Transcriptomic insights into the tripartite plant–pathogen–mycoparasite interaction reveal novel mechanisms involving fungal secretomes and plant amino acid metabolism

Transcriptomic insights into the tritrophic plant–pathogen–mycoparasite interaction reveal coordinated reprogramming fungal secretomes and plant amino acid metabolism | 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 Transcriptomic insights into the tritrophic plant–pathogen–mycoparasite interaction reveal coordinated reprogramming fungal secretomes and plant amino acid metabolism View ORCID Profile Kazuya Maeda , View ORCID Profile Mariko Kouda , Mai Ohara , View ORCID Profile Takumi Kawase , View ORCID Profile Koki Saito , View ORCID Profile Eishin Iwao , View ORCID Profile Hirotoshi Sushida , View ORCID Profile Tomoko Suzuki , View ORCID Profile Takuya Sumita , View ORCID Profile Yuichiro Iida doi: https://doi.org/10.1101/2025.11.24.690098 Kazuya Maeda 1 Laboratory of Plant Pathology, Faculty of Agriculture, Setsunan University , Nagaotoge-cho 45-1, Hirakata, Osaka 573-0101, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kazuya Maeda Mariko Kouda 1 Laboratory of Plant Pathology, Faculty of Agriculture, Setsunan University , Nagaotoge-cho 45-1, Hirakata, Osaka 573-0101, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mariko Kouda Mai Ohara 1 Laboratory of Plant Pathology, Faculty of Agriculture, Setsunan University , Nagaotoge-cho 45-1, Hirakata, Osaka 573-0101, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Takumi Kawase 1 Laboratory of Plant Pathology, Faculty of Agriculture, Setsunan University , Nagaotoge-cho 45-1, Hirakata, Osaka 573-0101, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Takumi Kawase Koki Saito 1 Laboratory of Plant Pathology, Faculty of Agriculture, Setsunan University , Nagaotoge-cho 45-1, Hirakata, Osaka 573-0101, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Koki Saito Eishin Iwao 1 Laboratory of Plant Pathology, Faculty of Agriculture, Setsunan University , Nagaotoge-cho 45-1, Hirakata, Osaka 573-0101, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Eishin Iwao Hirotoshi Sushida 2 Institute of Food Research, National Agriculture and Food Research Organization (NARO) , 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hirotoshi Sushida Tomoko Suzuki 3 Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University , Mejirodai 2-8-1, Bunkyo-ku, Tokyo 112-8681, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tomoko Suzuki Takuya Sumita 4 Department of biological resources management, School of Environmental Science, The University of Shiga Prefecture , Hasaka-cho 2500, Hikone, Shiga 522-8533 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Takuya Sumita Yuichiro Iida 1 Laboratory of Plant Pathology, Faculty of Agriculture, Setsunan University , Nagaotoge-cho 45-1, Hirakata, Osaka 573-0101, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yuichiro Iida For correspondence: yuichiro.iida{at}setsunan.ac.jp Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Summary The tomato– Cladosporium fulvum (syn. Fulvia fulva ) pathosystem has served as a model for the gene-for-gene concept of effectors and resistance proteins, but this binary framework does not include the potential influence of other microbial participants. Here we describe the dramatic changes in gene expression of all members of the tritrophic interaction among tomato, C. fulvum , and mycoparasitic fungus Hansfordia pulvinata . Transcriptomic analyses of the mycoparasite H. pulvinata during parasitism of C. fulvum on tomato on planta and in vitro revealed a dramatic upregulation of genes encoding small secreted proteins during mycoparasitism, notably, a Nep1-like protein (HpNlp1) lacked typical necrosis-inducing activity but induced the accumulation of antifungal compounds inhibiting spore germination of C. fulvum . Similarly, in C. fulvum parasitized by H. pulvinata , effector genes were highly expressed. Strikingly, effector protein Ecp2 was found to share structural similarity with pathogen killer toxin 4 proteins and had broad-spectrum antifungal activity, indicating a dual function in fungal competition and Cf-ECP2 -mediated plant resistance. In tomato plants infected by C. fulvum parasitized by H. pulvinata , primary metabolism and defense-related genes were exclusively activated. These results suggest that in the tritrophic interaction, the mycoparasite simultaneously suppressed the pathogen and induced plant resistance. This study uncovers a multilayered molecular network in which the mycoparasite coordinates pathogen suppression and plant defense within the tritrophic interaction. INTRODUCTION The biotrophic filamentous fungus Cladosporium fulvum Cooke [syn. Fulvia fulva (Cooke) Cif.] causes tomato leaf mold, a serious economic threat due to reducing fruit yield and quality and occasionally killing tomato plants ( Solanum lycopersicum L.) ( Thomma et al ., 2005 ; de Wit et al ., 2009 ). The fungus enters tomato leaves through open stomata in high humidity, colonizes the apoplast, and secretes numerous effector proteins to suppress plant immunity and facilitate infection ( Mesarich et al ., 2023 ). Commercial tomato cultivars have thus been bred with Cf resistance genes encoding receptor-like proteins (RLPs) with extracytoplasmic leucine-rich repeats from wild relatives ( Mesarich et al ., 2023 ). These RLPs recognize the corresponding C. fulvum effector proteins as virulence factors via a gene-for-gene relationship and trigger a hypersensitive response to restrict the growth of C. fulvum ( Mesarich et al ., 2023 ). However, C. fulvum rapidly evolves new races through mutation or loss of effector genes, thereby evading plant immunity ( Iida et al ., 2015 ; de la Rosa et al ., 2024 ). The extensive use of resistant cultivars carrying only one or a few Cf genes has consequently led to the emergence of C. fulvum races that can overcome the resistance of the tomato plants ( Luderer et al ., 2002 ; Mesarich et al ., 2014 ). To date, over 100 effector genes have been identified in C. fulvum ( Mesarich et al ., 2018 ; Zaccaron & Stergiopoulos, 2024 ). Most encode small cysteine-rich proteins that contain an N-terminal signal peptide for secretion. The identified avirulence (Avr) and extracellular proteins (Ecp) effectors—such as Avr2, Avr4, Avr4E, Avr5, Avr9B, Avr9C, Ecp1, Ecp2, Ecp4, Ecp5, and Ecp6—exemplify the complex coevolution between fungal virulence factors and their cognate Cf immune receptors ( Wulff et al ., 2009 ; de la Rosa et al ., 2024 ). Several effectors have been functionally characterized. Avr2 acts as a cysteine protease inhibitor that targets the apoplastic cysteine proteases Rcr3 and Pip1, thereby interfering with basal defense mechanisms conserved across solanaceous plants ( Rooney et al ., 2005 ; Shabab et al ., 2008 ; Kourelis et al ., 2020 ). Ecp6 suppresses chitin-triggered immunity by sequestering chitin oligomers released from the fungal cell wall, thus evading recognition by host pattern-recognition receptors ( de Jonge et al ., 2010 ). In contrast, Ecp2 induces cell death in both tomato and nonhost plants, suggesting a dual role as a potential virulence and avirulence factor ( de Kock et al ., 2004 ; de Wit et al ., 2012 ). Notably, the Ecp2 effector contains an Hce2 (homolog of Cladosporium fulvum Ecp2 ) domain that is widely conserved across diverse fungal and bacterial taxa, implying a fundamental role for this domain in microbe–plant interactions ( Stergiopoulos et al ., 2012 ). However, the biochemical function of Ecp2 remains unclear. Of additional concern is that C. fulvum has developed resistance to fungicides ( Yan et al ., 2008 ; Watanabe et al ., 2017 ). The extensive breakdown of the resistance conferred by single Cf resistance genes and the emergence of fungicide-resistant strains emphasize the need for sustainable disease management strategies beyond conventional breeding and chemical approaches. To better understand the molecular basis of disease suppression, previous studies have primarily focused on the C. fulvum –tomato interaction within the framework of the gene-for-gene model, but tritrophic interactions involving additional microorganisms remain largely unexplored. Hansfordia pulvinata (Berk. & M.A. Curtis) S. Hughes (syn. Dicyma pulvinata ) is a mycoparasitic fungus serendipitously discovered on tomato leaves infected with C. fulvum in a greenhouse ( Iida et al ., 2018 ). It parasitizes C. fulvum and overgrows lesions, effectively suppressing disease progression, and has broad mycoparasitic activity against all known C. fulvum races ( Iida et al ., 2018 ). H. pulvinata recognizes C. fulvum hyphae, coils around them, and directly penetrates the hyphae, then forms intercellular structures within the host fungus ( Peresse & Le Picard, 1980 ; Maeda et al ., 2025 ). It also produces an eremophilane-type sesquiterpenoid, 13-deoxyphomenone (also known as sporogen-AO1), which has antifungal activity against C. fulvum ( Tirilly et al ., 1983 ; Maeda et al ., 2025 ). The compound was initially identified as a sporulation-inducing factor in the koji mold Aspergillus oryzae (Tanaka et al ., 1984a, 1984b). Comparative genomics recently revealed that the biosynthetic gene cluster for 13-deoxyphomenone ( DPH ) in H. pulvinata shares high synteny with that in Aspergillus species, suggesting horizontal gene transfer from an ancestral Aspergillus lineage ( Maeda et al ., 2025 ). Subsequently, H. pulvinata appears to have retained this gene cluster to support its mycoparasitic lifestyle, repurposing the endogeni c sporogenesis function in Aspergillus species into exogenic antifungal activity that inhibits spore germination and hyphal elongation of C. fulvum . However, the molecular mechanisms underlying its mycoparasitism remain largely unknown. Since its mycoparasitism is not restricted to the phyllosphere ( Maeda et al ., 2025 ), comparative analyses of fungal–fungal interactions under different mycoparasitic conditions could provide new insights on the molecular basis of fungal host recognition and leaf-surface biocontrol mechanisms. The C. fulvum –tomato interaction has long served as a valuable model for dissecting gene-for-gene dynamics in plant–pathogen systems ( Mesarich et al. 2023 .). Recent chromosome-scale genome assemblies of multiple C. fulvum races have substantially advanced our understanding of its genome structure and effector repertoire ( Zaccaron et al ., 2022 ; Zaccaron & Stergiopoulos, 2024 ). Nevertheless, the molecular basis governing fungal–fungal recognition and the tritrophic interactions with tomato plants remain largely uncharacterized. Here, we present transcriptomic evidence revealing how the mycoparasitic fungus H. pulvinata modulates both C. fulvum and tomato responses, providing molecular insights into the interplay among plant, pathogen and mycoparasite. These findings underscore the potential of the foliar mycoparasitic fungus as a promising biocontrol agent within complex plant–microbe ecosystems. MATERIALS AND METHODS Culture conditions for fungal strains and plants, treatments and assays Fungal strains H. pulvinata 414-3 and C. fulvum CF301 were cultured on half-strength potato dextrose agar (PDA; BD Difco, Sparks, NJ, USA) or minimal medium (MM) agar (15 g sucrose, 5 g ammonium tartrate, 1 g NH 4 NO 3 , 1 g KH 2 PO 4 , 0.5 g MgSO 4 ×7H 2 O, 0.1 g NaCl, 0.1 g CaCl 2 ×H 2 O, 25 μL 0.2 mg mL −1 biotin, 15 g agar and 1 mL trace elements per liter) at 25 °C in the dark for 1 ( H. pulvinata ) and 2 weeks ( C. fulvum ) ( Maeda et al ., 2025 ). Spores were collected in distilled water and adjusted to 1 × 10 6 spores mL −1 using a hemocytometer. For in vitro assays, 100 μL of each spore suspension was spread onto nylon membranes on PDA and incubated for 1 ( H. pulvinata ) or 2 weeks ( C. fulvum ) (hereafter “ in vitro Hp ” and “ in vitro Cf ” conditions; Fig. S1a ). For establishing the mycoparasitism in vitro , membranes bearing C. fulvum colonies were transferred to water agar (nutrient-poor) and sprayed with 1 mL of H. pulvinata spore suspension (1 × 10 6 spores mL −1 ); plates were then incubated at 25 °C in the dark for 2 weeks ( in vitro Cf/Hp ; Fig. S1a ). Seedlings of tomato ( Solanum lycopersicum ) cv. Moneymaker (lacking all known Cf resistance genes) were grown at 25 °C with 16 h light/8 h dark. Four-week-old plants were either grown in high humidity as healthy controls for another 2 weeks (Healthy leaf; Fig. S1a ) or sprayed with approximately 3 mL of C. fulvum spore suspension (1 × 10 6 spores mL −1 ), then incubated in high humidity for 2 weeks until lesions developed (“ on planta Cf ” interaction; Fig. S1a ). For establishing the mycoparasitism on plants, plants with leaf mold lesions were sprayed with approximately 3 mL of H. pulvinata spore suspension (1 × 10⁶ conidia mL⁻¹) and incubated under high humidity at 25 °C for 2 weeks until lesions were overgrown by white mycelia of H. pulvinata (“ on planta Cf/Hp ”; Fig. S1a ). N. benthamiana plants were grown for 4 weeks at 25 °C with a 16 h light/8 h dark. Arabidopsis thaliana ecotype Col-0 seeds were surface-sterilized in 70% (v/v) ethanol for 1 min, rinsed three times with sterile distilled water, plated on Murashige and Skoog (MS) agar (4.33 g MS basal medium (Sigma-Aldrich, St. Louis, MO, USA) and 20 g sucrose per liter, pH 5.7), stratified at 4 °C in the dark for 1 month, then transferred to soil and grown for 5 weeks at 25 °C with 16 h light/8 h dark. Scanning electron microscopy (SEM) Details of the sample preparation and imaging procedures for SEM are described previously ( Maeda et al ., 2025 ). Nylon membranes with fungal cultures were cut into about 5-mm squares and fixed in 2.5% (v/v) glutaraldehyde in 100 mM cacodylate buffer (pH 7.4) overnight at 4 °C, then 1% (w/v) osmium tetroxide in 100 mM cacodylate buffer (pH 7.4) for 1 h at room temperature. Samples were then dehydrated with a graded ethanol series, critical-point dried in absolute ethanol using Leica EM CPD300 critical point dryer (Leica Microsystems, Wetzlar, Germany), then mounted on stubs, coated with platinum-palladium, and observed with a Hitachi SU-8220 scanning electron microscope (Tokyo, Japan). RNAseq and transcriptome analysis Three biological replicates were collected for each condition described above. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Poly(A) mRNA was enriched with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA), and libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB). Libraries were sequenced on an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) with the PE150 kit (150 bp × 2 paired-end), producing 2 or 4 Gb of data per sample. Raw read quality was assessed using FastQC Ver. 16.0.1 (bioinformatics.babraham.ac.uk/projects/fastqc). Low-quality bases and adapter sequences were removed using Trimmomatic Ver. 0.39 ( Bolger et al ., 2014 ). Clean reads were aligned to the respective reference genomes using HISAT2 Ver. 2.2.1; SAM files were converted to BAM files with SamTools Ver. 1.18 ( Li et al ., 2009 ; Kim et al ., 2019 ). Read counts were generated using Rsubread Ver. 2.20.0. Differentially expressed genes (DEGs) were identified with DESeq2 using thresholds of |log₂(Fold-change)| ≥ 1 and adjusted P -value (padj) ≤ 0.05 ( Love et al ., 2014 ). Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses were performed using TBtools-II Ver. 2.119 ( Aleksander et al ., 2023 ; Chen et al ., 2023 ; Kanehisa et al ., 2025 ). Data were visualized using SRplot ( Tang et al ., 2023 ). Bioinformatic analysis Orthologous coding sequences were identified using OrthoFinder Ver. 2.5.5 ( Emms & Kelly, 2019 ); 100 single-copy orthologs were selected for multiple sequence alignment. Sequence alignments were generated using MAFFT Ver. 7.526 and subsequently trimmed with TrimAl Ver. 1.4 ( Katoh and Standley, 2013 ; Capella-Gutiérrez et al ., 2009 ). Maximum-likelihood phylogenetic trees were constructed with RAxML Ver. 8.2.12 using 1,000 bootstrap replicates and the GTRCAT or PROTCATAUTO model ( Stamatakis, 2014 ). The consistency of tree topology was assessed using approximately unbiased (AU) tests ( Shimodaira, 2002 ) with 10,000 bootstrap replicates as previously described ( Maeda et al ., 2025 ). Signal peptides were predicted with SignalP-6.0 ( Teufel et al ., 2022 ), candidate effectors with EffectorP-fungi 3.0 ( Sperschneider & Dodds, 2022 ), and subcellular localization with LOCALIZER ( Sperschneider et al ., 2017 ). Secondary metabolite biosynthetic gene clusters were identified using the fungal version of antiSMASH ( Blin et al ., 2023 ). GPI-anchored proteins were predicted with NetGPI 1.1 ( Gíslason et al ., 2021 ), and carbohydrate-active enzymes (CAZymes) were annotated using DIAMOND ( Zhang et al ., 2018 ). Default parameters were used for all analyses unless otherwise specified. For sequence similarity comparison, the EMBOSS Needle tool was used for pairwise alignments ( Madeira et al ., 2024 ). Protein domains and motifs were predicted using the Pfam database and the MEME Suite, respectively ( Bailey et al ., 2015 ; Mistry et al ., 2021 ). The 3D structures of proteins were predicted using ColabFold Ver. 1.5.5 ( Mirdita et al ., 2022 ). Five models were generated with three iterative recycles each, and the models were ranked according to their predicted local distance difference test (pLDDT) scores. The top-ranked models were refined by AMBER energy minimization to resolve steric clashes and improve stereochemical geometry. The resulting relaxed structures were used for subsequent structural analyses. The structure of Ecp2 was analyzed using the Dali server to retrieve structural homologs based on a non-redundant dataset clustered at 25% sequence identity (PDB25), and structures were compared with TM-align ( Zhang & Skolnick, 2005 ; Holm, 2022 ). Plasmid construction and protein expression Necrosis-inducing protein 1-Like (NLP) protein coding sequences were synthesized (VectorBuilder, IL, USA), cloned into the pSfinx vector using a primer set (Table S1 ) and NEBuilder HiFi assembly kit (NEB). The NLP genes were digested with EcoRI and subcloned into the pPIC9K vector (Thermo Fisher Scientific, Waltham, MA, USA) for expression in Pichia pastoris GS115 strain according to the manufacturer’s instructions. Transformants were selected on YPD medium (Fujifilm Wako, Osaka, Japan) containing G418 (Fujifilm Wako) and confirmed by PCR using the primer set in Table S1 . Expression was induced by adding 5 mL of the pre-culture grown in BMGY (buffered minimal medium supplemented with 1% glycerol, 2% peptone, and 1% yeast extract, 1.34% amino acid–free yeast nitrogen base, 100 mM potassium phosphate buffer [pH 6.0], and 4 × 10⁻⁵% D-biotin) broth to 100 mL of BMMY (1.5 % [w/v] methanol instead of glycerol in BMGY), and incubating the culture at 30 °C and 300 rpm for 96 h. Supernatants were collected by centrifugation at 12,000 rpm for 10 min. The gene Ecp2 was PCR-amplified from genomic DNA of C. fulvum using a primer set (Table S1 ) and cloned into pET-28b vector (Merck, Darmstadt, Germany) to express an N-terminal His 6 -tagged protein in Escherichia coli strain BL21(DE3). Expression was induced in LB medium with 1 mM IPTG at 16 °C for 16 h. Cells were resuspended with 50 mM KPi buffer (pH 7.4) and disrupted by sonication on ice (10 min: 2 s on–4 s off). Insoluble fractions were solubilized in 10 mL of denaturing buffer (6 M guanidine hydrochloride, 10 mM Tris, 10 mM β-mercaptoethanol, pH 8.0) for 1 h at room temperature. Proteins were purified in a Ni-affinity column HisTrap HP (Cytiva, Medemblik, The Netherlands), concentrated using Vivaspin 6 (GE Healthcare, IL, USA) and quantified with a Qubit fluorometer (Thermo Fisher Scientific). BL21(DE3) carrying empty pET-28b served as a negative control. Western blot analysis All protein samples were heated at 95 °C for 10 min in sample buffer (1% SDS, 1% 2-mercaptoethanol, 10 mM Tris–HCl, 20% (v/v) glycerol, pH 6.8) for denaturation. The proteins were separated in 12% SDS-polyacrylamide gel and transferred onto PVDF membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% skim milk in TBS-T for 1 h before antibody incubation. Anti-His and anti-HA monoclonal antibodies (Cell Signaling Technology, Danvers, MA, USA) were diluted in Can Get Signal solution 1 (1:10,000) (TOYOBO, Osaka, Japan) and used as primary antibodies to detect Ecp2 and NLPs, respectively. Horseradish peroxidase-conjugated anti-rabbit antibody was diluted with Can Get Signal solution 2 (1:2,000) (TOYOBO) for use as the secondary antibody. Signals were visualized using an ECL western detection kit (GE Healthcare, Chicago, IL, USA) and captured using a ChemiDoc imaging system (Bio-Rad). Functional assay of NLPs pSfinx plasmids were introduced into Agrobacterium tumefaciens strain GV3101 via electroporation ( Ma et al ., 2012 ). Agrobacterium suspensions were adjusted to OD 600 = 0.3 in infiltration buffer (2.03 g MgCl 2 ×6H 2 O, 1.95 g MES, 500 μL of 200 mM acetosyringone per liter, pH 5.6) and used to infiltrate leaves on 4-week-old plants of N. benthamiana by syringe pressure. Leaves were monitored for necrosis 14 days after infiltration. Necrosis was visualized by trypan blue staining ( Ma et al ., 2012 ) and quantified using ilastik (Ver. 1.4.1) ( Berg et al ., 2019 ). P. pastoris culture supernatant containing NLP proteins or BM medium (BMGY without glycerol) was mixed at a 1:1 ratio with C. fulvum spore suspension (1 × 10 6 spores mL −1 ). A 40 µL drop of each mixture was placed on a hydrophobic glass slide and incubated at room temperature for 24 h. After incubation, germination of 200 spores per treatment was assessed using a Nikon E600 light microscope (Tokyo, Japan). Culture supernatants containing NLP proteins were collected from P. pastoris and used to syringe-infiltrate N. benthamiana leaves. Twenty-four hours after infiltration, apoplastic fluid was collected by vacuum infiltration with distilled water, followed by centrifugation (1,000 rpm, 10 min) ( Joosten, 2012 ). Equal volumes (10 µL) of C. fulvum spore suspension and apoplast fluid were then incubated at room temperature for 24 h. A light microscope (Nikon) was then used to assess 200 spores for germination. Synthetic peptides of nlp24 (GenScript Biotech, Nanjing, China) were prepared in 500 nM stock solutions in dimethyl sulfoxide. Reactive oxygen species (ROS) were detected as previously described with slight modifications ( Yang et al ., 2022 ). Leaf disks (0.125 cm²) of N. benthamiana were floated on sterile distilled water for 24 h, then treated with 10 μL of a substrate mixture (5 μL of 10 mM L-012 [Fujifilm Wako] and 5 μL of protein or peptide). Luminescence was recorded every 2 min for 40 min using a Tristar3 Multimode Reader (Berthold Technologies, Bad Wildbad, Germany). Functional assays of Ecp2 The abaxial side of Cf-ECP2 tomato leaves was infiltrated with purified Ecp2 protein (final concentration 1 μM) using a syringe. Leaves were incubated at 25 °C with 16 h light/8 h dark for 6 d and checked for a hypersensitive response (HR). In vitro antifungal activity was assayed as previously described ( Chavarro-Carrero et al ., 2024 ). Briefly, fungal spores were suspended in 5% PDB containing 15 μM Ecp2 or empty vector control (1 × 10⁴ spores mL −1 ), then 100 μL of the samples (three wells per sample) was incubated in wells of 96-well plates at 25 °C for 4 days. For each well, five fields of view were photographed ( N = 15). Fungal growth was quantified and compared with the control using the Hybrid Cell Count module of the BZ-800X (KEYENCE, Osaka, Japan). Statistical analyses R version 4.4.2 ( R Core Team, 2024 ) was used for all analyses. The proportion of necrosis relative to total leaf area in N. benthamiana was assessed using Dunnett’s test, with the empty vector as the control ( N = 3). Spore germination rate of C. fulvum treated with NLPs was analyzed by one-way ANOVA followed by Tukey’s HSD test ( N = 3). The spore germination rate of C. fulvum in apoplastic fluid from N. benthamiana leaves treated with NLPs was compared using the Mann–Whitney U test, with the buffer as the control ( N = 12). The relative growth rate of fungi and yeast treated with Ecp2 was compared using Student’s t -test, with the empty vector as the control ( N = 15). RESULTS Overview of RNA-seq datasets RNA was extracted from healthy control leaves, leaves infected with C. fulvum ( on planta Cf ), and leaves with C. fulvum lesions overgrown by H. pulvinata ( on planta Cf/Hp ) ( Fig. 1a ; Fig. S1a ). Because H. pulvinata parasitizes C. fulvum on leaf surfaces and on nutrient-poor water agar ( Iida et al ., 2018 ; Maeda et al ., 2025 ), fungal RNA was also extracted from in vitro cultures of H. pulvinata alone ( in vitro Hp ), C. fulvum alone ( in vitro Cf ), and dual cultures of C. fulvum and H. pulvinata ( in vitro Cf/Hp ) on water agar ( Fig. 1a ; Fig. S1a ). Typical mycoparasitic growth characterized by white mycelium of H. pulvinata was observed at 14 days after inoculation for the on planta Cf/Hp and in vitro Cf/Hp conditions ( Fig. 1a ). Microscopic observation confirmed that H. pulvinata penetrated C. fulvum hyphae ( Fig. 1b ), and that C. fulvum entered tomato leaves through stomata (Fig. S1b ) as previously described ( Maeda et al ., 2025 ). Also, hyphae of H. pulvinata elongated on the leaf surface and entered stomata in the presence of C. fulvum (Fig. S1b ). Raw reads were aligned to the reference genome sequences of H. pulvinata , C. fulvum and tomato plants using HISAT2. The proportion of uniquely mapped reads varied across samples, ranging from 10.3 % and 88.1 % (Table S2 ), reflecting the multi organismal nature of several samples. Principal component analysis (PCA) of normalized expression values revealed clear clustering of biological replicates by treatment for each organism, indicating high within-treatment reproducibility (Fig. S1c ). In the transcriptome analysis, we identified DEGs using adjusted p -value (padj) ≤ 0.05 and |log 2 Fold-change| ≥ 1.0. Functional enrichment of DEGs was performed using KEGG pathway annotation to reveal biological processes that are positively and negatively regulated during mycoparasitism. Download figure Open in new tab Fig. 1. Cladosporium fulvum parasitized by mycoparasitic fungus Hansfordia pulvinata . (a) Representative images of a healthy tomato leaf, lesions caused by C. fulvum ( on planta Cf ), and lesions covered by white mycelium of H. pulvinata ( on planta Cf/Hp ). Tomato plants were inoculated with C. fulvum , and lesions developed after 2 weeks. H. pulvinata was then inoculated onto the diseased leaves, and white mycelia appeared on the lesions a week after the second inoculation. In vitro cultures of H. pulvinata ( in vitro Hp ) and C. fulvum ( in vitro Cf ) on nylon membranes on water agar after transfer from PDA. H. pulvinata parasitized C. fulvum colonies on water agar ( in vitro Cf/Hp ). (b) Scanning electron micrographs of hyphae of C. fulvum and H. pulvinata in in vitro Cf and Cf/Hp conditions. H. pulvinata hyphae have penetrated C. fulvum hyphae in the in vitro Cf/Hp condition. Bar indicates 10 μm. Transcriptomic reprogramming in H. pulvinata during parasitisation of C. fulvum Transcriptome analysis of H. pulvinata during parasitisation of C. fulvum in vitro ( in vitro Cf/Hp ) and on planta ( on planta Cf/Hp ) and the control ( in vitro Hp ) was used to identify DEGs involved in mycoparasitism based on gene annotation (Table S3 ). During parasitisation of C. fulvum in vitro ( in vitro Cf/Hp ), 1,336 genes were significantly upregulated and 738 genes were downregulated ( Fig. 2a ), whereas 1,793 were upregulated and 2,086 were downregulated during parasitisation of C. fulvum grown on tomato leaves ( on planta Cf/Hp ). A core set of 891 upregulated DEGs was shared between the in vitro Cf/Hp and the on planta Cf/Hp conditions and a core set of 557 genes was downregulated and were mapped to 21 upregulated and 25 downregulated KEGG pathways ( Fig. 2b ), suggesting in H. pulvinata a conserved transcriptional program was activated during parasitisation of C. fulvum both in vitro and on planta . Download figure Open in new tab Fig. 2. Differentially expressed genes (DEGs) and enriched KEGG pathways in mycoparasite Hansfordia pulvinata . (a) UpSet plot of DEGs in the in vitro and on planta mycoparasitic conditions ( in vitro Cf/Hp and on planta Cf/Hp ; Fig. 1a ). Connected dots indicate shared DEGs; Black single dots represent condition-specific DEGs. Vertical bars show the number of up- and downregulated genes; colored horizontal bars indicate the total number of DEGs detected in each interaction. (b) Bubble plot of KEGG pathway enrichment analysis based on the 891 and 557 shared DEGs shown in (a). Keys: color of symbols indicates significance level of enrichment (–log adjusted P ); size of symbol indicates the number of enriched DEGs in each pathway; the symbol indicates the functional class of the enriched DEGs. KEGG enrichment indicated reprogramming of primary and secondary metabolism in H. pulvinata during parasitisation. KEGG pathways involved in secondary metabolism, including steroids, terpenoids, and polyketides, were strongly upregulated. Genome mining prediction revealed 33 putative secondary metabolite biosynthetic gene clusters in H. pulvinata —12 non-ribosomal peptide synthetases (NRPSs), 10 type I polyketide synthases (T1PKSs), 7 terpene synthetases, 3 fungal ribosomally synthesized and post-translationally modified peptides (fungal-RiPPs), and an isocyanide—all of which underwent a marked transcriptional change during parasitisation of C. fulvum (Table S4 ). Deoxyphomenone biosynthesis ( DPH ) genes were also upregulated, consistent with H. pulvinata producing the antifungal sesquiterpenoid 13-deoxyphomenone during parasitisation (Table S4 ) ( Tirilly et al ., 1983 ; Maeda et al ., 2025 ). Interestingly, multiple amino acid metabolism pathways were consistently downregulated, despite H. pulvinata growing vegetatively during parasitisation ( Fig. 2b ). Given the central role of amino acids for protein synthesis and vegetative growth, this KEGG pattern suggested that H. pulvinata may acquire amino acids derived from C. fulvum or tomato plants to supplement its amino acid pool during mycoparasitism. Expression profile and functional analysis of small secreted proteins in H. pulvinata A comparison of the genome structures of H. pulvinata with those of fungi and the mycoparasite Trichoderma atroviride revealed that H. pulvinata had the smallest number of genes encoding carbohydrate-active enzymes (CAZymes), and the highest number of genes encoding secreted proteins (Fig. S3 ; Table S3 ). To analyze the secretome of H. pulvinata , we filtered 891 DEGs commonly upregulated in both mycoparasitic interactions ( in vitro Cf/Hp and on planta Cf/Hp ) and identified 165 genes encoding small secreted proteins ( Fig. 3a ). Although most of them could not be annotated for function, 27 were annotated as genes previously reported to be associated with pathogenicity in plant pathogenic fungi, including glycoside hydrolase, T2 family ribonuclease, EPL1-like protein (orthologous to Ecp45 effector protein in C. fulvum ), hydrophobin, necrosis-inducing protein, KP4-type killer toxin proteins, cutinase-like protein, superoxide dismutase protein, endoglucanase, and metalloprotease (Table S5 ). We focused on a necrosis inducing protein (DIP_10004627), designated HpNLP1 , which is an Nep1-like protein (NLP) homologous to PsojNIP, because HpNLP1 showed a particularly high –log 10 padj, indicating a high level of significance ( Fig. 3b ; Fig. S4 ). NLPs represent a widespread family of virulence-associated proteins in plant pathogens that trigger necrosis and generation of ROS in host plants ( Seidl & Van den Ackerveken, 2019 ). Expression of HpNLP1 increased 34-fold and 14-fold during parasitisation of C. fulvum in vitro and on planta , respectively (Table S5 ). The NLP gene HpNLP2 (DIP_10008029) is also present in H. pulvinata , but its expression was unchanged during parasitisation of C. fulvum . Download figure Open in new tab Fig. 3. A Nep1-like protein (NLP) secreted from the mycoparasite Hansfordia pulvinata induces a plant-mediated toxicity against spores of Cladosporium fulvum . (a) Workflow for identifying small secreted proteins synthesized in H. pulvinata during mycoparasitism. (b) MA plot for expression levels of 165 selected genes. Cutoff thresholds are log 2 Fold-change (±1.0) for in vitro Cf/Hp interaction and log Mean (0.3). (c) Phylogenetic tree constructed based on NLP amino acid sequences using the maximum likelihood method with 1,000 bootstrap replicates. Type I and Type II have two and four cysteine residues, respectively. Proteins analyzed in this study are in bold. (d) Multiple sequence alignment of NLP using MAFFT. Conserved residues are highlighted in orange. (e) Transient expression assays of NLPs in Nicotiana benthamiana via agroinfiltration. Chlorosis and necrosis were assessed 14 days after infiltration by trypan blue staining. Similar results were obtained in three independent experiments; representative results are in Supplementary Fig. S5c. (f) Percentage germination of conidia of C. fulvum after 24-h treatment with apoplastic fluid from N. benthamiana leaves that had been infiltrated with different NLPs or buffer. Asterisks indicate significant differences between treatments determined using Dunnett’s or the Mann–Whitney U test. Phylogenetic analysis placed HpNlp1 and HpNlp2 within the type II and fungal type I clades, respectively, in NLPs ( Fig. 3c ). HpNlp1 contained four cysteine residues predicted to form two disulfide bonds between positions 39–65 and 82–87 (Fig. S4a and b ). Multiple sequence alignment showed that both HpNlp1 and HpNlp2 contained the conserved region I and the C-terminal heptapeptide GHRHDWE motif; this conserved 24-amino-acid peptide (nlp24) is recognized as a microbe- or pathogen-associated molecular pattern (MAMP/PAMP) ( Oome et al ., 2014 ). The tryptophan (W) and proline (P) residues essential for necrosis-inducing activity in the conserved region I ( Zhou et al ., 2012 ) were conserved in HpNlp2 but replaced by tyrosine (Y) and glutamic acid (E) in HpNlp1 ( Fig. 3d ), which was the only NLP strongly expressed during mycoparasitism. In the heptapeptide motif of HpNlp1, the second histidine (H) was replaced by asparagine (N) ( Fig. 3d ). Structural superposition of PsojNIP and HpNlp1 yielded a TM-score of 0.86 and a root mean square deviation (RMSD) of 1.93 Å, demonstrating that despite mutations in the nlp24-like regions of HpNlp1, the global protein structure was largely preserved, as indicated by these similar values compared to the PsojNIP (Fig. S4c ). Recombinant proteins PsojNIP, HpNlp1 and HpNlp2 were produced in P. pastoris and used to infiltrate Nicotiana benthamiana leaves. Western blot analysis of HpNlp1 demonstrated an additional band larger than the predicted molecular weight, suggesting dimer formation (Fig. S5a and b ). As previously reported, PsojNIP induced necrosis and chlorosis ( Qutob et al ., 2002 ), whereas HpNlp1 and HpNlp2 did not trigger visible necrosis or ROS accumulation ( Fig. 3e ; Fig. S5c and d ). The high expression of HpNLP1 gene under in vitro Cf/Hp condition, in the absence of the plant, suggested that HpNlp1 might directly suppress C. fulvum growth. Contrary to this expectation, no such direct antifungal activity was detected against C. fulvum spore germination and hyphal elongation (Fig. S5e ). Remarkably, however, apoplastic fluid—where C. fulvum develops—collected from N. benthamiana leaves that had been infiltrated with HpNlp1 strongly suppressed C. fulvum spore germination, whereas the protein with a point mutantation (HpNlp1 N138H ) failed to elicit antifungal activity ( Fig. 3f ). Neither of the nlp24 peptides of HpNlp1 and HpNlp1 N138H induced RLP23-mediated resistance (Fig. S5f ). These findings together demonstrated that HpNlp1 diverges from typical necrosis-inducing NLPs in plant pathogens, inducing the accumulation of apoplastic antifungal substances that suppress C. fulvum spore germination instead of promoting host cell death. Transcriptomic responses of pathogen C. fulvum parasitized by H. pulvinata Next, we investigated the DEGs of C. fulvum through KEGG pathway analysis after tomato was infected by C. fulvum ( on planta Cf ), dual infection on tomato leaves ( on planta Cf/Hp ), and dual culture on agar medium ( in vitro Cf/Hp ) ( Fig. 1a ). DEGs in C. fulvum were identified during parasitisation relative to the in vitro Cf control, in which the fungus was cultured on water agar. A total of 1,902 genes were upregulated and 1,783 genes were downregulated when C. fulvum infected tomato leaves ( on planta Cf ) ( Fig. 4a ). Under the two mycoparasitic conditions, 2,195 upregulated and 2,435 downregulated genes were detected on planta Cf/Hp , and 1,479 upregulated and 845 downregulated were detected in vitro Cf/Hp . We identified 363 upregulated and 158 downregulated genes shared between the two mycoparasitic conditions ( in vitro Cf/Hp and on planta Cf/Hp ), indicating a conserved transcriptional response to H. pulvinata attack. KEGG enrichment analysis assigned these shared DEGs to seven upregulated and three downregulated pathways, revealing strong activation of protein folding and processing pathways in C. fulvum during parasitisation by H. pulvinata ( Fig. 4b ). Conversely, genes involved in amino acid metabolism were consistently downregulated, as was also observed in H. pulvinata , suggesting coordinated suppression of primary metabolism in this fungus-fungus interaction. Download figure Open in new tab Fig. 4. Differentially expressed genes (DEGs) and enriched KEGG pathways in the pathogen Cladosporium fulvum . (a) UpSet plot showing DEGs identified in the on planta Cf, in vitro Cf/Hp, and on planta Cf/HP conditions (see Fig. 1A ). Connected dots indicate shared DEGs; Black single dots represent condition-specific DEGs. Vertical bars show the numbers of up- and downregulated genes; colored horizontal bars indicate the total number of DEGs detected in each condition. (b) Bubble plot of KEGG pathway enrichment analysis based on the 363 and 158 shared DEGs shown in (a). Keys: color of symbols indicates significant level of enrichment (–log adjusted P ); size of symbol indicates the number of enriched DEGs in each pathway; the symbol indicates the functional class of the enriched DEGs. In C. fulvum , protein folding and processing pathways were strongly activated when parasitized by H. pulvinata ( Fig. 4a ). These responses are indicative of endoplasmic reticulum (ER) stress and accumulation of misfolded proteins, phenomena often triggered by exposure to antifungal compounds ( Chaillot et al ., 2015 ). Similar to the results in the mycoparasite H. pulvinata , the downregulation of secondary metabolism-related genes suggested that C. fulvum reallocated resources from the production of secondary metabolites to survive and mitigate stress. The expression profiles of DEGs detected in C. fulvum were further analyzed based on functional classification as previously reported by Zaccaron & Stergiopoulos (2024) . Among the 42 genes associated with secondary metabolite biosynthesis, 15 (35.7 %) and 18 (42.8 %) were downregulated in the on planta Cf and on planta Cf/Hp interaction, respectively ( Fig. 5a ; Table S6 ). Thirteen genes, including PKS6 (CLAFUR5_12905), a key enzyme in cladofulvin biosynthesis ( Griffiths et al ., 2018 ), were consistently downregulated in both interactions. Download figure Open in new tab Fig. 5. Effector genes in the pathogen Cladosporium fulvum were highly expressed during mycoparasitism by Hansfordia pulvinata. (a) Functional classification of DEGs identified for the on planta Cf , in vitro Cf/Hp and on planta Cf/Hp interactions. Red: upregulated, stable: grey, blue: downregulated. The number of genes assigned to each category is in parentheses. (b) Heatmap showing expression profiles of effector genes. The red-to-blue gradient represents up- and downregulated expression, respectively. Genes with an adjusted P -value ≥0.05 are indicated in gray as not detected (ND). Red arrowhead indicates Ecp2 . (c) Antimicrobial activity prediction for 56 effector genes upregulated during the in vitro Cf/Hp interaction (determined using the AMAPEC program). Symbols denote protein location predicted by EffectorP-fungi 3.0. In contrast, the category of small secreted proteins had the highest number of upregulated DEGs, including numerous known or predicted effectors ( Fig. 5a ). An expression heatmap of effector genes showed that, as expected, the majority of the 106 effector genes were highly expressed during infection of tomato by C. fulvum ( on planta Cf ) ( Fig. 5b ). Expression of effector genes are generally low on rich synthetic media ( Mesarich et al ., 2014 ); however surprisingly, 56 effector genes were upregulated in the in vitro coculture of C. fulvum and H. pulvinata ( in vitro Cf/Hp ) ( Fig. 5b ). On tomato leaves, effector expression in C. fulvum remained high regardless of the presence of H. pulvinata (Fig. S6 ). These results suggested that C. fulvum effectors may play a defensive role, acting as countermeasures against mycoparasitic attack by H. pulvinata or are upregulated due to nitrogen limitation caused by parasitisation by H. pulvinata. To investigate this possibility, we conducted an AMAPEC analysis, which predicts antimicrobial proteins based on 3D-structural signatures ( Mesny et al ., 2024 ). Of the 56 effector genes that were upregulated under the in vitro Cf/Hp condition, 34 were predicted to possess antimicrobial activity, suggesting that many effectors may act as antifungal proteins ( Fig. 5c ; Table S7 ). Among these, we focused on the effector Ecp2, which is known to trigger a hypersensitive response (HR) in tomato cultivars carrying the Cf-ECP2 resistance gene. Structural and functional similarity between C. fulvum Ecp2 effector and antifungal killer toxin 4 proteins The Alphafold2-predicted structure of Ecp2 yielded high confidence scores, with a pLDDT of 91.3 and a pTM of 0.872, indicating reliable local and global structures ( Fig. 6a ). Structural similarity search using the DALI server identified two killer toxin 4 (KP4)-like proteins, ZtKP4 in Zymoseptoria tritici and UmVKP4 in Ustilago maydis , despite their low amino acid sequence similarity (2.9 to 36.9%) (Table S8 ). Comparative structural analysis showed that the overall arrangement of the α-helices and multiple β-sheets were conserved among these proteins ( Fig. 6a and S7a ). The TM-scores for Ecp2 relative to ZtKP4 and UmVKP4 were 0.81 and 0.59, respectively, supporting that Ecp2 had the same structural fold as the KP4 protein family according to the SCOP and CATH classification criteria ( Fig. 6b ). Download figure Open in new tab Fig. 6. Sequence dissimilarity and structural similarity between Cladosporium fulvum effector Ecp2 and killer toxin KP4 proteins. (a) Predicted 3D structure of Ecp2 generated using AlphaFold2. The color spectrum from blue to red indicates the N- to C-terminal positions of residues. (b) Structural comparison of Ecp2 with ZtKP4 and UmVKP4 proteins. The superposition of the aligned proteins was evaluated using TM-align, and the TM-score and RMSD values are shown. (c) Heatmap showing sequence similarity among Ecp2, ZtKP4, and UmVKP4 proteins as identified by BLASTp analysis. (d) Conserved motifs of the proteins identified by MEME motif analysis. (e) Representative tomato leaf from 4-week-old tomato plant carrying the Cf-ECP2 resistance gene at 6 days after infiltration with purified Ecp2 protein produced by Escherichia coli . White dotted outline: area infiltrated with infiltrated with 1 μM Ecp2; black dotted outline: infiltrated with control protein purified from E. coli harboring an empty vector (EV). Numbers in parentheses indicate the number of leaves with a hypersensitive response (HR) relative to the number of total leaves. (f) In vitro antifungal activity of Ecp2 against C. fulvum , H. pulvinata , Alternaria sp., Colletotrichum orbiculare , and Saccharomyces cerevisiae . Spores were cultured on 5% potato dextrose agar with or without Ecp2. Relative hyphal length was determined 4 days after treatment. Bars = 250 μm. Significant differences between EV and Ecp2 treatments were assessed using Student’s t-test, and p -values are indicated. To explore the evolutionary relationships among these KP4-like proteins, we searched for homologs of Ecp2, ZtKP4, and UmVKP4. Nineteen Ecp2 homologs were identified across three fungal classes, while only one ZtKP4 homolog was detected in Zymoseptoria tritici (syn. Mycosphaerella graminicola ) (Table S9 ). A total of 188 UmVKP4 homologs were found in 10 classes. Based on sequence similarity, these proteins clustered into two groups; Ecp2 and ZtKP4 were placed in the same clade ( Fig. 6c ). Motif analysis revealed distinct conserved amino acid motifs in each group, and UmVKP4 group lacked an Hce2 domain ( Fig. 6d ). While Ecp2 and ZtKP4 shared two conserved disulfide bonds, UmVKP4 had five, suggesting differences in structural stability and folding despite their shared overall architecture (Fig. S7b ). These results indicated that Ecp2 and UmVKP4 protein groups likely originated from two distinct evolutionary lineages. Given that Ecp2 is structurally and phylogenetically close to ZtKP4, which has antifungal activity ( de Guillen et al ., 2024 ), we investigated the antifungal potential of Ecp2. Infiltration of Cf-ECP2 tomato leaves with recombinant Ecp2 (Fig. S7c ) triggered a specific HR ( Fig. 6e ). At the high concentrations used, the active Ecp2 protein significantly inhibited hyphal growth in diverse fungi: C. fulvum , H. pulvinata , Alternaria sp. isolated from tomato fruits, the plant pathogen Colletotrichum orbiculare and the yeast Saccharomyces cerevisiae ( Fig. 6f ). Together, these results demonstrated that Ecp2 has a dual role: inducing Cf-ECP2 -mediated immunity in plant while inhibiting fungal growth. H. pulvinata exhibited marginally higher resistance than the other tested fungi and yeast to Ecp2. Enrichment analysis of DEGs and KEGG pathways in tomato plants We also analyzed differential gene expression in tomato leaves inoculated with C. fulvum and inoculated with C. fulvum followed by H. pulvinata , each compared with uninoculated control leaves ( Fig. 1a ). Infection by C. fulvum alone led to 3,231 upregulated and 4,980 downregulated genes, whereas in the presence of H. pulvinata ( on planta Cf/Hp ), 3,465 genes were upregulated and 5,679 downregulated ( Fig. 7a ). Among these, 1,619 up- and 2,153 downregulated genes were uniquely responsive to H. pulvinata , mapping to 35 and 33 enriched KEGG pathways, respectively ( Fig. 7b ). Pathways associated with plant–pathogen interaction, phytoalexin biosynthesis (stilbenoid, diarylheptanoid and gingerol), biosynthesis of unsaturated fatty acids, other secondary metabolites, phenylpropanoid biosynthesis and plant hormone signal transduction were induced when H. pulvinata parasitized C. fulvum on leaves ( Fig. 7b ). Genes encoding calcium-dependent protein kinases, PR1, RIN4, MAP kinase, WRKY22 and defensins were highly expressed (Fig. S8a ; Table S10 ). These results indicated that H. pulvinata triggers plant immunity during parasitisation of C. fulvum on tomato leaves. In contrast, pathways associated with photosynthesis antenna proteins and porphyrin metabolism, which is involved in chlorophyll biosynthesis, were upregulated during infection by C. fulvum alone, but significantly downregulated when parasitized by H. pulvinata ( Fig. 7b ; Fig. S8b and c ), indicating that H. pulvinata triggered pronounced plant resistance responses during parasitisation of C. fulvum . Download figure Open in new tab Fig. 7. Differentially expressed genes (DEGs) and enriched KEGG pathways in tomato plants. (a) UpSet plot of DEGs in the on planta Cf and on planta Cf/Hp conditions (see Fig. 1a ). Healthy leaves were used as a control. Connected dots indicate shared DEGs; Black single dots represent condition-specific DEGs. Vertical bars show the number of up- and downregulated genes; colored horizontal bars indicate the total number of DEGs detected in each interaction. (b) Bubble plot of KEGG pathway enrichment analysis based on the 1,619 and 2,153 DEGs shown in (a). Keys: color of symbols indicates significance level of enrichment (–log adjusted P ); size of symbol indicates the number of enriched DEGs in each pathway; the symbol indicates the functional class of the enriched DEGs. Stars indicate pathways highlighted in (c). (c) KEGG pathways involved in amino acid biosynthesis in tomato. Strikingly, 13 amino acid biosynthesis and metabolism pathways were strongly activated in the on planta Cf/Hp condition ( Fig. 7b and c ). Although amino acid metabolism was broadly repressed in both C. fulvum and H. pulvinata during mycoparasitism ( Fig. 2b ; Fig. 4b ), it was upregulated in tomato ( Fig. 7b and c ). The pronounced activation of amino acid biosynthesis pathways in tomato suggests that the plant supplies amino acids to both the pathogen C. fulvum and its mycoparasite H. pulvinata during the tritrophic interaction. DISCUSSION In this study, we analyzed gene expression in tomato plants, the phytopathogenic fungus C. fulvum , and its mycoparasite H. pulvinata alone or during their bitrophic and tritrophic interaction to capture coordinated and distinct responses. We thus discovered previously unrecognized aspects of molecular crosstalk and revealed how the transcriptome of each organism was dynamically reprogrammed during these interactions. Evolutionary and functional insights into an atypical NLP in the biocontrol mycoparasite H. pulvinata The identification of 165 secreted small proteins upregulated during mycoparasitism highlights the central role of the secretome of H. pulvinata . Among them, HpNlp1, an atypical NLP, lacked both necrosis-inducing activity or RLP23 receptor-mediated response in plants, unlike canonical NLPs such as PsojNIP ( Qutob et al ., 2006 ; Böhm et al ., 2014 ; Oome et al ., 2014 ). Instead, HpNlp1 accumulated antifungal substances in the apoplast and inhibited spore germination of C. fulvum . Although H. pulvinata cannot grow independently on tomato leaves ( Iida et al ., 2018 ), it follows the hyphae of C. fulvum that enter the stomata of tomato leaves (Fig. S1b ), indicating that the two fungi co-occur in the tomato apoplast. Furthermore, the functional divergence of the NLPs between the pathogen and mycoparasite indicates the evolutionary plasticity of NLP family. Despite its structural similarity to PsojNIP, HpNlp1 has distinct biological functions, suggesting that subtle amino acid modifications can drive functional divergence within the NLP family. If H. pulvinata induced a strong necrotic or immune response in tomato, the host fungus C. fulvum would decline, limiting its own nutrient supply. Therefore, the high upregulation of a noncytotoxic NLP such as HpNlp1 during mycoparasitism is evolutionarily reasonable. Notably, the HpNLP1 gene was expressed in H. pulvinata when parasiting its host on planta and in vitro , suggesting that it recognizes C. fulvum itself rather than plant-derived signals. Although HpNlp1 did not directly inhibit C. fulvum growth, it indirectly promoted the accumulation of antifungal metabolites, suggesting coevolution of H. pulvinata with not only its fungal host C. fulvum but also tomato plants. KEGG enrichment analysis indicated that H. pulvinata activated multiple secondary metabolic pathways, particularly those related to terpenoids, polyketides, steroids and DPH biosynthetic gene cluster, during mycoparasitism. These findings agree with previous findings that H. pulvinata produces 13-deoxyformenone, an eremophilane-type sesquiterpenoid, which is fungistatic to C. fulvum ( Maeda et al ., 2025 ). The production of antifungal substances triggered by HpNlp1 in the tomato apoplast was correlated with the induction of genes encoding defensins and phytoalexins, which were expressed only when C. fulvum was parasitized by H. pulvinata on tomato leaves. Given that similar noncytotoxic NLPs from the biocontrol oomycete Pythium oligandrum also induce plant defensins ( Yang et al ., 2022 ), HpNlp1 may represent a conserved mechanism among biocontrol agents that modulate plant immunity without causing cell death. H. pulvinata may employ a two-tiered strategy to preserve the spores of its host fungus C. fulvum , combining a direct antifungal sesquiterpenoid with an indirect, noncytotoxic protein, HpNlp1. Ecp2 effector as a novel antifungal protein in the pathogen C. fulvum KP4 family proteins, broadly conserved in phytopathogenic fungi, are antifungal but do not interact with the plant. For instance, U. maydis UmVKP4 suppresses the growth of susceptible strains of the same species by inhibiting calcium-dependent pathways that regulate the cell cycle and morphogenesis ( Gu et al ., 1995 ; Gage et al ., 2002 ). Z. tritici ZtKP4 has fungicidal activity even against itself ( de Guillen et al ., 2024 ). Our structural modeling showed that C. fulvum Ecp2 shared similarity with KP4 proteins and had broad-spectrum antifungal activity ranging from filamentous fungi to yeast, including self-inhibition similar to that of ZtKP4. The β1–β2 loop in Ecp2 should functionally correspond to the β3–β4 loop of UmVKP4, which mediates interactions with intracellular proteins ( Gu et al ., 1995 ). However, Ecp2 lacked the key lysine residue (Lys42) that UmVKP4 requires for full antifungal activity ( Gage et al ., 2001 ), suggesting a similar mechanism underlying antifungal action but involving an alternative molecular interface. During the arms race between tomato and C. fulvum , a KP4-like antifungal protein may have been diverted to Ecp2 effector. However, the structural basis underlying CfECP2 -mediated immune recognition and how Ecp2 acquired the dual ability to interact with both fungal and plant targets remain unresolved. Ecp2 was strongly expressed even during in vitro mycoparasitism, suggesting a dual role as both a virulence factor against plants and an antifungal protein in microbial competition. Similar dual functionality is seen in the root endophyte Serendipita spp., where a chitinase encoding effector is induced during both plant colonization and microbial interaction ( Eichfeld et al ., 2024 ). The vascular wilt fungus Verticillium dahliae secretes antimicrobial effectors such as VdAve1 and VdAMP2 to gain an ecological advantage in the soil biome ( Snelders et al ., 2020 ). The soil-borne white root rot pathogen Rosellinia necatrix , which is phylogenetically related to V. dahliae , secretes antimicrobial effector proteins during host plant colonization ( Chavarro-Carrero et al ., 2024 ). While the rhizosphere harbors highly diverse and competitive microbial communities, the diversity of the microbial community in the endosphere such as the apoplastic space is generally relatively low ( Snelders et al ., 2022 ) because colonization of the apoplast is typically restricted to specialized microbes that can secrete effectors to suppress host immunity ( Rocafort et al ., 2020 ). In fact, despite previous predictions that some apoplastic effectors produced by C. fulvum may have antimicrobial activity, no direct evidence had been found so far ( Mesarich et al ., 2018 ). Our finding indicates the potential for C. fulvum not only to utilize effectors for efficient colonization of tomato leaves but also to compete with/inhibit other microbes in the endosphere and phyllosphere. Although H. pulvinata was moderately tolerant of Ecp2, the molecular basis for this tolerance during mycoparasitism remains unclear. The Hce2 (Homologs of C. fulvum Ecp2) superfamily is widely conserved across the Ascomycota and Basidiomycota , suggesting that its origin predates the divergence of the Dikarya lineage ( Stergiopoulos et al ., 2012 ). In contrast, U. maydis UmVKP4, which shares structural similarity with Ecp2, lacks the Hce2 domain and shared sequence motifs, and differs in the number of disulfide bonds (Fig. S7b ). These findings suggest that UmVKP4 represents a case of convergent evolution, resulting in structural resemblance to Ecp2 and ZtKP4. Molecular evolutionary analyses suggest that the amino acid substitution underlying Hce2 function diversification was fixed early in evolution ( Stergiopoulos et al ., 2012 ). Ancestral Hce2 proteins may initially act as antimicrobial factors to provide competitive advantages in microbe-rich habitats, and have been co-opted as an effector to inhibit plant immunity. Mutations in effector genes that allow C. fulvum to evade Cf resistance in plants may increase its susceptibility to antagonistic microbes such as H. pulvinata . This hypothesis suggests that mycoparasitic biocontrol agents can circumvent the classical arms race between plant and pathogen, providing a sustainable disease management. Potential metabolic trade-offs in amino acid pathways between fungi and tomato plants The physiological changes found in tomato plants highlight the dynamic interplay between microbial interplay and plant defense responses. The expression of genes in multiple amino acid biosynthetic pathways were enhanced only when H. pulvinata parasitized C. fulvum on tomato leaves. Amino acids are key mediators of plant–microbe interactions, functioning as signaling molecules that activate defense and as nutritional sources that are exploited by pathogens ( Moormann et al ., 2022 ; Tünnermann et al ., 2022 ). Indeed, infection by C. fulvum leads to the accumulation of free amino acids in the apoplast ( Solomon & Oliver, 2001 ). Because the acquisition of plant-derived amino acids is energetically advantageous to microbes, many plant pathogens modulate expression of genes encoding amino acid transporters to optimize uptake ( Sonawala et al ., 2018 ). Thus, the regulation of amino acid assimilation and transport in plants can be a decisive factor shaping the outcome of plant–pathogen interactions. In contrast, amino acid biosynthetic pathways in H. pulvinata and C. fulvum were downregulated under mycoparasitic conditions, suggesting that tomato-derived amino acids could be shared or sequentially utilized by these fungi ( Fig. 8 ). In this metabolic trade-off, tomato plants might invest in amino acids that benefit H. pulvinata , while receiving biocontrol protection in return. Although amino acids are fundamental primary metabolites during fungal vegetative growth, the metabolic profile of H. pulvinata , which exhibited vigorous hyphal growth during mycoparasitism, deviated from this conventional paradigm. Because H. pulvinata parasitizes C. fulvum , it likely becomes the recipient of these plant-derived nutrients via its host C. fulvum , forming a potential metabolic trade-off in the tritrophic interaction. In this context, the activation of amino acid biosynthesis in tomato may provide a nutritional foundation that ultimately supports colonization and biocontrol activity by H. pulvinata . Although C. fulvum may also acquire amino acids from plants, these resources are likely reallocated to H. pulvinata , the ultimate beneficiary of the tritrophic interaction. Similar strategies have been reported for the biocontrol fungus Pseudozyma flocculosa, active against powdery mildews, which transiently suppresses photosynthesis to promote amino acid release ( Laur et al ., 2018 ). The mycoparasitic fungus Trichoderma erinaceum exploits environmental amino acids to enhance the synthesis of antifungal secondary metabolites derived from amino acids ( Guo et al ., 2020 ). Together, these results suggested that shifts in amino acid metabolism enable H. pulvinata to harness pathogen-induced plant responses to its own benefit. Download figure Open in new tab Fig. 8. Overview of the tritrophic interaction among tomato plants, pathogen Cladosporium fulvum , and mycoparasite Hansfordia pulvinata . C. fulvum secretes effector proteins that function as virulence factors in susceptible tomato cultivars and antifungal proteins active against other fungi, but act as avirulence factors upon recognition by Cf -resistant cultivars. H. pulvinata parasitizes C. fulvum and induces the production of apoplastic antifungal substances in tomato plants via the Nep1-like protein HpNlp1. H. pulvinata also activates the biosynthesis of the antifungal sesquiterpene 13-deoxyphomenone during mycoparasitism. Other plant defense responses support this biocontrol effect. Pathways related to amino acid biosynthesis in the three organisms suggest that nutrients from the plant are supplied to H. pulvinata . Mycoparasitism by H. pulvinata modulates plant immunity and pathogen growth In conclusion, the mycoparasite H. pulvinata actively reprogrammes gene expression and physiological responses in the pathogen C. fulvum and its tomato host to establish a stable tritrophic interaction ( Fig. 8 ). H. pulvinata induced expression of the 13-deoxyphomenone biosynthetic gene cluster in tomato plants; antimicrobial substances accumulated in the apoplast and directly suppressed C. fulvum , thus enhancing plant resistance. In turn, C. fulvum secretes effectors such as Ecp2 that exhibites roles in both fungal competition and plant immunity. During the tritrophic interaction in tomato, amino acid metabolism and defense response pathways are reprogrammed, orchestrating distinct strategies against beneficial and pathogenic microbes. Collectively, these findings underscore the dynamic interplay in plant–pathogen–mycoparasite interactions, demonstrating that H. pulvinata simultaneously strengthens tomato immunity and represses C. fulvum virulence via direct antagonism and modulation of plant metabolic pathways. COMPETING INTERESTS The authors declare that they have no commercial or financial relationships that could be construed as a potential conflict of interest. AUTHOR CONTRIBUTIONS YI designed the study and managed the research funds. KM, HS and TaS performed RNA-seq and analyzed the data. KM, MK, MO, and TK analyzed NLPs. ToS conducted electron microscopy and EI developed a microscopic observation protocol. KM and KS studied the Ecp2 protein. KM and YI wrote the manuscript. All authors reviewed and approved the final version. DATA AVAILABILITY Genomic sequences for C. fulvum strain Race5_Kim and H. pulvinata strain 414-3 are available as accessions PRJNA565804 and PRJDB8178 at NCBI BioProject, respectively ( Sushida et al ., 2019 ; Zaccaron & Stergiopoulos, 2024 ). Tomato genome data were obtained from the Solanaceae Genomics Network ( Hosmani et al ., 2019 ). Additional genomes are accessible via JGI MycoCosm ( mycocosm.jgi.doe.gov ) and the DDBJ/EMBL/GenBank repository ( www.ncbi.nlm.nih.gov ). Custom scripts and supporting datasets were deposited at Figshare (doi.org/10.6084/m9.figshare.30265009). SUPPORTING INFORMATION Fig. S1 Experimental treatments and principal component analysis (PCA) of transcripts from the mycoparasite Hansfordia pulvinata (Hp), the pathogen Cladosporium fulvum (Cf), and tomato plant, Solanum lycopersicum . Fig. S2 Secondary metabolite biosynthesis gene clusters in the mycoparasite Hansfordia pulvinata . Fig. S3 Phylogenetic analysis and functional characterization of proteins in fungal species. Fig. S4 Structure of HpNlp1. Fig. S5 Responses in plant leaves triggered by HpNlp1 protein. Fig. S6 Heat map and hierarchical clustering of effector genes expressed in Cladosporium fulvum . Fig. S7 Structural comparative analysis of Ecp2 with ZtKP4 and UmVKP4. Fig. S8 Differentially expressed genes (DEGs) in KEGG pathways detected in tomato plants. Table S1 Primer sequences used in this study. Table S2 Summary statistics of RNA-seq data and mapping results. Table S3 Functional annotation of all genes in Hansfordia pulvinata genome. Table S4 Predicted gene clusters involved in secondary metabolite biosynthesis in Hansfordia pulvinata . Table S5 Small secreted protein-coding genes (≤300 aa) in Hansfordia pulvinata . Table S6 Key genes encoding secondary metabolism enzymes in Cladosporium fulvum . Table S7 Effector genes identified in Cladosporium fulvum . Table S8 Structural homologs of Ecp2 protein identified from PDB25 using the Dali server. Table S9 Species possessing homologs of Ecp2 and KP4 proteins. Table S10 Log 2 fold-change for defensin-like genes in tomato plants. ACKNOWLEDGEMENTS This work was supported by a Grant-in-Aid for Scientific Research from JSPS (20H02993 and 24K08919) and the Research and Implementation Promotion Program through Open Innovation Grants (JPJ011937) from the Project of the Bio-oriented Technology Research Advancement Institution (BRAIN) (Y. Iida). We are grateful to K. Ikeda for providing fungal strains, M.H.A.J. Joosten for Cf-ECP2 tomato seeds, D. Takemoto for the psojNIP gene, F. Takken for pSfinx, and Y. Kubo for the C. orbiculare strain. We thank Y. Kubo, M.Z. Fanani and S. Kodama for valuable suggestions, C. Tanaka for laboratory management, P.J.G.M. de Wit for critical reading of the manuscript. Funder Information Declared Japan Society for the Promotion of Science, https://ror.org/00hhkn466 , 20H02993 , 24K08919 Bio-oriented Technology Research Advancement Institution, https://ror.org/02f62sy66 , JPJ011937 Footnotes The text and figures have been revised for clarity. https://doi.org/10.6084/m9.figshare.30265009 REFERENCES ↵ Bailey TL , Johnson J , Grant CE , Noble WS . 2015 . The MEME Suite . Nucleic Acids Research . 2015 Jul 1; 43 ( W1 ): W39 – 49 . doi: 10.1093/nar/gkv416 . Epub 2015 May 7. PMID: 25953851 ; PMCID: PMC4489269 . OpenUrl CrossRef PubMed ↵ Berg S , Kutra D , Kroeger T , Straehle CN , Kausler BX , Haubold C , Schiegg M , Ales J , Beier T , Rudy M , et al. 2019 . ilastik: interactive machine learning for (bio)image analysis . Nature Methods . 2019 Dec; 16 ( 12 ): 1226 – 1232 . doi: 10.1038/s41592-019-0582-9 . Epub 2019 Sep 30. PMID: 31570887 . OpenUrl CrossRef PubMed ↵ Blin K , Shaw S , Augustijn HE , Reitz ZL , Biermann F , Alanjary M , Fetter A , Terlouw BR , Metcalf WW , Helfrich EJN , et al. 2023 . antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualisation . Nucleic Acids Research . 2023 Jul 5; 51 ( W1 ): W46 – W50 . doi: 10.1093/nar/gkad344 . PMID: 37140036 ; PMCID: PMC10320115 . OpenUrl CrossRef PubMed ↵ Böhm H , Albert I , Oome S , Raaymakers TM , Van den Ackerveken G , Nürnberger T. 2014 . A conserved peptide pattern from a widespread microbial virulence factor triggers pattern-induced immunity in Arabidopsis . PLOS Pathogens . 2014 Nov 6; 10 ( 11 ): e1004491 . doi: 10.1371/journal.ppat.1004491 . PMID: 25375108 ; PMCID: PMC4223075 . OpenUrl CrossRef PubMed ↵ Bolger AM , Lohse M , Usadel B . 2014 . Trimmomatic: a flexible trimmer for Illumina sequence data . Bioinformatics . 2014 Aug 1; 30 ( 15 ): 2114 – 20 . doi: 10.1093/bioinformatics/btu170 . Epub 2014 Apr 1. PMID: 24695404 ; PMCID: PMC4103590 . OpenUrl CrossRef PubMed Web of Science ↵ Capella-Gutiérrez S , Silla-Martínez JM , Gabaldón T . 2009 . trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses . Bioinformatics . Aug 1; 25 ( 15 ): 1972 – 3 . doi: 10.1093/bioinformatics/btp348 . Epub 2009 Jun 8. PMID: 19505945 ; PMCID: PMC2712344 . OpenUrl CrossRef PubMed Web of Science ↵ Chaillot J , Tebbji F , Remmal A , Boone C , Brown GW , Bellaoui M , Sellam A . 2015 . The Monoterpene Carvacrol Generates Endoplasmic Reticulum Stress in the Pathogenic Fungus Candida albicans . Antimicrobial Agents and Chemotherapy . 2015 Aug; 59 ( 8 ): 4584 – 92 . doi: 10.1128/AAC.00551-15 . Epub 2015 May 26. PMID: 26014932 ; PMCID: PMC4505245 . OpenUrl Abstract / FREE Full Text ↵ Chavarro-Carrero EA , Snelders NC , Torres DE , Kraege A , López-Moral A , Petti GC , Punt W , Wieneke J , García-Velasco R , López-Herrera CJ , et al. 2024 . The soil-borne white root rot pathogen Rosellinia necatrix expresses antimicrobial proteins during host colonization . PLOS Pathogens . 2024 Jan 18; 20 ( 1 ): e1011866 . doi: 10.1371/journal.ppat.1011866 . PMID: 38236788 ; PMCID: PMC10796067 . OpenUrl CrossRef PubMed ↵ Chen C , Wu Y , Li J , Wang X , Zeng Z , Xu J , Liu Y , Feng J , Chen H , He Y , et al. 2023 . TBtools-II: A “one for all, all for one” bioinformatics platform for biological big-data mining . Molecular Plant . 2023 Nov 6; 16 ( 11 ): 1733 – 1742 . doi: 10.1016/j.molp.2023.09.010 . Epub 2023 Sep 22. PMID: 37740491 . OpenUrl CrossRef PubMed ↵ Eichfeld R , Mahdi LK , De Quattro C , Armbruster L , Endeshaw AB , Miyauchi S , Hellmann MJ , Cord-Landwehr S , Peterson D , Singan V , et al. 2024 . Transcriptomics reveal a mechanism of niche defense: two beneficial root endophytes deploy an antimicrobial GH18-CBM5 chitinase to protect their hosts . New Phytologist . 2024 Nov; 244 ( 3 ): 980 – 996 . doi: 10.1111/nph.20080 . Epub 2024 Sep 3. PMID: 39224928 . OpenUrl CrossRef PubMed ↵ Emms DM , Kelly S . 2019 . OrthoFinder: phylogenetic orthology inference for comparative genomics . Genome Biology . 2019 Nov 14; 20 ( 1 ): 238 . doi: 10.1186/s13059-019-1832-y . PMID: 31727128 ; PMCID: PMC6857279 . OpenUrl CrossRef PubMed ↵ Gage MJ , Bruenn J , Fischer M , Sanders D , Smith TJ . 2001 . KP4 fungal toxin inhibits growth in Ustilago maydis by blocking calcium uptake . Molecular Microbiology . 2001 Aug; 41 ( 4 ): 775 – 85 . doi: 10.1046/j.1365-2958.2001.02554.x . PMID: 11532143 . OpenUrl CrossRef PubMed ↵ Gage MJ , Rane SG , Hockerman GH , Smith TJ . 2002 . The virally encoded fungal toxin KP4 specifically blocks L-type voltage-gated calcium channels . Molecular Pharmacology . 2002 Apr; 61 ( 4 ): 936 – 44 . doi: 10.1124/mol.61.4.936 . PMID: 11901234 . OpenUrl Abstract / FREE Full Text ↵ Gene Ontology Consortium ; Aleksander SA , Balhoff J , Carbon S , Cherry JM , Drabkin HJ , Ebert D , Feuermann M , Gaudet P , Harris NL , et al. 2023 . The Gene Ontology knowledgebase in 2023 . Genetics . 2023 May 4; 224 ( 1 ): iyad031 . doi: 10.1093/genetics/iyad031 . PMID: 36866529 ; PMCID: PMC10158837 . OpenUrl CrossRef PubMed ↵ Gíslason MH , Nielsen H , Almagro Armenteros JJ , Johansen AR . 2021 . Prediction of GPI-anchored proteins with pointer neural networks . Current Research in Biotechnology . 2021; 3 : 6 – 13 . doi: 10.1016/j.crbiot.2021.01.001 . OpenUrl CrossRef ↵ Griffiths S , Mesarich CH , Overdijk EJR , Saccomanno B , de Wit PJGM , Collemare J. 2018 . Down-regulation of cladofulvin biosynthesis is required for biotrophic growth of Cladosporium fulvum on tomato . Molecular Plant Pathology . 2018 Feb; 19 ( 2 ): 369 – 380 . doi: 10.1111/mpp.12527 . Epub 2017 Feb 6. PMID: 27997759 ; PMCID: PMC6638085 . OpenUrl CrossRef PubMed ↵ Gu F , Khimani A , Rane SG , Flurkey WH , Bozarth RF , Smith TJ . 1995 . Structure and function of a virally encoded fungal toxin from Ustilago maydis : a fungal and mammalian Ca2+ channel inhibitor . Structure . 1995 Aug 15; 3 ( 8 ): 805 – 14 . doi: 10.1016/s0969-2126(01)00215-5 . PMID: 7582897 . OpenUrl CrossRef PubMed ↵ de Guillen K , Mammri L , Gracy J , Padilla A , Barthe P , Hoh F , Lahfa M , Rouffet J , Petit-Houdenot Y , Kroj T , et al. 2024 . Zymoseptoria tritici effectors structurally related to killer proteins UmV-KP4 and UmV-KP6 are toxic to fungi, and define extended protein families in fungi . bioRxiv . 2024 Oct 14:2024.10.14.618152. doi: 10.1101/2024.10.14.618152 . OpenUrl Abstract / FREE Full Text ↵ Guo YW , Gong BQ , Yuan J , Li HJ , Mahmud T , Huang Y , Li JF , Yang DP , Lan WJ . 2020 . l-Phenylalanine Alters the Privileged Secondary Metabolite Production in the Marine-Derived Fungus T richoderma erinaceum F1-1 . Journal of Natural Products . 2020 Jan 24; 83 ( 1 ): 79 – 87 . doi: 10.1021/acs.jnatprod.9b00710 . Epub 2019 Dec 30. PMID: 31886665 . OpenUrl CrossRef PubMed ↵ Holm L . 2022 . Dali server: structural unification of protein families . Nucleic Acids Research . 2022 Jul 5; 50 ( W1 ): W210 – W215 . doi: 10.1093/nar/gkac387 . PMID: 35610055 ; PMCID: PMC9252788 . OpenUrl CrossRef PubMed ↵ Hosmani PS , Flores-Gonzalez M , van de Geest H , Maumus F , Bakker LV , Schijlen E , van Haarst J , Cordewener J , Sanchez-Perez G , Peters S , et al. 2019 . An improved de novo assembly and annotation of the tomato reference genome using single-molecule sequencing, Hi-C proximity ligation and optical maps . bioRxiv . 2019; 767764 . doi: 10.1101/767764 OpenUrl Abstract / FREE Full Text ↵ Iida Y , Ikeda K , Sakai H , Nakagawa H , Nishi O , Higashi Y . 2018 . Evaluation of the potential biocontrol activity of Dicyma pulvinata against Cladosporium fulvum , the causal agent of tomato leaf mold . Plant Pathology . 2018; 67 ( 9 ): 1883 – 1890 . doi: 10.1111/ppa.12916 . OpenUrl CrossRef ↵ Iida Y , van ’t Hof P , Beenen H , Mesarich C , Kubota M , Stergiopoulos I , Mehrabi R , Notsu A , Fujiwara K , Bahkali A , et al. 2015 . Novel Mutations Detected in Avirulence Genes Overcoming Tomato Cf Resistance Genes in Isolates of a Japanese Population of Cladosporium fulvum . PLoS One . 2015 Apr 22; 10 ( 4 ): e0123271 . doi: 10.1371/journal.pone.0123271 . PMID: 25902074 ; PMCID: PMC4406682 . OpenUrl CrossRef PubMed ↵ de Jonge R , van Esse HP , Kombrink A , Shinya T , Desaki Y , Bours R , van der Krol S , Shibuya N , Joosten MH , Thomma BP. 2010 . Conserved fungal LysM effector Ecp6 prevents chitin-triggered immunity in plants . Science . 2010 Aug 20; 329 ( 5994 ): 953 – 5 . doi: 10.1126/science.1190859 . PMID: 20724636 . OpenUrl Abstract / FREE Full Text ↵ Joosten MH . 2012 . Isolation of apoplastic fluid from leaf tissue by the vacuum infiltration-centrifugation technique . Methods in Molecular Biology . 2012; 835 : 603 – 10 . doi: 10.1007/978-1-61779-501-5_38 . PMID: 22183681 . OpenUrl CrossRef PubMed ↵ Kanehisa M , Furumichi M , Sato Y , Matsuura Y , Ishiguro-Watanabe M . 2025 . KEGG: biological systems database as a model of the real world . Nucleic Acids Research . 2025 Jan 6; 53 ( D1 ): D672 – D677 . doi: 10.1093/nar/gkae909 . PMID: 39417505 ; PMCID: PMC11701520 . OpenUrl CrossRef PubMed ↵ Katoh K. , Standley DM . 2013 . MAFFT multiple sequence alignment software version 7: improvements in performance and usability . Molecular biology and evolution. Apr ; 30 ( 4 ): 772 – 80 . doi: 10.1093/molbev/mst010 . Epub 2013 Jan 16. PMID: 23329690 ; PMCID: PMC3603318 . OpenUrl CrossRef PubMed Web of Science ↵ Kim D , Paggi JM , Park C , Bennett C , Salzberg SL . 2019 . Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype . Nature Biotechnology . 2019 Aug; 37 ( 8 ): 907 – 915 . doi: 10.1038/s41587-019-0201-4 . Epub 2019 Aug 2. PMID: 31375807 ; PMCID: PMC7605509 . OpenUrl CrossRef PubMed ↵ de Kock MJ , Iskandar HM , Brandwagt BF , Laugé R , de Wit PJGM , Lindhout P. 2004 . Recognition of Cladosporium fulvum Ecp2 elicitor by non-host Nicotiana spp. is mediated by a single dominant gene that is not homologous to known Cf -genes . Molecular Plant Pathology . 2004 Sep 1; 5 ( 5 ): 397 – 408 . doi: 10.1111/j.1364-3703.2004.00239.x . PMID: 20565616 . OpenUrl CrossRef PubMed Web of Science ↵ Kourelis J , Malik S , Mattinson O , Krauter S , Kahlon PS , Paulus JK , van der Hoorn RAL. 2020 . Evolution of a guarded decoy protease and its receptor in solanaceous plants . Nature Communications . 2020 Sep 2; 11 ( 1 ): 4393 . doi: 10.1038/s41467-020-18069-5 . Erratum in: Nature Communications . 2024 Mar 13;15(1):2278. doi: 10.1038/s41467-024-46611-2. PMID: 32879321 ; PMCID: PMC7468133 . OpenUrl CrossRef PubMed ↵ Laur J , Ramakrishnan GB , Labbé C , Lefebvre F , Spanu PD , Bélanger RR . 2018 . Effectors involved in fungal-fungal interaction lead to a rare phenomenon of hyperbiotrophy in the tritrophic system biocontrol agent-powdery mildew-plant . New Phytologist . 2018 Jan; 217 ( 2 ): 713 – 725 . doi: 10.1111/nph.14851 . Epub 2017 Oct 18. PMID: 29044534 ; PMCID: PMC6079639 . OpenUrl CrossRef PubMed ↵ Li H , Handsaker B , Wysoker A , Fennell T , Ruan J , Homer N , Marth G , Abecasis G , Durbin R ; 1000 Genome Project Data Processing Subgroup . 2009 . The Sequence Alignment/Map format and SAMtools . Bioinformatics . 2009 Aug 15; 25 ( 16 ): 2078 – 9 . doi: 10.1093/bioinformatics/btp352 . Epub 2009 Jun 8. PMID: 19505943 ; PMCID: PMC2723002 . OpenUrl CrossRef PubMed Web of Science ↵ Love MI , Huber W , Anders S . 2014 . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 . Genome Biology . 2014; 15 ( 12 ): 550 . doi: 10.1186/s13059-014-0550-8 . PMID: 25516281 ; PMCID: PMC4302049 . OpenUrl CrossRef PubMed ↵ Luderer R , Takken FL , de Wit PJGM , Joosten MH. 2002 . Cladosporium fulvum overcomes Cf-2 -mediated resistance by producing truncated AVR2 elicitor proteins . Molecular Microbiology . 2002 Aug; 45 ( 3 ): 875 – 84 . doi: 10.1046/j.1365-2958.2002.03060.x . PMID: 12139631 . OpenUrl CrossRef PubMed Web of Science ↵ Ma L , Lukasik E , Gawehns F , Takken FL . 2012 . The use of agroinfiltration for transient expression of plant resistance and fungal effector proteins in Nicotiana benthamiana leaves . Methods in Molecular Biology . 2012; 835 : 61 – 74 . doi: 10.1007/978-1-61779-501-5_4 . PMID: 22183647 . OpenUrl CrossRef PubMed ↵ Madeira F , Madhusoodanan N , Lee J , Eusebi A , Niewielska A , Tivey ARN , Lopez R , Butcher S . 2024 . The EMBL-EBI Job Dispatcher sequence analysis tools framework in 2024 . Nucleic Acids Research . 2024 Jul 5; 52 ( W1 ): W521 – W525 . doi: 10.1093/nar/gkae241 . PMID: 38597606 ; PMCID: PMC11223882 . OpenUrl CrossRef PubMed ↵ Maeda K , Sumita T , Nishi O , Sushida H , Higashi Y , Nakagawa H , Suzuki T , Iwao E , Fanani MZ , Nishiya Y , Iida Y . 2025 . Adaptive evolution of sesquiterpene deoxyphomenone in mycoparasitism by Hansfordia pulvinata associated with horizontal gene transfer from Aspergillus species . mBio . 2025 Apr 9; 16 ( 4 ): e0400724 . doi: 10.1128/mbio.04007-24 . Epub 2025 Mar 20. PMID: 40111082 ; PMCID: PMC11980549 . OpenUrl CrossRef PubMed ↵ Mesarich CH , Griffiths SA , van der Burgt A , Okmen B , Beenen HG , Etalo DW , Joosten MH , de Wit PJGM . 2014 . Transcriptome sequencing uncovers the Avr5 avirulence gene of the tomato leaf mold pathogen Cladosporium fulvum . Molecular Plant-Microbe Interactions . 2014 Aug; 27 ( 8 ): 846 – 57 . doi: 10.1094/MPMI-02-14-0050-R . PMID: 24678832 . OpenUrl CrossRef PubMed ↵ Mesarich CH , Ӧkmen B , Rovenich H , Griffiths SA , Wang C , Karimi Jashni M , Mihajlovski A , Collemare J , Hunziker L , Deng CH , et al. 2018 . Specific Hypersensitive Response-Associated Recognition of New Apoplastic Effectors from Cladosporium fulvum in Wild Tomato . Molecular Plant-Microbe Interactions . 2018 Jan; 31 ( 1 ): 145 – 162 . doi: 10.1094/MPMI-05-17-0114-FI . Epub 2017 Nov 16. PMID: 29144204 . OpenUrl CrossRef PubMed ↵ Mesarich CH , Barnes I , Bradley EL , de la Rosa S , de Wit PJGM , Guo Y , Griffiths SA , Hamelin RC , Joosten MHAJ , Lu M , et al. 2023 . Beyond the genomes of Fulvia fulva (syn. Cladosporium fulvum ) and Dothistroma septosporum : New insights into how these fungal pathogens interact with their host plants . Molecular Plant Pathology . 2023 May; 24 ( 5 ): 474 – 494 . doi: 10.1111/mpp.13309 . Epub 2023 Feb 15. PMID: 36790136 ; PMCID: PMC10098069 . OpenUrl CrossRef PubMed ↵ Mesny F , Wolf V , López-Moral A , Kraege A , Punt W , Park J , Zhu J , Sato Y , Thomma BPHJ. 2024 . Plant-associated fungi co-opt ancient antimicrobials for host manipulation . bioRxiv . Preprint posted online 2024 Jan 4. doi: 10.1101/2024.01.04.574150 . OpenUrl Abstract / FREE Full Text ↵ Mirdita M , Schütze K , Moriwaki Y , Heo L , Ovchinnikov S , Steinegger M . 2022 . ColabFold: making protein folding accessible to all . Nature Methods . 2022 Jun; 19 ( 6 ): 679 – 682 . doi: 10.1038/s41592-022-01488-1 . Epub 2022 May 30. PMID: 35637307 ; PMCID: PMC9184281 . OpenUrl CrossRef PubMed ↵ Mistry J , Chuguransky S , Williams L , Qureshi M , Salazar GA , Sonnhammer ELL , Tosatto SCE , Paladin L , Raj S , Richardson LJ , et al. 2021 . Pfam: The protein families database in 2021 . Nucleic Acids Research . 2021 Jan 8; 49 ( D1 ): D412 – D419 . doi: 10.1093/nar/gkaa913 . PMID: 33125078 ; PMCID: PMC7779014 . OpenUrl CrossRef PubMed ↵ Moormann J , Heinemann B , Hildebrandt TM . 2022 . News about amino acid metabolism in plant-microbe interactions . Trends in Biochemical Sciences . 2022 Oct; 47 ( 10 ): 839 – 850 . doi: 10.1016/j.tibs.2022.07.001 . Epub 2022 Aug 1. PMID: 35927139 . OpenUrl CrossRef PubMed ↵ Oome S , Raaymakers TM , Cabral A , Samwel S , Böhm H , Albert I , Nürnberger T , Van den Ackerveken G. 2014 . Nep1-like proteins from three kingdoms of life act as a microbe-associated molecular pattern in Arabidopsis . Proceedings of the National Academy of Sciences, USA . 2014 Nov 25; 111 ( 47 ): 16955 – 60 . doi: 10.1073/pnas.1410031111 . Epub 2014 Nov 3. PMID: 25368167 ; PMCID: PMC4250136 . OpenUrl Abstract / FREE Full Text ↵ Peresse M , Le Picard D. 1980 . Hansfordia pulvinata , mycoparasite destructeur du Cladosporium fulvum . Mycopathologia 1980. 71 : 23 – 30 . OpenUrl ↵ Qutob D , Kamoun S , Gijzen M . 2002 . Expression of a Phytophthora sojae necrosis-inducing protein occurs during transition from biotrophy to necrotrophy . The Plant Journal . 2002 Nov; 32 ( 3 ): 361 – 73 . doi: 10.1046/j.1365-313x.2002.01439.x . PMID: 12410814 . OpenUrl CrossRef PubMed Web of Science ↵ Qutob D , Kemmerling B , Brunner F , Küfner I , Engelhardt S , Gust AA , Luberacki B , Seitz HU , Stahl D , Rauhut T , et al. 2006 . Phytotoxicity and innate immune responses induced by Nep1-like proteins . The Plant Cell . 2006 Dec; 18 ( 12 ): 3721 – 44 . doi: 10.1105/tpc.106.044180 . Epub 2006 Dec 28. PMID: 17194768 ; PMCID: PMC1785393 . OpenUrl Abstract / FREE Full Text ↵ R Core Team R . 2024 . R: a language and environment for statistical computing . Vienna, Austria : R Foundation for Statistical Computing . ↵ Rocafort M , Fudal I , Mesarich CH . 2020 . Apoplastic effector proteins of plant-associated fungi and oomycetes . Current Opinion in Plant Biology . 2020 Aug; 56 : 9 – 19 . doi: 10.1016/j.pbi.2020.02.004 . Epub 2020 Apr 2. PMID: 32247857 . OpenUrl CrossRef PubMed ↵ Rooney HC , Van’t Klooster JW , van der Hoorn RA , Joosten MH , Jones JD , de Wit PJGM . 2005 . Cladosporium Avr2 inhibits tomato Rcr3 protease required for Cf-2 -dependent disease resistance . Science . 2005 Jun 17; 308 ( 5729 ): 1783 – 6 . doi: 10.1126/science.1111404 . Epub 2005 Apr 21. Erratum in: Science. 2005 Oct 7;310(5745):54. PMID: 15845874 . OpenUrl Abstract / FREE Full Text ↵ de la Rosa S , Schol CR , Ramos Peregrina Á , Winter DJ , Hilgers AM , Maeda K , Iida Y , Tarallo M , Jia R , Beenen HG , et al. 2024 . Sequential breakdown of the Cf-9 leaf mold resistance locus in tomato by Fulvia fulva . New Phytologist . 2024 Aug; 243 ( 4 ): 1522 – 1538 . doi: 10.1111/nph.19925 . Epub 2024 Jun 24. PMID: 38922927 . OpenUrl CrossRef PubMed ↵ Seidl MF , Van den Ackerveken G. 2019 . Activity and Phylogenetics of the Broadly Occurring Family of Microbial Nep1-Like Proteins . Annual Review of Phytopathology . 2019 Aug 25; 57 : 367 – 386 . doi: 10.1146/annurev-phyto-082718-100054 . Epub 2019 Jul 5. PMID: 31283435 . OpenUrl CrossRef PubMed ↵ Shabab M , Shindo T , Gu C , Kaschani F , Pansuriya T , Chintha R , Harzen A , Colby T , Kamoun S , van der Hoorn RA. 2008 . Fungal effector protein AVR2 targets diversifying defense-related cys proteases of tomato . The Plant Cell . 2008 Apr; 20 ( 4 ): 1169 – 83 . doi: 10.1105/tpc.107.056325 . Epub 2008 Apr 30. PMID: 18451324 ; PMCID: PMC2390736 . OpenUrl CrossRef PubMed ↵ Shimodaira H . 2002 . An approximately unbiased test of phylogenetic tree selection . Syst Biol. Jun ; 51 ( 3 ): 492 – 508 . doi: 10.1080/10635150290069913 . PMID: 12079646 . OpenUrl CrossRef PubMed Web of Science ↵ Snelders NC , Rovenich H , Petti GC , Rocafort M , van den Berg GCM , Vorholt JA , Mesters JR , Seidl MF , Nijland R , Thomma BPHJ. 2020 . Microbiome manipulation by a soil-borne fungal plant pathogen using effector proteins . Nature Plants . 2020 Nov; 6 ( 11 ): 1365 – 1374 . doi: 10.1038/s41477-020-00799-5 . Epub 2020 Nov 2. PMID: 33139860 . OpenUrl CrossRef PubMed ↵ Snelders NC , Rovenich H , Thomma BPHJ . 2022 . Microbiota manipulation through the secretion of effector proteins is fundamental to the wealth of lifestyles in the fungal kingdom . FEMS Microbiology Reviews . 2022 Sep 2; 46 ( 5 ): fuac022 . doi: 10.1093/femsre/fuac022 . PMID: 35604874 ; PMCID: PMC9438471 . OpenUrl CrossRef PubMed ↵ Stamatakis A . 2014 . RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies . Bioinformatics . May 1; 30 ( 9 ): 1312 – 3 . doi: 10.1093/bioinformatics/btu033 . Epub 2014 Jan 21. PMID: 24451623 ; PMCID: PMC3998144 . OpenUrl CrossRef PubMed Web of Science ↵ Solomon PS , Oliver RP . 2001 . The nitrogen content of the tomato leaf apoplast increases during infection by Cladosporium fulvum . Planta . 2001 Jun; 213 ( 2 ): 241 – 9 . doi: 10.1007/s004250000500 . PMID: 11469589 . OpenUrl CrossRef PubMed Web of Science ↵ Sonawala U , Dinkeloo K , Danna CH , McDowell JM , Pilot G . 2018 . Review: Functional linkages between amino acid transporters and plant responses to pathogens . Plant Science . 2018 Dec; 277 : 79 – 88 . doi: 10.1016/j.plantsci.2018.09.009 . Epub 2018 Sep 19. PMID: 30466603 . OpenUrl CrossRef PubMed ↵ Sperschneider J , Dodds PN . 2022 . EffectorP 3.0: Prediction of Apoplastic and Cytoplasmic Effectors in Fungi and Oomycetes . Molecular Plant-Microbe Interactions . 2022 Feb; 35 ( 2 ): 146 – 156 . doi: 10.1094/MPMI-08-21-0201-R . Epub 2022 Feb 1. PMID: 34698534 . OpenUrl CrossRef PubMed ↵ Sperschneider J , Catanzariti AM , DeBoer K , Petre B , Gardiner DM , Singh KB , Dodds PN , Taylor JM . 2017 . LOCALIZER: subcellular localization prediction of both plant and effector proteins in the plant cell . Scientific Reports . 2017 Mar 16; 7 : 44598 . doi: 10.1038/srep44598 . PMID: 28300209 ; PMCID: PMC5353544 . OpenUrl CrossRef PubMed ↵ Stergiopoulos I , Kourmpetis YA , Slot JC , Bakker FT , De Wit PJ , Rokas A. 2012 . In silico characterization and molecular evolutionary analysis of a novel superfamily of fungal effector proteins . Molecular Biology and Evolution . 2012 Nov; 29 ( 11 ): 3371 – 84 . doi: 10.1093/molbev/mss143 . Epub 2012 May 23. PMID: 22628532 . OpenUrl CrossRef PubMed Web of Science ↵ Sushida H , Sumita T , Higashi Y , Iida Y . 2019 . Draft Genome Sequence of Dicyma pulvinata Strain 414-3, a Mycoparasite of Cladosporium fulvum , Causal Agent of Tomato Leaf Mold. Microbiology Resource Announcements . 2019 Aug 29; 8 ( 35 ): e00655 – 19 . doi: 10.1128/MRA.00655-19 . PMID: 31467096 ; PMCID: PMC6715866 . OpenUrl CrossRef PubMed Tanaka S , Wada K , Katayama M , Marumo S . 1984 . Isolation of Sporogen-AO1, a sporogenic substance, from Aspergillus oryzae . Agricultural and biological chemistry . 1984; 48 ( 12 ): 3189 – 3191 . doi: 10.1271/bbb1961.48.3189 . OpenUrl CrossRef Tanaka S , Wada K , Marumo S , Hattori H . 1984 . Structure of sporogen-ao 1, a sporogenic substance of Aspergillus oryzae . Tetrahedron Letters . 1984; 25 ( 51 ): 5907 – 5910 . doi: 10.1016/S0040-4039(01)81717-2 . OpenUrl CrossRef ↵ Tang D , Chen M , Huang X , Zhang G , Zeng L , Zhang G , Wu S , Wang Y . 2023 . SRplot: A free online platform for data visualization and graphing . PLoS One . 2023 Nov 9; 18 ( 11 ): e0294236 . doi: 10.1371/journal.pone.0294236 . PMID: 37943830 ; PMCID: PMC10635526 . OpenUrl CrossRef PubMed ↵ Teufel F , Almagro Armenteros JJ , Johansen AR , Gíslason MH , Pihl SI , Tsirigos KD , Winther O , Brunak S , von Heijne G , Nielsen H. 2022 . SignalP 6.0 predicts all five types of signal peptides using protein language models . Nature Biotechnology . 2022 Jul; 40 ( 7 ): 1023 – 1025 . doi: 10.1038/s41587-021-01156-3 . Epub 2022 Jan 3. PMID: 34980915 ; PMCID: PMC9287161 . OpenUrl CrossRef PubMed ↵ Thomma BP , VAN Esse HP , Crous PW , DE Wit PJ . 2005 . Cladosporium fulvum (syn. Passalora fulva ), a highly specialized plant pathogen as a model for functional studies on plant pathogenic Mycosphaerellaceae . Molecular Plant Pathology . 2005 Jul 1; 6 ( 4 ): 379 – 93 . doi: 10.1111/j.1364-3703.2005.00292.x . PMID: 20565665 . OpenUrl CrossRef PubMed Web of Science ↵ Tirilly Y , Kloosterman J , Sipma G , Kettenes-Van Den Bosch JJ . 1983 . A fungitoxic sesquiterpene from Hansfordia pulvinata . Phytochemistry . 1983; 22 ( 9 ): 2082 – 2083 . doi: 10.1016/0031-9422(83)80051-X . OpenUrl CrossRef ↵ Tünnermann L , Colou J , Näsholm T , Gratz R . 2022 . To have or not to have: expression of amino acid transporters during pathogen infection . Plant Molecular Biology . 2022 Jul; 109 ( 4-5 ): 413 – 425 . doi: 10.1007/s11103-022-01244-1 . Epub 2022 Feb 1. PMID: 35103913 ; PMCID: PMC9213295 . OpenUrl CrossRef PubMed ↵ Watanabe H , Horinouchi H , Muramoto Y , Ishii H . 2017 . Occurrence of azoxystrobin-resistant isolates in Passalora fulva , the pathogen of tomato leaf mold disease . Plant Pathology . 2017; 66 ( 9 ): 1472 – 1479 . doi: 10.1111/ppa.12701 . OpenUrl CrossRef ↵ de Wit PJGM , van der Burgt A , Ökmen B , Stergiopoulos I , Abd-Elsalam KA , Aerts AL , Bahkali AH , Beenen HG , Chettri P , Cox MP , et al. 2012 . The genomes of the fungal plant pathogens Cladosporium fulvum and Dothistroma septosporum reveal adaptation to different hosts and lifestyles but also signatures of common ancestry . PLOS Genetics . 2012; 8 ( 11 ): e1003088 . doi: 10.1371/journal.pgen.1003088 . Epub 2012 Nov 29. Erratum in: PLoS Genet. 2015 Dec 22;11(12):e1005775. doi: 10.1371/journal.pgen.1005775. PMID: 23209441 ; PMCID: PMC3510045 . OpenUrl CrossRef PubMed ↵ de Wit PJGM , Joosten MHAJ , Thomma BHPJ , Stergiopoulos I . 2009 . Gene for gene models and beyond: the Cladosporium fulvum –tomato pathosystem. Plant relationships . Berlin , Heidelberg: Springer ; 2009. p. 135 – 156 . (The Mycota; vol 5 ). doi: 10.1007/978-3-540-87407-2_7 . OpenUrl CrossRef ↵ Wulff BB , Chakrabarti A , Jones DA . 2009 . Recognitional specificity and evolution in the tomato- Cladosporium fulvum pathosystem . Molecular Plant-Microbe Interactions . 2009 Oct; 22 ( 10 ): 1191 – 202 . doi: 10.1094/MPMI-22-10-1191 . PMID: 19737093 . OpenUrl CrossRef PubMed Web of Science ↵ Yan L , Chen J , Zhang C , Ma Z . 2008 . Molecular characterization of benzimidazole-resistant isolates of Cladosporium fulvum . Fems Microbiology Letters . 2008 Jan; 278 ( 2 ): 242 – 8 . doi: 10.1111/j.1574-6968.2007.00999.x . PMID: 18096020 . OpenUrl CrossRef PubMed ↵ Yang K , Chen C , Wang Y , Li J , Dong X , Cheng Y , Zhang H , Zhai Y , Ai G , Song Q , et al. 2022 . Nep1-Like Proteins From the Biocontrol Agent Pythium oligandrum Enhance Plant Disease Resistance Independent of Cell Death and Reactive Oxygen Species . Frontiers in Plant Science . 2022 Mar 4; 13 : 830636 . doi: 10.3389/fpls.2022.830636 . PMID: 35310640 ; PMCID: PMC8931738 . OpenUrl CrossRef PubMed ↵ Zaccaron AZ , Stergiopoulos I . 2024 . Analysis of five near-complete genome assemblies of the tomato pathogen Cladosporium fulvum uncovers additional accessory chromosomes and structural variations induced by transposable elements effecting the loss of avirulence genes . BMC Biology . 2024 Jan 29; 22 ( 1 ): 25 . doi: 10.1186/s12915-024-01818-z . PMID: 38281938 ; PMCID: PMC10823647 . OpenUrl CrossRef PubMed ↵ Zaccaron AZ , Chen LH , Samaras A , Stergiopoulos I . 2022 . A chromosome-scale genome assembly of the tomato pathogen Cladosporium fulvum reveals a compartmentalized genome architecture and the presence of a dispensable chromosome . Microbial Genomics . 2022 Apr; 8 ( 4 ): 000819 . doi: 10.1099/mgen.0.000819 . PMID: 35471194 ; PMCID: PMC9453070 . OpenUrl CrossRef PubMed ↵ Zhang H , Yohe T , Huang L , Entwistle S , Wu P , Yang Z , Busk PK , Xu Y , Yin Y . 2018 . dbCAN2: a meta server for automated carbohydrate-active enzyme annotation . Nucleic Acids Research . 2018 Jul 2; 46 ( W1 ): W95 - W101 . doi: 10.1093/nar/gky418 . PMID: 29771380 ; PMCID: PMC6031026 . OpenUrl CrossRef PubMed ↵ Zhang Y , Skolnick J . 2005 . TM-align: a protein structure alignment algorithm based on the TM-score . Nucleic Acids Research . 2005 Apr 22; 33 ( 7 ): 2302 – 9 . doi: 10.1093/nar/gki524 . PMID: 15849316 ; PMCID: PMC1084323 . OpenUrl CrossRef PubMed Web of Science ↵ Zhou BJ , Jia PS , Gao F , Guo HS . 2012 . Molecular characterization and functional analysis of a necrosis- and ethylene-inducing, protein-encoding gene family from Verticillium dahliae . Molecular Plant-Microbe Interactions . 2012 Jul; 25 ( 7 ): 964 – 75 . doi: 10.1094/MPMI-12-11-0319 . PMID: 22414440 . OpenUrl CrossRef PubMed Web of Science View the discussion thread. Back to top Previous Next Posted February 25, 2026. Download PDF Supplementary Material Data/Code 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 Transcriptomic insights into the tritrophic plant–pathogen–mycoparasite interaction reveal coordinated reprogramming fungal secretomes and plant amino acid metabolism 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 Transcriptomic insights into the tritrophic plant–pathogen–mycoparasite interaction reveal coordinated reprogramming fungal secretomes and plant amino acid metabolism Kazuya Maeda , Mariko Kouda , Mai Ohara , Takumi Kawase , Koki Saito , Eishin Iwao , Hirotoshi Sushida , Tomoko Suzuki , Takuya Sumita , Yuichiro Iida bioRxiv 2025.11.24.690098; doi: https://doi.org/10.1101/2025.11.24.690098 Share This Article: Copy Citation Tools Transcriptomic insights into the tritrophic plant–pathogen–mycoparasite interaction reveal coordinated reprogramming fungal secretomes and plant amino acid metabolism Kazuya Maeda , Mariko Kouda , Mai Ohara , Takumi Kawase , Koki Saito , Eishin Iwao , Hirotoshi Sushida , Tomoko Suzuki , Takuya Sumita , Yuichiro Iida bioRxiv 2025.11.24.690098; doi: https://doi.org/10.1101/2025.11.24.690098 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41937) Biophysics (21452) Cancer Biology (18589) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15156) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)