The Predicted Mannosyltransferase GT69-2 Antagonizes RFW-1 To Regulate Cell Fusion in Neurospora crassa

ABSTRACT Filamentous fungi undergo somatic cell fusion to create a syncytial, interconnected hyphal network which confers a fitness benefit during colony establishment. However, barriers to somatic cell fusion between genetically different cells have evolved that reduce invasion by parasites or exploitation by maladapted genetic entities (cheaters). Here, we identified a predicted mannosyltransferase, glycosyltransferase family 69 protein (GT69-2) that was required for somatic cell fusion in Neurospora crassa. Cells lacking GT69-2 prematurely ceased chemotropic signaling and failed to complete cell wall dissolution and membrane merger in pairings with wild-type cells or between Δgt69-2 cells (self fusion). However, loss-of-function mutations in the linked regulator of cell fusion and cell wall remodeling-1 (rfw-1) locus suppressed the self-cell-fusion defects of Δgt69-2 cells, although Δgt69-2 Δrfw-1 double mutants still failed to undergo fusion with wild-type cells. Both GT69-2 and RFW-1 localized to the Golgi apparatus. Genetic analyses indicated that RFW-1 negatively regulates cell wall remodeling-dependent processes, including cell wall dissolution during cell fusion, separation of conidia during asexual sporulation, and conidial germination. GT69-2 acts as an antagonizer to relieve or prevent negative functions on cell fusion by RFW-1. In Neurospora species and N. crassa populations, alleles of gt69-2 were highly polymorphic and fell into two discrete haplogroups. In all isolates within haplogroup I, rfw-1 was conserved and linked to gt69-2. All isolates within haplogroup II lacked rfw-1. These data indicated that gt69-2/rfw-1 are under balancing selection and provide new mechanisms regulating cell wall remodeling during cell fusion and conidial separation. IMPORTANCE Cell wall remodeling is a dynamic process that balances cell wall integrity versus cell wall dissolution. In filamentous fungi, cell wall dissolution is required for somatic cell fusion and conidial separation during asexual sporulation. In the filamentous fungus Neurospora crassa, allorecognition checkpoints regulate the cell fusion process between genetically different cells. Our study revealed two linked loci with transspecies polymorphisms and under coevolution, rfw-1 and gt69-2, which form a coordinated system to regulate cell wall remodeling during somatic cell fusion, conidial separation, and asexual spore germination. RFW-1 acts as a negative regulator of these three processes, while GT69-2 functions antagonistically to RFW-1. Our findings provide new insight into the mechanisms involved in regulation of fungal cell wall remodeling during growth and development.

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Identification and Characterization of LFD-2, a Predicted Fringe Protein Required for Membrane Integrity during Cell Fusion in Neurospora crassa

Eukaryotic Cell ◽

10.1128/ec.00233-14 ◽

2015 ◽

Vol 14 (3) ◽

pp. 265-277 ◽

Cited By ~ 6

Author(s):

Javier Palma-Guerrero ◽

Jiuhai Zhao ◽

A. Pedro Gonçalves ◽

Trevor L. Starr ◽

N. Louise Glass

Keyword(s):

Neurospora Crassa ◽

Somatic Cell ◽

Cell Fusion ◽

Cell Lysis ◽

Extracellular Calcium ◽

Data Set ◽

Content Type ◽

Calcium Dependent ◽

Somatic Cell Fusion ◽

Dependent Cell

ABSTRACTThe molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungusNeurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. InN. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ΔPrm1or Δlfd-1germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, forlatefusiondefect-2) that showed a calcium-dependent cell lysis phenotype.lfd-2encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter,fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion.

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The ham-2 Locus, Encoding a Putative Transmembrane Protein, Is Required for Hyphal Fusion in Neurospora crassa

Genetics ◽

10.1093/genetics/160.1.169 ◽

2002 ◽

Vol 160 (1) ◽

pp. 169-180

Author(s):

Qijun Xiang ◽

Carolyn Rasmussen ◽

N Louise Glass

Keyword(s):

Neurospora Crassa ◽

Somatic Cell ◽

Molecular Mechanism ◽

Cell Fusion ◽

Deletion Mutant ◽

Transmembrane Protein ◽

Wild Type ◽

Vegetative Incompatibility ◽

Hyphal Fusion ◽

Aerial Hyphae

Abstract Somatic cell fusion is common during organogenesis in multicellular eukaryotes, although the molecular mechanism of cell fusion is poorly understood. In filamentous fungi, somatic cell fusion occurs during vegetative growth. Filamentous fungi grow as multinucleate hyphal tubes that undergo frequent hyphal fusion (anastomosis) during colony expansion, resulting in the formation of a hyphal network. The molecular mechanism of the hyphal fusion process and the role of networked hyphae in the growth and development of these organisms are unexplored questions. We use the filamentous fungus Neurospora crassa as a model to study the molecular mechanism of hyphal fusion. In this study, we identified a deletion mutant that was restricted in its ability to undergo both self-hyphal fusion and fusion with a different individual to form a heterokaryon. This deletion mutant displayed pleiotropic defects, including shortened aerial hyphae, altered conidiation pattern, female sterility, slow growth rate, lack of hyphal fusion, and suppression of vegetative incompatibility. Complementation with a single open reading frame (ORF) within the deletion region in this mutant restored near wild-type growth rates, female fertility, aerial hyphae formation, and hyphal fusion, but not vegetative incompatibility and wild-type conidiation pattern. This ORF, which we named ham-2 (for hyphal anastomosis), encodes a putative transmembrane protein that is highly conserved, but of unknown function among eukaryotes.

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The Cell Wall Integrity MAPK pathway controls actin cytoskeleton assembly during fungal somatic cell fusion

10.1101/2020.08.12.246843 ◽

2020 ◽

Author(s):

Antonio Serrano ◽

Hamzeh H. Hammadeh ◽

Natalie Schwarz ◽

Ulrike Brandt ◽

André Fleißner

Keyword(s):

Cell Wall ◽

Actin Cytoskeleton ◽

Somatic Cell ◽

Cell Fusion ◽

Rho Gtpase ◽

Cell Wall Integrity ◽

Physical Contact ◽

Functional Link ◽

Somatic Cell Fusion ◽

Gtpase Rac

AbstractSomatic cell fusion is widely studied in the filamentous fungus Neurospora crassa. The interaction of genetically identical germlings is mediated by a signaling mechanism in which the cells take turns in signal-sending and receiving. The switch between these physiological states is represented by the alternating membrane recruitment of the SO protein and the MAPK MAK-2. This dialog-like behavior is observed until the cells establish physical contact, when the cell-wall-integrity MAK-1 is recruited to the contact area to control the final steps of the cell fusion process. This work revealed, for the first-time, an additional MAK-1-function during the tropic growth phase. Specific inhibition of MAK-1 during tropic-growth resulted in disassembly of the actin-aster, and mislocalization of SO and MAK-2. Similar defects were observed after the inhibition of the Rho-GTPase RAC-1, suggesting a functional link between them, being MAK-1 upstream of RAC-1. In contrast, after inhibition of MAK-2, the actin-aster stayed intact, however, its subcellular localization became instable within the cell-membrane. Together these observations led to a new working model, in which MAK-1 promotes the formation and stability of the actin-aster, while MAK-2 controls its positionning and cell growth directionality.Summary statementThe CWI MAPK MAK-1 pathway controls actin cytoskeleton assembly at the cell tips through activation of the Rho-GTPase RAC-1 exclusively on somatic cell fusion.

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Use of1H Nuclear Magnetic Resonance To Measure Intracellular Metabolite Levels during Growth and Asexual Sporulation in Neurospora crassa

Eukaryotic Cell ◽

10.1128/ec.00231-10 ◽

2011 ◽

Vol 10 (6) ◽

pp. 820-831 ◽

Cited By ~ 12

Author(s):

James D. Kim ◽

Kayla Kaiser ◽

Cynthia K. Larive ◽

Katherine A. Borkovich

Keyword(s):

Nuclear Magnetic Resonance ◽

Magnetic Resonance ◽

Neurospora Crassa ◽

Nutrient Sensing ◽

Wild Type ◽

Content Type ◽

Asexual Sporulation ◽

Significant Difference ◽

High Sucrose ◽

Nuclear Magnetic

ABSTRACTConidiation is an asexual sporulation pathway that is a response to adverse conditions and is the main mode of dispersal utilized by filamentous fungal pathogens for reestablishment in a more favorable environment. Heterotrimeric G proteins (consisting of α, β, and γ subunits) have been shown to regulate conidiation in diverse fungi. Previous work has demonstrated that all three of the Gα subunits in the filamentous fungusNeurospora crassaaffect the accumulation of mass on poor carbon sources and that loss ofgna-3leads to the most dramatic effects on conidiation. In this study, we used1H nuclear magnetic resonance (NMR) to profile the metabolome ofN. crassain extracts isolated from vegetative hyphae and conidia from cultures grown under conditions of high or low sucrose. We compared wild-type and Δgna-3strains to determine whether lack ofgna-3causes a significant difference in the global metabolite profile. The results demonstrate that the global metabolome of wild-type hyphae is influenced by carbon availability. The metabolome of the Δgna-3strain cultured on both high and low sucrose is similar to that of the wild type grown on high sucrose, suggesting an overall defect in nutrient sensing in the mutant. However, analysis of individual metabolites revealed differences in wild-type and Δgna-3strains cultured under conditions of low and high sucrose.

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Conflict, Competition, and Cooperation Regulate Social Interactions in Filamentous Fungi

Annual Review of Microbiology ◽

10.1146/annurev-micro-012420-080905 ◽

2020 ◽

Vol 74 (1) ◽

pp. 693-712 ◽

Cited By ~ 2

Author(s):

A. Pedro Gonçalves ◽

Jens Heller ◽

Adriana M. Rico-Ramírez ◽

Asen Daskalov ◽

Gabriel Rosenfield ◽

...

Keyword(s):

Social Interactions ◽

Somatic Cell ◽

Filamentous Fungi ◽

Cell Fusion ◽

Balancing Selection ◽

Kin Recognition ◽

Competitive Interactions ◽

Competition And Cooperation ◽

Somatic Cell Fusion ◽

Tactile Cues

Social cooperation impacts the development and survival of species. In higher taxa, kin recognition occurs via visual, chemical, or tactile cues that dictate cooperative versus competitive interactions. In microbes, the outcome of cooperative versus competitive interactions is conferred by identity at allorecognition loci, so-called kind recognition. In syncytial filamentous fungi, the acquisition of multicellularity is associated with somatic cell fusion within and between colonies. However, such intraspecific cooperation entails risks, as fusion can transmit deleterious genotypes or infectious components that reduce fitness, or give rise to cheaters that can exploit communal goods without contributing to their production. Allorecognition mechanisms in syncytial fungi regulate somatic cell fusion by operating precontact during chemotropic interactions, during cell adherence, and postfusion by triggering programmed cell death reactions. Alleles at fungal allorecognition loci are highly polymorphic, fall into distinct haplogroups, and show evolutionary signatures of balancing selection, similar to allorecognition loci across the tree of life.

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The Cell Wall Integrity Pathway Contributes to the Early Stages of Aspergillus fumigatus Asexual Development

Applied and Environmental Microbiology ◽

10.1128/aem.02347-19 ◽

2020 ◽

Vol 86 (7) ◽

Cited By ~ 1

Author(s):

Marina Campos Rocha ◽

João Henrique Tadini Marilhano Fabri ◽

Isabelle Taira Simões ◽

Rafael Silva-Rocha ◽

Daisuke Hagiwara ◽

...

Keyword(s):

Cell Wall ◽

Aspergillus Fumigatus ◽

Filamentous Fungi ◽

Cell Wall Integrity ◽

Asexual Development ◽

Remarkable Feature ◽

Susceptible Host ◽

Content Type ◽

Cell Wall Integrity Pathway ◽

Cell Wall Remodeling

ABSTRACT Aspergillus fumigatus is a major cause of human disease. The survival of this fungus is dependent on the cell wall organization and function of its components. The cell wall integrity pathway (CWIP) is the primary signaling cascade that controls de novo synthesis of the cell wall in fungi. Abundant conidiation is a hallmark in A. fumigatus, and uptake of conidia by a susceptible host is usually the initial event in infection. The formation of conidia is mediated by the development of fungus-specific specialized structures, conidiophores, which are accompanied by cell wall remodeling. The molecular regulation of these changes in cell wall composition required for the rise of conidiophore from the solid surface and to disperse the conidia into the air is currently unknown. Here, we investigated the role of CWIP in conidiation. We show that CWIP pkcAG579R, ΔmpkA, and ΔrlmA mutants displayed reduced conidiation during synchronized asexual differentiation. The transcription factor RlmA directly regulated the expression of regulators of conidiation, including flbB, flbC, brlA, abaA, and rasB, as well as genes involved in cell wall synthesis and remodeling, and this affected the chitin content in aerial hyphae. Phosphorylation of RlmA and MpkA was increased during asexual differentiation. We also observed that MpkA physically associated with the proteins FlbB, FlbC, BrlA, and RasB during this process, suggesting another level of cross talk between the CWIP and asexual development pathways. In summary, our results support the conclusion that one function of the CWIP is the regulation of asexual development in filamentous fungi. IMPORTANCE A remarkable feature of the human pathogen Aspergillus fumigatus is its ability to produce impressive amounts of infectious propagules known as conidia. These particles reach immunocompromised patients and may initiate a life-threatening mycosis. The conidiation process in Aspergillus is governed by a sequence of proteins that coordinate the development of conidiophores. This process requires the remodeling of the cell wall so that the conidiophores can rise and withstand the chains of conidia. The events regulating cell wall remodeling during conidiation are currently unknown. Here, we show that the cell wall integrity pathway (CWIP) components RlmA and MpkA directly contribute to the activation of the conidiation cascade by enabling transcription or phosphorylation of critical proteins involved in asexual development. This study points to an essential role for the CWIP during conidiation and provides further insights into the complex regulation of asexual development in filamentous fungi.

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α-1,6-Mannosylation of N-Linked Oligosaccharide Present on Cell Wall Proteins Is Required for Their Incorporation into the Cell Wall in the Filamentous Fungus Neurospora crassa

Eukaryotic Cell ◽

10.1128/ec.00134-10 ◽

2010 ◽

Vol 9 (11) ◽

pp. 1766-1775 ◽

Cited By ~ 39

Author(s):

Abhiram Maddi ◽

Stephen J. Free

Keyword(s):

Cell Wall ◽

Neurospora Crassa ◽

Mutant Cell ◽

Cell Wall Protein ◽

Cell Wall Proteins ◽

Wild Type ◽

Content Type ◽

Fold Reduction ◽

Total Lack ◽

Mutant Cells

ABSTRACT The enzyme α-1,6-mannosyltransferase (OCH-1) is required for the synthesis of galactomannans attached to the N-linked oligosaccharides of Neurospora crassa cell wall proteins. The Neurospora crassa och-1 mutant has a tight colonial phenotype and a defective cell wall. A carbohydrate analysis of the och-1 mutant cell wall revealed a 10-fold reduction in the levels of mannose and galactose and a total lack of 1,6-linked mannose residues. Analysis of the integral cell wall protein from wild-type and och-1 mutant cells showed that the mutant cell wall had reduced protein content. The och-1 mutant was found to secrete 18-fold more protein than wild-type cells. Proteomic analysis of the proteins released by the mutant into the growth medium identified seven of the major cell wall proteins. Western blot analysis of ACW-1 and GEL-1 (two glycosylphosphatidylinositol [GPI]-anchored proteins that are covalently integrated into the wild-type cell wall) showed that high levels of these proteins were being released into the medium by the och-1 mutant. High levels of ACW-1 and GEL-1 were also released from the och-1 mutant cell wall by subjecting the wall to boiling in a 1% SDS solution, indicating that these proteins are not being covalently integrated into the mutant cell wall. From these results, we conclude that N-linked mannosylation of cell wall proteins by OCH-1 is required for their efficient covalent incorporation into the cell wall.

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Ultrastructure and Morphology of the Neurospora Crassa Cell Wall Mutants, Slime And Slime X, Using Tem and Sem

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100090257 ◽

1976 ◽

Vol 34 ◽

pp. 54-55

Author(s):

Karen S. Howard ◽

H. D. Braymer ◽

M. D. Socolofsky ◽

S. A. Milligan

Keyword(s):

Cell Wall ◽

Neurospora Crassa ◽

Wild Type ◽

Morphological Comparison ◽

Small Areas ◽

Solid Media ◽

Thin Sectioning ◽

X Cells ◽

Mutant Cells ◽

Isolated Cell

The recently isolated cell wall mutant slime X of Neurospora crassa was prepared for ultrastructural and morphological comparison with the cell wall mutant slime. The purpose of this article is to discuss the methods of preparation for TEM and SEM observations, as well as to make a preliminary comparison of the two mutants.TEM: Cells of the slime mutant were prepared for thin sectioning by the method of Bigger, et al. Slime X cells were prepared in the same manner with the following two exceptions: the cells were embedded in 3% agar prior to fixation and the buffered solutions contained 5% sucrose throughout the procedure.SEM: Two methods were used to prepare mutant and wild type Neurospora for the SEM. First, single colonies of mutant cells and small areas of wild type hyphae were cut from solid media and fixed with OSO4 vapors similar to the procedure used by Harris, et al. with one alteration. The cell-containing agar blocks were dehydrated by immersion in 2,2-dimethoxypropane (DMP).

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