GH25 lysozyme mediates tripartite interkingdom interactions and microbial competition on the plant leaf surface

GH25 lysozyme mediates tripartite interkingdom interactions and microbial competition on the plant leaf surface | 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 GH25 lysozyme mediates tripartite interkingdom interactions and microbial competition on the plant leaf surface Zarah Sorger , Priyamedha Sengupta , Klara Beier-Heuchert , Jaqueline Bautor , View ORCID Profile Jane E. Parker , View ORCID Profile Eric Kemen , View ORCID Profile Gunther Doehlemann doi: https://doi.org/10.1101/2025.04.04.647216 Zarah Sorger 1 Institute for Plant Sciences and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne , Cologne, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Priyamedha Sengupta 1 Institute for Plant Sciences and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne , Cologne, Germany 2 Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Campus UAB (Cerdanyola del Valles) , 08193, Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: g.doehlemann{at}uni-koeln.de priyamedhasg{at}gmail.com Klara Beier-Heuchert 1 Institute for Plant Sciences and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne , Cologne, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jaqueline Bautor 3 Department of Plant-Microbe Interactions, Max Planck Institute for Plant Breeding Research , Cologne 50829, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jane E. Parker 3 Department of Plant-Microbe Interactions, Max Planck Institute for Plant Breeding Research , Cologne 50829, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jane E. Parker Eric Kemen 4 Department of Microbial Interactions, IMIT/ZMBP, University of Tübingen , Tübingen, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Eric Kemen Gunther Doehlemann 1 Institute for Plant Sciences and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne , Cologne, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gunther Doehlemann For correspondence: g.doehlemann{at}uni-koeln.de priyamedhasg{at}gmail.com Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Microbial communities inhabiting plants have emerged as crucial factors in regulating plant health and defense against disease-causing pathogens. The basidiomycete yeast Moesziomyces bullatus ex. Albugo on Arabidopsis ( MbA) releases Glycoside Hydrolase 25 (GH25) protein which regulates the leaf microbiome by antagonizing an oomycete A. laibachii biotrophic pathogen MbA. Application of both MbA and GH25 protein rescued fresh shoot weight of A. thaliana upon A. laibachii infection, showing its potential in plant protection. Tripartite interaction assays did not reveal antagonistic activity of GH25 towards other plant pathogenic oomycetes or fungi besides A. laibachii . We identified a core set of bacteria are closely associated with A. laibachii and established that GH25 inhibits members of this core group. Among A. laibachii- associated bacteria that were inhibited by GH25, Curtobacterium sp . could override the inhibition of A. laibachii by MbA . We describe a tripartite antagonistic interaction in which bacterium and oomycete protect each other from growth inhibition by MbA. Curtobacterium sp ., in turn, exhibits specific inhibition of A. laibachii -associated bacteria that are not targeted by MbA but themselves antagonize A. laibachii . Our study reveals an inter-kingdom interaction network in which a GH25 lysozyme shapes the antagonistic relationship between yeast, a pathogenic oomycete and an oomycete-associated bacterium. Introduction Pathogenic microorganisms such as bacteria, fungi, oomycetes and viruses cause various plant diseases. In particular, ooomycete and fungal pathogens infect various agronomically important crops; thereby, posing a threat to global food security. For example, the oomycete Phytophthora infestans caused the Irish famine (1845) through potato late blight disease, while other species of Phytophthora , namely P. ramorum, P. capsici and P. sojae are known to infect oak trees, solanaceous crops, and soybean, respectively ( Kamoun et al., 2015 ). Botrytis cinerea , a fungal pathogen with a broad host range, can cause severe economic loses with the disease control being heavily dependent on fungicide application ( De Angelis et al., 2022 ; Weiberg et al., 2013 ). In addition, climate change is predicted to exacerbate plant disease outbreaks through the emergence and evolution of pathogenic strains, potentially affecting both natural biodiversity and agricultural systems ( Singh et al., 2023 ). Microbial antagonism or biological control of pathogens by the action of beneficial microorganisms has emerged as a more environmentally sustainable approach towards plant protection. For example, biological control of Phytophthora has been reported by inhibitory activities of potato associated bacteria ( De Vrieze et al., 2019 ), or by volatiles secreted from bacteria ( Gfeller et al., 2022 ) and fungi ( Oubaha et al., 2021 ). Therefore, a thorough knowledge on the mechanism of microbial antagonism is necessary to attain successful crop protection. While several modes of action for microbial antagonists have been described, such as triggering plant immune response, hyper-parasitism on pathogens or secretion of secondary metabolites ( Köhl et al., 2019 ), competition by associated microbiota may be a crucial criterion for effective pathogen control and restoration of plant health (Cernava, 2024). Among such interactions, Albugo laibachii , the causal agent of white rust, has emerged as a microbial hub that promotes disease-associated microbiota in the Arabidopsis phyllosphere, in contrast to the health-associated communities found in uninfected plants ( Mahmoudi et al., 2024 ). Microbiota inhabiting plants secrete a range of glycoside hydrolases (GH) which aid nutrient acquisition and competition with other microbes, or can be virulence factors to colonize a host ( Bradley et al., 2022 ). Antagonistic yeasts such as Trichoderma were reported to produce a variety of hydrolytic enzymes to antagonize fungal pathogens in crop plants (reviewed by Freimoser et al., 2019 ). For example, chitinase from T. asperellum PQ34 inhibited growth of fungal pathogens Sclerotium rolfsii and Colletotrichum ( Bradley et al., 2022 ; Loc et al., 2020 ). Additionally, cellulose encoding genes from T. harzianum were found to elicit plant immunity against pathogen F. graminearum by upregulating the production of DIMBOA and other defense related genes in maize roots ( Saravanakumar et al., 2018 ). Glycoside hydrolases are one of the largest and most diverse groups among hydrolytic enzymes with 172 families and 18 different GH clans ( Henrissat and Davies, 1997 ). In phytopathogenic fungi, GH families have been characterized mostly with respect to their suppression of plant immunity and pathogenic virulence. For example, secreted GHs from oomycete P. sojae and fungi Verticillium dahliae, M. oryzae and F. oxysporum can act as microbe-associated molecular patterns (MAMPs) ( Gui et al., 2017 ; Ma et al., 2015 ; Zhang et al., 2021 ) or release damage associated molecular patterns (DAMPs) leading to the activation of pattern triggered immunity (PTI) ( Bradley et al., 2022 ). In contrast, deletion of cell wall hydrolyzing enzymes encoded by GH families in B. cinerea, M. oryzae and A. alternata impairs fungal virulence ( Brito et al., 2007 ; Ma et al., 2015 ; Yu et al., 2018 ; Zhang et al., 2021 ). Glycoside hydrolases can also be involved in microbial antagonism of pathogens. For example, deletion of the GH18 chitinase gene in the fungus Clonostachys rosea reduced inhibitory activity against B. cinerea and Rhizoctonia solani , although biocontrol of B. cinerea was not compromised ( Bradley et al., 2022 ; Tzelepis et al., 2015 ). More recently, Glycoside Hydrolase 30 (GH30) produced by the bacterium Bacillus paralicheniformis triggered plant defense responses via programmed cell death and restricted phytopathogens such as Sclerotinia sclerotiorum, P. capisici and tobacco mosaic virus ( Yu et al., 2024 ). In addition, GH19 chitinase from Chitinilyticum aquatile CSC-1 was recombinantly expressed in E. coli and displayed inhibition of fungal growth in several Fusarium species, suggesting its role in biological control of phytopathogenic fungi ( Yang et al., 2024 ). In previous work ( Eitzen et al., 2021 ) we described how the basidiomycete yeast, Moesziomyces bullatus ex. Albugo on Arabidopsis ( MbA) regulates the Arabidopsis thaliana phyllosphere microbiota by inhibiting the white rust pathogen Albugo laibachii through a secreted Glycoside Hydrolase 25 (GH25) protein. The GH25 family of hydrolases, characterized by a DXE motif active site, cleaves the β-1,4-glycosidic linkage between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) in the bacterial peptidoglycan ( Rau et al., 2001 ). However, the biological function of GH25 in fungi remains poorly understood. In this study, we functionally investigated how GH25 antagonizes A. laibachii . We examined the effect of MbA and MbA _GH25 against other plant pathogens and found that GH25 acts specifically against A. laibachii through an indirect mechanism involving an associated bacterium of the genus Curtobacterium . We extended our analysis by functionally characterizing GH25 orthologs from the plant pathogenic fungi Ustilago maydis and Rhizoctonia solani in the antagonism towards A. laibachii and suggest a possible mechanism of evolutionary adaptation of GH25 specificity. Results MbA limits growth of Albugo laibachii through GH25 activity In a previous study ( Eitzen et al., 2021 ), we explored how a basidiomycete yeast, Moesziomyces bullatus ex Albugo on Arabidopsis ( MbA ) antagonizes the oomycete Albugo laibachii in the A. thaliana phyllosphere by secreting Glycoside Hydrolase 25. Recombinantly produced MbA _GH25 also reduced A. laibachii growth in-planta , which revealed the importance of GH25 in the antagonism of this oomycete pathogen. In this study, we aimed to clarify how GH25 mediates the observed antagonism between MbA and A. laibachii . We first quantified A. laibachii infection symptoms in 3-week-old A. thaliana seedlings grown in the gnotobiotic plate system with and without application of MbA ( Fig. 1A ). Although MbA significantly reduced the white rust infection symptom at 14 dpi, for a more quantitative assessment, the relative fungal biomass A. laibachii at 10 dpi was analyzed using the oomycete internal transcribed spacer (ITS) 5.8s sequence normalized to the A. thaliana housekeeping gene ef1-α. We observed that A. laibachii biomass was also significantly reduced in the presence of MbA ( Fig. 1B ). Additionally, a protective effect of MbA on the plant was evaluated by measuring the fresh shoot weight of A. thaliana seedlings following different treatments ( Fig. 1C ). We observed that the reduced fresh shoot weight during oomycete infection could be reversed by pre-treating the plants with MbA or by adding recombinantly produced MbA _GH25 to the yeast deletion strain MbA Δgh25 ( Fig. 1C ). Download figure Open in new tab Figure 1: MbA restricts A. laibachii growth and recovers A. thaliana fresh shoot weight during infection. A) Addition of MbA reduces A. laibachii induced leaf disease symptoms of A. thaliana at 14 dpi. The number of infected plants (n) were scored across 3 biological replicates (unpaired t-test, p value =0.02). B) Relative quantification of A. laibachii biomass in response to MbA treatment by qPCR. The oomycete internal transcribed spacer (ITS) 5.8 s was normalized to A. thaliana EF1-α gene to quantify the amount of A. laibachii DNA in the samples at 10 dpi (unpaired t-test, p value =0.03). Error bars indicate SD. C) Fresh shoot weight measured at 11dpi showed A. thaliana seedlings to be reduced in growth upon infection with A. laibachii compared to mock treated seedlings. Addition of purified MbA _GH25 protein to the gh25 deletion mutant of MbA ( MbA Δ gh25 ) led to a higher recovery of fresh shoot weight in A. laibachii infected seedlings. The number of seedlings (N) were measured across 3 biological replicates. One-way ANOVA and Tukey’ HSD (multiple comparisons of means; 95% family-wise confidence level) was performed to find significant difference between treatments. Representative image of A. thaliana seedlings is added next to each treatment. Since MbA _GH25 plays a key role in oomycete inhibition, we analyzed the evolutionary conservation of GH25 in the fungal kingdom. Previously, we showed that the DXE active site motif is highly conserved in the GH25 amino acid sequence of different fungal groups such as Basidiomycetes, Ascomycetes and Chytrids ( Eitzen et al., 2021 ). A newly constructed phylogenetic tree including various fungal species showed that the GH25 of Ustilaginales, including MbA and U. maydis , clustered together with a high degree of sequence similarity. However, evolutionarily more diverse ascomycete fungi, including several plant pathogens, also have significantly conserved GH25 orthologs ( Fig. 2A ). We recombinantly expressed orthologues from MbA, U. maydis and Rhizoctonia solani in FB1 strain of U. maydis . The recombinant strains were inoculated onto A. thaliana seedlings prior to A. laibachii infection and infection symptoms on leaves were scored at 11dpi. FB1 strains overexpressing active GH25s from MbA or U. maydis significantly inhibited A. laibachii infection in-planta , as opposed to GH25 from R. solani ( Fig. 2B ). Therefore, inhibitory activities of GH25 from MbA and U. maydis are functionally conserved compared to GH25 from the more distantly related fungal species R. solani . Download figure Open in new tab Figure 2: Overexpression of GH25 orthologues from MbA and U. maydis restricts A. laibachii growth compared to GH25 from distantly related R. solani : A) Phylogenetic tree of GH25 orthologues. Protein sequences were aligned using MAFFT version 7 multiple alignment program for amino acid of nucleotide sequences with default parameters. Phylogenetic tree construction was based on the alignment using Phylio.io. B) Infection assay of A. laibachii on A. thaliana with pre-treatment of U. maydis wild type strain FB1 and recombinant FB1 strains overexpressing GH25 orthologues from MbA, U. maydis ( Um ) and R. solani ( Rs ) (FB1_pOtef_* MbA/Um/Rs *_GH25) in active or mutant version (D124A). % of A. laibachii infected leaves were scored at 11 dpi across 3 biological replicates in non-sterile condition. One-way ANOVA and Tukey’s multiple comparisons test (alpha 0.05) was performed to find significant difference between treatments. N=number of seedlings analyzed. No evidence of plant immunity activation or direct inhibition of oomycete and fungal plant pathogens by MbA GH25 To elucidate the mechanism of the observed Mba antagonism towards A. laibachii , we explored whether MbA _GH25 impacts plant immunity. To this end, 2.5-week-old A. thaliana seedlings were treated with purified recombinant MbA _GH25, MbA _GH25 (D124A) or heat-inactivated (Hi) MbA _GH25 proteins as a negative control. The broadly recognized bacterial PAMP flagellin 22 (flg22), was used as a defense-inducing positive control in the elicitor assay. Thirty minutes after treatments, seedlings were harvested to check for activation of several defense marker genes. Flg22 treatment induced the expression of defense genes WRKY53, WRKY33, WRKY30 , and FRK1 compared to other treatments ( Fig. S1 ). No difference in defense gene expression levels was detected between active and inactive (mutated and heat-inactivated) versions of the MbA _GH25 protein ( Fig. S1 ). Therefore, we concluded that the enzymatic activity of MbA _GH25 likely does not induce plant defense in A. thaliana . Since the MbA _GH25 mediated antagonism of A. laibachii does not depend on an induction of the plant PTI system, we tested whether MbA _GH25 directly targets the oomycete. The cell walls of oomycetes are mainly composed of β-1,3, and β-1,6 glucans ( Aronson et al., 1967 ), with varying levels of N-acetyl Glucosamine (NAG)-the building blocks of bacterial peptidoglycan ( Mélida et al., 2013 ). Therefore, to determine whether MbA_ GH25 directly targets A. laibachii cell wall we used commercially available Laminarin (seaweed polysaccharide with ß-1,3 linked glucan bonds and ß-1,6 linked side chains) as a substrate to monitor for release of oligosaccharides upon addition of purified MbA _GH25 protein via Thin Layer Chromatography. This approach showed no activity of GH25 on Laminarin ( Fig. S2A ). Additionally, we isolated A. laibachii cell walls from harvested zoospores ( Mélida et al., 2013 ), and looked for activation of MbA gh25 gene expression. However, no significant induction of the gh25 expression was detected in growing MbA cultures ( Fig. S2B ). To determine whether the antagonistic activity of MbA /GH25 is specific to A. laibachii , we tested for possible interactions with the oomycete pathogen Hyaloperenospora arabidopsidis ( Hpa; biotrophic pathogen, adapted to A. thaliana ) and Phytophtora infestans (hemibiotrophic pathogen of solanaceous plants, not adapted to A. thaliana ) as well as the fungal pathogen Botrytis cinerea (nectrotrophic pathogen, generalist). To test for Mba /GH25 effects on Hpa infection, 2.5-week-old A. thaliana seedlings (Col-0 and Col-0 eds1-12 ( Ordon et al., 2017 )) were pre-treated with a growing culture (OD 600nm =0.8-1) of MbA and MbA Δgh25, followed by spray inoculation of Hpa (15*10 4 spores/ml) 2 days later. Purified MbA GH25 or MbA GH25(D124A) was mixed directly with Hpa spores (6µM conc.) and sprayed on seedlings. No significant differences in Hpa sporulation (release of Hpa spores per gram of A. thaliana at 5 dpi) were observed among the different treatments in both A. thaliana wild-type Col-0 and the Hpa -hypersusceptible mutant eds1-12 ( Fig. 3A ). Hpa sporulation was significantly higher in the eds1-12 mutant line compared to Col-0, indicating that Hpa infection conditions on A. thaliana were suitable. ( Fig. 3A ). Furthermore, relative gh25 expression levels were quantified in MbA upon interaction with Hpa on A. thaliana and no significant change in gh25 expression was observed ( Fig. S3A ). Thus, the presence of the basidiomycete yeast or its active and inactive hydrolase did not impact leaf infection Hpa . Download figure Open in new tab Figure 3: GH25-mediated inhibition of A. laibachii is not conserved against other pathogenic oomycetes and fungi. A ) MbA and purified MbA _GH25 do not affect Hpa infection in A. thaliana Col-0 and eds1-12 mutant (Col-0 background). Experiments were conducted in 3 biological replicates (consisting of 3 technical replicates). Quantification of Hpa spores *10 4 / g of leaves was performed at 5 dpi. B) Interaction of P. infestans with MbA strains and U. maydis strains: Quantification of necrotic area on leaf surface was performed by ImageJ and percentage of necrosis calculated in 3 biological replicates (N=number of leaves analyzed). C) In planta droplet infection assay was performed by dropping 5 µl of spore suspension in detached A. thaliana leaves, which were pretreated with mock (water), MbA, MbA )GH25, MbA + A. laibachii or A. laibachii . Lesion size quantification was performed using ImageJ across 3 biological replicates (N= number of lesions analyzed). One-way ANOVA and Tukey’s multiple comparisons test (alpha 0.05) was performed to find significant difference between treatments. In-planta interaction assays of P. infestans with MbA were performed using detached leaves of 5-week-old N. benthamiana . We observed that necrotic lesions caused by P. infestans were not reduced upon its co-inoculation in the presence of MbA and MbA Δgh25 ( Fig. 3B ). In addition, the MbA gh25 gene was recombinantly expressed in the smut pathogen U. maydis (SG200_pOtef: MbA GH25) and tested for interaction with P. infestans in the detached leaf assay. However, co-inoculation of the recombinant strain SG200_pOtef: MbA GH25 did not limit the development of necrotic lesions ( Fig. 3B ). Confrontation assays in axenic culture between MbA strains and P. infestans were carried out on Rye-Sucrose Agar (RSA) plates. However, although no zone of inhibition was observed between the two interacting microorganisms, P. infestans was restricted from growing over the area already colonized by yeast strains ( Fig. S3B ). To investigate the inhibitory effect of MbA on a necrotrophic fungus, Botrytis cinerea was tested by using both in-vitro and in-planta assays. During the axenic confrontation assay, no inhibition of B. cinerea could be observed upon confrontation with MbA, MbA Δgh25 and purified MbA _GH25 protein ( Fig. S3C ). However, the lesion size of B. cinerea was significantly reduced after pretreatment of A. thaliana with both MbA and MbA Δgh25, indicating an inhibition of B. cinerea by MbA that is independent of GH25 activity ( Fig. 3C ). Thus, the observed antagonistic activity of MbA via GH25 appears to specifically act against A. laibachii . In summary, we found no evidence for a GH25 stimulating activity on plant immunity or a GH25 direct antimicrobial activity against any of the tested eukaroytic plant pathogens. Therefore, we hypothesized that GH25-mediated inhibition of A. laibachii is mediated by an indirect interaction involving additional microbes. Bacteria associated with Albugo laibachii are targeted by GH25 Recently, Goossens et al. (2023) investigated the presence of several bacterial isolates closely associated with H. arabidopsidis on A. thaliana . The bacterial population thrived during Hpa infection and was actively recruited by the plant to limit disease progression ( Goossens et al., 2023 ). Here, we tested whether A. laibachii could be associated with a bacterial community. In previous gnotobiotic A. laibachii infection assays, oomycete zoospores were treated with an antibiotic cocktail (streptomycin, kanamycin, rifampicin, and geneticin) prior to inoculation of A. thaliana seedlings. To determine whether any bacterial isolates remained after the antibiotic treatment, we harvested zoospores of A. labachii NC14 strain and plated them on King’s B medium after treatment with the antibiotic cocktail. This treatment resulted in the isolation of a set of A. laibachii- associated bacteria, comprising mainly Pseudomonas sp., Microbacterium sp. and Curtobacterium sp. ( Table S2 ). We then tested the individual A. laibachii associated bacterial strains in one-to-one interactions with MbA , purified MbA _GH25 protein or a recombinant U. maydis strain constitutively expressing MbA _GH25 ( Fig. 4 , S4 ). MbA inhibited two of the Pseudomonas strains, while the recombinant MbA _GH25 strain inhibited two of the Curtobacterium strains, although halo formation was more pronounced in the case of MbA ( Fig. S4 ). Purified MbA _GH25 inhibited both Stapylococcus sp. and Curtobacterium isolates. In contrast, the catalytically inactive MbA _GH25 (D124A) protein did not cause any inhibition, indicating that the antibacterial activity is dependent on GH25 enzymatic activity ( Fig. 4A-D ). Thus, A. laibachii is associated with a community of bacteria of which Stapylococcus sp. and Curtobacterium sp. are inhibited by MbA / GH25 activity. Next, we tested whether these microbial interactions are linked with the antagonistic interplay of MbA and A. laibachii on the leaf surface. Download figure Open in new tab Figure 4: Albugo laibachii associated bacteria are targeted by MbA _GH25. A) Heatmap showing overview of the confrontation assay between A. laibachii associated bacteria and purified recombinant MbA _GH25/ MbA _GH25(D124A) protein across 3 independent replicates. Curtobacterium sp. (#1 and #2) and Staphylococcus sp. were inhibited by MbA _GH25 active protein. B) Zone of inhibition of both isolates of Curtobacterium sp. and Staphylococcus sp. by purified MbA _GH25 was analyzed compared to buffer control and mutated MbA _GH25 (D124A) across 3 independent replicates. Representative image of bacterial inhibition added next to respective treatments as indicated by the presence or absence of halo formation surrounding the agar well, 3 days after application of recombinant protein (0.8ug/ul). Error bars show SD. One-way ANOVA and Tukey’s multiple comparisons test (alpha 0.05) was performed to find significant difference between treatments. An antagonistic interplay between bacteria, an oomycete and a fungal yeast To elucidate an eventual biological role of the observed microbial interplay, the tripartite interactions of MbA, A. laibachii and either Curtobacterium sp . or Staphylococcus sp . on Arabidopsis leaves were investigated. While Pseudomonas sp . and Staphylococcus sp . had no, or marginal negative impact on Albugo laibachii , oomycete infection was strongly influenced by presence of Curtobacterium sp . ( Fig. 5A-C ). Staphylococcus sp. inhibited A. laibachii infection both in the presence and absence of MbA , similar to Curtobacterium sp. #1 ( Fig. 5C ). In contrast, Pseudomonas sp. had no inhibitory effect on A. laibachii infection and partially suppressed the inhibition of A. laibachii by MbA ( Fig. 5C ), suggesting that Pseudomonas sp. positively affects A. laibachii colonization. Interestingly, the two strains of Curtobacterium sp. showed contrasting behavior in the tripartite interaction: Curtobacterium sp. #1 reduced A. laibachii infection similarly to MbA ( Fig. 5A ). In contrast, Curtobacterium sp. #2 did not significantly affect A. laibachii infection. Moreover, co-inoculation almost completely restored A. laibachii infection in presence of MbA; i.e. Curtobacterium sp. #2 protected A. laibachii from antagonism by MbA ( Fig. 5B ). Download figure Open in new tab Figure 5: Curtobacterium sp. #2 suppresses MbA inhibition of A. laibachii . A-C) Infection assay of A. laibachii on A. thaliana with pre-treatment of MbA and different bacteria; A) Curtobacterium sp. #1, B) Curtobacterium sp. #2, C) Staphylococcus sp. and Pseudomonas sp. Pretreatment was performed 2 days prior to A. laibachii infection. Plants were scored at 11 dpi. One-way ANOVA and Tukey’s multiple comparisons test (alpha 0.05) was performed to find significant difference between treatments. D) Relative quantification of Curtobacterium sp. #2 biomass in response to A. laibachii and MbA . 23S rRNA specific region in Curtobacterium sp. was normalized to A. thaliana EF1-α to quantify the amount of Curtobacterium DNA in the samples at 11dpi. E) Relative quantification of MbA biomass in response to A. laibachii and Curtobacterium sp. GH25 (g2490) as a single-copy gene was normalized to A. thaliana EF1-α to quantify the amount of MbA DNA in the samples at 11dpi. Unpaired t-test carried out to find significant difference between treatments. Both Curtobacterium sp. #2 and MbA biomass was significantly reduced upon interaction with one another. F) Relative quantification of MbA gh25 expression in response to A. laibachii and Curtobacterium sp. #2, normalized to MbA housekeeping gene ppi at 3dpi. One-way ANOVA and Tukey’s multiple comparisons test (alpha 0.05) was performed to find significant difference between treatments. Next, we performed a qPCR approach to quantify the growth of the interacting microbes on the plant surface. This revealed that MbA biomass was reduced in the presence of Curtobacterium sp . 2 ( Fig. 5E ). Also growth of Curtobacterium sp. #2 was also significantly reduced in presence of MbA ( Fig. 5F ), reflecting a bidirectional antagonism between Curtobacterium sp. #2 and MbA . Notably, the MbA -mediated inhibition of Curtobacterium sp. #2 was rescued by the presence of A. laibachii , suggesting a protective effect of A. laibachii on Curtobacterium sp. #2 . An interesting observation was made regarding the transcriptional induction of MbA_gh25 . In our previous study ( Eitzen et al., 2021 ), we had initially identified gh25 as a candidate gene based on its transcriptional activation on the plant surface specifically in the presence of A. laibachii . Here we find that gh25 expression in MbA was significantly upregulated in the presence of both A. laibachii and Curtobacterium sp. # 2. However, also confrontation of MbA with Curtobacterium sp. # 2 alone resulted in transcriptional activation of gh25 . In contrast, antibiotic-treated A. laibachii alone did not induce gh25 expression in MbA ( Fig. 5D ). We concluded from the above assays that it is not A. laibachii but its associated Curtobacterium sp. # 2, which directly antagonizes MbA , that is the trigger of gh25 expression. Next we investigated whether the observed strain-specific inhibitory activity of GH25 was restricted to MbA _GH25 or might be a conserved property of GH25 proteins across clades. To test this, we performed confrontation assays with the FB1 strains overexpressing GH25 orthologs derived from MbA, U. maydis or R. solani (see Fig. 2A ) and Curtobacterium sp. #2 . Consistent with GH25 antagonism of A. laibachii infection, GH25 from MbA and U . maydis inhibited Curtobacterium sp. #2 ( Fig. S5A ). In contrast, GH25 from R. solani had no effect on Curtobacterium sp. #2 , whereas a bacterial control strain ( Priestia sp.) was inhibited by enzymatically active versions of all three GH25 orthologs ( Fig. S5B ). This result suggests an ortholog-dependent target specificity of GH25, which may reflect a host-specific adaptation. Finally, we tested whether the protective effect of Curtobacterium sp. #2 on A. laibachii can be attributed to its modulation of the leaf microbiome. We examined potential cross-inhibition within the microbial community by spreading one bacterium as a lawn and applying treatment bacteria to observe any resulting halo formation ( Fig. S6A ). This showed that Curtobacterium sp. #1 and #2, as well as Pseudomonas sp. #1 and #2, inhibited most of the associated bacteria that were not targeted by GH25 ( Fig. 6A ). Furthermore, when these bacteria were used as a lawn, the inhibited bacteria were unable to grow, suggesting strong inhibitory potential ( Fig. S6B ). Based on these findings, we hypothesized that Curtobacterium sp. and Pseudomonas sp. might promote A. laibachii growth through their inhibitory effects on other microbes. To test this, we conducted an infection assay using Microbacterium sp. #2 and Chryseobacterium sp., two bacteria that not inhibited by GH25 but are targeted by both Pseudomonas sp. and Curtobacterium sp. We found that both Microbacterium sp. #2 and Chryseobacterium sp. displayed an inhibitory effect on A. laibachii and were unable to alleviate inhibition of A. laibachii by MbA ( Fig. 6B ). In addition, they rescued the growth inhibition of A. thaliana caused by A. laibachii ( Fig. S6C ). Taken together, our findings reveal a tripartite, inter-kingdom interaction between bacteria, oomycete and yeast, in which a GH25 lysozyme shapes a highly specific antagonism between MbA, A. laibachii and Curtobacterium sp .. We propose that this interplay between organisms on the leaf enables a balanced competition and maintenance of microbial niches in the plant phyllosphere ( Fig. 7 ). Download figure Open in new tab Figure 6: Cross-inhibition events within the associated bacterial community shape infection outcome. A) Inhibition assays between bacteria were conducted on square PD plates. 250 µl lawn bacterium was plated out and 5 µl competitor/treatment bacterium spotted on top. After 3-4 days halo area was measured and analyzed using ImageJ. 3-4 biological were performed for each bacterial pair tested. Mean of replicates is used for plotting. B) Infection assay of A. laibachii on A. thaliana with pre-treatment of MbA and Microbacterium sp._#2 and Chryseobacterium sp. Pretreatment was performed 2 days prior to A. laibachii infection. Plants were scored at 11 dpi. One-way ANOVA and Tukey’s multiple comparisons test (alpha 0.05) was performed to find significant difference between treatments. Download figure Open in new tab Figure 7: Model of the inter kingdom interactions shaped by MbA GH25. We depict how white rust infection in A. thaliana , caused by oomycete A. laibachii is inhibited by basidiomycete yeast MbA through antagonism of oomycete associated bacteria. A) Albugo laibachii is closely associated with a bacterial consortium in the phyllosphere of A. thaliana . Particularly, Curtobacterium sp. and Staphylococcus sp. are positively correlated with the host infection caused by A. laibachii . B) Basidiomycete yeast MbA produces Glycoside Hydrolase 25 protein to target Curtobacterium sp. #2, which leaves A. laibachii unprotected from competition with MbA , leading to a reduction of white rust infection in A. thaliana . Discussion We previously described antagonism by the basidiomycete yeast MbA of an oomycete plant pathogen A. laibachii through an MbA-secreted GH25 lysozyme ( Eitzen et al., 2021 ). Here, we demonstrate that the observed antagonism between MbA and A. laibachii is mediated through an A. laibachii associated Curtobacterium sp. in a strain-specific manner. We tested two different modes of antagonism, including a direct action of the GH25 on the oomycete cell wall, as well as an indirect inhibition of infection via the activation of plant immune responses. Simultaneously, we found that MbA and Glycoside Hydrolase 25 were unable to antagonize oomycete pathogens Hpa and P. infestans . Although both A. laibachii and Hpa are foliar pathogens in natural A. thaliana populations, Hpa is less abundant and more affected by hormonal alterations in the host ( Ruhe et al., 2016 ). For instance, while A. laibachii growth is unperturbed in ABA and SA mutant lines of A. thaliana, Hpa growth is restricted on JA accumulating ABA mutant line and increased on sid2-2 mutant, defective in Isochorismate Synthase 1 (precursor in SA signaling) ( Ruhe et al., 2016 ). Nevertheless, A. candida is known to suppress broad spectrum host innate immunity and increase susceptibility of the plant to downey mildew infection ( Cooper et al., 2008 ; Prince et al., 2017 ). Several reports have shown Hpa disease symptoms to be controlled by the presence of bacteria. Berendsen et al. (2018) reported three bacterial taxa ( Stenotrophomonas sp., Xanthomonas sp., and Microbacterium sp. to become enriched in the A. thaliana rhizosphere upon infection with Hpa . More interestingly, in a study by Almario et al. (2022) , although Hpa comprised the core taxa, the sampled leaves were asymptomatic due to the presence of plant beneficial bacterium such as Sphingomonas and Variovorax . Whereas, for P. infestans , a fungal endophyte, Monosporacus sp. inhibited Phytophthora in culture conditions and not in planta ( de Vries et al., 2018 ). Since both Hpa and A. laibachii are obligate biotrophic pathogens, their survival depends on the living host and therefore maintaining a stable equilibrium in the host-microbial community is crucial ( Agler et al., 2016 ; Ruhe et al., 2016 ). Exploring microbiota associated with pathogens can offer insights into disease emergence and proliferation ( Kong and Hong, 2016 ). For example, A. laibachii infections help P. infestans to colonize and sporulate on Arabidopsis ( Belhaj et al., 2017 ), which could result from Albugo infections in Brassicaceae promoting a host jump by certain pathogens ( Thines, 2014 ). Albugo candida has been shown to directly influence the plant microbiome by releasing proteins and peptides with antimicrobial activity into the apoplast ( Gómez-Pérez et al., 2023 ). Moreover, microbiome analysis during the interaction between A. thaliana and A. laibachii revealed the formation of a disease associated microbial community (DCom) in infected plants, distinct from the health-associated microbial community (HCom) found in uninfected controls ( Mahmoudi et al., 2024 ). Furthermore, co-inoculation with HCom members more effectively suppressed A. laibachii infection than co-inoculation with DCom-associated microbes ( Mahmoudi et al., 2024 ). Therefore, we were intrigued to explore how associated bacteria of A. laibachii might be a key component in the MbA _GH25 mediated inhibition of A. laibachii . To this end, we isolated 14 bacterial members from A. laibachii Nc14 spores and tested for interaction with MbA and MbA _GH25. During the confrontation assays, we found the Pseudomonas sp. strains to be inhibited by MbA , and not MbA _GH25, which indicates that the antimicrobial activity against Pseudomonas is independent of GH25 secretion. The MbA induced bacterial inhibition could be potentially caused by bio-surfactants or secondary metabolites as described previously for related yeasts ( Morita et al., 2007 ). Interestingly, both Curtobacterium sp . isolates were inhibited by GH25 protein, as well as by recombinant U. maydis strains overexpressing GH25. However, only Curtobacterium sp. #2 rescued the inhibition of A. laibachii by MbA , reflecting a high level of specificity. Moreover, both Curtobacterium sp. #2 and MbA significantly antagonize each other in-planta , indicating a bi-directional negative interaction. Finally, gh25 gene expression in MbA was upregulated in presence of Curtobacterium sp. #2 rather than A. laibachii . Furthermore, we found Curtobacterium sp . and Pseudomonas sp . to be key microbes in shaping the microbiome associated with A. laibachii , by inhibiting most isolated associated bacteria that are not targeted by GH25. Taken together, our observations reveal a tripartite antagonism between MbA, Curtobacterium sp. #2 and A. laibachii , which results in reshaping of the microbial community associated to A. laibachii . Together with results that demonstrate a preference of A. labachii towards DCom microbes ( Mahmoudi et al., 2024 ) and the general role of A. laibachii as a hub microbe in shaping the microbial community ( Agler et al., 2016 ) one could hypothezised that the association with Curtobacterium sp. #2 aids A. laibachii in maintaining the stability of its niche. Unlike Hpa , which is associated with bacteria inhibiting the pathogens infection ( Goossens et al., 2023 ), A. laibachii thus might have developed mechanisms to maintain a microbiome including protective microbes. Simultaneously, we tested GH25 orthologues from U. maydis and R. solani in the antagonism towards A. laibachii . We found that GH25 from closely related basidiomycetes MbA and U. maydis were able to inhibit both A. laibachii in-planta and Curtobacterium sp. #2 during in vitro confrontation assays, as opposed to that from phylogenetically more distant plant pathogen R. solani . Therefore, these results indicate a host-specific adaptation of GH25 orthologues across the fungal kingdom and underline the importance of Curtobacterium sp. #2 inhibition for successful A. laibachii inhibition in-planta. It has been shown that filamentous plant pathogens secrete effectors to modulate the plant microbiota ( Flores-Nunez and Stukenbrock, 2024 ; Snelders et al., 2022 , 2018). For example, the secretion of phytotoxic ribonucleases by fungal pathogens Zymoseptoria tritici ( Kettles et al., 2018 ) and U. maydis ( Ökmen et al., 2023 )can modify the leaf microbiome of their respective hosts. In case of GH25 specificity, one could hypothesize that adaptation to competing bacteria in the leaf phyllosphere might contribute to niche adaptation in pathogenic fungi, but also increases fitness of beneficial fungi such as MbA to establish itself in an epiphytic lifestyle in association with highly competitive A. laibachii . In conclusion, competition with associated microbes of plant pathogens can be a key component in effective disease control and plant health restoration (Cernava, 2024). Our study reveals a tripartite interkingdom interaction between the yeast MbA , the oomycete A. laibachii , and the bacterium Curtobacterium sp. #2 on the plant surface. We show that MbA and Curtobacterium sp. #2 exhibit mutual antagonism, with A. laibachii providing a protective effect on Curtobacterium sp. #2 and vice versa. Our data further suggests a host-specific adaptation of GH25 orthologues in microbial competition and direct targeting of specific bacteria leading to a reshaping of the microbial community structure. Here, we propose a model depicting the cross-kingdom interaction between bacteria, oomycete and yeast orchestrated by GH25 ( Fig. 7 ). Materials and methods Growth conditions for microbial strains MbA and U. maydis strains were grown in liquid YEPS light medium at 22°C and 28°C respectively in a rotary shaker (200 rpm) and maintained on PD agar plates. Albugo laibachii associated bacterial strains ( Table S2 ) were grown in King’s B liquid media at 22°C overnight in a rotary shaker (200 rpm). Pichia pastoris KM71H-OCH was used for recombinant protein expression as described in Eitzen et al. (2021) . Plant infection assays Albugo laibachii infections in Arabidopsis thaliana were performed as described in ( Eitzen et al., 2021 ). A. laibachii infections of the orthologue experiment were performed in non-sterile conditions. Fresh shoot weight was taken at 11 dpi and roots were removed for the measurements. For Hyaloperenospora arabidopsidis ( Hpa ) infection assays, Hpa isolate Noco2 ( van der Biezen et al., 2002 )was sprayed onto 3-week-old Arabidopsis thaliana Col-0 and Col eds1-12 mutant ( Ordon et al., 2017 ) seedlings, two days after spraying with MbA strains and placed in a controlled environment under 10-h light/14-h dark regime at 22°C and 60% relative humidity. At 5dpi, plant fresh weight was determined and seedlings were resuspended in 5ml of ddH 2 O to release the conidiospores of Hpa . Conidiospores were counted under the light microscope using a Neubauer Chamber. N. benthamiana plants were cultivated in a growth chamber with 16 h of light and 8 h of darkness at 22°C for 4 weeks. Subsequently, the 3rd or 4th leaf was detached and placed on moist tissue paper. Spores from Phytophthora infestans strain 88069 were harvested by addition of ddH 2 O to mycelium growing on plate. After 3-4 h incubation at 4°C, the zoospores were released by scratching the mycelium with a sterile tip. The zoospore concentration was adjusted to 10 5 spores/ml of water.10µl of P. infestans spore suspension was dropped on detached leaves. After 6 days, the necrotic lesions were evaluated using ImageJ. For B. cinerea infection assay, B. cinerea B05 . 10 was grown 10 days on HA plates (10 g malt extract, 4 g glucose, 4 g yeast extract, 15 g agar per liter, pH 5.5). Spores were harvested and introduced at a concentration of 1x10 5 conidia/ml to 10 ml Gamborg semi-solid inoculation medium at approcimately 37°C. The medium was incubated for 19h, 25°C, 200 rpm. 5 µl of the suspension was dropped on detached 6-week-old A. thaliana leaves (pretreated with MbA strains and A. laibachii as described in Eitzen et al. (2021) . Lesion size was evaluated at 72hpi with ImageJ. Isolation of A. laibachii associated bacteria A. laibachii spore suspension was prepared as described in in ( Eitzen et al., 2021 ). After antibiotic treatment of the spores, spore solution was diluted 1:10, 1:100 and 1:1000 and 50 µl of each dilution and the undiluted suspension were plated on LB media. Plates were incubated at room temperature and after 3-5 days single colonies were picked. The identity of the isolated strains was determined via 16S rRNA sequencing using 16S rRNA primer pairs ( Table S1 ). For each isolated bacterial strain, a colony PCR was performed with 16S rRNA primer pairs and purified PCR fragments were sequenced at the Eurofins sequencing facility (Germany). See Table S2 for an overview of the isolated bacteria and the respective sequences. Microbial confrontation assays For bacterial confrontation assays, MbA / U. maydis cultures grown to OD600nm 1.0 were dropped (10ul) in four quadrants of a PD Agar plate, spread previously with 100µl of bacterial culture with an OD600nm of 0.6. 20 µl of GH25 protein (0.8µg/µl) was applied to a hole created in the centre of the plate (d=4mm). For bacterial cross-inhibition assays, square PD plates were used. Bacteria were diluted to an OD600nm of 0.8 in 10 mM MgCl2 and 250 µl of a lawn bacterium was spread. 5 µl of competitor bacteria were dropped on the plate. Plates were incubated for 2-4 days at 22°C. For P. infestans confrontation assay, RSA plates supplemented with 100µg/ml Ampicillin were used. A mycelial plug of P. infestans was placed in the centre of the plate and growing culture of MbA (10µl) was placed as droplets on two corners of the plate. For B. cinerea confrontation assays, MbA strains were grown to an OD600:1.0 and 10 µl were dropped in the centre of an PD plate and incubated overnight at 22°C. 20 µl of MbA_GH25 (0.8µg/µl) was applied to a hole created in the centre of the plate (d=4mm). B. cinerea B05 . 10 plugs were placed on two corners of the plate with the same distance to the competitor. Inhibition was evaluated at 3-4 dpi. Elicitor assays 2.5-week-old Col-0 Arabidopsis thaliana seedlings on liquid MS media were hand-infiltrated with purified recombinant proteins MbA _GH25, MbA _GH25(D124A), heat-killed MbA _GH25 in 2µM concentration. Bacterial PAMP, flg22 (50nM conc.) was used as a positive control. Leaves were harvested 30 minutes after treatment and frozen in liquid nitrogen. RNA and cDNA extracted from harvested tissue was used to perform qPCR of different marker genes. (modified from protocol of ( Cao et al., 2014 )). Nucleic acid methods Total RNA extraction from plant samples were performed with Trizol Reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions followed by treatment with Turbo DNA-Free Kit (Ambion/Applied Biosystems; Darmstadt, Germany) to remove any DNA contamination in the extracted RNA. Synthesis of cDNA was performed using First Strand cDNA Synthesis Kit (Thermo Fisher Scientific; Darmstadt, Germany) according to recommended instruction starting with a concentration of 10 µg RNA. RT-qPCR oligonucleotide pairs were designed with Primer3 Plus. Relative expression levels of marker genes were analyzed with GoTaq® qPCR Master Mix in a Bio-Rad iCycler system using the following program: 2 min at 95 °C followed by 45 cycles of 30 s at 95 °C, 30 s at 61 °C and 30 s 72 °C. Plasmid Prep Kit (QIAGEN, Venlo, The Netherlands) was used for isolation of plasmid DNA from bacteria after the principle of alkaline lysis. Genomic DNA was isolated using phenol–chloroform extraction protocol ( Ruhe et al., 2016 ). Construction of plasmids All plasmids were constructed using either restriction enzyme mediated ligation using T4 DNA ligase or Gibson assembly (New England Biolabs; Frankfurt a.M., Germany). All GH25 orthologue gene expression constructs ( p123-pUmOtef::MbA_GH25-2xHA, p123-pUmPit2:: MbA_GH25(D124A)2xHA , p123pUmOtef::Umaydis_GH252xHA, p123pUmOtef::Umaydis_G H25(D124A)-2xHA , p123pUmOtef::Rsolani_GH252xHA and p123pUmOtef::Rsolani_GH25(D122A)-2xHA) were constructed using Gibson assembly. Rhizoctonia solani GH25 was di-codon optimized for expression in U. maydis and synthesized gene was obtained from BioCat GmbH, Heidelberg. GH25 active site mutants created by using QuickChange XL Site-Directed Mutagenesis Kit from Agilent Technology according to the manufacturer’s instructions. E. coli transformation was performed using heat shock according to standard molecular biology methods ( Sambrook et al., 1989 ). Vector constructs and oligonucleotide sequences are mentioned in Table S1. Generated constructs were verified at the Eurofins sequencing facility (Germany). Fungal Transformation and Validation The heterologous gene expression constructs (Table S1) were introduced in SG200 or FB1 (GH25 orthologue overexpression) by ip integration, using protoplasts according to ( Kämper, 2004 ). Generated strains were further analyzed by southern blot to validate the integration and copy number (data not shown). Phylogenetic analysis For phylogenetic tree construction, all selected GH25 orthologues (see Eitzen et al., (2021) ) were aligned using MAFFT Multiple Sequence Alignment Software Version 7. Subsequently, a phylogenetic tree was constructed using NJ (Neighbor-Joining) with default settings and 100 bootstrap replications using Phyl.io ( Robinson et al., 2016 ). Statistical analysis For statistical analysis, to identify significant differences between treatments, an analysis of variance (ANOVA) model with Tukey’s HSD test was used for multiple comparisons. Graphpad Prism (10.4.1) was used to generate data plots. ImageJ 1.53K version (Wayne Rasband and Contributors National Institute of Health, USA) was used to calculate necrotic lesions of P. infestans, B. cinerea and zone of inhibition by fungal and yeast strains. Fiji (2.16.0) was used to analyse area of inhibition zones in bacterial confrontation assay. Di-Codon Optimization of R. solani GH25 was performed using Python 3.x (Python Software Foundation, 2023). Data availability Source data are provided in this paper. All data supporting the findings of this study that are not directly available within the paper (and its supplementary data) will be upon reasonable request available from the corresponding authors (GD, PS). Author contributions GD, ZS and PS designed the experiments with input from JP and EK. ZS, PS, KBH and JB conducted the experiments. PS, ZS and GD wrote the manuscript with contributions from all authors. Competing interests The authors declare no competing interests. Supplementary Information Supplementary Figure S1: Testing plant defense marker gene expression by MbA_GH25 Supplementary Figure S2: Interaction of A. laibachii cell wall with MbA_GH25 Supplementary Figure S3: Interaction of MbA and MbA _GH25 with oomycetes and fungi Supplementary Figure S4: Inhibition of A. laibachii associated bacteria Supplementary Figure S5 : Inhibition of A. laibachii associated bacteria by GH25 orthologs Supplementary Figure S6 : Connection of cross-inhibition events on-plate and A. laibachii inhibition on-planta Supplementary Table S1 : Information on Constructs & Primer used Supplementary Table S2: Information on bacterial strains used Acknowledgements This project has received from the Cluster of Excellence on Plant Sciences (CEPLAS) funded under Germany’s Excellence Strategy—EXC 2048/1—project ID: 390686111 and the DFG priority program SPP2125 ‘DECRyPT’. 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Share GH25 lysozyme mediates tripartite interkingdom interactions and microbial competition on the plant leaf surface Zarah Sorger , Priyamedha Sengupta , Klara Beier-Heuchert , Jaqueline Bautor , Jane E. Parker , Eric Kemen , Gunther Doehlemann bioRxiv 2025.04.04.647216; doi: https://doi.org/10.1101/2025.04.04.647216 Share This Article: Copy Citation Tools GH25 lysozyme mediates tripartite interkingdom interactions and microbial competition on the plant leaf surface Zarah Sorger , Priyamedha Sengupta , Klara Beier-Heuchert , Jaqueline Bautor , Jane E. 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