FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2971352197> ?p ?o ?g. }

• W2971352197 endingPage "3017.e3" @default.
• W2971352197 startingPage "3006" @default.
• W2971352197 abstract "•Conspecific cooperation and cell fusion are crucial for microbial development•Allorecognition upon contact blocks cell fusion of genetically non-identical cells•Incompatible partners cannot switch from communication to cell wall dissolution mode•The independent appearance of polymorphic cwr alleles suggests convergent evolution Somatic cell fusion and conspecific cooperation are crucial social traits for microbial unicellular-to-multicellular transitions, colony expansion, and substrate foraging but are also associated with risks of parasitism. We identified a cell wall remodeling (cwr) checkpoint that acts upon cell contact to assess genetic compatibility and regulate cell wall dissolution during somatic cell fusion in a wild population of the filamentous fungus Neurospora crassa. Non-allelic interactions between two linked loci, cwr-1 and cwr-2, were necessary and sufficient to block cell fusion: cwr-1 encodes a polysaccharide monooxygenase (PMO), a class of enzymes associated with extracellular degradative capacities, and cwr-2 encodes a predicted transmembrane protein. Mutations of sites in CWR-1 essential for PMO catalytic activity abolished the block in cell fusion between formerly incompatible strains. In Neurospora, alleles cwr-1 and cwr-2 were highly polymorphic, fell into distinct haplogroups, and showed trans-species polymorphisms. Distinct haplogroups and trans-species polymorphisms at cwr-1 and cwr-2 were also identified in the distantly related genus Fusarium, suggesting convergent evolution. Proteins involved in chemotropic processes showed extended localization at contact sites, suggesting that cwr regulates the transition between chemotropic growth and cell wall dissolution. Our work revealed an allorecognition surveillance system based on kind discrimination that inhibits cooperative behavior in fungi by blocking cell fusion upon contact, contributing to fungal immunity by preventing formation of chimeras between genetically non-identical colonies. Somatic cell fusion and conspecific cooperation are crucial social traits for microbial unicellular-to-multicellular transitions, colony expansion, and substrate foraging but are also associated with risks of parasitism. We identified a cell wall remodeling (cwr) checkpoint that acts upon cell contact to assess genetic compatibility and regulate cell wall dissolution during somatic cell fusion in a wild population of the filamentous fungus Neurospora crassa. Non-allelic interactions between two linked loci, cwr-1 and cwr-2, were necessary and sufficient to block cell fusion: cwr-1 encodes a polysaccharide monooxygenase (PMO), a class of enzymes associated with extracellular degradative capacities, and cwr-2 encodes a predicted transmembrane protein. Mutations of sites in CWR-1 essential for PMO catalytic activity abolished the block in cell fusion between formerly incompatible strains. In Neurospora, alleles cwr-1 and cwr-2 were highly polymorphic, fell into distinct haplogroups, and showed trans-species polymorphisms. Distinct haplogroups and trans-species polymorphisms at cwr-1 and cwr-2 were also identified in the distantly related genus Fusarium, suggesting convergent evolution. Proteins involved in chemotropic processes showed extended localization at contact sites, suggesting that cwr regulates the transition between chemotropic growth and cell wall dissolution. Our work revealed an allorecognition surveillance system based on kind discrimination that inhibits cooperative behavior in fungi by blocking cell fusion upon contact, contributing to fungal immunity by preventing formation of chimeras between genetically non-identical colonies. Complex multicellularity results from a developmental program that leads to differentiation of specialized structures and requires intercellular social cooperation [1Knoll A.H. The multiple origins of complex multicellularity.Annu. Rev. Earth Planet. Sci. 2011; 39: 217-239Crossref Scopus (301) Google Scholar]. In multicellular organisms, conspecific cooperation enhances adaptation to environmental variations due to the communal nature of produced goods (e.g., nutrients in a fungal colony). However, cooperation can cause conflict due to transmission of infectious elements and genotypes that negatively impact cellular fitness [2Bastiaans E. Aanen D.K. Debets A.J. Hoekstra R.F. Lestrade B. Maas M.F. Regular bottlenecks and restrictions to somatic fusion prevent the accumulation of mitochondrial defects in Neurospora.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2014; 369: 20130448Crossref PubMed Scopus (16) Google Scholar, 3Bastiaans E. Debets A.J. Aanen D.K. Experimental demonstration of the benefits of somatic fusion and the consequences for allorecognition.Evolution. 2015; 69: 1091-1099Crossref PubMed Scopus (23) Google Scholar, 4Biella S. Smith M.L. Aist J.R. Cortesi P. Milgroom M.G. Programmed cell death correlates with virus transmission in a filamentous fungus.Proc. Biol. Sci. 2002; 269: 2269-2276Crossref PubMed Scopus (103) Google Scholar, 5Fernàndez-Busquets X. Körnig A. Bucior I. Burger M.M. Anselmetti D. Self-recognition and Ca2+-dependent carbohydrate-carbohydrate cell adhesion provide clues to the cambrian explosion.Mol. Biol. Evol. 2009; 26: 2551-2561Crossref PubMed Scopus (32) Google Scholar, 6Zhang D.X. Spiering M.J. Dawe A.L. Nuss D.L. Vegetative incompatibility loci with dedicated roles in allorecognition restrict mycovirus transmission in chestnut blight fungus.Genetics. 2014; 197: 701-714Crossref PubMed Scopus (54) Google Scholar]. In this regard, high genetic relatedness and cooperation correlate positively to prevent exploitation of communal goods by cheaters [7Aanen D.K. Debets A.J. de Visser J.A. Hoekstra R.F. The social evolution of somatic fusion.BioEssays. 2008; 30: 1193-1203Crossref PubMed Scopus (43) Google Scholar, 8Bastiaans E. Debets A.J. Aanen D.K. Experimental evolution reveals that high relatedness protects multicellular cooperation from cheaters.Nat. Commun. 2016; 7: 11435Crossref PubMed Scopus (42) Google Scholar, 9Diggle S.P. Griffin A.S. Campbell G.S. West S.A. Cooperation and conflict in quorum-sensing bacterial populations.Nature. 2007; 450: 411-414Crossref PubMed Scopus (573) Google Scholar, 10Kuzdzal-Fick J.J. Fox S.A. Strassmann J.E. Queller D.C. High relatedness is necessary and sufficient to maintain multicellularity in Dictyostelium.Science. 2011; 334: 1548-1551Crossref PubMed Scopus (80) Google Scholar]. The interconnected fungal mycelium is a prototype of a complex multicellular body. It operates as a polarized syncytium that expands via tip elongation and somatic cell-cell fusion [11Glass N.L. Rasmussen C. Roca M.G. Read N.D. Hyphal homing, fusion and mycelial interconnectedness.Trends Microbiol. 2004; 12: 135-141Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 12Fischer M.S. Glass N.L. Communicate and fuse: how filamentous fungi establish and maintain an interconnected mycelial network.Front. Microbiol. 2019; 10: 619Crossref PubMed Scopus (46) Google Scholar]. In Neurospora crassa, cell fusion between genetically identical cells (either germinated asexual spores [germlings] or between hyphae within a single colony) is a fitness character, as fusion mutants show a lag in colony development [3Bastiaans E. Debets A.J. Aanen D.K. Experimental demonstration of the benefits of somatic fusion and the consequences for allorecognition.Evolution. 2015; 69: 1091-1099Crossref PubMed Scopus (23) Google Scholar, 13Richard F. Glass N.L. Pringle A. Cooperation among germinating spores facilitates the growth of the fungus, Neurospora crassa.Biol. Lett. 2012; 8: 419-422Crossref PubMed Scopus (34) Google Scholar]. Fusion between genetically non-identical cells results in a heterokaryotic syncytia that contains organelles of dissimilar genetic backgrounds. Although heterokaryon formation has been postulated to increase fitness in fungal populations [3Bastiaans E. Debets A.J. Aanen D.K. Experimental demonstration of the benefits of somatic fusion and the consequences for allorecognition.Evolution. 2015; 69: 1091-1099Crossref PubMed Scopus (23) Google Scholar], it is often precluded by allorecognition systems that either reduce somatic cell fusion between genetically distinct cells or cause cell death of fusion compartments [8Bastiaans E. Debets A.J. Aanen D.K. Experimental evolution reveals that high relatedness protects multicellular cooperation from cheaters.Nat. Commun. 2016; 7: 11435Crossref PubMed Scopus (42) Google Scholar, 14Gonçalves A.P. Heller J. Daskalov A. Videira A. Glass N.L. Regulated forms of cell death in fungi.Front. Microbiol. 2017; 8: 1837Crossref PubMed Scopus (60) Google Scholar, 15Heller J. Clavé C. Gladieux P. Saupe S.J. Glass N.L. NLR surveillance of essential SEC-9 SNARE proteins induces programmed cell death upon allorecognition in filamentous fungi.Proc. Natl. Acad. Sci. USA. 2018; 115: E2292-E2301Crossref PubMed Scopus (41) Google Scholar, 16Heller J. Zhao J. Rosenfield G. Kowbel D.J. Gladieux P. Glass N.L. Characterization of greenbeard genes involved in long-distance kind discrimination in a microbial eukaryote.PLoS Biol. 2016; 14: e1002431Crossref PubMed Scopus (39) Google Scholar]. These allorecognition systems reduce transmission of mycoviruses, senescence plasmids, crippled mitochondria, and defective nuclei between fungal colonies [2Bastiaans E. Aanen D.K. Debets A.J. Hoekstra R.F. Lestrade B. Maas M.F. Regular bottlenecks and restrictions to somatic fusion prevent the accumulation of mitochondrial defects in Neurospora.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2014; 369: 20130448Crossref PubMed Scopus (16) Google Scholar, 3Bastiaans E. Debets A.J. Aanen D.K. Experimental demonstration of the benefits of somatic fusion and the consequences for allorecognition.Evolution. 2015; 69: 1091-1099Crossref PubMed Scopus (23) Google Scholar, 4Biella S. Smith M.L. Aist J.R. Cortesi P. Milgroom M.G. Programmed cell death correlates with virus transmission in a filamentous fungus.Proc. Biol. Sci. 2002; 269: 2269-2276Crossref PubMed Scopus (103) Google Scholar, 6Zhang D.X. Spiering M.J. Dawe A.L. Nuss D.L. Vegetative incompatibility loci with dedicated roles in allorecognition restrict mycovirus transmission in chestnut blight fungus.Genetics. 2014; 197: 701-714Crossref PubMed Scopus (54) Google Scholar, 17Debets F. Yang X. Griffiths A.J. Vegetative incompatibility in Neurospora: its effect on horizontal transfer of mitochondrial plasmids and senescence in natural populations.Curr. Genet. 1994; 26: 113-119Crossref PubMed Scopus (92) Google Scholar]. To examine cooperation during the acquisition of multicellularity, we assessed fusion dynamics between wild Neurospora isolates that showed chemotropic interactions and identified a cell wall dissolution arrest phenotype following contact [11Glass N.L. Rasmussen C. Roca M.G. Read N.D. Hyphal homing, fusion and mycelial interconnectedness.Trends Microbiol. 2004; 12: 135-141Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 12Fischer M.S. Glass N.L. Communicate and fuse: how filamentous fungi establish and maintain an interconnected mycelial network.Front. Microbiol. 2019; 10: 619Crossref PubMed Scopus (46) Google Scholar]. A population genomics analysis led to identification of cell wall remodeling checkpoint loci (cwr-1 and cwr-2), whose allelic specificity regulates whether strains can transit from communication to cell wall dissolution and cell fusion. The cwr allorecognition checkpoint displays signs of balancing selection and convergent evolution in distinct fungal genera and allows cells to undergo kind recognition, presumably to avoid cooperation with disadvantageous partners during development of syncytial, multinucleate colonies. Previously, we reported that allelic specificity at determinant of communication (doc) loci determines pre-contact kind recognition in N. crassa by regulating chemotropic behavior prior to somatic cell fusion [16Heller J. Zhao J. Rosenfield G. Kowbel D.J. Gladieux P. Glass N.L. Characterization of greenbeard genes involved in long-distance kind discrimination in a microbial eukaryote.PLoS Biol. 2016; 14: e1002431Crossref PubMed Scopus (39) Google Scholar]. The wild-type strains, FGSC2489 and JW258, are unable to establish chemotropic interactions prior to somatic cell fusion because they belong to different doc haplogroups (CGH1 and CGH2, respectively). However, these two strains can mate and produce progeny able to communicate with only one of their parents [16Heller J. Zhao J. Rosenfield G. Kowbel D.J. Gladieux P. Glass N.L. Characterization of greenbeard genes involved in long-distance kind discrimination in a microbial eukaryote.PLoS Biol. 2016; 14: e1002431Crossref PubMed Scopus (39) Google Scholar]. We evaluated whether post-chemotropic interactions were affected in progeny of the FGSC2489 × JW258 cross by staining germlings with FM4-64 and assessing cell fusion frequency when paired with an FGSC2489 strain expressing cytoplasmic GFP. Of these progeny, 62% underwent chemotropic interactions, cell fusion, and cytoplasmic mixing with FGSC2489 (Figure 1A, top; Video S1), and 38% did not show cell fusion and cytoplasmic mixing after chemotropic interactions and cell contact with FGSC2489 cells (Figure 1A, bottom; Video S2). Of progeny that underwent chemotropic interactions with JW258 cells, 52% showed cytoplasmic mixing with JW258, and 48% did not (Figure S1A). Over time, arrested cells redirected their growth, indicating that fusion was irreversibly blocked (Figure 1A, bottom). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI1Nzg3ODE0MDFlNzRiYzNjMGIwZDNhMDQ3OTJlMWFkMyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NjAxMzI5fQ.DxA_ayYqjbqrbioGTuWQCXlm-4slpQ4A0soXM8U2idYVh7igMQv9UivBgv4jwIja_UVy1elSRtKGoKeB7IPgXE0drE0Y6Qt5Tye37DMsPtA0OHOme5Cy9SLIGO12u9fjHB5VPfYExzJpuDgqoVAkWFJueO-_GvvJfdhbKsSkg9TPXP0jlBEpD4-wIc2TBel-eR0h8cSqMXc6O2MxlYRrSk-H6824mfpVQZJmODUFChSyYUD3Eim3KTTVZnVQg39-mcYaodkm8xwierlKf9WAc6Ij_asYAWhw6DxOYtOkayW39iCQYI1O6axbnGTu8RA2vgCbzCC-VOQr60tANyVw8w Download .mp4 (1.64 MB) Help with .mp4 files Video S1. Successful Germling Fusion and Concomitant Cytoplasmic Mixing, Related to Figure 1Compatible fusion between free GFP-expressing FGSC2489 and FM4-64-stained Seg11. An arrowhead marks the moment of contact between the two cells (defined as time 0). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJhOGFiYjAwYThlMDcyNmQxZTM0Mzk2N2Q3MzY2NDgzMyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NjAxMzI5fQ.IPLH8yDMsZ6FiOSGeb4gNKkBzN1EPJaqxoNWcyoBZvavkEJqfpKD6kvqAGvcBhRfL4T4YyLl4K8YD1wSppBRt01nHCwzfxJqbNLTbkDb9Uov2V63szENosoIbUqUHgoO641CGqiGIw1OvQEnAl24e9qn79nzsV3a0bF7p1DlZ7QlupiCEBOJRLpCdy2agUzktPOPFQ_k5YGgJpPkr6XE16H94epulaPGIe0b_8HcgEd3AeREH4pI4bMgpCcvJuOLoBpbFYZqd1oNMey96gq19_V-_7OSfvGZtvHikKdC5kN-5mfVK1v7hdJMEsJvV5aO0MmJor5NZe1eGliekl1SWw Download .mp4 (3.57 MB) Help with .mp4 files Video S2. Fusion Arrest upon Cell-Cell Contact, Related to Figure 1Incompatible fusion attempt and subsequent arrest state between free GFP-expressing FGSC2489 and FM4-64-stained Seg3. An arrowhead marks the moment of contact between the two cells (defined as time 0). To quantify cell fusion frequencies, we utilized a flow cytometry method based on a robust post-fusion death response mediated by genetic differences at sec-9 [15Heller J. Clavé C. Gladieux P. Saupe S.J. Glass N.L. NLR surveillance of essential SEC-9 SNARE proteins induces programmed cell death upon allorecognition in filamentous fungi.Proc. Natl. Acad. Sci. USA. 2018; 115: E2292-E2301Crossref PubMed Scopus (41) Google Scholar]. Isogenic germlings (identical sec-9 alleles) undergo cell fusion at high frequency and display low basal cell death levels (Figure 1B; Table S1), and germlings containing alternate sec-9 alleles (but otherwise isogenic) show similar fusion frequencies but high post-fusion death rates [15Heller J. Clavé C. Gladieux P. Saupe S.J. Glass N.L. NLR surveillance of essential SEC-9 SNARE proteins induces programmed cell death upon allorecognition in filamentous fungi.Proc. Natl. Acad. Sci. USA. 2018; 115: E2292-E2301Crossref PubMed Scopus (41) Google Scholar]. Thus, introducing alternate sec-9 alleles in otherwise isogenic strains allowed us to use cell death as a proxy for fusion frequency. A program that allowed for automatic and unbiased gating and analysis of flow cytometry samples was generated (Figure S2). To assess cell fusion frequencies in progeny from the FGSC2489 × JW258 cross, we mixed FGSC2489 cells with Seg11 (progeny that undergoes chemotropic interactions and cell fusion with FGSC2489) or with Seg3 (progeny that undergoes chemotropic interactions but is blocked in cell fusion with FGSC2489). The relatively high death frequencies in FGSC2489 + Seg11 pairings and low death rates in FGSC2489 + Seg3 pairings indicated successful and blocked cell fusion, respectively (Figure 1B). Transmission electron microscopy was used to assess whether cell fusion arrest observed for FGSC2489 + Seg3 pairings was due to failure in cell wall dissolution or in membrane merger. In samples of FGSC2489 cells alone or Seg3 cells alone, cell fusion was easily observed as indicated by dissolution of cell walls and plasma membrane at contact points (Figure 1C). In contrast, in mixtures of FGSC2489 + Seg3 cells, a high frequency of cell-cell contact sites showed an increase in cell wall material, consistent with a block in cell fusion during cell wall dissolution. To examine this phenotype further, we stained mixtures of FGSC2489 + Seg11 (compatible) versus FGSC2489 + Seg3 (incompatible) cells with the cell wall dye calcofluor white. Incompatible pairs of FGSC2489 + Seg3 germlings displayed significantly higher accumulation of dye at contact sites as compared to FGSC2489 + Seg11 pairs (Figure 1D). These data suggested that a cellular checkpoint is triggered upon cell-cell contact between genetically different cells that aborts fusion before cell wall dissolution is initiated. Members of a mitogen-activated protein kinase (MAPK) signaling complex (HAM-5/NRC-1/MEK-2/MAK-2) are recruited to fusion tips in germlings (termed conidial anastomosis tubes or CATs) [18Roca M.G. Arlt J. Jeffree C.E. Read N.D. Cell biology of conidial anastomosis tubes in Neurospora crassa.Eukaryot. Cell. 2005; 4: 911-919Crossref PubMed Scopus (130) Google Scholar] and to tips of fusion hyphae [12Fischer M.S. Glass N.L. Communicate and fuse: how filamentous fungi establish and maintain an interconnected mycelial network.Front. Microbiol. 2019; 10: 619Crossref PubMed Scopus (46) Google Scholar]. The MAK-2 complex assembles and disassembles at CAT tips every 8–10 min. A second protein complex bearing SOFT also assembles and disassembles at CAT tips but perfectly out of phase with the MAK-2 complex [19Fleissner A. Leeder A.C. Roca M.G. Read N.D. Glass N.L. Oscillatory recruitment of signaling proteins to cell tips promotes coordinated behavior during cell fusion.Proc. Natl. Acad. Sci. USA. 2009; 106: 19387-19392Crossref PubMed Scopus (117) Google Scholar]. When FGSC2489 cells expressing either MAK-2-GFP or SOFT-GFP were paired with compatible Seg11 cells, MAK-2 and SOFT oscillated to CATs during chemotropic interactions and disappeared during cell wall dissolution and membrane merger (Figures 2A and 2B ). In contrast, in arrested cell pairs (FGSC2489 + Seg3), MAK-2 and SOFT continued to oscillate at CAT tips long after cell contact (Figures 2A and 2B). These data indicated that germlings with a cell fusion block were unable to switch from communication to cell wall dissolution and membrane merger mode. To identify the causative locus of cell fusion arrest, we performed bulk segregant analysis (BSA) of progeny from the FGSC2489 × JW258 cross. After whole-genome resequencing, a region spanning approximately 1 Mb on chromosome V was identified that showed 100% SNP segregation between FGSC2489 fusion-compatible and FGSC2489 fusion-incompatible pools of progeny DNA (Figure S1B). Using genomic sequences from 26 N. crassa isolates [15Heller J. Clavé C. Gladieux P. Saupe S.J. Glass N.L. NLR surveillance of essential SEC-9 SNARE proteins induces programmed cell death upon allorecognition in filamentous fungi.Proc. Natl. Acad. Sci. USA. 2018; 115: E2292-E2301Crossref PubMed Scopus (41) Google Scholar, 16Heller J. Zhao J. Rosenfield G. Kowbel D.J. Gladieux P. Glass N.L. Characterization of greenbeard genes involved in long-distance kind discrimination in a microbial eukaryote.PLoS Biol. 2016; 14: e1002431Crossref PubMed Scopus (39) Google Scholar, 20Zhao J. Gladieux P. Hutchison E. Bueche J. Hall C. Perraudeau F. Glass N.L. Identification of allorecognition loci in Neurospora crassa by genomics and evolutionary approaches.Mol. Biol. Evol. 2015; 32: 2417-2432Crossref PubMed Scopus (31) Google Scholar], we identified three linked genes (NCU01380, NCU01381, and NCU01382), whose alleles showed high sequence diversity among individuals in this population and fell into six discrete haplogroups (Figure 3A). For example, the amino acid identity between FGSC2489 and JW228 for NCU01380, NCU01381, and NCU01382 was 75.2%, 73.0%, and 40.3%, respectively (Figure 3B). In contrast, NCU01379 showed 99.5% identity between FGSC2489 and JW228 (Figure 3B). The six haplogroups were completely conserved between alleles of NCU01380, NCU01381, and NCU01382 but only partially conserved for NCU01379 (Figure 3A), indicating recombination between NCU01379 and NCU01380-NCU01382. The high sequence diversity observed for NCU01380, NCU01381, and NCU01382 is a property of genes involved in allorecognition, such as self-incompatibility loci in plants [21Fujii S. Kubo K. Takayama S. Non-self- and self-recognition models in plant self-incompatibility.Nat. Plants. 2016; 2: 16130Crossref PubMed Scopus (122) Google Scholar] and heterokaryon incompatibility loci in fungi [20Zhao J. Gladieux P. Hutchison E. Bueche J. Hall C. Perraudeau F. Glass N.L. Identification of allorecognition loci in Neurospora crassa by genomics and evolutionary approaches.Mol. Biol. Evol. 2015; 32: 2417-2432Crossref PubMed Scopus (31) Google Scholar, 22Hall C. Welch J. Kowbel D.J. Glass N.L. Evolution and diversity of a fungal self/nonself recognition locus.PLoS ONE. 2010; 5: e14055Crossref PubMed Scopus (43) Google Scholar, 23Muirhead C.A. Glass N.L. Slatkin M. Multilocus self-recognition systems in fungi as a cause of trans-species polymorphism.Genetics. 2002; 161: 633-641PubMed Google Scholar, 24Paoletti M. Vegetative incompatibility in fungi: from recognition to cell death, whatever does the trick.Fungal Biol. Rev. 2016; 30: 152-162Crossref Scopus (35) Google Scholar]. To test whether NCU01380, NCU01381, and/or NCU01382 were required for cell fusion arrest, we assessed the phenotype of mutants bearing deletions of NCU01380, NCU01381, or NCU01382. Individual deletion strains ΔNCU01380, ΔNCU01381, and ΔNCU01382 were blocked in cell fusion with Seg3 cells (Figures 4A and 4B ), a phenotype identical to parental strain FGSC2489. However, a strain bearing a deletion of all three genes (ΔNCU01380ΔNCU01381ΔNCU01382) underwent cell fusion with both FGSC2489 and Seg3 cells (Figures 3C and 4B). To further test the hypothesis that allelic differences at NCU01380, NCU01381, and NCU01382 are causal for cell fusion arrest, we determined whether the block in cell fusion between wild isolates and FGSC2489 was dependent on NCU01380–NCU01382. First, we used strain JW196 (same haplogroup as FGSC2489; Figure 3A), and as predicted, JW196 fused with FGSC2489. However, cell fusion was blocked between FGSC2489 and wild isolates from the other haplogroups (JW228, JW199, JW258, D111, and JW242; Figure S3). But when JW228, JW199, JW258, D111, or JW242 were paired with the triple-deletion strain ΔNCU01380ΔNCU01381ΔNCU01382, cell fusion was restored (Figure S3). These data confirmed that genetic differences at NCU01380, NCU01381, and NCU01382 were responsible for cell fusion arrest. To determine whether all three genes were required for cell fusion arrest, we constructed double mutant strains ΔNCU01380 ΔNCU01381, ΔNCU01381 ΔNCU01382, and ΔNCU01380 ΔNCU01382. The double mutants ΔNCU01380 ΔNCU01381 and ΔNCU01381 ΔNCU01382 arrested during fusion in pairings with Seg3 cells. However, the ΔNCU01380 ΔNCU01382 double mutant underwent cell fusion with both FGSC2489 and Seg3 cells (Figures 4A and 4B). The triple mutant transformed with a GFP-tagged NCU01381 allele also underwent cell fusion with both FGSC2489 and Seg3 germlings (Figures 4A and 4B). These data indicated that allelic differences at NCU01380 and NCU01382, but not NCU01381, were required for the block in cell fusion. We named NCU01380 cell wall remodeling checkpoint-1 (cwr-1) and NCU01382 cell wall remodeling checkpoint-2 (cwr-2). The morphological or growth phenotypes of any of the mutant combinations (including triple-deletion strain Δcwr-1ΔNCU01381Δcwr-2) were not significantly different from the parental FGSC2489 strain (Figure 3D). However, although triple mutant germlings underwent chemotropic interactions and self-fusion, they required significantly more time to fuse after contact, in comparison to FGSC2489 germlings (Figure 3E). These data indicated that cwr-1, NCU01381, and cwr-2 contributed to the efficiency of self-fusion events during cell wall dissolution. To determine whether CWR-1 or CWR-2 were sufficient to induce cell fusion arrest, we cloned cwr-1, cwr-2, and NCU01381 alleles from JW228 (incompatible with FGSC2489; Figure S3). The cwr-1JW228, cwr-2JW228, and NCU01381JW228 alleles were transformed individually into the Δcwr-1ΔNCU01381Δcwr-2 mutant. Homokaryotic strains bearing cwr-1JW228, cwr-2JW228, or NCU01381JW228 alleles were paired with FGSC2489, and fusion frequency was assessed microscopically and by flow cytometry. Consistent with cwr-1 and cwr-2 playing an essential role in a cell wall remodeling checkpoint, expression of either cwr-1JW228 or cwr-2JW228 alleles in the triple deletion strain was sufficient to induce cell fusion arrest with an otherwise isogenic parental strain FGSC2489 (Figures 4C and 4D). These data also indicated that incompatibility functioned in trans (between cells), as a strain carrying only one allele at cwr-1 or cwr-2 was blocked in cell fusion with an incompatible strain. cwr-1 encodes a predicted polysaccharide monooxygenase (PMO), with a signal peptide, a linker region rich in glycine and serine residues, and a carbohydrate-binding domain (Figure 3B). PMOs are an auxiliary activity (AA) within the carbohydrate-active enZYmes database [25Span E.A. Marletta M.A. The framework of polysaccharide monooxygenase structure and chemistry.Curr. Opin. Struct. Biol. 2015; 35: 93-99Crossref PubMed Scopus (60) Google Scholar]. CWR-1 shows substantial homology to a chitin-active copper-dependent AA11 PMO from Aspergillus oryzae [26Hemsworth G.R. Henrissat B. Davies G.J. Walton P.H. Discovery and characterization of a new family of lytic polysaccharide monooxygenases.Nat. Chem. Biol. 2014; 10: 122-126Crossref PubMed Scopus (266) Google Scholar] (Figure S4A). Of the 22 PMOs in N. crassa, four fell into the AA11 subtype, including NCU00822, NCU05932, NCU05404, and CWR-1 (Figure S4B). cwr-2 encodes a protein containing two DUF3433 (domain of unknown function) and eight predicted transmembrane regions (Figure 3B). The PMO portion of CWR-1 contained conserved amino acid residues associated with catalytic activity, including the histidine brace and a hydrogen-bonding network (Figure S4A) [25Span E.A. Marletta M.A. The framework of polysaccharide monooxygenase structure and chemistry.Curr. Opin. Struct. Biol. 2015; 35: 93-99Crossref PubMed Scopus (60) Google Scholar]. To test whether PMO catalytic activity was essential for cell fusion arrest, an allele with a substitution of Y159A (cwr-1 Y159A) was constructed (Figures 4E and 4F). This tyrosine is strictly conserved among AA11 PMOs and, when mutated in other AA families, leads to loss in catalytic activity [27Harris P.V. Welner D. McFarland K.C. Re E. Navarro Poulsen J.C. Brown K. Salbo R. Ding H. Vlasenko E. Merino S. et al.Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family.Biochemistry. 2010; 49: 3305-3316Crossref PubMed Scopus (593) Google Scholar]. Although introduction of cwr-1FGSC2489 into the triple-deletion mutant restored cell fusion arrest in pairings with incompatible JW199 cells, a triple-deletion mutant carrying cwr-1 Y159A underwent cell fusion. These data indicated that PMO catalytic activity is required for triggering the cell wall remodeling checkpoint. Our data indicated that cwr-1 or cwr-2 were sufficient for triggering cell fusion arrest in cells with incompatible cwr alleles. To investigate whether allelic versus non-allelic interactions were important for conferring this block, we expressed incompatible cwr-1/cwr-2 alleles in a single strain. The cwr-1JW228 and cwr-2JW228 alleles were introduced into FGSC2489 (i.e., containing cwr-1FGSC2489 cwr-2FGSC2489); strains co-expressing incompatible cwr-1 and cwr-2 alleles produced shorter aerial hyphae and asexual spores that were paler than parental FGSC2489 spores (Figures 5A, 5B, and S5A). This phenotype was not observed when cwr-1JW228 and cwr-2JW228 alleles were co-expressed in the triple deletion mutant (Δcwr-1ΔNCU01381Δcwr-2; Figure S5A). Introduction of a cwr-1 allele from a different haplogroup (cwr-1D111) into FGSC2489 also resulted in abnormal growth (Figures 5A and 5B). W" @default.
• W2971352197 created "2019-09-05" @default.
• W2971352197 creator A5001850906 @default.
• W2971352197 creator A5018625809 @default.
• W2971352197 creator A5019962582 @default.
• W2971352197 creator A5033319200 @default.
• W2971352197 creator A5039561796 @default.
• W2971352197 creator A5053941734 @default.
• W2971352197 creator A5056577532 @default.
• W2971352197 creator A5072187410 @default.
• W2971352197 creator A5086045650 @default.
• W2971352197 date "2019-09-01" @default.
• W2971352197 modified "2023-10-15" @default.
• W2971352197 title "Allorecognition upon Fungal Cell-Cell Contact Determines Social Cooperation and Impacts the Acquisition of Multicellularity" @default.
• W2971352197 cites W1778113906 @default.
• W2971352197 cites W1931626364 @default.
• W2971352197 cites W1984529935 @default.
• W2971352197 cites W1990155217 @default.
• W2971352197 cites W1992058072 @default.
• W2971352197 cites W2000096092 @default.
• W2971352197 cites W2007289706 @default.
• W2971352197 cites W2008515795 @default.
• W2971352197 cites W2009280448 @default.
• W2971352197 cites W2009914881 @default.
• W2971352197 cites W2011485738 @default.
• W2971352197 cites W2012988322 @default.
• W2971352197 cites W2013786215 @default.
• W2971352197 cites W2030825540 @default.
• W2971352197 cites W2032061457 @default.
• W2971352197 cites W2041099435 @default.
• W2971352197 cites W2041431867 @default.
• W2971352197 cites W2043331015 @default.
• W2971352197 cites W2060081344 @default.
• W2971352197 cites W2063908784 @default.
• W2971352197 cites W2075132251 @default.
• W2971352197 cites W2076198589 @default.
• W2971352197 cites W2084311881 @default.
• W2971352197 cites W2091448229 @default.
• W2971352197 cites W2091513159 @default.
• W2971352197 cites W2099407265 @default.
• W2971352197 cites W2099540110 @default.
• W2971352197 cites W2100516574 @default.
• W2971352197 cites W2108877607 @default.
• W2971352197 cites W2108998914 @default.
• W2971352197 cites W2110418787 @default.
• W2971352197 cites W2111211467 @default.
• W2971352197 cites W2112050613 @default.
• W2971352197 cites W2112358579 @default.
• W2971352197 cites W2119109707 @default.
• W2971352197 cites W2142529984 @default.
• W2971352197 cites W2144100913 @default.
• W2971352197 cites W2149813126 @default.
• W2971352197 cites W2152112359 @default.
• W2971352197 cites W2157186150 @default.
• W2971352197 cites W2160378127 @default.
• W2971352197 cites W2167474377 @default.
• W2971352197 cites W2168107110 @default.
• W2971352197 cites W2179113683 @default.
• W2971352197 cites W2311203695 @default.
• W2971352197 cites W2341058059 @default.
• W2971352197 cites W2346052203 @default.
• W2971352197 cites W2519821319 @default.
• W2971352197 cites W2542682147 @default.
• W2971352197 cites W2758554095 @default.
• W2971352197 cites W2767122808 @default.
• W2971352197 cites W2788464082 @default.
• W2971352197 cites W2803061241 @default.
• W2971352197 cites W2925408623 @default.
• W2971352197 cites W2952656033 @default.
• W2971352197 doi "https://doi.org/10.1016/j.cub.2019.07.060" @default.
• W2971352197 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/31474536" @default.
• W2971352197 hasPublicationYear "2019" @default.
• W2971352197 type Work @default.
• W2971352197 sameAs 2971352197 @default.
• W2971352197 citedByCount "41" @default.
• W2971352197 countsByYear W29713521972019 @default.
• W2971352197 countsByYear W29713521972020 @default.
• W2971352197 countsByYear W29713521972021 @default.
• W2971352197 countsByYear W29713521972022 @default.
• W2971352197 countsByYear W29713521972023 @default.
• W2971352197 crossrefType "journal-article" @default.
• W2971352197 hasAuthorship W2971352197A5001850906 @default.
• W2971352197 hasAuthorship W2971352197A5018625809 @default.
• W2971352197 hasAuthorship W2971352197A5019962582 @default.
• W2971352197 hasAuthorship W2971352197A5033319200 @default.
• W2971352197 hasAuthorship W2971352197A5039561796 @default.
• W2971352197 hasAuthorship W2971352197A5053941734 @default.
• W2971352197 hasAuthorship W2971352197A5056577532 @default.
• W2971352197 hasAuthorship W2971352197A5072187410 @default.
• W2971352197 hasAuthorship W2971352197A5086045650 @default.
• W2971352197 hasBestOaLocation W29713521971 @default.
• W2971352197 hasConcept C104317684 @default.
• W2971352197 hasConcept C144024400 @default.
• W2971352197 hasConcept C1491633281 @default.
• W2971352197 hasConcept C18903297 @default.
• W2971352197 hasConcept C200315206 @default.
• W2971352197 hasConcept C207936829 @default.
• W2971352197 hasConcept C2993273237 @default.

• next