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• W4313037811 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Organisms require the ability to differentiate themselves from organisms of different or even the same species. Allorecognition processes in filamentous fungi are essential to ensure identity of an interconnected syncytial colony to protect it from exploitation and disease. Neurospora crassa has three cell fusion checkpoints controlling formation of an interconnected mycelial network. The locus that controls the second checkpoint, which allows for cell wall dissolution and subsequent fusion between cells/hyphae, cwr (cell wall remodeling), encodes two linked genes, cwr-1 and cwr-2. Previously, it was shown that cwr-1 and cwr-2 show severe linkage disequilibrium with six different haplogroups present in N. crassa populations. Isolates from an identical cwr haplogroup show robust fusion, while somatic cell fusion between isolates of different haplogroups is significantly blocked in cell wall dissolution. The cwr-1 gene encodes a putative polysaccharide monooxygenase (PMO). Herein we confirm that CWR-1 is a C1-oxidizing chitin PMO. We show that the catalytic (PMO) domain of CWR-1 was sufficient for checkpoint function and cell fusion blockage; however, through analysis of active-site, histidine-brace mutants, the catalytic activity of CWR-1 was ruled out as a major factor for allorecognition. Swapping a portion of the PMO domain (V86 to T130) did not switch cwr haplogroup specificity, but rather cells containing this chimera exhibited a novel haplogroup specificity. Allorecognition to mediate cell fusion blockage is likely occurring through a protein–protein interaction between CWR-1 with CWR-2. These data highlight a moonlighting role in allorecognition of the CWR-1 PMO domain. Editor's evaluation This fundamental study identifies an important role for a lytic polysaccharide monooxygenase in allorecognition in the filamentous fungus Neurospora crassa, which is independent of the catalytic activity of this remarkable class of proteins. The study's findings are compelling, combining microscopy with genetics and biochemistry. The study will be of great interest to fungal biologists and microbiologists, as well as biochemists studying carbohydrate-active enzymes. https://doi.org/10.7554/eLife.80459.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Allorecognition is the ability of a cell to recognize self or kin and has widespread importance for many organisms that have multicellular organization. Allorecognition in some form is utilized by the social amoeba, Dictyostelium discoideum (Kundert and Shaulsky, 2019; Kuzdzal-Fick et al., 2011), the bacterium, Proteus mirabilis (Gibbs et al., 2008; Gibbs and Greenberg, 2011), gymnosperms (Pandey, 1960), slime molds (Clark, 2003; Shaulsky and Kessin, 2007), and invertebrates, such as Botryllus schlosseri (Detomaso et al., 2005; Rosengarten and Nicotra, 2011; Yoshito et al., 2008) or the cnidarian Hydractinia symbiolongicarpus (Rosengarten and Nicotra, 2011). In Drosophila melanogaster, the dscam gene, which contains >19,000 splicing isoforms, uses differential splicing and homo-dimer binding to recognize and not synapse with itself (Wojtowicz et al., 2004; Wojtowicz et al., 2007). In vertebrate species, the major histocompatibility complex (MHC) is crucial for the immune response and allows cells to identify infected cells or cells that are no longer kin for destruction (Afzali et al., 2008; Marino et al., 2016). Although not strictly multicellular, most filamentous fungi grow as an interconnected hyphal network that shares cytoplasm, nuclei, and nutrients, and utilizes allorecognition machinery to regulate this process (Gonçalves and Glass, 2020). This syncytial organization of filamentous fungi allows an interconnected colony to thrive in an environment that is heterogeneous for nutrients and habitats (Anna et al., 2012). The formation of an interconnected mycelial network is a process that is controlled by communication and cell fusion checkpoints to help ensure that cells with higher genetic identity form a colony (Gonçalves et al., 2020; Gonçalves et al., 2020). Without checkpoints, cell fusion can occur between genetically unrelated colonies and cells, allowing ‘cheater’ nuclei to steal resources (Bastiaans et al., 2016; Grum-Grzhimaylo et al., 2021) or enabling the spread of cytoplasmic diseases such as mycoviruses (Zhang and Nuss, 2016) or selfish genetic elements (Debets et al., 2012), which are spread via cell fusion throughout fungal populations. The filamentous ascomycete species, Neurospora crassa, has three cell fusion checkpoints that function in allorecognition. The first checkpoint regulates chemotropic interactions between hyphae and/or germinated asexual spores (germlings) and is controlled by the determinant of communication (doc) locus that senses chemosignaling (Heller et al., 2016). Cells with identical allelic specificity at the doc locus undergo chemotropic interactions, while cells with alternate doc allelic specificity show greatly reduced interactions. The second checkpoint is regulated by the cell wall remodeling locus, cwr, which contains the two linked genes, cwr-1 (UniProtKB Q1K703) and cwr-2 (UniProtKB Q1K701) (Gonçalves et al., 2019). Cells/hyphae with identical allelic specificity at doc and cwr undergo chemotropic interactions and, upon contact, undergo cell wall dissolution, membrane merger, and cytoplasmic mixing. However, if cells have identical doc specificity, but differ in cwr allelic specificity, hyphae/cells undergo chemotropic interactions, but upon contact, cells remain adhered and fail to undergo cell wall deconstruction and membrane merger at the point of contact (Gonçalves et al., 2019). The final checkpoint is a post-fusion checkpoint. Following chemotrophic interactions (doc alleles with identical allelic specificity) and cell fusion (cwr alleles with identical allelic specificity), if cells/hyphae differ in specificity at several post-fusion allorecognition loci, the fusion cell is compartmentalized and undergoes rapid cell death (Daskalov et al., 2019; Heller et al., 2018; Rico-Ramírez et al., 2022). Recent data in N. crassa showed that one of these post-fusion allorecognition loci has functional and structural similarity to mammalian gasdermin and confers rapid cell death via a pyroptotic-like mechanism (Corinne et al., 2022; Daskalov and Glass, 2022; Daskalov et al., 2020; Rico-Ramírez et al., 2022) while in the related filamentous fungus, Podospora anserina, a different post-fusion cell death allorecognition locus has functional similarities to necroptosis (Saupe, 2011; Saupe, 2020). The cwr-1 gene is predicted to encode a chitin-active polysaccharide monooxygenase (PMO) in the CAZy (Carbohydrate-Active enZYme) database designation of Auxiliary Activity 11 (AA11) family (Levasseur et al., 2013). The cwr-2 gene encodes a predicted protein that contains eight transmembrane regions with two annotated domains of unknown function (PF11915). Within populations of N. crassa, cwr-1/cwr-2 alleles show severe linkage disequilibrium and fall into six different haplogroups (HGs). Cells/germlings containing only CWR-1 from HG1 are blocked in cell fusion with isogenic cells containing only CWR-2 from a different haplogroup (Gonçalves et al., 2019). These data indicate that incompatible CWR-1-CWR-2 function in trans when present in different interacting cells and are necessary and sufficient to trigger the cell fusion block. PMOs, alternatively referred to as lytic polysaccharide monooxygenases (LPMOs), have been studied extensively for over a decade (Phillips et al., 2011; Vaaje-Kolstad et al., 2010). These proteins were first discovered as auxiliary redox enzymes that greatly enhance cellulose degradation in combination with glycosyl hydrolases (Harris et al., 2010). Beyond just cellulolytic activity, other PMOs have been demonstrated to show activity on other polysaccharides such as chitin (Hemsworth et al., 2014), starch (Vu et al., 2014b; Lo Leggio et al., 2015; Vu et al., 2019; Vu and Marletta, 2016), xylans (Couturier et al., 2018; Hüttner et al., 2019), and various hemicelluloses (Agger et al., 2014; Monclaro et al., 2020). PMOs have been of fundamental interest because they catalyze the hydroxylation of a strong C–H bond and their utility in industrial biofuel and nanocellulose applications (Johansen, 2016; Moreau et al., 2019). However, as more PMO families have been identified, other roles have been noted (Vandhana et al., 2022; Hangasky et al., 2020), including in the life cycle of insect viruses (Chiu et al., 2015), insect development (Sabbadin et al., 2018), fungal development (Fu et al., 2014; Gonçalves et al., 2019; Maddi et al., 2012), and in symbiotic associations between bacteria and arthropods (Distel et al., 2011; Pinheiro et al., 2015). A family of related proteins termed X325 have a similar fold and bind copper but are not functional PMOs. Instead one member of this family has a unique biological role in copper transport (Garcia-Santamarina et al., 2020). All PMOs contain a secretion signal peptide that is cleaved, leaving the mature protein with an N-terminal histidine residue that binds a single copper atom (Phillips et al., 2011). Of the four other proteins characterized in the AA11 family, only a chitin-degradation role has been demonstrated (Hemsworth et al., 2014; Rieder et al., 2021; Støpamo et al., 2021; Wang et al., 2018a; Wang et al., 2018b). Three of the four proteins have a similar protein architecture composed of an N-terminal PMO domain followed by a glycine serine (GS)-rich disordered linker and an X278 domain at the C-terminus. The X278 domain has been compared to CBM sequences (Hemsworth et al., 2014). The recently published Alphafold structure in UniProt (UniProtKB Q1K703) shows two aromatic residues on the same side of a small predominant beta-sheet domain that may be positioned to bind polysaccharides. If this is a true CBM, it likely binds chitin as it is also found in GH18 chitinases (Hemsworth et al., 2014). In this study, we assessed whether CWR-1 from each of the six different haplogroups has PMO activity and defined the substrate and products of that PMO activity. We also assessed whether CWR-1 proteins from each haplogroup formed unique products that would explain CWR-1 allelic specificity. By the further construction of PMO domain chimeras, we evaluated whether a polymorphic region of the PMO domain (LS and LC loop) was a factor in triggering allorecognition in interactions with CWR-2. Our data indicates that CWR-1 has chitin PMO activity, but that catalytic activity was not required for cell fusion blockage, highlighting an evolved and additional function of this PMO in allorecognition. Results The PMO domain of CWR-1 is sufficient to confer an allorecognition checkpoint A multiple sequence alignment (MSA) of characterized AA11s revealed that three of the four characterized PMOs maintain a three-part architecture, while the other is a single-domain PMO containing only a catalytic domain (Figure 1—figure supplement 1). The three-domain architecture includes an N-terminus AA11 PMO domain Hemsworth et al., 2014; Levasseur et al., 2013 followed by an extended GS-rich linker region that is likely disordered, and a C-terminal X278 domain proposed to be a chitin-binding domain; this domain is present in other predicted AA11 proteins and GH18 chitinases (Gonçalves et al., 2019; Hemsworth et al., 2014; Figure 1A). AA11 proteins, similar to all other PMOs, contain a signal peptide at the N-terminus (predicted by SignalP) that directs the protein to the ER and that is subsequently cleaved during translocation, leaving a histidine residue at the N-terminus. Figure 1 with 6 supplements see all Download asset Open asset Characterization of the polysaccharide monooxygenase (PMO) domain from CWR-1 from a haplogroup 1 strain (FGSC2489) and functional dissection of CWR-1 domains in vivo. (A) A schematic diagram depicting the series of truncated cwr-1 constructs studied. SP indicates signal peptide; GS linker indicates the glycine/serine-rich region that connects the PMO catalytic domain to the presumptive chitin-binding module, X278. (B) Cell fusion assays between Δcwr-1Δ81Δcwr-2 germlings alone or bearing either HG1 cwr-1 (FGSC2489) or truncated versions cwr-1ΔGS, cwr-1ΔGSΔX278 (all expressing cytoplasmic GFP) and subsequently paired with FM4-64-stained Δcwr-1Δ81Δcwr-2 germlings expressing an HG3 cwr-2 allele (from JW258). (see Figure 1—figure supplement 3 for fusion rates with all haplogroups [HGs] with HG1 and fusion controls). (C) Quantification of cell fusion frequencies shown in (B) of Δcwr-1Δ81Δcwr-2 (GFP) germlings (ΔΔΔ), or ΔΔΔ germlings bearing HG1 cwr-1HG1 or ΔΔΔ germlings bearing truncated versions cwr-1ΔG/S or cwr-1ΔGSΔX278 and paired with FM4-64-stained ΔΔΔ germlings expressing an HG3 cwr-2 allele. Experiments were performed in biological triplicate, assessing fusion of 100 germling pairs for each replicate. A one-way ANOVA followed by Tukey’s post-hoc test was used for statistical analysis, error bars represent SD, *p<0.05, ****p<0.0001. Individual p-values are reported in Figure 1—source data 4. (D) Schematic depiction of the E. coli expression constructs. pelB indicates the signal peptide, the PMO domain from an HG1 strain (FGSC2489), and a thrombin-cleavable hexahistidine tag showing cleavage site at the indicated triangle (see Figure 1—figure supplement 4 for protein gel and MS of purified protein). (E) Initial characterization of the PMO domain from HG1 strain (FGSC2489). This PMO exhibited C1-oxiding activity on chitin in the presence of ascorbate, reduced oxygen in the absence of chitin, and bound one copper atom per protein, all properties consistent with previously characterized AA11 PMOs. ICP analyses were done in technical triplicate, each datapoint in the oxygen reduction assay represents a biological replicate. All HPAEC-PAD assays were done in at least biological triplicate with a typical trace shown. Asc. means ascorbate. Black arrows denote peaks that elute in the region of the method corresponding to C1-oxidized oligosaccharides. The copper stoichiometry and oxygen reduction assay error bars are SEM where n=3. Source data for this figure can be found in Figure 1—source data 1, Figure 1—source data 2, and Figure 1—source data 3. See Figure 1—figure supplement 5 for oxidized standards and Figure 1—figure supplement 6 for MS/MS spectra on PMO products from α-chitin. Figure 1—source data 1 HPAEC-PAD source data. Blank subtracted HPAEC-PAD traces. The samples were prepared as described in ‘Materials and methods'. The time and nC response are listed in the table. https://cdn.elifesciences.org/articles/80459/elife-80459-fig1-data1-v2.xlsx Download elife-80459-fig1-data1-v2.xlsx Figure 1—source data 2 ICP source data. The amount of bound copper that was calculated for each polysaccharide monooxygenase (PMO) construct. The amount of copper was determined using ICP-MS and then divided by the protein concentration to generate a ratio. The protein concentration was determined using the absorbance at 280 nm with a NanoDrop UV-Vis with extinction coefficients predicted by Benchling (all Cys oxidized). The results from the technical triplicates are listed. https://cdn.elifesciences.org/articles/80459/elife-80459-fig1-data2-v2.xlsx Download elife-80459-fig1-data2-v2.xlsx Figure 1—source data 3 Horseradish peroxidase (HRP)-oxygen reduction assay source data. The slopes from monitoring polysaccharide monooxygenase (PMO)-catalyzed oxygen reduction using an HRP-coupled assay. A PMO sample (2 µM) was incubated at room temperature with 100 µM Amplex red, 1.3 µM HRP (Sigma), 2 mM ascorbate in 50 mM MOPS pH 7.0. The change in absorbance at 540 nm vs. time (min) is listed for each biological replicate in this table. https://cdn.elifesciences.org/articles/80459/elife-80459-fig1-data3-v2.xlsx Download elife-80459-fig1-data3-v2.xlsx Figure 1—source data 4 p-Values. Fusion test of the truncated versions of CWR-1 paired with cwr-2HG3; ΔΔΔ. https://cdn.elifesciences.org/articles/80459/elife-80459-fig1-data4-v2.xlsx Download elife-80459-fig1-data4-v2.xlsx CWR-1 is a member of the three-region architecture cluster, and many fungi contain both AA11 PMO architectures in their genome. To more fully examine the relationships of predicted AA11 proteins, we performed sequence similarity network (SSN) analysis using the EFI-EST BLAST feature with the CWR-1HG1 sequence as the search peptide, which yielded ~1700 sequences. The three characterized AA11 proteins and CWR-1 that contained the GS linker and X278 domain were found in a single cluster (blue) (Figure 1—figure supplement 2). The single AA11 domain enzyme (AfAA11A) clustered separately into the red cluster. Since these domain architectures are clustered separately, this suggests a different biological function in fungi (Figure 1—figure supplement 2). This is not solely due to the difference in domain organization, for example, starch PMOs with different architectures cluster together at ~40% sequence ID (Vu et al., 2019). To determine which domain of CWR-1 was involved in allorecognition and the fusion checkpoint, a series of truncations were generated in the HG1 (FGSC2489) cwr-1 allele: full-length cwr-1, removal of the GS linker, and removal of the GS linker and X278 domain (Figure 1A). Full-length and truncated constructs were inserted at the his-3 locus under the native promoter (1111 bp) and transformed into a strain of N. crassa bearing a deletion of cwr-1 (NCU01380) and cwr-2 (NCU01382) (Δcwr-1Δ81Δcwr-2) and expressing cytoplasmic GFP (Gonçalves et al., 2019). These strains were assessed for cell fusion in pairings with isogenic strains containing the cwr-2 allele from each of the HG strains (HG1-6). The cwr-1HG1 strain gave the strongest cell fusion block in pairings with cwr-2HG3 germlings (Figure 1—figure supplement 3A and B). This cwr-2HG3 strain was therefore used to test strains harboring the cwr-1HG1 truncations that lacked the GS linker or the GS linker and the X278 domain. Strains containing any of the cwr-1 truncations showed a block in cell fusion with the cwr-2HG3 strain, including the truncation strain with only the catalytic PMO domain (Figure 1B and C). As a control, all of the truncation strains were also paired with a permissive mutant lacking cwr-1 and cwr-2 (Δcwr-1Δ81Δcwr-2); all showed robust cell fusion frequencies (Figure 1—figure supplement 3C and D). These data showed that the PMO catalytic domain was sufficient to cause cell fusion arrest at the CWR allorecognition checkpoint. Given that the PMO catalytic domain alone was responsible for conferring allorecognition, we performed experiments to probe the activity for this domain. The PMO catalytic domain of the cwr-1HG1 allele was expressed in Escherichia coli using the periplasmic expression pelB system, which cleaves scarlessly during expression to ensure an N-terminal histidine with a C-terminal cleavable hexahistidine tag (Figure 1D) and subsequently purified using Ni-IMAC. The protein ran slightly higher (~26–27 kDa) on an SDS-PAGE gel than the predicted size of 22.6 kDa (Figure 1—figure supplement 4A); the mass was validated with whole-protein mass spectrometry. The spectrum revealed a deconvoluted mass of 22,624 Da, which corresponds to the exact mass of the protein with three disulfide bonds (22,630 – 3 × (2 Da for each disulfide)) (Figure 1—figure supplement 4B). The CWR-1HG1 PMO domain bound ~0.7 equivalents of copper after reconstitution and was shown to reduce oxygen to hydrogen peroxide in the presence of ascorbate (Figure 1E). The activity of the CWR-1HG1 protein was then tested with β-chitin as a substrate. New peaks between 18 and 22 min that corresponded to C1-oxidized products were only observed in the presence of both ascorbate and the CWR-1HG1 PMO domain (Figure 1E). C1-oxidized standards were generated from chitooligosaccharides (Figure 1—figure supplement 5A) using the AA3, chitin-C1-oxidizing enzyme, ChitO (from Fusarium graminearum) (Figure 1—figure supplement 5B). The ChitO-C1-oxidized oligosaccharides eluted at timepoints similar to the new peaks from CWR-1HG1 PMO at around 18–22 min (Figure 1—figure supplement 5B, Figure 1E). To further validate the CWR-1HG1 C1 regioselectivity, tandem MS/MS was performed on the crude reaction from PMOHG1 action on α-chitin in the presence of ascorbate. The resulting spectra were consistent with C1 oxidation (Figure 1—figure supplement 6). These data were in line with all other characterized AA11 PMOs that exhibit C1-oxidative activity on both α-chitin and β-chitin. Our data showed that the PMO domain of CWR-1HG1 was the driver for fusion arrest at the cwr allorecognition checkpoint. We therefore compared sequences of the PMO domain bioinformatically from the six different CWR-1 haplogroups. The phylogenetic tree showed identical clades to a tree created using full-length CWR-1 sequences (Gonçalves et al., 2019; Figure 2A). The intra-haplogroup similarity was very high with >95% sequence identity across the PMO domain for alleles within a haplogroup (Supplementary file 1e). All six PMO haplogroups contained the residues essential for PMO activity; two histidines in the histidine brace and residues implicated in the catalytic mechanism, including a tyrosine involved in the secondary coordination sphere and other conserved hydrogen-bonding residues (Span et al., 2017). Of note, the regions that have the most inter-haplogroup PMO domain differences corresponded to the AA9 nomenclature of LC, LS, and L2 loops (Danneels et al., 2018; Liu et al., 2018), which have been shown to play a role in substrate binding and specificity. There were also minor differences in the L8 loop located opposite the substrate-binding surface of the PMO domain (Figure 2B). The differences between the different haplogroup PMO domains were more apparent when a homology model was constructed using the crystal structure of AoAA11 (PDB: 4MAI) as a template (Figure 2C). Figure 2 Download asset Open asset Comparison of CWR-1 polysaccharide monooxygenase (PMO) domains. In all panels, blue indicates cwr-1 predicted proteins from HG1 isolates, red indicates HG2, light blue indicates HG3, orange indicates HG4, purple indicates HG5, and green indicates HG6 isolates. (A) A phylogenetic tree was constructed using the predicted CWR-1 PMO domain from 26 wild-type isolates. The phylogenetic tree was made using PhyML Phylogenetic Maximum Likelihood and edited in MEGA11. (B). Alignment of the six CWR-1 PMO domain protein sequences from representative isolates from HG1-6. The red arrows show the two histidine residues of the histidine brace that are involved in copper coordination. The blue arrow shows the position of the tyrosine involved in the secondary coordination sphere. L2, LS, and LC correspond to loops that exhibited sequence variation between the CWR-1 PMO domains among the six different haplogroups. (C) A SwissProt homology model of the PMO domain from the six haplogroups. There are apparent differences in the outer loops, whereas the core of the protein is not predicted to have significant differences between haplogroups. The four most affected loops are the loops that correspond to AA9 LS, L2, LC, and L8 loops (middle panel). A portion of the LS loop (left panel) has the most striking differences between haplogroups, and a portion of the LC loop (right panel) contains the second-best region for allelic differences. It is possible that the product of CWR-1 from each of the haplogroups was either generating a different product profile or recognized a different substrate. Mutations along substrate binding loops can change the regioselectivity of PMOs (C1 vs. C4) partially or entirely, and these residues can determine soluble substrate preferences (Courtade et al., 2018; Danneels et al., 2018; Liu et al., 2018; Vu et al., 2014a). The two regions that exhibited the most prominent differences were the LC and LS loops, which was consistent with the hypothesis that a substrate/product(s) could be the signal for the cwr allorecognition checkpoint (Figure 2C). Our in vivo data indicated that the PMO domain from cwr-1HG1 was sufficient to confer the cwr allorecognition checkpoint. We therefore tested whether the full-length CWR-1 protein from each haplogroup was sufficient for fusion arrest and for conferring allelic specificity. Full-length cwr-1 alleles from each haplogroup were expressed in a Δcwr-1Δ81Δcwr-2 strain and confronted against cwr-1HG1 cwr-2HG1 germlings. As expected, strains bearing cwr-1 from any of the five different haplogroups showed significantly lower fusion rates as compared to a strain carrying cwr-1HG1 and paired with cwr-1HG1 cwr-2HG1 germlings (Figure 3A and D). As a control to ensure fusion machinery is not impaired, strains bearing the six different cwr-1 haplotypes showed high fusion rates with the Δcwr-1Δ81Δcwr-2 strain (Figure 3—figure supplement 1A and C). Figure 3 with 1 supplement see all Download asset Open asset The polysaccharide monooxygenase (PMO) domain in CWR-1 functions to confer the allorecognition fusion checkpoint. (A) Micrographs of the dominant fusion events between germlings expressing cwr-1 alleles from each haplogroup in a Δcwr-1Δ81Δcwr-2 GFP background when paired with cwr-1HG1 cwr-2HG1 germlings (FGSC2489) stained with FM4-64. (B) CWR-1 chimeras with the PMO domain from the different cwr haplogroups (cwr-1HG2 from D111, cwr-1HG3 from JW258, cwr-1HG4 from JW242, cwr-1HG5 from P4476, and cwr-1HG6 from JW228), with the glycine serine linker and X278 domains from cwr-1HG1 (from FGSC2489) are schematically shown. (C) Micrographs of the dominant fusion events between germlings expressing the chimeric CWR-1 proteins paired with cwr-1HG1 cwr-2HG1 germlings stained with FM4-64. (D) Quantification of cell fusion frequencies depicted in (A) and (C). The experiments were performed in biological triplicate, assessing fusion of 100 germling pairs for each replicate. A one-way ANOVA followed by Tukey’s post-hoc test was used for statistical analysis, error bars represent SD, *p<0.05, ****p<0.0001. Individual p-values are reported in Figure 3—source data 1. (E) Micrographs of the dominant fusion event between GFP germlings expressing the chimeric CWR-1 proteins paired with a Δcwr-1 cwr-2HG1 strain stained with FM4-64. (F) Quantification of cell fusion frequencies between 100 germlings depicted in (E). See Figure 3—figure supplement 1 for control experiments. Figure 3—source data 1 p-Values. Figure 3D. Fusion test of the different CWR-1 full-length and CWR-1 chimeras paired with cwr-1HG1cwr-2HG1. https://cdn.elifesciences.org/articles/80459/elife-80459-fig3-data1-v2.xlsx Download elife-80459-fig3-data1-v2.xlsx To determine whether the PMO domain from the CWR-1 proteins from all six different haplogroups was essential for allorecognition, chimeric constructs containing the N-terminal PMO domain from each HG (2–5) were fused to the GS linker region and X278 domain of cwr-1HG1 (Figure 3B) in a Δcwr-1Δ81Δcwr-2 strain (which also expressed cytoplasmic GFP). The resulting germlings were paired with a cwr-1HG1 cwr-2HG1 strain, a Δcwr-1Δ81Δcwr-2 mutant, and a Δcwr-1 cwr-2HG1 strain. The fusion percentages between germlings carrying any of the HG2-5 cwr-1 chimeric constructs were low when paired with a cwr-1HG1 cwr-2HG1 strain or with the Δcwr-1 cwr-2HG1 strain, but were high when paired with the permissive Δcwr-1Δ81Δcwr-2 mutant (Figure 3C–F, Figure 3—figure supplement 1B and C). No significant differences were observed in percentages of fusion when either full-length cwr-1 from the different haplogroups or with chimeric cwr-1 alleles with the PMO domain from the different haplogroups were paired with a cwr-1HG1 cwr-2HG1 germlings (Figure 3D). These experiments showed that allorecognition is mediated in trans between a cell containing cwr-1 and a cell containing cwr-2 from the different haplogroups and provide strong evidence that the PMO domain from the six different haplogroups conferred the allorecognition fusion checkpoint. PMO catalytic activity is not required for the allorecognition fusion checkpoint Our data above indicated that the PMO domain from each CWR-1 haplogroup was required to confer the allorecognition fusion checkpoint. These data suggested that the CWR-1 PMO domain from each haplogroup might generate a unique product distribution that would confer allorecognition. To test this hypothesis, the PMO domain from the remaining five CWR-1 haplogroups was expressed in a heterologous system, and the protein was purified using the same strategy for the expression of the PMO domain from the HG1 strain (FGSC2489) (Figure 1D and E). The CWR-1 proteins from HG2-6 ran slightly high (approximately 1–2 kDa higher than expected) on SDS-PAGE (Figure 1—figure supplement 4A) and were confirmed to be the correct mass (Figure 1—figure supplement 4B), where each deconvoluted mass was consistent with three disulfides present on the protein (±1 Da) similar to the HG1 protein. All six PMOs from the different haplogroups oxidatively degraded both the α- and β- alloforms of chitin (Figure 4A and B), with β-chitin producing more oxidized fragments than α-chitin. Additionally, each of the six CWR-1 PMO proteins from the six different haplogroups generated C1-oxidized products, bound ~1 equivalent of copper and reduced oxygen at similar rates (Figure 4C and D). None of the other substrates tested showed activity (Supplem" @default.
• W4313037811 created "2023-01-06" @default.
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• W4313037811 date "2022-07-26" @default.
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• W4313037811 title "Decision letter: A moonlighting function of a chitin polysaccharide monooxygenase, CWR-1, in Neurospora crassa allorecognition" @default.
• W4313037811 doi "https://doi.org/10.7554/elife.80459.sa1" @default.
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