Identification of regions required for allelic specificity at the cell wall remodeling allorecognition checkpoint in Neurospora crassa

Identification of regions required for allelic specificity at the cell wall remodeling allorecognition checkpoint in Neurospora crassa | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Identification of regions required for allelic specificity at the cell wall remodeling allorecognition checkpoint in Neurospora crassa Adriana M. Rico-Ramirez , View ORCID Profile N. Louise Glass doi: https://doi.org/10.1101/2025.01.17.633681 Adriana M. Rico-Ramirez 1 Department of Plant and Microbial Biology, University of California , Berkeley, Berkeley, CA 94720, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: Lglass{at}berkeley.edu amricoramirez{at}gmail.com N. Louise Glass 1 Department of Plant and Microbial Biology, University of California , Berkeley, Berkeley, CA 94720, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for N. Louise Glass For correspondence: Lglass{at}berkeley.edu amricoramirez{at}gmail.com Abstract Full Text Info/History Metrics Preview PDF Abstract Allorecognition is the ability of organisms/cells to differentiate self from non-self. In the fungus Neurospora crassa , allorecognition systems serve as checkpoints to restrict germling/hyphal fusion between genetically incompatible strains. The c ell w all r emodeling ( cwr ) checkpoint functions after chemotrophic interactions and is triggered upon cell/hyphal contact, regulating cell wall dissolution and subsequent cell fusion. The cwr region consists of two linked loci, cwr-1 and cwr-2 , that are under severe linkage disequilibrium. Phylogenetic analysis of wild N. crassa populations showed that cwr-1/cwr-2 alleles fall into six different haplogroups (HGs). Strains containing deletions of cwr-1 and cwr-2 will fuse with previously HG incompatible cells, indicating cwr negatively regulates cell fusion. CWR-1 encodes a polysaccharide monooxygenase (PMO) domain that oxidatively degrades chitin; the PMO domain is sufficient to cause fusion arrest and confers allelic specificity by interacting in trans with CWR-2, a predicted transmembrane protein. However, the catalytic activity of CWR-1 is not required for triggering a block in cell fusion. The L2 and LC regions of the CWR-1 PMO domain show high levels of structural variability between different HGs. CWR-1 chimeras containing a LC region from a different HG were sufficient to trigger a cell fusion block, but not quite at wild type levels, suggesting that the complete PMO structure is necessary for allorecognition. Modeling of the transmembrane protein CWR-2 revealed allelic variability in the two major extracellular domains (ED2/ED4). Chimeras of CWR-2 with swapped ED2 or ED4 or ED2/ED4 domains from different cwr-2 haplogroups also altered allelic specificity. Summary Allorecognition or nonself recognition enables fungi to distinguish genetically different individuals, thereby regulating cooperation to form mycelial networks. This study focused on the cell wall remodeling checkpoint ( cwr ), where genetic differences in two genes, cwr-1 and cwr-2 , triggers allorecognition. Upon cell contact, CWR-1 in one cell functions in trans with CWR-2 in a second cell to confer a block in cell fusion. Chimeric proteins were created and tested to pinpoint domains involved in allelic specificity. For CWR-1, the L2 and LC domains are critical, while for CWR-2, the ED2 and ED4 domains have an important role in regulating cell fusion. Download figure Open in new tab Graphical Abstract Introduction Allorecognition is defined as the capability of the organism to discern self from non-self. Allorecognition processes are highly specific, and their control depends on numerous genetic variables. In nature, there are many examples of species that use these systems, from vertebrates to bacteria. In vertebrates, the Major Histocompatibility Complex (MHC) is involved in nonself recognition, thereby triggering an immune response, particularly during organ and tissue transplantation ( A fzali et al . 2008 ; M arino et al . 2016 ; C harmetant et al . 2024 ). The amoeba Dictyostelium discoideum uses allorecognition systems to restrict formation of aggregates between genetically different cells, which is required for the formation of multicellular sporulation structures under starvation conditions ( K essin 2001 ; K atoh -K urasawa et al . 2024 ). In marine invertebrates, such as the colonial ascidian Botryllus schlosseri and cnidarian Hydractinia symbiolongicarpus , allorecognition restricts fusion of colonies to self and close kin, thus mediating the outcome of contested space in populations ( R osengarten and N icotra 2011 ; K aradge et al . 2015 ; H uene et al . 2022 ; R odriguez -V albuena et al . 2024 ). In filamentous fungi, allorecognition systems during vegetative growth are crucial for enabling fusion between cells (germlings and/or hyphae) that are genetically identical/similar, ensuring successful colony development and asexual reproduction. To achieve this, fungi have checkpoints that regulate the cell fusion process to avoid fusing with an unrelated individual that can potentially transfer detrimental elements, such as mycoviruses and debilitated organelles ( D ebets et al . 1994 ; V an D iepeningen et al . 1997 ; D ebets and G riffiths 1998 ). In Neurospora crassa, three checkpoints that regulate cell fusion at different stages have been characterized: pre-contact, contact-fusion and post-fusion systems ( G oncalves et al . 2020 ). At the pre-contact checkpoint, chemotropic interactions between cells/hyphae are regulated by the “determinant of communication” or doc locus. This checkpoint ensures that cells/hyphae with doc alleles of identical specificity undergo chemotropic interactions that result in cell fusion. If cells have different allelic specificity at the doc locus, chemotropic interactions are disrupted, and cell fusion is rare ( H eller et al . 2016 ; G oncalves et al . 2020 ). After chemotropic interactions and upon cell/hyphal contact, the next checkpoint is mediated by the “cell wall remodeling” or cwr locus, which is composed of two linked genes, cwr-1 and cwr-2 . If cells/hyphae have different allelic specificity at cwr-1/cwr-2, cell fusion is blocked at the cell wall dissolution stage (Supplementary Figure S1) ( G oncalves et al . 2019 ; D etomasi et al . 2022 ). The third checkpoint functions post-fusion and results in regulated cell death of the fusion cells/hyphae ( R ico -R amirez et al . 2022 ). Genetic differences at post-fusion loci, such as rcd-1 “regulator of cell death”, which is a homolog of mammalian gasdermin ( D askalov et al . 2019 ; D askalov et al . 2020 ), sec-9/plp-1 ( H eller et al . 2018 ; R ico -R amirez et al . 2022 ), or het loci ( S aupe 2000 ; G lass and K aneko 2003 ; G oncalves et al . 2017 ) triggers rapid vacuolization and cell death of the fusion cells or hyphal compartments. Previous calculations of allorecognition systems described in N. crassa that regulate cell fusion estimate ∼2,000,000 different genotypes in recombining populations, ensuring fusion only between near genetically identical individuals ( G oncalves et al . 2020 ). Phylogenetic analysis of sequences from a wild population of N. crassa ( E llison et al . 2011 ; P alma -G uerrero et al . 2013 ) revealed that the linked cwr-1 and cwr-2 alleles exhibit severe linkage disequilibrium and fall into six discrete haplogroups (HGs) (Supplementary Figure S2). Within each HG, the protein sequence of cwr-1 and cwr-2 alleles are completely conserved ( G oncalves et al . 2019 ). Other filamentous ascomycete fungi, such as Fusarium fujikuroi, F. verticillioides and N. discreta , also have linked cwr-1/cwr-2 loci that fell into discrete HGs in population samples ( G oncalves et al . 2019 ). In N. crassa , allorecognition triggered by genetic differences at the cwr loci functions in trans , such that a cell/hyphae containing cwr-1 from one HG shows a cell fusion block when in contact with a cell/hyphae containing cwr-2 from a different HG (Supplementary Figure S1a) ( G oncalves et al . 2019 ; D etomasi et al . 2022 ). Cells/hyphae that express cwr-1 and cwr-2 alleles from the same HG complete cell fusion. Strain bearing deletions of cwr-1 and cwr-2 ( Δcwr-1 Δcwr-2) lose allorecognition capacity and undergo cell fusion with strains from any of the six cwr HGs. These data indicate that the cwr allorecognition functions to negatively regulate cell wall breakdown and remodeling associated with cell fusion. cwr-1 encodes a lytic polysaccharide monooxygenase (PMO) domain classified in the CAZY (Carbohydrate-Active enZYmes) as Auxiliary Activity Family 11 (AA11) ( H emsworth et al . 2014 ), with a glycine/serine linker and a potential chitin-binding domain (X278) at the C-terminus. The X278 domain is found in other predicted AA11 proteins and GH18 chitinases ( H emsworth et al . 2014 ; G oncalves et al . 2019 ). CWR-1 also contains an N-terminal histidine residue that is part of the histidine brace required for the binding a copper atom ( P hillips et al . 2011 ; D etomasi et al . 2022 ). The PMO domain of CWR-1 exhibits C1-oxiding activity on chitin and has been shown to be necessary and sufficient to confer allorecognition. However, mutations that abolish the catalytic activity of the PMO domain do not affect allorecognition and the cell fusion block, indicating that CWR-1 has a moonlighting function as an allorecognition locus ( D etomasi et al . 2022 ). Strains that only have cwr-1 from one HG are blocked in cell fusion when interacting in trans with a second cell/hyphae that contains only cwr-2 from a different HG ( G oncalves et al . 2019 ). cwr-2 is predicted to encode a plasma membrane protein with eight transmembrane domains, including two domains of unknown function, DUF3433 (Pfam PF11915) ( G oncalves et al . 2019 ). The aim of this study was to clarify regions of CWR-1 and CWR-2 that confer allelic specificity in allorecognition leading to the cell fusion block. To achieve this, various cwr-1 and cwr-2 chimeras were designed based on structural analyses, which revealed regions important for regulating CWR allelic specificity. Materials and methods Strains and culture conditions Strains are listed in Supplementary Table S1 and have been deposited at the Fungal Genetics Stock Center ( https://www.plantpath.k-state.edu/research-services/fungal-genetics-stock-center/ ). Strains were grown in Vogel’s minimal medium (VMM) ( V ogel 1956 ), with 1.5 % agar added for solid medium along with the necessary supplements. L-histidine (L-histidine hydrochloride monohydrate, 98%, Acros Organics) was added at a final concentration of 0.5 mg/mL to support the growth of histidine-auxotrophic strains. For crosses Westergaard’s synthetic cross-medium was employed ( W estergaard and M itchell 1947 ). Recombinant DNA techniques and plasmid constructions Genes were amplified using genomic DNA of N. crassa strains, FGSC 2489, P4471, D111 (FGSC 8871), JW258, JW228 ( E llison et al . 2011 ; P alma -G uerrero et al . 2013 ) (Supplementary Table S1) as a template. PCR reactions were performed in MiniAmp Plus Thermal Cycler using Q5 High-Fidelity DNA polymerase (2000 U/mL) (NEB) according to the manufacturer’s instructions. The DNA fragments amplified were purified using Monarch DNA Gel extraction Kit (T1020S, NEB). Primers used in this study are reported in the Supplementary Table S2. All plasmids used are listed in Supplementary Table S3. All constructions were performed using Gibson Assembly ® Master Mix (E2611S, NEB) following the manufacturer’s instructions. The constructions generated were transformed into NEB 5-alpha Competent E. coli (NEB #C2987) for propagation and storage. The plasmid DNA was extracted with Monarch Plasmid Miniprep Kit (T1010S, NEB), in accordance with the manufacturer’s instructions. The cwr-1 HG1 or cwr-1 HG6 chimeras were made using plasmid DNA PMF::his-3::Pcwr-1-cwr-1-Tcwr-1 (from FGSC 2489; HG1) or PMF::his-3-Ptef-1-cwr-1-Tcwr-1 (JW228; HG6) as a template, respectively. Chimera LC HG1 is under the regulation of the P cwr-1 HG1 promoter and chimera LC HG6 is under the regulation of the P tef-1 promoter. The cwr-2 HG1 chimeras were made using plasmid DNA PMF::his-3-Ptef-1-cwr-2-V5-Tccg-1 (P4471; HG1) as a template for ED2 and ED4 domain swapping from cwr-2 from a HG6 (JW228) strain. The cwr-2 HG3 chimeras plasmid PMF::his-3-Ptef-1-cwr-2-V5-Tccg-1 (JW258; HG3) as a template for swapping the ED2 and ED4 domains from cwr-2 HG2 strain D111. The cwr-1 HG1 and cwr-1 HG1 chimeric constructs were directed to the his-3 locus ( M argolin 1997 ) under the regulation of the native P cwr-1 HG1 promoter. The cwr-1 HG6 , cwr-2 HG3 and cwr-2 HG6 chimeric constructs and cwr-1 HG1 , cwr-1 HG2 , cwr-1 HG3 , cwr-1 HG6 , cwr-2 HG1 , cwr-2 HG2 , cwr-2 HG3 and cwr-2 HG6 constructs were targeted to his-3 locus under the regulation of the P tef-1 promoter. All chimeric strains and strains expressing a single cwr-1 or cwr-2 allele were constructed in the triple delete background Δcwr-1; ΔNCU01381; Δcwr-2 ( ΔΔΔ ) ( G oncalves et al . 2019 ); deletion of the NCU01381 gene has no influence on allorecognition ( G oncalves et al . 2019 ). For the evaluation of the cwr-2 HG1 chimeras, strains expressing cwr-1 HG1 under the P tef-1 promoter was used as one of the control strains. This strategy ensured that all strains expressing individual alleles and the cwr-2 HG1 chimeras were under the regulation of the same promoter. Supplementary Figure S3 compares cell fusion rates of strains expressing the cwr-1 HG1 allele under different promoters and at different loci, paired with either cwr-2 HG1 or cwr-2 HG6 ; comparable fusion rates were obtained among all these strains. Transformation of Neurospora crassa, crosses and selection of homokaryons his-3 conidia from the cwr triple delete background were subjected to transformation with Pac I (R0547S, NEB), Nde I (R0111S, NEB) or Ssp I-HF (R3132S, NEB) linearized plasmids. Following established protocols ( M argolin 1997 ), transformation was conducted through electroporation using a Bio-Rad Pulse controller plus and Bio-Rad gene pulser II. Electroporation was performed using 1 mm gap cuvettes (Bio-Rad Gene Pulser/MicroPulser Cuvette; Bio-Rad) at 1.5 kV, 600 ohm, 25 μF. For each transformation, 30 histidine (His + ) prototroph transformants were chosen and subsequently transferred to tubes containing VMM with Hygromycin B (10687010, 50 mg/mL; Thermo Fisher Scientific), at a final concentration of 200 μg/mL for selection. Integration of the constructs in the selected transformants was validated using the Phire Plant Direct PCR Kit (#F-130WH, Thermo Fisher Scientific) and Sanger sequencing. Selected heterokaryotic transformants were used as a female parent and cultivated on Westergaard’s synthetic crossing medium ( W estergaard and M itchell 1947 ) at 25 °C in constant light until the emergence of the protoperithecia. Strains were fertilized with a conidial suspension of the male parental strain, either FGSC 9716 /his-3 or FGSC 2489 GFP/his-3 (his-3 csr-1::Pccg-1-gfp) (Supplementary Table S1). Ascospores were subjected to heat shock at 60 °C for 40 minutes, followed by inoculation onto agar plates (VMM with 1.5% agar and mixture of 20% sorbose, 0.5% fructose, 0.5% glucose) and an overnight incubation at 30 °C. Germinated ascospores were transferred to VMM ( V ogel 1956 ) slants. Homokaryotic selections were made based on histidine (His + ) prototrophy, resistance to Hygromycin B (Hyg + ), and/or to Cyclosporin A (Cyclosporin + ). Cyclosporin A (30024, Sigma) was employed at a final concentration of 5 μg/mL for selection purposes. PCR analysis was performed to confirm the presence of the construct using the Phire Plant Direct PCR Kit (#F-130WH, Thermo Fisher Scientific) and subsequent Sanger sequencing. RNA isolation and cDNA synthesis To extract RNA from the strains FGSC 2489, D111 (FGSC 8871), JW258, JW228 (Supplementary Table S1), 1×10 8 conidia of each strain were resuspended in 100 mL of liquid VMM ( V ogel 1956 ) and grown under constant light at 30 °C, 220 rpm, for 4 hr. Germlings were filtered using nitrocellulose membranes (0.45 µm) (#88018 Thermo Fisher Scientific), then added into 2 mL screw-cap tubes with 0.25g of 0.5 mm Zirconia/silica beads (BioSpec Products 11079105Z, #NC0450473, Fisher scientific) on dry ice. 1 mL TRIzol (#15596026, Thermo Fisher Scientific) was added to the frozen mycelia. Tubes were bead-beated at maximum speed for 1 min, 0.2 mL chloroform was added and vortexed. Subsequently, the samples were centrifugated for 15 min at 20000g and 4°C. 450 µL of the supernatant was transferred to a new Eppendorf tube, and 500 µL of isopropanol was added. The samples were gently shaken for 10 min at room temperature, followed by another centrifugation step at 20000g, 4°C for 10 min. The supernatant was discarded, and the pellet was washed with 75% ethanol, followed by another centrifugation step. The supernatant was discarded, and the pellet was dried and then resuspended in 87.5 µL of RNAse-free water and incubated at 60°C for 10 min. The samples were submitted to DNase digestion using RNase-Free DNase Set (#79254, QIAGEN), following the manufacturer’s instructions. Subsequently the RNA was cleaned using RNeasy Mini Kit (#74104, QIAGEN). For cDNA synthesis was used the ProtoScript ® II First Strand cDNA synthesis kit (E6560S, NEB). Germling-fusion assays and microscopy analysis An aliquot of 200 μL of fresh conidia were stained with 40 μL of FM4-64 (styryl dye N-(3-triethylammoniumpropyl)–4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide, 514/670 nm absorption/emission, at a final concentration of 16.5 μM in ddH 2 O from a stock solution in DMSO of 16.5 mM (Invitrogen). Conidia were incubated a room temperature for 15 min in the dark and centrifuged in a microcentrifuge at 5000 rpm for 2 min, the supernatant was discarded, conidia were resuspended in 1 mL ddH 2 O and centrifuged; the procedure was repeated two times. The pellet was resuspended in 100 μL of sterile ddH 2 O and the number of conidia was counted using a hematocytometer. The concentration of the conidial suspension was adjusted to 3×10 7 conidia/mL. An aliquot of 45 μL of the conidial suspension stained with FM4-64 was mixed with 45 μL of the conidia from strains expressing cytoplasmic GFP. 80 μL was plated on VMM agar plates (60 mm × 15 mm) and subsequently incubated for 3.5 hr at 30°C. Agar rectangles measuring approximately 3 cm × 2 cm were excised and examined using a ZEISS Axioskop 2 MOT microscope. Images were taken employing a Q IMAGING FAST1394 COOLED MONO 12 BIT microscope camera (RETIGA 2000R SN:Q31594, 01-RET-2000R-F-M-12-C) with a Ph3 ×40/1.30 ∞/0.17 Plan-Neofluar oil immersion objective and processed using iVision-Mac Scientific Image Processing Bio Vision Technologies (iVision 4.5.6r4). The cell fusion percentage of germling pairings was determined by examining the cytoplasmic merging of GFP into germlings stained with FM4-64. Images were captured across a minimum of 15 fields (for each DIC, Blue, Green, Red filter), documenting at least 100 germling-contact events, with three biological replicates. All images underwent further processing using Fiji (ImageJ2, version 2.14.0/1.54f; Build: c89e8500e4) ( S chindelin et al . 2012 ). ANOVA statistical analyses were conducted using GraphPad Prism 10 (version 10.2.2) to identify differences in data across various strains and genotypes. A one-way ANOVA was applied when examining a single independent variable, whereas a two-way ANOVA was used for analyses involving two independent variables. Tukey’s test or Šídák’s test were subsequently performed for multiple comparisons to pinpoint significant differences. The p-values and 95% confidence intervals for each analysis are provided in the Supplementary Table S4. The images and figures were edited using Adobe Photoshop 2023 (Version 23.5.5). Prediction of protein structure and analysis using ColabFold ColabFold V1.5.5 AlphaFold2 ( J umper et al . 2021 ; M irdita et al . 2022 ), an online software that uses MMseqs2 (Many-against-Many sequence searching) to predict protein structures, was utilized to predict the three-dimensional structures of: PMO domain from the six different HGs, the CWR-1 chimeras and CWR-2 from HG1, HG2, HG3, HG6 and CWR-2 chimeras. ( https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb ). For visualization of the predicted structures and their structural superposition, the PyMOL Molecular Graphics System Version 2.5.4 (Copyright (C) Schrödinger, LLC) was used. The Root Mean Square Deviation (RMSD) values were obtained using the align plugin in the PyMOL program ( https://pymolwiki.org/index.php/Align ), with default settings applied: 5 cycles of outlier rejection and a cutoff of 2. Illustrations Digital illustrations were created using Adobe Illustrator 2023. Results The PMO LC domain is the most variable region between the six CWR-1 haplogroups The PMO structure of the six different HGs was modeled using ColabFold ( M irdita et al . 2022 ), which showed a slightly different structure of the variable regions of the PMO domain among the six different HGs compared to previous SwissProt predictions ( D etomasi et al . 2022 ). A comparison of the predicted three-dimensional PMO structures revealed two regions with the most structural variation among members of the different six CWR-1 HGs: L2 and LC ( Figure 1a, b and c ). Members within a CWR-1 HG showed no amino acid variation in either the L2 or LC regions ( G oncalves et al . 2019 ) (Supplementary Figure S2). In the L2 region, there were noticeable conformational variations among the loops, particularly between amino acids 26 and 36 (PCQNTGGGY, using HG1 as a reference) ( Figure 1a ). In contrast, the LC region shows more pronounced structural differences in the loops, as well as variations in the positioning of the β-sheet secondary structure. Notably, only the PMO domain from HG1 and HG2 strains retain an α-helix at the end of the LC region ( Figure 1a ). Download figure Open in new tab Figure 1. Model of the polysaccharide monooygenase (PMO) catalytic domain of CWR-1 from a member from each of the six different haplogroups (HGs). a. The models identify structural differences between members of the different CWR-1 HGs, specifically the L2 and LC regions. The two panels on the left display different views of these two regions. b. Alignment of representative sequences from one member of each of the six different HGs focused on the L2 region. c. Alignment of the amino acid sequences corresponding to LC region, using a representative sequence from a member from each HG. Conserved amino acids are highlighted in gray. The sequences used for modeling and alignment were from isolates FGSC 2489 (HG1), D111 (HG2), JW258 (HG3), JW242 (HG4), P4476 (HG5), and JW228 (HG6) ( E llison et al . 2011 ; P alma -G uerrero et al . 2013 ). These sequences are representative of members of each of the respective HGs. No significant amino acid sequence differences are present in CWR-1 between members of the same HG ( G oncalves et al . 2019 ). Alignment was performed using MAFFT version 7 ( https://mafft.cbrc.jp/alignment/server/ ) and edited with SnapGene Viewer (Version 7.2.1). Prediction of crystal structure analysis using ColabFold V1.5.5 AlphaFold2 ( J umper et al . 2021 ; M irdita et al . 2022 ). To assess the similarities between the different conformational structures of the PMO domains from the six different CWR-1 HGs, quantitative data were obtained by calculating the Root Mean Square Deviation (RMSD). The RMSD provides a numerical measure of the structural similarity between two aligned atomic configurations, expressed in angstroms (Å) ( K absch 1976 ; K ufareva and A bagyan 2012 ). Values closer to zero indicate higher similarity, while larger values suggest greater structural deviations. Supplementary Table S5 shows the results of the comparisons of the conformational structures of the PMO domain from each of the six HGs. With overall RMSD values ranging from 0.171 Å to 0.281 Å, the deviations are minimal, suggesting only slight structural differences. For specific regions, the L2 and LC regions show slightly higher variability (0.208 Å to 0.448 Å and 0.151 Å to 0.494 Å, respectively), but still within a range that signifies strong similarity. RMSD values below 1 Å are generally considered very similar ( C hothia and L esk 1986 ), indicating minimal conformational differences across the PMO domain HG structures. We first assessed fusion percentages between strains with wild type cwr-1 and cwr-2 alleles from the most divergent HGs (HG1 and HG6; Supplementary Figure S2). These cwr-1/cwr-2 alleles from members of the different HGs were targeted to the his-3 locus in the triple-deletion strain, Δcwr-1 ΔNCU01381 Δcwr-2 ( ΔΔΔ ). This mutant ( ΔΔΔ ) is capable of undergoing cell fusion with both cwr compatible and incompatible strains due to the absence of the genes responsible for triggering allorecognition at the cell fusion checkpoint ( G oncalves et al . 2019 ). Fusion assays were conducted using one strain expressing cytoplasmic GFP and the partner strain stained with the FM4-64 ( H ickey et al . 2002 ; H ickey et al . 2004 ). If cell fusion occurred, GFP migrated to the cell stained with the FM4-64, resulting in a pink tint against a green background. However, if no cell fusion occurred, germlings were observed in physical contact, but transfer of GFP fluorescence did not occur ( Figure 2a ). Download figure Open in new tab Figure 2. cwr-1 and cwr-2 from different HGs function in trans to trigger allorecognition and fusion block at the cell wall remodeling checkpoint. a. Diagram to show experimental design to test cell fusion percentages where germlings from one HG express cytoplasmic GFP while germlings from another HG are stained with the dye FM4-64 (left panel). If germlings contain compatible cwr-1 and cwr-2 alleles, germlings undergo cell fusion, and the migration of cytoplasmic GFP protein into the opposite germling stained with FM4-64 is observed (middle panel). If germlings contain incompatible cwr-1 and cwr-2 alleles, allorecognition is triggered and cell fusion is blocked; no transfer of GFP between germlings is observed (right panel). b . Micrographs showing the most common observed events in fusion tests. Germlings from a wild type HG1 strain ( cwr-1 HG1 and cwr-2 HG1 ) expressing cytoplasmic GFP paired with germlings harboring cwr-1 HG1 or cwr-2 HG1 (compatible combinations) or with germlings harboring cwr-1 HG6 or cwr-2 HG6 (incompatible combinations). All strains were constructed in the triple delete background Δcwr-1; ΔNCU01380; Δcwr-2 ( ΔΔΔ ) and the cwr-1 or cwr-2 alleles were targeted to the his-3 locus. c . Micrographs showing fusion tests between a strain bearing cwr-1 HG1 ; ΔΔΔ paired with germlings that were either cwr-2 HG1 ; ΔΔΔ or cwr-2 HG6 ; ΔΔΔ. d. Micrographs showing fusion tests between germlings carrying cwr-1 HG6 ; ΔΔΔ paired with cwr-2 HG1 ; ΔΔΔ or cwr-2 HG6 ; ΔΔΔ germlings. e. Quantification of cell fusion events shown in panel B. f . Quantification of cell fusion events shown in panel C. g . Quantification of cell fusion events shown in panel D. Fusion tests were performed in biological triplicate, assessing fusion interactions of 100 germling pairs for each replicate. Individual p-values are reported in Supplementary Table S4. To evaluate cell fusion percentages of strains bearing different cwr-1 or cwr-2 alleles, we first paired them with a HG1 strain (FGSC 2489), which carries both cwr-1 HG1 and cwr-2 HG2 alleles at the native locus. When the cwr-1 HG1 cwr-2 HG1 strain was paired with strains bearing either cwr-1 HG1 or cwr-2 HG1 , high cell fusion percentages were observed, reaching 83% and 86%, respectively ( Figure 2e ). These data indicated that the engineered strains were fusion proficient. We then assessed fusion in strains bearing the most distantly related cwr-1/cwr-2 alleles: HG1 paired with HG6 (Supplementary Figure S2). In contrast to fusion percentages between strains bearing cwr-1 and cwr-2 from the same HG, fusion percentages of cwr-1 HG1 cwr-2 HG1 + cwr-1 HG6 and cwr-1 HG1 cwr-2 HG1 + cwr-2 HG6 pairings were very low (6% and 11%, respectively; Figure 2b and e ). In wild isolates, allorecognition is triggered by two non-allelic cwr interactions: cwr-1 in cell 1 with cwr-2 in cell 2 and cwr-2 in cell 1 with cwr-1 in cell 2 (Supplementary Figure S1a), potentially leading to a more robust allorecognition response. We therefore engineered strains with cwr-1 or cwr-2 alleles targeted to the his-3 locus in the ΔΔΔ background; these engineered strains were completely isogenic except for cwr-1 or cwr-2. In pairings between strains with compatible alleles, such as a cwr-1 HG1 + cwr-2 HG1 or cwr-1 HG6 + cwr-2 HG6 pairings, a high percentage of fusion was observed (81% and 90%, respectively) ( Figure 2c , d, f and g). However, pairings between cwr-1 HG1 + cwr-2 HG6 strains showed a fusion percentage of 27% ( Figure 2c and f ; Supplementary Figure S3b), which was a significantly higher fusion percentage than of cwr-1 HG1 cwr-2 HG1 + cwr-2 HG6 pairings (11%). Similarly, cwr-1 HG6 + cwr-2 HG1 pairings showed a fusion percentage of 24%, also a significantly higher fusion percentage than pairings between cwr-1 HG1 cwr-2 HG1 + cwr-1 HG6 strains (6%) ( Figure 2d, e and g ; Supplementary Figure S1c). To determine whether fusion behavior was restricted to pairings between HG6 and HG1 strains, we also assessed interactions between strains bearing cwr alleles from a HG2 and HG3 strains; cwr-1/cwr-2 in HG2 and HG3 are more closely related than HG1 and HG6 alleles (Supplementary Figure S2). Pairings between cwr-1 HG3 + cwr-2 HG2 strains showed a fusion percentage of 52%, while cwr-1 HG2 + cwr-2 HG3 pairings exhibited a fusion percentage of 24% (Supplementary Figure S1b). These observations indicate that one cwr-1/cwr-2 HG non-allelic combination may confer a greater degree of fusion block than other combinations (Supplementary Figure S1b, c). In pairings with the cwr-1 HG1 cwr- 2 HG1 (FGSC 2489) strain, fusion percentages were reduced, with a fusion rate of 14% between cwr-1 HG1 cwr- 2 HG1 + cwr-1 HG2 , 8% in pairings between cwr-1 HG1 cwr-2 HG1 + cwr-2 HG2 , 15% in pairings between cwr-1 HG1 cwr- 2 HG1 + cwr-1 HG3 and 8% in pairings between cwr-1 HG1 cwr- 2 HG1 + cwr-2 HG3 (Supplementary Figure S1d). These data indicated that strains that contained only one cwr non-allelic interaction did not show as robust allorecognition response as in pairings with a wild type strain, where cwr-1 and cwr-2 are at their native locus. It is possible that cwr-1 and cwr-2 at the native locus functions better than targeted insertions of cwr-1 or cwr-2 at the his-3 locus, or that interactions between CWR-1 and CWR-2 within the same cell may be playing a role to enhance the cell fusion block. Examining the impact of the L2 and LC regions on allorecognition at the cell fusion checkpoint We hypothesized that the L2 and LC regions that were divergent between cwr-1 haplogroups, but identical within members of the same haplogroup, would be involved in cwr allelic specificity ( Figure 1b, c ). To test this hypothesis, chimeras were constructed using cwr-1 from HG1, where the L2 (H35-A61) and LC (N182-V229) regions of the PMO domain were exchanged with their homologous counterparts from HG6 (L2: Q35-S67, LC: D183-K230). Three chimeras cwr-1 HG1 L2 HG6 , cwr-1 HG1 LC HG6 and cwr-1 HG1 L2 HG6 LC HG6 were constructed ( Figure 3a ). The structural conformation of the chimeric PMO domains were also assessed using ColabFold ( M irdita et al . 2022 ); the three-dimensional structures displayed a conformation very similar to the predicted PMO HG1 domain but with notable variations in the L2 HG6 and LC HG6 regions ( Figure 3b ). Strains carrying the three different chimeras, cwr-1 HG1 L2 HG6 , cwr-1 HG1 LC HG6 or cwr-1 HG1 L2 HG6 LC HG6 , were first tested for cell fusion with the triple deletion strain ( ΔΔΔ ); all three chimeras showed robust cell fusion percentages (∼82%), validating that the constructs did not disrupt the cell fusion process ( Figure 3c ). Download figure Open in new tab Figure 3. Evaluation of the effects on allorecognition at the cell fusion check point of the CWR-1 HG1 PMO chimeras, with L2 HG6 or LC HG6 or L2 HG6 /LC HG6 . a . Schematic of CWR-1 HG1/HG6 chimeric constructs. The PMO (polysaccharide monooxygenase) domain, GS linker (glycine, serine linker) and X278 domain (predicted chitin binding domain) are indicated. The L2 or LC regions, or both, of the CWR-1 HG1 PMO domain were replaced by the corresponding regions from the PMO domain of HG6. b . The three panels show the predicted protein structures of the HG1/HG6 CWR-1 chimeras ( cwr-1 HG1 L2 HG6 , cwr-1 HG1 LC HG6 and cwr-1 HG1 L2 HG6 LC HG6 ) generated by ColabFold ( M irdita et al . 2022 ) and overlapped with the PMO of the CWR-1 HG1 (grey). c . Quantification of cell fusion events between strains harboring the CWR-1 HG1 chimeras (bearing L2, LC or L2/LC from a cwr-1 HG6 allele) paired with a strain lacking both cwr-1 and cwr-2 ( ΔΔΔ ). All chimeric constructs were targeted to the his-3 locus in the ΔΔΔ background. d . Quantification of cell fusion events between strains harboring the CWR-1 HG1 chimeras (bearing L2, LC or L2/LC from a cwr-1 HG6 allele) paired with germlings harboring either cwr-2 HG1 or cwr-2 HG6 alleles in the ΔΔΔ background. A two-way ANOVA followed by Tukey’s post-hoc test was used for statistical analysis, error bars represent SD (Standard deviation), ****p<0.0001, ns: not significant. e . Quantification of cell fusion events between strains harboring the CWR-1 HG1 chimeras (bearing L2, LC or L2/LC from a cwr-1 HG6 allele) paired with cwr-1 HG1 cwr-2 HG1 germlings (FGSC 2489). As controls, germlings expressing either cwr-1 HG1 or cwr-1 HG6 were paired with cwr-1 HG1 cwr-2 HG1 germlings (FGSC 2489). A one-way ANOVA followed by Tukey’s post-hoc test was used for statistical analysis, error bars represent SD (Standard deviation), **p<0.01, ****p<0.0001, ns: not significant. The cell fusion test experiments were performed in biological triplicate, assessing fusion of 100 germling pairs for each replicate. Individual p-values are reported in Supplementary Table S4. Strains expressing the cwr-1 HG1 L2 HG6 + cwr-2 HG1 pairings showed a slight reduction in cell fusion percentages (72%), as compared to cell fusion percentages of 89% in control pairings ( cwr-1 HG1 + cwr-2 HG1 ) ( Figure 2d ). The cwr-1 HG1 LC HG6 + cwr-2 HG1 pairings displayed a more pronounced reduction in cell fusion (51%). Swapping both L2 and LC ( cwr-1 HG1 L2 HG6 LC HG6 + cwr- 2 HG1 ) pairings resulted in a more significant reduction in cell fusion (36% fusion) and which was indistinguishable from the control pairings between cwr-1 HG1 + cwr-2 HG6 strains ( Figure 3d ). Significantly, both the cwr-1 HG1 LC HG6 + cwr-2 HG6 and cwr-1 HG1 L2 HG6 LC HG6 + cwr-2 HG6 pairings showed a significant increase in fusion percentages (67% and 78%, respectively) ( Figure 3d ). Fusion percentages of the cwr-1 HG1 L2 HG6 LC HG6 + cwr-2 HG1 and cwr-1 HG1 L2 HG6 LC HG6 + cwr-2 HG6 pairings were indistinguishable from the control pairings between cwr-1 HG6 + cwr-2 HG1 and cwr-1 HG6 + cwr-2 HG6 . Thus, swapping the L2 and LC region from a HG6 strain into the PMO domain of a HG1 strain switched its specificity to a HG6 strain. These data strongly support the involvement of the L2 and particularly the LC region in cwr-1 allelic specificity and allorecognition. The fusion percentages of the cwr-1 HG1 L2 HG6 , cwr-1 HG1 LC HG6 and cwr-1 HG1 L2 HG6 LC HG6 chimeric strains were also assessed when paired with the cwr-1 HG1 cwr-2 HG1 strain (FGSC 2489). The cwr-1 HG1 cwr-2 HG1 + cwr-1 HG1 L2 HG6 chimera showed a slightly reduced fusion percentage (70%) as compared to the fusion percentage between cwr-1 HG1 cwr-2 HG1 + cwr-1 HG1 strains (84%) ( Figure 2e ). However, pairings between the cwr-1 HG1 cwr-2 HG1 + cwr-1 HG1 LC HG6 showed a significant reduction in fusion percentages (26%), while the cwr-1 HG1 cwr-2 HG1 + cwr-1 HG1 L2 HG6 LC HG6 pairings displayed and even more reduced fusion (16%). To determine whether the cwr-1 L2 and LC allelic swaps were symmetrical in function, chimeras were constructed with a cwr-1 HG6 allele with the L2 HG6 (Q35-S67) and LC HG6 (D183-K230) regions replaced with L2 HG1 (H35-S66) and LC HG1 (N182-V229) regions from the PMO domain of cwr-1 HG1 ( Figure 4a ). The chimeric proteins were modeled, and the overlapping analysis showed that swapping the two motifs, L2 HG1 and LC HG1 , did not significantly affect the conformational structure of cwr-1 HG6 PMO domain ( Figure 4b ). The three chimeras ( cwr-1 HG6 L2 HG1 ; cwr-1 HG6 LC HG1 ; cwr-1 HG6 L2 HG1 LC HG1 ) were first tested for cell fusion by pairing them with the triple-deletion strain and all showed high percentages of cell fusion ( Figure 4c ). Download figure Open in new tab Figure 4. Evaluation of the effects on allorecognition and cell fusion at the cell wall remodeling checkpoint of the CWR-1 HG6 PMO chimeras. a . Schematic of the CWR-1 HG6 L2 HG1 , LC HG1 and L2 HG1 /LC HG1 chimeras. PMO (polysaccharide monooxygenase domain), GS linker (glycine, serine linker domain) and X278 domain (predicted chitin binding domain) are indicted. b . The three panels show the predicted structures of the CWR-1 HG6 L2 HG1 , CWR-1 HG6 LC HG1 and CWR-1 HG6 L2 HG1 LC HG1 chimeric proteins generated by ColabFold ( M irdita et al . 2022 ) and overlapped with the PMO of the CWR-1 HG6 (grey). c . Quantification of cell fusion events of cwr-1 HG6 L2 HG1 , cwr-1 HG6 LC HG1 or cwr-1 HG6 L2 HG1 LC HG1 strains paired with a strain lacking both cwr-1 and cwr-2 ( ΔΔΔ ). All chimeric constructs were targeted to the his-3 locus in the ΔΔΔ background. d . Quantification of cell fusion events between germlings carrying the cwr-1 HG6 L2 HG1 , cwr-1 HG6 LC HG1 or cwr-1 HG6 L2 HG1 LC HG1 chimeras paired with germlings bearing cwr-2 HG1 or cwr-2 HG6 alleles targeted to the his-3 locus in the ΔΔΔ background. Two-way ANOVA followed by Tukey’s post-hoc test was used for statistical analysis, error bars represent SD (Standard deviation), ****p<0.0001, ns: not significant. e. Quantification of fusion events of germlings carrying the cwr-1 HG6 L2 HG1 , cwr-1 HG6 LC HG1 or cwr-1 HG6 L2 HG1 LC HG1 chimeras with the cwr-1 HG1 cwr-2 HG1 strain (FGSC 2489) or Δcwr-1 HG1 cwr-2 HG1 germlings. A Two-way ANOVA followed by Tukey’s post-hoc test was used for statistical analysis, error bars represent SD (Standard deviation), ***p<0.001, ****p<0.0001, ns: not significant. Cell fusion tests were performed in biological triplicate, assessing fusion of 100 germling pairs for each replicate. Individual p-values are reported in Supplementary Table S4. The cwr-1 HG6 L2 HG1 + cwr-2 HG1 pairings showed a fusion percentage of 41%, but a high fusion percentage of 82% in cwr-1 HG6 L2 HG1 + cwr-2 HG6 pairings ( Figure 4d ). In contrast, the cwr-1 HG6 LC HG1 + cwr-2 HG6 pairings showed a fusion percentage of 38%, while cwr-1 HG6 LC HG1 + cwr-2 HG1 pairings showed a fusion percentage of 58% ( Figure 4d ). Pairings between cwr-1 HG6 L2 HG1 LC HG1 + cwr-2 HG6 showed a fusion percentage of only 12%, a value that was indistinguishable to that of control pairings between cwr-1 HG1 + cwr-2 HG6 . In cwr-1 HG6 L2 HG1 LC HG1 + cwr-2 HG1 pairings, the fusion percentage was 89%, a value indistinguishable to the control pairings ( cwr-1 HG1 + cwr-2 HG1 ) ( Figure 4d ). These data indicated that swapping both the L2 and LC regions of cwr-1 HG6 with L2 and LC region from a cwr-1 HG1 strain completely switched cwr-1 allelic specificity to a HG1 strain. As additional controls, the cwr-1 HG6 L2 HG1 , cwr-1 HG6 LC HG1 and cwr-1 HG6 L2 HG1 LC HG1 chimeric strains were also paired with the wild type cwr-1 HG1 cwr-2 HG1 strain and a mutant strain Δcwr-1 HG1 cwr-2 HG1 that expresses cwr-2 HG1 from the native locus. As shown in Figure 4e , the cwr-1 HG1 cwr-2 HG1 + cwr-1 HG6 L2 HG1 LC HG1 and Δcwr-1 HG1 cwr-2 HG1 + cwr-1 HG6 L2 HG1 LC HG1 pairings showed high fusion percentages (75% and 66%), indicating that swapping the L2 and LC region of the PMO domain has an important role in allelic specificity of cwr-1 . In the fusion tests with the engineered strains, the LC region has a more significant influence than the L2 region for switching allelic specificity ( Figure 3d and Figure 4d ). However, in fusion tests with the wild type ( cwr-1 HG1 cwr-2 HG1 ) and the cwr-1 deletion strain ( Δcwr-1 HG1 cwr-2 HG1 ) , where the cwr-2 alleles are at the native locus, both the L2 and LC regions were required for switching allelic specificity ( Figure 4e ). The LC region alone causes an alteration in allelic specificity at the cell fusion checkpoint To assess whether the LC region alone can affect allorecognition at the cell wall remodeling checkpoint, the LC regions of HG1 and HG6 were first modeled independently using ColabFold, comparing each three-dimensional model with the corresponding predicted structure of the PMO domain for HG1 and HG6. The predicted structures of the LC regions for both HG1 and HG6 exhibited variations in their three-dimensional conformations compared to the LC structure within an intact PMO domain. These changes included the absence of a beta sheet ( Figure 5a and b , arrows in green) and an alpha helix in the architecture of LC from the HG1 PMO domain ( Figure 5a , pink arrow). Two chimeric constructs were designed, LC HG1 and LC HG6 , by incorporating the signal peptide of CWR-1, the LC region of the PMO domain, the glycine and serine linker domain and the X278 domain ( Figure 5c ). The RMSD values were calculated for both chimeras ( K ufareva and A bagyan 2012 ). The comparison of the three-dimensional structure of the LC HG1 chimera with PMO HG1 resulted in an RMSD of 1.306 Å, while the comparison of LC HG6 with PMO HG6 resulted in a RMSD of 1.305 Å (Supplementary Table S5). These values suggest that the chimeras retain a similar overall structure to the original PMO region, although with some differences in structural conformation. RMSD values around 1 Å generally indicate high structural similarity, with minor but noticeable conformational changes ( C hothia and L esk 1986 ). Download figure Open in new tab Figure 5. LC domain alone is not sufficient to trigger full allorecognition to block cell fusion. a . Overlap of the structural conformation between the PMO domain of HG1 and the LC domain HG1. b . Overlap of the structural conformation between the PMO domain of HG6 and the LC domain HG6. The predicted structures for a and b were generated by ColabFold ( M irdita et al . 2022 ). The pink arrow indicates the missing alpha helix in LC HG1 , while the green arrow indicates the missing beta sheet in both LC structures. c . Schematic showing the LC HG1 and LC HG6 constructs. The LC HG1 and LC HG6 constructs have a deletion of the PMO domain and contain the GS linker and the predicted X278 chitin binding domain. All chimeric constructs are targeted to his-3 locus. d . Quantification of fusion events of LC HG1 and LC HG6 chimeric strains paired with the strains indicated in the graphic. A two-way ANOVA followed by Šídák’s multiple comparison test was used for statistical analysis, error bars represent SD (Standard deviation), *p<0.05, ***p<0.001, ns: not significant. Cell fusion tests were performed in biological triplicate, assessing fusion events between 100 germling pairs for each replicate. Individual p-values are reported in Supplementary Table S4. The LC HG1 -GS-X278 and LC HG6 -GS-X278 chimeric strains were paired with the ΔΔΔ mutant and fusion percentages were high (86%) ( Figure 5d ). Pairings between cwr-1 HG1 cwr-2 HG1 + LC HG1 -GS-X278 showed a 93% fusion percentage, while cwr-1 HG1 cwr-2 HG1 + LC HG6 -GS-X278 pairings showed a lower fusion percentage (74%). Pairings between Δcwr-1 HG1 cwr-2 HG1 + LC HG1 -GS-X278 pairings showed a fusion percentage of 95%, while Δcwr-1 HG1 cwr-2 HG1 + LC HG6 GS-X278 pairings had a fusion percentage of 76% ( Figure 5d ), a statistically significant difference. In LC HG1 -GS-X278 + cwr-2 HG6 pairings, a significant difference (p<0.05) in fusion percentage was observed (81%) as compared to a 93% fusion percentage in LC HG6 -GS-X278 + cwr-2 HG6 pairings. These results suggest that the LC region alone can trigger allorecognition and a cell fusion block to a degree, thus reducing cell fusion when paired with strains expressing incompatible cwr-1 alleles. Assessing the effect of the ED2 and ED4 extracellular domains of CWR-2 on allorecognition at the cell fusion checkpoint CWR-2 is a predicted transmembrane protein composed of four extracellular domains (ED), five cytoplasmic domains (CD), and eight transmembrane domains. It contains two domains of unknown function (DUF3433) (Supplementary Figure S4). We initially experienced difficulty in cloning cwr-2 alleles from some of the haplogroups, suggesting that the annotation for the cwr-2 ORF might be different in members of the different cwr-2 HGs. We therefore isolated RNA from strains from different cwr-2 HGs, which was subjected to RT-PCR to identify the cwr-2 introns (see Materials and Methods). Comparative analysis of cwr-2 sequences showed that members of HG1, HG2, and HG3 each contained three introns, while members in HG6 exhibited an additional intron positioned within the coding region for the extracellular domain 4 (ED4) (Supplementary Figure S5). CWR-2 is a large protein that differs in size among the CWR-2 haplogroups: 1217 amino acids (aa) in CWR-2 HG1 isolates, 1220 aa in CWR-2 HG2 isolates, 1216 aa in CWR-2 HG3 isolates, 1218 aa in CWR-2 HG4 isolates, 1233 aa in CWR-2 HG5 isolates, and 1273 aa in CWR-2 HG6 isolates. The spatial structure of CWR-2, as predicted by ColabFold ( M irdita et al . 2022 ), provided a clear depiction of the conformational structure in space of CWR-2 and its complexity, and shows that two of the extracellular domains, ED2 and ED4, are extensive and prominent in comparison to the ED1 and ED3 extracellular domains (Supplementary Figure S6). The transmembrane domains of CWR-2 are composed of alpha helices composed of approximately 21 amino acids. The cytoplasmic domains are composed of a combination of alpha helices, beta sheets, and loops and are arranged in close proximity, making it difficult to differentiate them. However, the CD5 domain in HG6 is notably larger compared to members of the other HGs, containing approximately 40 additional amino acids (Supplementary Figure S4). To identify regions important for CWR-2 allelic specificity, we first examined members of the most distant haplogroups, cwr-2 HG1 and cwr-2 HG6 (Supplementary Figure S2). We hypothesized that ED2 and ED4 extracellular domains of CWR-2, due to their large size, sequence diversity and orientation towards the cell wall, might play a crucial role in allorecognition. The overlay of CWR-2 HG1 and CWR-2 HG6 structures revealed significant differences in these domains ( Figure 6a ). A structural comparison between the two CWR-2 proteins generated an RMSD value of 1.690 Å, indicating that CWR-2 HG1 and CWR-2 HG6 share similar three-dimensional structures, with some variations across the entire proteins. Analyzing the ED2 and ED4 domains separately resulted in RMSD values of 0.730 Å and 0.650 Å, respectively, indicating closer similarity in these regions compared to the entire CWR-2 HG1 and CWR-2 HG6 proteins. For the design of the cwr-2 HG1/HG6 chimeras, the extracellular domains ED2 HG1 (G164-R496) and ED4 HG1 (N746-A1100) were exchanged for the corresponding ED2 HG6 (G164-R500) and ED4 HG6 (F760-A1113) domains of CWR-2 HG6 , generating three different chimeras: cwr-2 HG1 ED2 HG6 , cwr-2 HG1 ED4 HG6 , and cwr-2 HG1 ED2 HG6 ED4 HG6 ( Figure 6b ). Download figure Open in new tab Figure 6. Dissecting the influence of two extracellular domains of CWR-2 in allorecognition at the cell wall remodeling checkpoint. a . Predicted three-dimensional structure of CWR-2 HG1 and CWR-2 HG6 using ColabFold ( M irdita et al . 2022 ), with a merged image of both structures. The lower panels show a close-up of the CWR-2 HG1 ED2 and ED4 domains, the CWR-2 HG6 ED2 and ED4 domains and merged image. b . Schematic of CWR-2 HG1 chimeras bearing ED2 HG6 , ED4 HG6 or ED2 HG6 /ED4 HG6 regions. Constructs were targeted to the his-3 locus. c . Cell fusion tests of CWR-2 HG1 ED2 HG6 , CWR-2 HG1 ED4 HG6 and CWR-2 HG1 ED2 HG6 /ED4 HG6 chimeric strains paired a strain lacking cwr-1 and cwr-2 ( ΔΔΔ ). d . Cell fusion tests cwr-2 HG1 ED2 HG6 , cwr-2 HG1 ED4 HG6 and cwr-2 HG1 ED2 HG6 /ED4 HG6 chimeric strains and a strain carrying either cwr-1 HG1 or cwr-1 HG6 alleles. All alleles were targeted to the his-3 locus. e . Cell fusion tests of cwr-2 HG1 ED2 HG6 , cwr-2 HG1 ED4 HG6 and cwr-2 HG1 ED2 HG6 /ED4 HG6 chimeric strains paired with cwr-1 HG1 cwr-2 HG1 (FGSC 2489) germlings or with germlings expressing cwr-1 HG1 at the native locus ( cwr-1 HG1 Δcwr-2 HG1 ). A Two-way ANOVA followed by Tukey’s post-hoc test was used for statistical analysis. Error bars represent SD (Standard deviation), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. Cell fusion test experiments were performed in biological triplicate, assessing the fusion of 100 germling pairs for each replicate. Individual p-values are reported in Supplementary Table S4. All cwr-2 chimeric strains were first paired with the ΔΔΔ mutant; all showed fusion percentages of around 80% ( Figure 6c ). In contrast, cell fusion percentages in pairings between the cwr-1 HG1 + cwr-2 HG1 ED2 HG6 and cwr-1 HG1 + cwr-2 HG1 ED2 HG6 ED4 HG6 strains showed fusion percentages of 44% and 46%, respectively ( Figure 6d ). Fusion percentages between cwr-1 HG6 + cwr-2 HG1 ED2 HG6 and cwr-1 HG6 + cwr-2 HG1 ED2 HG6 ED4 HG6 were higher (59% and 62%, respectively). These data suggested that the inclusion of ED2 from HG6 affected allelic specificity of an otherwise CWR-2 HG1 strain. In contrast, the cwr-1 HG1 + cwr-2 HG1 ED4 HG6 chimeras showed fusion percentages of 60% and 41% in pairings with a cwr-1 HG6 strain, indicating that the E4 domain affected fusion, but did not significantly alter cwr-2 allelic specificity ( Figure 6d ). These data suggest that the ED2 HG6 domain of CWR-2 is an important domain for HG6 allorecognition. The cwr-2 HG1 chimeras were also paired with the cwr-1 HG1 cwr-2 HG1 (FGSC 2489) strain as well as a cwr-1 HG1 Δcwr-2 HG1 mutant ( cwr-1 is at the native locus in this strain). In cwr-1 HG1 cwr-2 HG1 + cwr-2 HG1 ED2 HG6 and cwr-1 HG1 cwr-2 HG1 + cwr-2 HG1 ED4 HG6 pairings, the fusion percentage of ∼48% was obtained ( Figure 6e ). Pairings where both the ED2 and ED4 regions were swapped ( cwr-1 HG1 cwr-2 HG1 + cwr-2 HG1 ED2 HG6 ED4 HG6 ) showed a lower fusion percentage of 25% ( Figure 6e ). Fusion was higher in pairings with a cwr-2 deletion strain ( cwr-1 HG1 Δcwr-2 HG1 + cwr-2 HG1 ED2 HG6 and cwr-1 HG1 Δcwr-2 HG1 + cwr-2 HG1 ED4 HG6 ) (63% and 67%, respectively), versus pairings between cwr-1 HG1 Δcwr-2 HG1 + cwr-2 HG1 ED2 HG6 ED4 HG6 (fusion percentages of 35%) ( Figure 6e ). These data indicated that the cwr-2 HG1 ED2 HG6 ED4 HG6 chimera functioned more efficiently in switching allelic specificity when paired with cwr-1 HG1 cwr-2 HG1 (FGSC 2489) and cwr -1 HG6 Δcwr-2 HG1 strains, both of which possess the cwr-1 allele at the native locus. Examining the impact of the ED2 and ED4 domains of CWR-2 between two closely related haplogroups (HG3 and HG2) on allorecognition at the cell wall remodeling fusion checkpoint Data from the chimeric swaps between cwr-2 HG1 and cwr-2 HG6 indicated that ED2/ED4 extracellular domains impacted cwr-2 allelic recognition. To test allelic specificity domains in cwr-2 haplogroups that are closely related, we constructed similar chimeras between cwr-2 HG2 and cwr-2 HG3 (Supplementary Figure S2). Modeling of the CWR-2 HG2 and CWR-2 HG3 by ColabFold ( M irdita et al . 2022 ) is shown in Figure 7a . The RMSD value for this CWR-2 protein pair was 0.950 Å, which is lower than the value of 1.690 Å obtained between HG1 and HG6 CWR-2 proteins. This value indicates that the overall three-dimensional structures of CWR-2 from HG2 and HG3 were quite similar or closely aligned structurally. The RMSD values for the ED2 and ED4 domains were 0.747 Å and 0.895 Å, respectively, slightly higher than those obtained for HG1 and HG6 (0.730 Å and 0.650 Å). The ED2 HG3 (G164-R495) and ED4 HG3 (F743-A1099) domains were replaced by the corresponding ED2 HG2 (G164-R495) and ED4 HG2 (F743-A1096) domains from HG2, resulting in the cwr-2 HG3 ED2 HG2 , cwr-2 HG3 ED4 HG2 , and cwr-2 HG3 ED2 HG2 ED4 HG2 chimeric strains ( Figure 7b ). All of the cwr-2 HG3/HG2 chimeras showed identical and high fusion percentages with the ΔΔΔ mutant lacking both cwr-1 and cwr-2 ( Figure 7c ). In control pairings, cwr-1 HG2 + cwr-2 HG3 showed a fusion percentage of 24%, while cwr-1 HG3 + cwr-2 HG2 pairings showed a fusion percentage of 53% ( Figure 7d ; Supplementary Figure S1b). These data support our findings that not all pairings of wild type cwr-1 + cwr-2 strains in reciprocal combinations have an identical fusion blockage effect. Cell fusion percentages were similar between the control pairing ( cwr-1 HG3 + cwr-2 HG2 ; 53%) and cwr-1 HG3 + cwr-2 HG3 ED2 HG2 , cwr-1 HG3 + cwr-2 HG3 ED4 HG2 , and cwr-1 HG3 + cwr-2 HG3 ED2/ED4 HG2 pairings (43%, 53% and 47% respectively) ( Figure 7d ). These data showed that the three chimeras exhibited fusion percentages comparable to the control cwr-1 HG3 + cwr-2 HG2 pairings (53%), indicating that the ED2, ED4 and ED2/ED4 domains play an important role in altering cwr-2 allelic specificity. In contrast, the cwr-1 HG2 + cwr-2 HG3 ED2 HG2 , cwr-1 HG2 + cwr-2 HG3 ED4 HG2 , and cwr-1 HG2 + cwr-2 HG3 ED2/ED4 HG2 pairings showed higher fusion percentages (77%, 72%, and 70%, respectively), close to the 80% fusion percentage observed in the control pairing cwr-1 HG2 + cwr-2 HG2 ( Figure 7d ). Statistical analysis showed no significant differences in the fusion percentages among the three CWR-2 chimeras. These results support a role for the CWR-2 ED2 and ED4 regions in two different HG pairings (HG1 + HG6 and HG2 + HG3) in determining allelic specificity, even between the closely related HG2 and HG3 strains. Download figure Open in new tab Figure 7. Examining the impact of two large extracellular domains of CWR-2 in allorecognition at the cell wall remodeling checkpoint. a . Predicted three-dimensional structure of CWR-2 from HG2 or HG3 using ColabFold ( M irdita et al . 2022 ) and merged image. Lower panels show a close-up of ED2 and ED4 and a merged image emphasizing conformational differences. b . Schematic of cwr-2 HG3 ED2 HG2 , ED4 HG2 , or ED2 HG2 /ED4 HG2 chimeras. Constructs were targeted to the his-3 locus and under the regulation of the P tef-1 promoter. c . The cwr-2 HG3 chimeric strains were paired a Δ cwr-1 Δ cwr-2 (ΔΔΔ) mutant. d . Analysis of the cell fusion events between the CWR-2 HG3 chimeric strains and cwr-1 HG3 or cwr-1 HG2 germlings. A two-way ANOVA followed by Tukey’s post-hoc test was used for statistical analysis. Error bars represent SD (Standard deviation), ***p<0.001, ****p<0.0001, ns: not significant. e . Evaluation of the cell fusion events of strains expressing the cwr-2 HG3 chimeras paired with cwr-1 HG1 cwr-2 HG1 (FGSC 2489) germlings. As controls, strains expressing cwr-2 HG3 or cwr-2 HG2 were paired with FGSC 2489. A one-way ANOVA followed by Tukey’s post-hoc test was used for statistical analysis. Error bars represent SD (standard deviation), *p<0.05, ****p<0.0001, ns: not significant. Cell fusion tests were performed in biological triplicate, assessing the fusion events of 100 germling pairs for each replicate. Individual p-values are reported in Supplementary Table S4. When paired with the wild type strain ( cwr-1 HG1 cwr-2 HG1 ; FGSC 2489), strains carrying cwr-2 HG2 or cwr-2 HG3 at the his-3 locus showed low fusion percentages (8% for both cases; Figure 7e ). A pairing between wild type and the cwr-2 HG3 ED2 HG2 chimera ( cwr-1 HG1 cwr-2 HG1 + cwr-2 HG3 ED2 HG2 ) also showed a low fusion percentage (5%). However, the cwr-1 HG1 cwr-2 HG1 + cwr-2 HG3 ED4 HG2 pairing showed a high fusion percentage (57%), while the cwr-1 HG1 cwr-2 HG1 + cwr-2 HG3 ED2/ED4 HG2 pairing showed an intermediate level of fusion (23%) ( Figure 7e ). These data suggest that the inclusion of the HG2 ED4 domain in an otherwise HG3 CWR-2 protein pushed allelic specificity towards compatibility with an CWR-1 HG1 strain, suggesting that this chimera may represent a novel CWR-2 specificity. Discussion The formation of an interconnected mycelial network via germling/hyphal fusion is a hallmark of filamentous fungi. Formation of this network involves a complex interplay of cell signaling that induces chemotropic interactions that ultimately result in cell fusion ( H erzog et al . 2015 ; F ischer and G lass 2019 ). The chemotropic signal for cell fusion appears to be highly conserved between the distantly related ascomycete fungi ( F leissner et al . 2022 ; H aj H ammadeh et al . 2022 ). Indeed, mutations in genes that disrupt hyphal fusion in N. crassa have a similar fusion defect when mutated in other filamentous ascomycete fungi, suggesting that signals and machinery for cell fusion are highly conserved ( S cott et al . 2018 ; G oncalves et al . 2020 ). In N. crassa , three checkpoints have been identified that affect cell fusion. Two of them, the communication checkpoint and the cell wall remodeling checkpoint, act prior to membrane merger and cytoplasmic mixing ( G oncalves and G lass 2020 ; G oncalves et al . 2020 ). Both of these checkpoints function to negatively regulate the fusion process, as strains with mutations in genes required for these checkpoints undergo chemotropic interactions (communication checkpoint) or cell wall breakdown and membrane merger (cell wall remodeling checkpoint) with strains they were formerly incompatible with ( H eller et al . 2016 ; G oncalves et al . 2019 ; D etomasi et al . 2022 ). The loci that regulate the pre- and post-fusion processes in filamentous fungi have hallmarks of being under balancing selection: alleles at these allorecognition loci are highly polymorphic, isolates group into haplogroups, allele frequency in populations are nearly equal and alleles often show trans-species polymorphisms ( K lein et al . 1998 ; G oncalves et al . 2020 ). Although a number of allorecognition loci have been identified in filamentous fungi, very few of them have had their molecular recognition mechanisms characterized and regions required for allelic specificity identified; exceptions include the post-fusion death loci, rcd-1 in N. crassa , which encodes a homolog of gasdermin ( D askalov et al . 2019 ; D askalov et al . 2020 ; L i et al . 2024 ) and het-s in Podospora anserina , which encodes a prion ( R iek and S aupe 2016 ; B ardin et al . 2021 ; S on 2024 ). The most well characterized cell fusion process in fungi is in the yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe ( M artin 2019 ; C lark -C otton et al . 2022 ; S ieber et al . 2023 ), which occurs during mating. In these species, secretory vesicles carrying cell wall degrading enzymes are localized by the actin cytoskeleton to the point of contact between mating cells ( C appellaro et al . 1998 ; P aterson et al . 2008 ; H uberman and M urray 2014 ). Regulated secretion of these enzymes is important to prevent premature cell wall digestion, which would result in cell lysis ( P hilips and H erskowitz 1997 ; M erlini et al . 2013 ; C lark -C otton et al . 2022 ). In N. crassa , cell wall breakdown and membrane merger are negatively regulated during vegetative cell fusion by CWR-1 and CWR-2, which, in incompatible cells, interact in trans to trigger a cell fusion block at the point of cell wall degradation. A key question in this context is how this recognition mechanism occurs. Our initial hypothesis was that CWR-1, which is a chitin polysaccharide monooxygenase, formed HG-specific chitin products in one cell, would be sensed by the transmembrane protein CWR-2 in the plasma membrane of the partner cell. However, this hypothesis was disproved as mutations in CWR-1 that abolish PMO activity were unaffected in the triggering allorecognition and a block in cell fusion ( D etomasi et al . 2022 ). We therefore turned our attention to regions of the PMO domain that were conserved within a haplogroup, but divergent between haplogroups and that regulated allelic specificity. Using ColabFold V1.5.5 AlphaFold2 ( J umper et al . 2021 ; M irdita et al . 2022 ), the superimposed conformational structures of the PMO domain from CWR-1 from the six different haplogroups highlighted two regions with major structural differences: the LC region and, to a lesser extent, the L2 region. The evaluation of the L2 and LC regions of the PMO domain by using chimeras from two distant HGs (HG1 and HG6) indicated that the L2 and LC regions were essential for conferring allelic specificity ( Figures 3 , 4 ; Supplemental Figure S7). Indeed, just the LC region of the PMO, attached to the glycine linker and X278 chitin binding domain was sufficient to alter fusion percentages to a degree ( Figure 5 ), even though there were structural differences that were lacking that are present in an intact PMO domain. The PMO domain regions that affected allelic specificity were reciprocal (i.e. L2 and LC were important for H1 and H6 specificity) further strengthening the support for this region in regulating CWR-1 allorecognition. It is unclear, however, from modeling data of L2 and LC how these regions functions in specificity on a molecular level. However, both the L2 and LC regions reside on the accessible regions of the PMO domain, suggesting that physical interaction, perhaps with CWR-2, may play a role. The structural prediction of the transmembrane protein CWR-2 helped to clarify the dimensionality of the protein inserted into the membrane and has a much more complex structural conformation than CWR-1. Given the architecture and extension of the two large extracellular domains ED2 and ED4 that were identical in amino acid sequence within a haplogroup, but divergent between haplogroups, we hypothesized that these domains would play a role in conferring selectivity in allorecognition. In distantly related haplogroups (HG1 and HG6), the ED2 domain appeared to have a more prominent role in driving allorecognition shifts. However, when paired with strains carrying the cwr-1 allele at the native locus ( cwr-1 cwr-2 and cwr-1 Δcwr-2 ), the chimera with both ED2 and ED4 domains replaced exhibited the most pronounced allelic specificity changes. In contrast, for closely related haplogroups (HG2 and HG3), all three chimeras (with replaced ED2, ED4, and ED2/ED4 domains) showed results similar to that of controls ( cwr-1 HG3 + cwr-2 HG2 and cwr-1 HG2 + cwr-2 HG2 ), suggesting that both domains are important for allelic specificity (Supplementary Figure S7). While our data does not conclusively establish whether ED2 or ED4 contributes to specificity more than the other, the high levels of cellular fusion observed within these closely related haplogroups may be attributed to their inherent similarities despite their haplogroup differences. Interestingly, when these chimeras were paired with the wild-type strain ( cwr-1 HG1 cwr-2 HG1 ), the chimera cwr-2 HG3 ED2 HG2 displayed the lowest fusion levels, highlighting the importance of the ED2 and aligning with the findings observed in chimeras from the more distant groups (HG1 and HG6). Evidence from chimeric strains of the CWR-2 indicates that the ED2 and ED4 domains are key determinants of allorecognition specificity. As with the L2 and LC regions of the PMO domain, the ED2 and ED4 were reciprocally important for CWR-2 allelic specificity, although the strength of this function was not as definitive as with CWR-1. Given the structural complexity of the protein, it is likely that other domains also play a role in this process. The alignment among the six different HGs (Supplementary Figure S4) showed that the cytoplasmic domain CD3 exhibits high amino acid variability across the six HGs as compared to other cytoplasmic domains. It is possible that CWR-2 may undergo conformational changes when activated that trigger an internal signal that involve haplogroup specific domains that face the cytoplasm. Upon contact, cells switch from undergoing chemotropic growth to cell wall breakdown and membrane merger ( H erzog et al . 2015 ; F ischer and G lass 2019 ; F leissner et al . 2022 ). This process must be carefully regulated to avoid cell lysis during mating and vegetative cell fusion ( J in et al . 2004 ; F leissner et al . 2009 ; P alma -G uerrero et al . 2014 ; P alma -G uerrero et al . 2015 ). Previously, it was reported that cells that are blocked at the cwr checkpoint show increased cell wall deposition and continued signaling of proteins associated with chemotropic growth ( G oncalves et al . 2019 ). These observations indicate that the cwr checkpoint prevents the switch from chemotrophic growth upon cell contact to cell wall deconstruction and membrane merger. It is unknown, even during yeast mating, how this transition is regulated. The simplest model for function of the cwr checkpoint is a physical interaction between CWR-1 and CWR-2 of different HGs that prevents this switch. Previous experiments where wild type compatible germlings were treated with CWR-1 purified from incompatible HGs did not change the cell fusion proficiency of these cells ( D etomasi et al . 2022 ), although there are obvious technical aspects of this experiment that were difficult to address. Our work described here on the deciphering the regions of CWR-1 and CWR-2 that regulate specificity provides some insight for further research should concentrate on elucidating the underlying mechanisms of the interactions between CWR-1 and CWR-2 and the signaling pathways involved in regulating cell fusion arrest in incompatible cells. Data Availability Statement All strains generated in this study are available at the Fungal Genetics Stock Center ( https://www.plantpath.k-state.edu/research-services/fungal-genetics-stock-center/ ). Primers used in this study are listed in Supplementary Table S2. The P-values for the cell fusion tests are reported in Supplementary Table S4. RMSD values of the predicted structures of CWR-1 and CWR-2 from the different HGs are reported in Supplementary Table S5. Conflict of Interest The authors declare that there is no conflict of interest. Acknowledgements This work was funded by a United States National Science Foundation Grant (MCB-1818283) to NLG. The authors are grateful to Dr. Tyler Detomasi for his invaluable suggestions for the development of the chimeras and to Maria Mercado for her assistance with lab work. References ↵ Afzali , B. , G. Lombardi and R. I. Lechler , 2008 Pathways of major histocompatibility complex allorecognition . Curr Opin Organ Transplant 13 : 438 – 444 . OpenUrl CrossRef PubMed ↵ Bardin , T. , A. Daskalov , S. Barrouilhet , A. Granger-Farbos , B. Salin et al. , 2021 Partial prion cross-seeding between fungal and mammalian amyloid signaling motifs . mBio 12 . ↵ Cappellaro , C. , V. Mrsa and W. Tanner , 1998 New potential cell wall glucanases of Saccharomyces cerevisiae and their involvement in mating . J Bacteriol 180 : 5030 – 5037 . OpenUrl Abstract / FREE Full Text ↵ Charmetant , X. , G. J. Pettigrew and O. Thaunat , 2024 Allorecognition Unveiled: Integrating recent breakthroughs into the current paradigm . Transpl Int 37 : 13523 . OpenUrl CrossRef PubMed ↵ Chothia , C. , and A. M. Lesk , 1986 The relation between the divergence of sequence and structure in proteins . EMBO J 5 : 823 – 826 . OpenUrl CrossRef PubMed Web of Science ↵ Clark-Cotton , M. R. , K. C. Jacobs and D. J. Lew , 2022 Chemotropism and cell-cell fusion in fungi . Microbiol Mol Biol Rev 86 : e0016521 . OpenUrl CrossRef PubMed ↵ Daskalov , A. , P. Gladieux , J. Heller and N. L. Glass , 2019 Programmed cell death in Neurospora crassa is controlled by the allorecognition determinant rcd-1 . Genetics 213 : 1387 – 1400 . OpenUrl Abstract / FREE Full Text ↵ Daskalov , A. , P. S. Mitchell , A. Sandstrom , R. E. Vance and N. L. Glass , 2020 Molecular characterization of a fungal gasdermin-like protein . Proc Natl Acad Sci U S A 117 : 18600 – 18607 . OpenUrl Abstract / FREE Full Text ↵ Debets , A. J. M. , and A. J. F. Griffiths , 1998 Polymorphism of het -genes prevents resource plundering in Neurospora crassa . Mycol. Res . 102 : 1343 – 1349 . OpenUrl CrossRef ↵ Debets , F. , X. Yang and A. J. Griffiths , 1994 Vegetative incompatibility in Neurospora: its effect on horizontal transfer of mitochondrial plasmids and senescence in natural populations . Curr Genet 26 : 113 – 119 . OpenUrl CrossRef PubMed ↵ Detomasi , T. C. , A. M. Rico-Ramirez , R. I. Sayler , A. P. Goncalves , M. A. Marletta et al. , 2022 A moonlighting function of a chitin polysaccharide monooxygenase, CWR-1, in Neurospora crassa allorecognition . Elife 11 . ↵ Ellison , C. E. , C. Hall , D. Kowbel , J. Welch , R. B. Brem et al. , 2011 Population genomics and local adaptation in wild isolates of a model microbial eukaryote . Proc Natl Acad Sci USA 108 : 2831 – 2836 . OpenUrl Abstract / FREE Full Text ↵ Fischer , M. S. , and N. L. Glass , 2019 Communicate and Fuse: How filamentous fungi establish and maintain an interconnected mycelial network . Front Microbiol 10 : 619 . OpenUrl CrossRef PubMed ↵ Fleissner , A. , S. Diamond and N. L. Glass , 2009 The Saccharomyces cerevisiae PRM1 homolog in Neurospora crassa is involved in vegetative and sexual cell fusion events but also has postfertilization functions . Genetics 181 : 497 – 510 . OpenUrl Abstract / FREE Full Text ↵ Fleissner , A. , A. G. Oostlander and L. Well , 2022 Highly conserved, but highly specific: Somatic cell-cell fusion in filamentous fungi . Curr Opin Cell Biol 79 : 102140 . OpenUrl CrossRef PubMed ↵ Glass , N. L. , and I. Kaneko , 2003 Fatal attraction: Nonself recognition and heterokaryon incompatibility in filamentous fungi . Eukaryot. Cell 2 : 1 – 8 . OpenUrl FREE Full Text ↵ Goncalves , A. P. , and N. L. Glass , 2020 Fungal social barriers: to fuse, or not to fuse, that is the question . Commun Integr Biol 13 : 39 – 42 . OpenUrl CrossRef PubMed ↵ Goncalves , A. P. , J. Heller , A. Daskalov , A. Videira and N. L. Glass , 2017 Regulated forms of cell death in fungi . Front Microbiol 8 : 1837 . OpenUrl CrossRef PubMed ↵ Goncalves , A. P. , J. Heller , A. M. Rico-Ramirez , A. Daskalov , G. Rosenfield et al. , 2020 Conflict, competition, and cooperation regulate social interactions in filamentous fungi . Annu Rev Microbiol 74 : 693 – 712 . OpenUrl CrossRef PubMed ↵ Goncalves , A. P. , J. Heller , E. A. Span , G. Rosenfield , H. P. Do et al. , 2019 Allorecognition upon fungal cell-cell contact determines social cooperation and impacts the acquisition of multicellularity . Curr Biol 29 : 3006 – 3017 e3003. OpenUrl CrossRef PubMed ↵ Haj Hammadeh , H. , A. Serrano , V. Wernet , N. Stomberg , D. Hellmeier et al. , 2022 A dialogue-like cell communication mechanism is conserved in filamentous ascomycete fungi and mediates interspecies interactions . Proc Natl Acad Sci U S A 119 : e2112518119 . OpenUrl CrossRef PubMed ↵ Heller , J. , C. Clave , P. Gladieux , S. J. Saupe and N. L. Glass , 2018 NLR surveillance of essential SEC-9 SNARE proteins induces programmed cell death upon allorecognition in filamentous fungi . Proc Natl Acad Sci U S A 115 : E2292 – E2301 . OpenUrl Abstract / FREE Full Text ↵ Heller , J. , J. Zhao , G. Rosenfield , D. J. Kowbel , P. Gladieux et al. , 2016 Characterization of greenbeard genes involved in long-distance kind discrimination in a microbial eukaryote . PLoS Biol 14 : e1002431 . OpenUrl CrossRef PubMed ↵ Hemsworth , G. R. , B. Henrissat , G. J. Davies and P. H. Walton , 2014 Discovery and characterization of a new family of lytic polysaccharide monooxygenases . Nat Chem Biol 10 : 122 – 126 . OpenUrl CrossRef PubMed ↵ Herzog , S. , M. R. Schumann and A. Fleissner , 2015 Cell fusion in Neurospora crassa . Curr Opin Microbiol 28 : 53 – 59 . OpenUrl CrossRef PubMed ↵ Hickey , P. C. , D. Jacobson , N. D. Read and N. L. Louise Glass , 2002 Live-cell imaging of vegetative hyphal fusion in Neurospora crassa . Fungal Genet Biol 37 : 109 – 119 . OpenUrl CrossRef PubMed Web of Science ↵ T. Savidge and C. Pothoulakis Hickey , P. C. , S. R. Swift , M. G. Roca and N. D. Read , 2004 Live-cell imaging of filamentous fungi using vital fluorescent dyes and confocal microscopy ., pp. 63 – 87 in Methods in Microbiology , edited by T. Savidge and C. Pothoulakis . Elsevier , London, U.K . ↵ Huberman , L. B. , and A. W. Murray , 2014 A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution . PLoS One 9 : e109780 . OpenUrl CrossRef PubMed ↵ Huene , A. L. , S. M. Sanders , Z. Ma , A. D. Nguyen , S. Koren et al. , 2022 A family of unusual immunoglobulin superfamily genes in an invertebrate histocompatibility complex . Proc Natl Acad Sci U S A 119 : e2207374119 . OpenUrl CrossRef PubMed ↵ Jin , H. , C. Carlile , S. Nolan and E. Grote , 2004 Prm1 prevents contact-dependent lysis of yeast mating pairs . Eukaryotic Cell 3 : 1664 – 1673 . OpenUrl Abstract / FREE Full Text ↵ Jumper , J. , R. Evans , A. Pritzel , T. Green , M. Figurnov et al. , 2021 Highly accurate protein structure prediction with AlphaFold . Nature 596 : 583 – 589 . OpenUrl CrossRef PubMed ↵ Kabsch , W ., 1976 A solution for the best rotation to relate two sets of vectors . Acta Crystallographica Section A 32 : 922 – 923 . OpenUrl CrossRef ↵ Karadge , U. B. , M. Gosto and M. L. Nicotra , 2015 Allorecognition proteins in an invertebrate exhibit homophilic interactions . Curr Biol 25 : 2845 – 2850 . OpenUrl CrossRef PubMed ↵ Katoh-Kurasawa , M. , P. Lehmann and G. Shaulsky , 2024 The greenbeard gene tgrB1 regulates altruism and cheating in Dictyostelium discoideum . Nat Commun 15 : 3984 . OpenUrl CrossRef PubMed ↵ Kessin , R. H ., 2001 Dictyostelium: Evolution, Cell Biology and the Development of Multicellularity . Cambridge University Press , Cambridge ↵ Klein , J. , A. Sato , S. Nagl and C. O’hUigin , 1998 Molecular trans-species polymorphism . Annu. Rev. Ecol. Syst . 29 : 1 – 21 . OpenUrl CrossRef Web of Science ↵ Kufareva , I. , and R. Abagyan , 2012 Methods of protein structure comparison . Methods Mol Biol 857 : 231 – 257 . OpenUrl CrossRef PubMed ↵ Li , Y. , Y. Hou , Q. Sun , H. Zeng , F. Meng et al. , 2024 Cleavage-independent activation of ancient eukaryotic gasdermins and structural mechanisms . Science 384 : adm9190 . OpenUrl CrossRef PubMed ↵ Margolin , B. S. , M. Freitag , and E.U. Selker 1997 Improved plasmids for gene targeting at the his-3 locus of Neurospora crassa by electroporation . Fungal Genetics Newsletter 34 : 34 – 36 OpenUrl ↵ Marino , J. , J. Paster and G. Benichou , 2016 Allorecognition by T lymphocytes and allograft rejection . Front Immunol 7 : 582 . OpenUrl PubMed ↵ Martin , S. G ., 2019 Molecular mechanisms of chemotropism and cell fusion in unicellular fungi . J Cell Sci 132 . ↵ Merlini , L. , O. Dudin and S. G. Martin , 2013 Mate and fuse: how yeast cells do it . Open Biol 3 : 130008 . OpenUrl CrossRef PubMed ↵ Mirdita , M. , K. Schutze , Y. Moriwaki , L. Heo , S. Ovchinnikov et al. , 2022 ColabFold: making protein folding accessible to all . Nat Methods 19 : 679 – 682 . OpenUrl CrossRef PubMed ↵ Palma-Guerrero , J. , C. R. Hall , D. Kowbel , J. Welch , J. W. Taylor et al. , 2013 Genome wide association identifies novel loci involved in fungal communication . PLoS Genet 9 : e1003669 . OpenUrl CrossRef PubMed ↵ Palma-Guerrero , J. , A. C. Leeder , J. Welch and N. L. Glass , 2014 Identification and characterization of LFD1, a novel protein involved in membrane merger during cell fusion in Neurospora crassa . Mol Microbiol 92 : 164 – 182 . OpenUrl CrossRef PubMed ↵ Palma-Guerrero , J. , J. Zhao , A. P. Goncalves , T. L. Starr and N. L. Glass , 2015 Identification and characterization of LFD-2, a predicted fringe protein required for membrane integrity during cell fusion in Neurospora crassa . Eukaryot Cell 14 : 265 – 277 . OpenUrl Abstract / FREE Full Text ↵ Paterson , J. M. , C. A. Ydenberg and M. D. Rose , 2008 Dynamic localization of yeast Fus2p to an expanding ring at the cell fusion junction during mating . J Cell Biol 181 : 697 – 709 . OpenUrl Abstract / FREE Full Text ↵ Philips , J. , and I. Herskowitz , 1997 Osmotic balance regulates cell fusion during mating in Saccharomyces cerevisiae . J Cell Biol 138 : 961 – 974 . OpenUrl Abstract / FREE Full Text ↵ Phillips , C. M. , W. T. Beeson , J. H. Cate and M. A. Marletta , 2011 Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa . ACS Chem Biol 6 : 1399 – 1406 . OpenUrl CrossRef PubMed Web of Science ↵ Rico-Ramirez , A. M. , A. P. Goncalves and N. Louise Glass , 2022 Fungal cell death: The beginning of the end . Fungal Genet Biol 159 : 103671 . OpenUrl CrossRef PubMed ↵ Riek , R. , and S. J. Saupe , 2016 The HET-S/s prion motif in the control of programmed cell death . Cold Spring Harb Perspect Biol 8 . ↵ Rodriguez-Valbuena , H. , J. Salcedo , O. De Their , J. F. Flot , S. Tiozzo et al. , 2024 Genetic and functional diversity of allorecognition receptors in the urochordate, Botryllus schlosseri . BioRxiv . ↵ Rosengarten , R. D. , and M. L. Nicotra , 2011 Model systems of invertebrate allorecognition . Curr Biol 21 : R82 – 92 . OpenUrl CrossRef PubMed ↵ Saupe , S. J ., 2000 Molecular genetics of heterokaryon incompatibility in filamentous ascomycetes . Microbiol Mol Biol Rev 64 : 489 – 502 . OpenUrl Abstract / FREE Full Text ↵ Schindelin , J. , I. Arganda-Carreras , E. Frise , V. Kaynig , M. Longair et al. , 2012 Fiji: an open-source platform for biological-image analysis . Nat Methods 9 : 676 – 682 . OpenUrl CrossRef PubMed Web of Science ↵ Scott , B. , K. Green and D. Berry , 2018 The fine balance between mutualism and antagonism in the Epichloe festucae-grass symbiotic interaction . Curr Opin Plant Biol 44 : 32 – 38 . OpenUrl CrossRef PubMed ↵ Sieber , B. , J. M. Coronas-Serna and S. G. Martin , 2023 A focus on yeast mating: From pheromone signaling to cell-cell fusion . Semin Cell Dev Biol 133 : 83 – 95 . OpenUrl CrossRef PubMed ↵ Son , M ., 2024 A story between s and S: [Het-s] prion of the fungus Podospora anserina . Mycobiology 52 : 85 – 91 . OpenUrl CrossRef PubMed ↵ van Diepeningen , A. D. , A. J. Debets and R. F. Hoekstra , 1997 Heterokaryon incompatibility blocks virus transfer among natural isolates of black Aspergilli . Curr Genet 32 : 209 – 217 . OpenUrl CrossRef PubMed ↵ Vogel , H. J ., 1956 A convenient growth medium for Neurospora . Microbiol. Genet. Bull . 13 : 42 – 46 . OpenUrl ↵ Westergaard , M. , and H. K. Mitchell , 1947 Neurospora V. A synthetic medium favoring sexual reproduction . Amer. J. Bot . 34 : 573 – 577 . OpenUrl CrossRef Web of Science View the discussion thread. Back to top Previous Next Posted January 22, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Identification of regions required for allelic specificity at the cell wall remodeling allorecognition checkpoint in Neurospora crassa Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Identification of regions required for allelic specificity at the cell wall remodeling allorecognition checkpoint in Neurospora crassa Adriana M. Rico-Ramirez , N. 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