated with activation of CTL1, PGX3 and MYB54 under salt stress. Moreover, constitutive expression of CTL1 in the gcn5 mutant restores salt tolerance and cell wall integrity. In addition, the expression of the wheat TaGCN5 gene in Arabidopsis gcn5 mutant plants complemented the salt tolerance and cell wall integrity phenotypes, suggesting that GCN5‐mediated salt tolerance is conserved between Arabidopsis and wheat. Taken together, our data indicate that GCN5 plays a key role in the preservation of salt tolerance via versatile regulation in plants.     

KRE genes are required for β‐1,6‐glucan synthesis,maintenance of capsule architecture and cell wall protein anchoring in Cryptococcus neoformans

The polysaccharide β‐1,6‐glucan is a major component of the cell wall of Cryptococcus neoformans, but its function has not been investigated in this fungal pathogen. We have identified and characterized seven genes, belonging to the KRE family, which are putatively involved in β‐1,6‐glucan synthesis. The H99 deletion mutants kre5Δ and kre6Δskn1Δ contained less cell wall β‐1,6‐glucan, grew slowly with an aberrant morphology, were highly sensitive to environmental and chemical stress and were avirulent in a mouse inhalation model of infection. These two mutants displayed alterations in cell wall chitosan and the exopolysaccharide capsule, a primary cryptococcal virulence determinant. The cell wall content of the GPI‐anchored phospholipase B1 (Plb1) enzyme, which is required for cryptococcal cell wall integrity and virulence, was reduced in kre5Δ and kre6Δskn1Δ. Our results indicate that KRE5, KRE6 and SKN1 are involved in β‐1,6‐glucan synthesis, maintenance of cell wall integrity and retention of mannoproteins and known cryptococcal virulence factors in the cell wall of C. neoformans. This study sets the stage for future investigations into the function of this abundant cell wall polymer.     

RHO1 and RHO2 share partially overlapping functions in the regulation of cell wall integrity and hyphal polarity in Neurospora crassa

Rho proteins are key regulators of cellular morphogenesis, but their function in filamentous fungi is poorly understood. By generating conditional rho‐1 mutants, we dissected the function of the essential GTPase RHO1 in cell polarization and maintenance of cell wall integrity in Neurospora crassa. We identified NCU00668/RGF1 as RHO1‐specific exchange factor, which controls actin organization and the cell wall integrity MAK1 MAP kinase pathway through the direct interaction of active RHO1 with the formin BNI1 and PKC1 respectively. The activity of RGF1 is controlled by an intramolecular interaction of its DEP and GEF domains that blocks the activation of the GTPase. Moreover, the N‐terminal region including the DEP domain of RGF1 interacts with the plasma membrane sensor NCU06910/WSC1, potentially to activate the cell wall integrity pathway. RHO1 also functions as regulatory subunit of the glucan synthase. N. crassa possesses a second GTPase, RHO2, that is highly homologous to RHO1. RHO2 is of minor importance for growth and does not interact with BNI1. Conditional rho‐1;rho‐2 double mutants display strong synthetic growth and cell polarity defects. We show that RHO2 does not regulate glucan synthase activity and the actin cytoskeleton, but physically interacts with PKC1 to regulate the cell wall integrity pathway.     

Zds1/Zds2–PP2ACdc55 complex specifies signaling output from Rho1 GTPase

Budding yeast Rho1 guanosine triphosphatase (GTPase) plays an essential role in polarized cell growth by regulating cell wall glucan synthesis and actin organization. Upon cell wall damage, Rho1 blocks polarized cell growth and repairs the wounds by activating the cell wall integrity (CWI) Pkc1–mitogen-activated protein kinase (MAPK) pathway. A fundamental question is how active Rho1 promotes distinct signaling outputs under different conditions. Here we identified the Zds1/Zds2–protein phosphatase 2A^Cdc55 (PP2A^Cdc55) complex as a novel Rho1 effector that regulates Rho1 signaling specificity. Zds1/Zds2–PP2A^Cdc55 promotes polarized growth and cell wall synthesis by inhibiting Rho1 GTPase-activating protein (GAP) Lrg1 but inhibits CWI pathway by stabilizing another Rho1 GAP, Sac7, suggesting that active Rho1 is biased toward cell growth over stress response. Conversely, upon cell wall damage, Pkc1–Mpk1 activity inhibits cortical PP2A^Cdc55, ensuring that Rho1 preferentially activates the CWI pathway for cell wall repair. We propose that PP2A^Cdc55 specifies Rho1 signaling output and that reciprocal antagonism between Rho1–PP2A^Cdc55 and Rho1–Pkc1 explains how only one signaling pathway is robustly activated at a time.     

N-acetylglucosamine-6-phosphate deacetylase (NagA) of Listeria monocytogenes EGD, an essential enzyme for the metabolism and recycling of amino sugars

The main aim of our study was to determine the physiological function of NagA enzyme in the Listeria monocytogenes cell. The primary structure of the murein of L. monocytogenes is very similar to that of Escherichia coli, the main differences being amidation of diaminopimelic acid and partial de-N-acetylation of glucosamine residues. NagA is needed for the deacetylation of N-acetyl-glucosamine-6 phosphate to glucosamine-6 phosphate and acetate. Analysis of the L. monocytogenes genome reveals the presence of two proteins with NagA domain, Lmo0956 and Lmo2108, which are cytoplasmic putative proteins. We introduced independent mutations into the structural genes for the two proteins. In-depth characterization of one of these mutants, MN1, deficient in protein Lmo0956 revealed strikingly altered cell morphology, strongly reduced cell wall murein content and decreased sensitivity to cell wall hydrolase, mutanolysin and peptide antibiotic, colistin. The gene products of operon 150, consisting of three genes: lmo0956, lmo0957, and lmo0958, are necessary for the cytosolic steps of the amino-sugar-recycling pathway. The cytoplasmic de-N-acetylase Lmo0956 of L. monocytogenes is required for cell wall peptidoglycan and teichoic acid biosynthesis and is also essential for bacterial cell growth, cell division, and sensitivity to cell wall hydrolases and peptide antibiotics.     

Role of the Guanine Nucleotide Exchange Factor Rom2 in Cell Wall Integrity Maintenance of Aspergillus fumigatus

Aspergillus fumigatus is a mold and the causal agent of invasive aspergillosis, a systemic disease with high lethality. Recently, we identified and functionally characterized three stress sensors implicated in the cell wall integrity (CWI) signaling of this pathogen, namely, Wsc1, Wsc3, and MidA. Here, we functionally characterize Rom2, a guanine nucleotide exchange factor with essential function for the cell wall integrity of A. fumigatus. A conditional rom2 mutant has severe growth defects under repressive conditions and incorporates all phenotypes of the three cell wall integrity sensor mutants, e.g., the echinocandin sensitivity of the Δwsc1 mutant and the Congo red, calcofluor white, and heat sensitivity of the ΔmidA mutant. Rom2 interacts with Rho1 and shows a similar intracellular distribution focused at the hyphal tips. Our results place Rom2 between the cell surface stress sensors Wsc1, Wsc3, MidA, and Rho1 and their downstream effector mitogen-activated protein (MAP) kinase module Bck1-Mkk2-MpkA.     

The transmembrane protein Sho1 cooperates with the mucin Msb2 to regulate invasive growth and plant infection in Fusarium oxysporum

In the vascular wilt pathogen Fusarium oxysporum, the mitogen‐activated protein kinase (MAPK) Fmk1 is essential for plant infection. The mucin‐like membrane protein Msb2 regulates a subset of Fmk1‐dependent functions. Here, we examined the role of the tetraspan transmembrane protein Sho1 as an additional regulator of the Fmk1 pathway and determined its genetic interaction with Msb2. Targeted Δsho1 mutants were generated in wild‐type and Δmsb2 backgrounds to test possible interactions between the two genes. The mutants were examined for hyphal growth under different stress conditions, phosphorylation of the MAPK Fmk1 and an array of Fmk1‐dependent virulence functions. Similar to Msb2, Sho1 was required for the activation of Fmk1 phosphorylation, as well as Fmk1‐dependent gene expression and invasive growth functions, including extracellular pectinolytic activity, cellophane penetration, plant tissue colonization and virulence on tomato plants. Δsho1 mutants were hypersensitive to the cell wall‐perturbing compound Calcofluor White, and this phenotype was exacerbated in the Δmsb2 Δsho1 double mutant. These results highlight that Sho1 and Msb2 have partially overlapping functions upstream of the Fmk1 MAPK cascade, to promote invasive growth and plant infection, as well as cell wall integrity, in F. oxysporum.     

TOR under stress: Targeting TORC1 by Rho1 GTPase

In the yeast Saccharomyces cerevisiae, small GTPase Rho1 controls polarized actin distribution and cell wall expansion in response to many different environmental and intracellular stimuli. Its activity is essential for cell survival and adaptation under various stress conditions. A recent study identified the TOR complex 1 (TORC1), a central regulator in cell growth and metabolism, as a direct target of the small GTPase. This novel crosstalk extends the signaling network of Rho1 into many TORC1-dependent processes and sheds light on how yeast cells coordinate polarized spatial expansion with mass increase.     

Rho1 GTPase and PKC Ortholog Pck1 Are Upstream Activators of the Cell Integrity MAPK Pathway in Fission Yeast

In the fission yeast Schizosaccharomyces pombe the cell integrity pathway (CIP) orchestrates multiple biological processes like cell wall maintenance and ionic homeostasis by fine tuning activation of MAPK Pmk1 in response to various environmental conditions. The small GTPase Rho2 positively regulates the CIP through protein kinase C ortholog Pck2. However, Pmk1 retains some function in mutants lacking either Rho2 or Pck2, suggesting the existence of additional upstream regulatory elements to modulate its activity depending on the nature of the environmental stimulus. The essential GTPase Rho1 is a candidate to control the activity of the CIP by acting upstream of Pck2, whereas Pck1, a second PKC ortholog, appears to negatively regulate Pmk1 activity. However, the exact regulatory nature of these two proteins within the CIP has remained elusive. By exhaustive characterization of strains expressing a hypomorphic Rho1 allele (rho1-596) in different genetic backgrounds we show that both Rho1 and Pck1 are positive upstream regulatory members of the CIP in addition to Rho2 and Pck2. In this new model Rho1 and Rho2 control Pmk1 basal activity during vegetative growth mainly through Pck2. Notably, whereas Rho2-Pck2 elicit Pmk1 activation in response to most environmental stimuli, Rho1 drives Pmk1 activation through either Pck2 or Pck1 exclusively in response to cell wall damage. Our study reveals the intricate and complex functional architecture of the upstream elements participating in this signaling pathway as compared to similar routes from other simple eukaryotic organisms.     

The synthetic genetic network around PKC1 identifies novel modulators and components of protein kinase C signaling in Saccharomyces cerevisiae

Budding yeast Saccharomyces cerevisiae contains one protein kinase C (PKC) isozyme encoded by the essential gene PKC1. Pkc1 is activated by the small GTPase Rho1 and plays a central role in the cell wall integrity (CWI) signaling pathway. This pathway acts primarily to remodel the cell surface throughout the normal life cycle and upon various environmental stresses. The pathway is heavily branched, with multiple nonessential branches feeding into and out of the central essential Rho1-Pkc1 module. In an attempt to identify novel components and modifiers of CWI signaling, we determined the synthetic lethal genetic network around PKC1 by using dominant-negative synthetic genetic array analysis. The resulting mutants are hypersensitive to lowered Pkc1 activity. The corresponding 21 nonessential genes are closely related to CWI function: 14 behave in a chemical-genetic epistasis test as acting in the pathway, and 6 of these genes encode known components. Twelve of the 21 null mutants display elevated CWI reporter activity, consistent with the idea that the pathway is activated by and compensates for loss of the gene products. Four of the 21 mutants display low CWI reporter activity, consistent with the idea that the pathway is compromised in these mutants. One of the latter group of mutants lacks Ack1(Ydl203c), an uncharacterized SEL-1 domain-containing protein that we find modulates pathway activity. Epistasis analysis places Ack1 upstream of Pkc1 in the CWI pathway and dependent on the upstream Rho1 GTP exchange factors Rom2 and Tus1. Overall, the synthetic genetic network around PKC1 directly and efficiently identifies known and novel components of PKC signaling in yeast.     

Arabidopsis CPR5 regulates ethylene signaling via molecular association with the ETR1 receptor

The plant hormone ethylene plays various functions in plant growth, development and response to environmental stress. Ethylene is perceived by membrane‐bound ethylene receptors, and among the homologous receptors in Arabidopsis, the ETR1 ethylene receptor plays a major role. The present study provides evidence demonstrating that Arabidopsis CPR5 functions as a novel ETR1 receptor‐interacting protein in regulating ethylene response and signaling. Yeast split ubiquitin assays and bi‐fluorescence complementation studies in plant cells indicated that CPR5 directly interacts with the ETR1 receptor. Genetic analyses indicated that mutant alleles of cpr5 can suppress ethylene insensitivity in both etr1‐1 and etr1‐2, but not in other dominant ethylene receptor mutants. Overexpression of Arabidopsis CPR5 either in transgenic Arabidopsis plants, or ectopically in tobacco, significantly enhanced ethylene sensitivity. These findings indicate that CPR5 plays a critical role in regulating ethylene signaling. CPR5 is localized to endomembrane structures and the nucleus, and is involved in various regulatory pathways, including pathogenesis, leaf senescence, and spontaneous cell death. This study provides evidence for a novel regulatory function played by CPR5 in the ethylene receptor signaling pathway in Arabidopsis.     

Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1

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 a pmt2 mutant and that this modification is important for signaling by Mid2.