Fungal Mst3-proteins are involved in fungal innate immunity needed for the recognition of bacteria surrounding the hyphae, as well as for plant pathogenicity

Fungal Mst3-proteins are involved in fungal innate immunity needed for the recognition of bacteria surrounding the hyphae, as well as for plant pathogenicity | 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 Fungal Mst3-proteins are involved in fungal innate immunity needed for the recognition of bacteria surrounding the hyphae, as well as for plant pathogenicity Shanshan Gong , Xiaorong Lin , Shenghua Liu , View ORCID Profile Stefan Olsson , Guodong Lu , View ORCID Profile Zonghua Wang , Ya Li doi: https://doi.org/10.1101/2025.11.25.690610 Shanshan Gong 1 State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University , Fuzhou, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xiaorong Lin 1 State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University , Fuzhou, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shenghua Liu 1 State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University , Fuzhou, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stefan Olsson 1 State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University , Fuzhou, China 2 Synthetic Biology Center, College of Future Technologies, Fujian Agriculture and Forestry University , Fuzhou, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stefan Olsson For correspondence: stefan.olsson.kvl{at}gmail.com liya-81{at}163.com Guodong Lu 1 State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University , Fuzhou, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zonghua Wang 1 State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University , Fuzhou, China 3 Institute of Oceanography, Minjiang University , Fuzhou, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Zonghua Wang Ya Li 1 State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University , Fuzhou, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: stefan.olsson.kvl{at}gmail.com liya-81{at}163.com Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Fungal innate immunity resembles mammalian innate immunity. It does not employ toll-like receptors (TLRs), but should employ endocytosis of non-fungal molecular patterns recognized by nuclear-localizing receptors (NLR). Downstream, both types of receptors are Mammalian Ste20 kinases (MSTs). We identified an MST3 ortholog in the plant pathogens Fusarium graminearum ( FgMST3 ) and Magnaporthe oryzae ( MoMST3 ). We knocked out both genes and investigated mutants using a standard panel of tests for growth, development, and pathogenicity for the respective fungi. Both ΔFgMST3 and ΔMoMST3 strains showed reduced pathogenicity. The deletions negatively affected conidia production and conidia germination but had little effect on growth rate. However, the two mutants reacted differently to various stress treatments, especially to Zn 2+ and gentamicin. In addition, we constructed an innate immunity reporter system for F. graminearum to detect less than 4-hour responses to non-self-molecular patterns (NSMP) like bacterial outer membrane vesicles (OMVs) and trace levels of sucrose, indicating plant. The reporter gene responses to OMVs of MST3 mutant strains are severely reduced. Our results indicate that both MoMst3 and FgMst3 are involved in fungal innate immunity downstream of unknown NLR proteins, motivating studies to identify genes for the NLR receptors. Finding such and investigating how they work and vary between fungal species and strains should be essential for understanding fungal biotic interactions with viruses, bacteria, plants, and animals. Introduction Sterile20 kinases (Ste20) are named after a yeast kinase that, when deleted, made yeast sterile ( Ramer and Davis 1993 ). A subclass of Ste20 kinases is Mammalian-like STe20 kinases (MSTs). These proteins are involved in non-self-signaling relaying signals from Toll-Like-Receptors (TLRs) on the cell membrane or from Nuclear Localizing Receptors (NLRs) intracellularly ( Strange et al. 2006 ; Shi and Zhou 2018 ). In mammals, the MSTs fall into two groups of GCKII and GCKIII, containing MST1/2 and MST3, respectively, depending on their structure ( Shi and Zhou 2018 ). Outside the MSTs, other Ste20 kinases have roles in cell volume sensing and regulation of Cl-transport. Yeast Ste20 controls a cell shrinkage-activated MAPK cascade that regulates organic osmolyte accumulation ( Strange et al. 2006 ). Fungi have no identified TLRs ( Soanes and Talbot 2010 ) but have a mammalian-like innate immunity ( Ipcho et al. 2016 ) that differs from plant innate immunity, possibly reflecting that in the early eukaryote evolution, plants split off from fungi-metazoans ( Burki 2014 ) and innate immunity is present already in Protista ( Sundaram et al. 2024 ). Since innate immunity also employs Nuclear Localized Receptors (NLRs) and which are possibly evolutionary older than TLRs and occur already in the first unicellular Eukaryotes ( Sundaram et al. 2024 ), and seem to be especially important in epithelia naturally colonized by bacteria as the gut ( Lavelle et al. 2010 ; Zhou et al. 2019 ; Wicherska-Pawłowska et al. 2021 ). In plants, it is unclear how it works, but innate immunity seems different in different tissues, and the normal TLR-triggered immunity is very limited in roots or the root epithelium ( Millet et al. 2010 ; Jacobs et al. 2013 ). Taken together, the innate immunity proteins in fungi recognizing microbial-associated molecular patterns are most likely NLRs of the STAND type that are similar to proteins in the inflammasome and could involve other repeats than just LRRs, like, for example, WD40 repeats (approx. 40 aa repeats ending with the amino acid WD, InterPro) ( Leipe et al. 2004 ; Paoletti et al. 2007 ; Paoletti and Saupe 2009 ; Daskalov et al. 2012 ). Considering all the above, orthologous fungal MST kinases, especially of the MST3 type, should be involved in non-self-signaling downstream of fungal NLR-like non-self-recognizing pattern recognition receptors (PRRs), triggering innate immunity responses and influencing fungal pathogenicity and interactions with bacteria. The plant is a non-self-entity as are bacteria that sheds molecular patterns in the phyllosphere/apoplast/rhizosphere that are different from what is present in fungi, like plant hormones, cellulose oligomer, pectin oligomers, sucrose and other non-self-molecular patterns (NSMP), that can be taken up through endocytosis and be presented to NLR type PRR receptors and most likely trigger inflammasome like proteins ( Li and Wu 2021 ). In line with this, sucrose has been shown to stimulate innate immunity-like responses in a fungus ( Ipcho et al. 2016 ). Thus, endocytosed NSMPs might be recognized by hitherto not identified NLR-type PRR receptors that are dependent on MST3 for downstream signaling. We selected 2 relatively closely related fungi, Fusarium graminearum and Magnaporthe oryzae, with different lifestyles that are both well-studied model pathogen fungi with established efficient transformation systems ( Miguel-Rojas et al. 2023 ), to investigate if MST orthologs are involved in pathogenesis. F. graminearum has to be able to handle bacteria on plant surfaces both above ground and especially below ground in the seedling rhizosphere ( Yang et al. 2011 ), while M. oryzae meets bacteria in the phyllosphere ( Sahu et al. 2021 ). Both fungi first infect as biotrophs and then, probably because of stresses inside the plant, switch to a necrotrophic lifestyle ( Miguel-Rojas et al. 2023 ). In the biotrophic/necrotrophic transition, plant innate immunity is strongly induced, although the pathogen copes and even enhances plant cell death, killing the plant cells, creating necroses. In the biotrophic stage, plant innate immunity is suppressed by the fungus ( Bhadauria et al. 2011 ). Fungal innate immunity signaling might consequently be especially important to the fungus in the biotrophic stage for identifying that it has entered a plant, to establish the interaction with the plant, and suppress plant immunity until the fungus has biomass and resources enough to cope with a switch to necrotrophy. We identified an MST3-like putative protein in each of the two fungi and could show that such MST3 orthologs can be found from Archaea across Eukaryotes. The deletion of both MST3-kinases had effects on growth, conidia production, conidia germination, stress phenotypes, and plant pathogenicity. Furthermore, we could confirm that the deletions had negative effects on fungal immunity responses triggered by bacterial NSMPs, indicating that MST3 signaling is important for fungal recognition of bacteria that might be pathogenic. It might also be important for sensing plant-produced NSMPs, signaling to the fungus that it is inside a plant cell or in the inner rhizosphere of a plant root or on a plant surface. Results Bioinformatics search for a putative MST3 gene in the genomes of both fungi In Rice, the BSR1, a kinase, works downstream of TLR receptors to relay signals in innate immunity ( Pei et al. 2024 ). Thus, we looked for a similar protein in F. graminearum and found one. We found that the closest human homologue to rice OsBsr1 is HsIrak4, which has a similar role in mammals, but both OsBsr1 and HsIrak4 are too short to have the same function as the closest homologue in F. graminearum . We then did a protein blast search in animal protein sequences at NCBI to search for a homologue to the protein sequence we had found in F. graminearum . We looked especially for experimentally well-described mammalian proteins since fungal innate immunity responses have similarities with innate immunity in mammals ( Ipcho et al. 2016 ). It was then that we realized that the F. graminearum gene could encode a well-conserved eukaryotic Mst3 kinase involved in relaying signals from NLRs to trigger innate immunity. The F. graminearum BSR1-like protein used as bait for a BLAST against human sequences gave one good candidate that is known as an Mst3 kinase in animals (E-value 5 x E-127. 67% identities and 82% positives and no gaps) so we named our bait gene FgMst3. In M. oryzae there is an almost Identical predicted protein to the FgMst3 (E-value 0.0. 62% identities and 71% positives and 12% gaps) and we named that MoMst3. Since Mst3 kinases seems very conserved in both fungi and mammals we downloaded potential orthologuos protein sequences for a range of organisms at further and further evolutionary distances from our two fungi and used the rice BSR1 and human Irak4 that are known to relay signals from TLR receptors as an outgroup. All the putative Mst3-like proteins from Archaea to Homo sapiens we found can potentially be involved in relaying signals from NLR receptors in innate immunity, since innate immunity is evolutionary ancient and appears to have evolved at the dawn of eukaryotes to recognize bacteria invading their cytoplasm ( Wein and Sorek 2022 ) ( Fig. 1 ). Download figure Open in new tab Figure 1. Phylogeny of MST3 protein homologues. The mammalian proteins are the 2 canonical MST3s from mouse and the human MST3 ( Qiu et al. 2023 ). The F. graminearum and Pyricularia oryzae (the teleomorph equivalent of Magnaporthe oryzae ) are boxed, as well as the human protein they are compared with using bioinformatics. The Mst3-like kinases are different from BSR1 and IRAK4 kinases that are known to relay signals from TLR cell surface receptors ( Pereira and Gazzinelli 2023 ; Pei et al. 2024 ) and appear truncated to only the N-terminal kinase part of the Mst3-like proteins ( Fig. S1 Alignment of the proteins in the phylogeny) The mammalian Mst3 proteins are known to have the kinase domain in the first half of the protein and both nuclear localization signal (NLS) and a possible nuclear export signal (NES) in the C-terminal end. In between the NLS and the NES is a caspase cleavage site for caspase in mammals ( Lee et al. 2004 ). For fungi, there should be a metacaspase cleavage site since fungal metacaspases cleave in a similar manner as Caspase 3 ( Thrane, Kaufmann, Stummann, et al. 2004 ; Carmona-Gutierrez et al. 2010 ). We used a prediction website to predict caspase/metacaspase cleavage sites ( https://scap.cbrc.pj.aist.go.jp/ScreenCap3/index.php for caspase cleavage) and alignment of the known NLS signal in the human protein to predict the putative NLS signal in the fungal sequences ( Fig. 2 Domain structure and see Supplemental File SF1 for the more detailed bioinformatics done to get the putative protein structure) Download figure Open in new tab Figure 2. Predicted domains and cleavage sites in the 2 fungal proteins and compared with known domains and the cleavage sites in human Mst3 (HsMst3). NLS=nuclear localization signal. NES?= suggested nuclear export signal based on a conserved alignment of the two fungal proteins with the 335-385 section of the HsMst3 implicated to contain a NES. The shorter C-terminal of the human protein contains the NES signal that is less well defined compared to the NLS, so we aligned the truncated C-terminal after a predicted caspase cleavage. In conclusion, cleavage by caspase at the first cleavage site of all 3 proteins should cleave off an NES signal. There is, however, a large extra chunk, approximately 200 AA, just after the predicted caspase cleavage site conserved in the fungal proteins, implying that these fungal Mst3 proteins could have additional functions in the cytoplasm after being cleaved off, but maybe also before cleavage. Deletion of genes in both fungi and the growth effects of the deletions on the growth and development of conidia The putative FgMST3 and MoMST3 genes were deleted successfully, and the resulting ΔFgmst3 and ΔFgMST3 strains were checked for possible ectopic integrations of the HPH gene used as a selection marker for deletions using GO-qPCR (see Materials and Methods). We constructed both FgMST3-GFP and MoMST3-GFP strains from these respective mutant strains ( Fig. S2 , S3 , S4 ). As extra control, we used RT-qPCR to show that no extra working copies of the two MST3 genes have been created or are present in the genome ( Fig. S5 ). We could also show that both FgMst3-GFP and MoMst3-GFP are mainly localized in the cytoplasm of the strains grown in vitro, as expected ( Fig. S6 and Fig. S7 ). Both mutant strains have a slower colony radial growth rate than their respective background strains, PH-1 and KU80, respectively, on all 4 media tested. The ΔFgMST3 strains grew relatively more slowly than the ΔMoMST3 strains ( Fig. 3 ). Download figure Open in new tab Figure 3. Growth effects of MST3 deletions on CM, CMII, SYM, and MM media. (A and C) Colony morphology (B and D) and colony diameter after 3- or 7-day incubation of F. graminearum or M. oryzae cultures, respectively. Error bars in (B and D) show SEMs. 3 stars indicate a P same <0.001 and 4 stars indicate a P same <0.0001 probability for the null hypothesis that these measurements are the same as for the respective background strain. Conidiation was strongly affected by the deletions of FgMST3 respective MoMST3 genes. Both mutant strains produced fewer conidia than their respective background strains and complementation strains. The conidia in ΔFgMST3 strains appeared a bit longer than conidia from PH-1, while there was no difference between ΔMoMST3 conidia and those from KU80 Fig. 4 ). Download figure Open in new tab Figure 4. F. graminearum and M. oryzae conidia morphology and conidia production Conidia morphology (A) and (B), and conidia production in CMC liquid medium (C) and (D). Error bars show SEMs, and 4 stars indicate P same <0.0001 probability for the null hypothesis that these measurements are the same as for the respective background strains. The effect of MST3 gene deletions on important fungal characteristics of special importance for plant pathogenicity that can be tested in vitro Conidial germination and rate of germination, as well as the tolerance of the fungal strains to stress treatments of the kind that a pathogenic fungus meets inside a plant, are such characteristics. In addition, there is a well-established appressorium formation assay available ( Lee and Dean 1994 ). Conidia of both the ΔFgMST3 and the ΔMoMST3 strains germinated well ( Fig. 5 A-D ) but faster than conidia from PH-1 and KU80 and reached the same level of germination. This faster increase in germination per day was for ΔFgMST3 strains between hours 2-4h and 2-4h as well as 6-8h for ΔMoMST3 strains ( Fig. 5B and Fig. 5D ). Appressorium formation from the conidia of ΔMoMST3 strains was always lower from 4 h to 12 h ( Fig. 5E ). Download figure Open in new tab Figure 5. Effect of FgMST3 and MoMST3 mutations on conidia germination and conidia germination dynamics. Conidia germination morphologies after different times of germination (A and C), cumulative germination dynamics (B and D), and appressoria formation from the germinated conidia. Error bars indicate SEMs, and *, **, ***, and **** symbolize P same <0.05, <0.01, < 0.001, and <0.0001, respectively, for the null hypothesis that these measurements are the same for their respective background strains at the same timepoint. A panel of standard stress treatment of relevance to the physiology of fungal plant pathogens were used to test whether the ΔFgMST3 and the ΔMoMST3 strains were affected in comparison to their background strains ( Fig. 6 ). The H 2 O 2 and SDS treatments both affect membrane stability, while the former also has effects on oxidizing DNA and proteins. CFW and CR treatments both have effects on cell wall integrity, while sorbitol and NaCl give osmotic and osmotic/ionic stresses, respectively Download figure Open in new tab Figure 6. F. graminearum and M. oryzae strains grown on a panel of stress-inducing media Colony morphology (A and C) on the different stress media and inhibition of colony diameter growth on the same media. The colony diameter for the background strains (PH-1 and Ku80) on CM without a stressing agent, minus the colony diameter on the stress medium, gives colony growth inhibition (B and D). The larger the value, the more inhibited by the stress medium. Error bars indicate SEMs, and *, **, ***, and **** symbolize P same <0.05, <0.01, <0.001and <0.0001, respectively, for the null hypothesis that these measurements are the same as for respective background strains. Treatments with H 2 O 2 , SDS, and NaCl had less effect on the ΔFgMST3 strain than on PH-1. Treatment with CR, on the other hand was more inhibiting at least for one of the ΔFgMST3 strains, and NaCl had less effect on the ΔFgMST3 strains ( Fig. 6B ). Contrary to for ΔFgMST3 strains, the ΔMoMST3 strains were more affected by H 2 O 2 than the control, thus less resistant, and it was also more strongly affected by CR ( Fig. 6D ). Taken together, it appears that M. oryzae and F. graminearum MST3 mutants react both similarly and differently to stresses, probably reflecting their respective biology as discussed below in the discussion. Effect of the two mutations on mutant strain pathogenicity Both fungi have an initial biotrophic stage that is essential for the spread of the fungus inside the plant tissue ( Miguel-Rojas et al. 2023 ). F. graminearum is mainly a problem when infecting wheat heads, causing head blight, but it also infects form soil, causing seedling blight ( Yang et al. 2011 ). Two standard pathogenicity assays were used, one for wheat coleoptiles and one for wheat heads. In both assays, the two ΔFgMST3 strains showed strongly reduced pathogenicity. The coleoptiles got smaller lesions ( Fig. 7 A-B ), and in the wheat head assay, it was obvious that the main difference was a faster spread from the initial infected flower in the PH-1 through the rachis connecting the flowers and to other flowers and developing seeds ( Fig. 7 C-D ). Taken together, the deletion of the FgMST3 gene negatively affected the F. graminearum pathogenicity on wheat. Download figure Open in new tab Figure 7. Pathogenicity test of F. graminearum strains on wheat coleoptiles and wheat heads (A) Wheat coleoptiles with lesions at 7DPI (B) Average percent lesion length of coleoptile lengths below the point of inoculum at 7DPI. (C) Wheat heads at 14DPI after inoculation at the 3-4 flowers from the base of the head. (D) Average percent diseased spikelets at 14DPI. Error bars indicate SEMs, and *, **, ***, and **** symbolize P same <0.05, <0.01, <0.001, and <0.0001, respectively, for the null hypothesis that these measurements are the same as for the PH-1. M. oryzae is a leaf pathogen of many grasses and can also infect wheat leaves from conidia germinating and forming an appressorium that penetrates the leaf epidermis, entering the plant and spreading between leaf cells as a biotroph. When the easily available soluble nutrients in a plant cytoplasm run out, the pathogen switches to the necrotrophic stage, killing the cell and degrading its components. The M. oryzae necrotrophic activity creates lesions of plant leaf cells dying by a combination of fungal activities and the plant’s own defense, triggering programmed cell death (PCD) of the plant cells ( Dickman and De Figueiredo 2013 ). The ΔMoMST3 where less pathogenic than KU80 in all three assays ( Fig. 8 A-F ) Download figure Open in new tab Figure 8. Lesion formation by M. oryzae strains on barley and rice leaves Conidia were dripped on excised barley leaves (A-B). Lesion appearance on the leaves at 3-5DPI. (A) Average percent lesion area of available leaf area (B). Conidia were sprayed on excised rice leaves (C-D). Lesion appearance on the leaves at 5-7DPI. (C) Average percent lesion area of available leaf area (D). Conidia were added to press-injured spots on excised rice leaves (E-F). Lesion appearance on the leaves at 15DPI (E). Average percent lesion area of available leaf area (F). Error bars indicate SEMs and *, **, ***, and **** symbolize P same <0.05, <0.01, <0.001and <0.0001, respectively, for the null hypothesis that these measurements are the same as for the KU80. Additional stress treatments by Zn and Gentamicin Uptake of both Zn and the weakly antifungal but strongly antibacterial compound gentamicin is dependent on the function of the fungal cell wall and cell membrane, as well as other processes in the fungus. Since the ΔFgMST3 strains and the ΔMoMST3 strains differed in their reaction to some of the stress treatments, and M. oryzae has melanized cell walls that F. graminearum lacks, we added assays for these stress treatments to investigate if these could show clearer differences between the deletion strains and their respective backgrounds. It turned out that ΔFgMST3 strains were less sensitive ( Fig. 9 A, B ) than ΔMoMST3 strains to Zn 2+ stress ( Fig. 9 C, D ). The same applied to gentamicin addition. It even appears that gentamicin is not stressful at all to F. graminearum, as both the PH-1 strain and the complement strain grow slightly better than controls without gentamicin. The largest positive effect of the gentamicin additions was for the two MST3 mutant strains. ( Fig. 10 A-B and Fig. 10 C-D ). Download figure Open in new tab Figure 9. Inhibition of growth by Zn 2+ for the different F. graminearum strains and M. oryzae strains. Colony morphology of F. graminearum strains in the stress medium (A) and inhibition of colony growth on the medium, colony diameter for PH-1 on control medium without stressing agent minus colony diameter on stress medium gives colony growth inhibition (the larger the value, the more inhibited by the stress medium) (B). Colony morphology of M. oryzae strains on the different stress media (C) and inhibition of colony growth on the same media, colony diameter for KU80 on control medium without stressing agent minus colony diameter on stress medium gives colony growth inhibition (the larger the value, the more inhibited by the stress medium (D). Error bars indicate SEMs and *, **, ***, and **** symbolize P same <0.05, <0.01, <0.001and <0.0001, respectively, for the null hypothesis that these measurements are the same as for the PH-1 or KU80 strains. Download figure Open in new tab Figure 10. Inhibition of growth by gentamicin + for the different F. graminearum strains. Colony morphology of F. graminearum strains in the stress medium (A) and inhibition of colony growth on the medium, colony diameter for PH-1 on control medium without stressing agent minus colony diameter on stress medium gives colony growth inhibition (the larger the value, the more inhibited by the stress medium) (B). Colony morphology of M. oryzae strains on the different stress media (C) and inhibition of colony growth on the same media, colony diameter for KU80 on control medium without stressing agent minus colony diameter on stress medium gives colony growth inhibition (the larger the value, the more inhibited by the stress medium (D). Error bars indicate SEMs and *, **, ***, and **** symbolize P same <0.05, <0.01, <0.001and <0.0001, respectively, for the null hypothesis that these measurements are the same as for the respective background strains. Effect of the MST3 mutations on fungal innate immunity A vacuolar iron siderophore transporter, FgMirA, had been identified as a potential innate immunity reporter gene strongly and quickly upregulated as an effector removing iron from the cytosol and transporting it into the vacuole for storage. Its activity can create a strong sink for the removal of iron from the environment when the fungus is challenged by NSAMPs from surrounding bacteria ( Ipcho et al. 2016 ). A fast restriction of bacterial access to iron is a classic function of innate immunity since iron is necessary for many enzymes for detoxification of ROS, as well as to withstand the ROS produced by innate immunity responses to kill invaders ( Ganz 2009 ). We first checked that the vacuolar FgMiraA protein (FGSG_00539) is rapidly upregulated in response to bacterial NSMPs. For that, we constructed a F. graminearum reporter strain expressing the FgMirA-GFP protein in the same way as we constructed the FgMst3-GFP complementation strain and tested the FgMirA-GFP localization and regulatory responses when the fungus became exposed to Esherichia coli outer membrane vesicles (OMVs) obtained from starving bacteria shedding OMVs, imitating the conditions in the soil rhizosphere where F. graminearum comes in contact with wheat roots and can cause seedling blight ( Yang et al. 2011 ). The OMVs are most likely internalized by fungal endocytosis for fungal NLRs to become exposed. It was clear from these experiments with the reporter strain that the FgMirA-GFP signal responds almost immediately (within 1h of exposure) to the E. coli NSAMPs ( Fig. S8 ). In addition tested the reporter system for a small amount of sucrose (0.1 mg/L) that can potentially serve as a plant NSAMP, more than a nutrient, telling the fungus that it has entered the plant inner rhizosphere or the plant apoplast ( Ipcho et al. 2016 ) ( Fig. S9 ). To be able to get responses from more fungal biomass with a simpler method, we tested the transcriptional response of the FgMirA gene to the same stimuli so that we can instead use fast transcriptional responses to NSAMPs relative to water control. As shown in Fig. S10 , the reporter gene shows clear responses to both NSAMPs although the response to the cocktail of bacterial NSAMPs on/in the OMVs were stronger than to the single plant NSAMP. We could then test if FgMst3 could be active in fungal innate immunity by testing the FgMirA reporter gene transcriptional responses to E. coli NSAMPs as OMVs of the PH-1 strain and compare that with the transcriptional responses to the same treatment of the two ΔFgMST3 strains ( Fig. 11 ). Already after 1 h, the transcription of FgMirA was on average 8 times higher in the PH-1 strain than in the ΔFgMST3 strains, and that difference rose to 60 times the expression in the ΔFgMST3 strains when exposed for 2 h to bacterial NSAMPs as OMVs. Download figure Open in new tab Figure 11. Fast reporter gene expression increases relative water controls for different F. graminearum strains after bacterial NSMPs addition. (A) Gene expression after NSMPs treatment for 1h relative to equal water treatment is about 8 times stronger and increases to 60 times stronger after 2 h (B). Error bars indicate SEMs, and *, **, ***, and **** symbolize P same <0.05, <0.01, < 0.001, and <0.0001, respectively, for the null hypothesis that these measurements are the same as for the PH-1 In conclusion, the deletion of the F. graminearum MST3 gene substantially decreases or even nullifies fungal innate immunity responses. Discussion The phylogeny of the different MST3-like protein sequences clusters mammalian sequences together with Dictyostelium and archaea, which could indicate that MST3s are old and from the origin of eukaryotic organisms ( Fig. 1 ). The yeast ste20 kinases seem to be a group that has evolved, maybe for different cellular roles. Yeasts are unicellular and might have lost the possibility of innate immunity, including inflammasome formation and triggering apoptosis to save the mycelium if pathogen invasion is severe in one hyphal compartment. Apoptosis is present but works differently in baker’s yeast and is executed by a metacaspase ( Mazzoni and Falcone 2008 ), and the classic highly conserved protein PARP needed to repair damaged DNA and is rapidly cleaved and inhibited by the apoptosis-activated caspase activities ( Chaitanya et al. 2010 ), is not present in yeasts as Saccharomyces cerevisiae ( Semighini et al. 2005 ). A PARP was, however, recently found in the environmentally highly genetically competent yeast Yarrowia lipolytica, which has a less-reduced number of genes in its genome compared to other ascomycetes, than is the case for S. cerevisiae ( Mamaev and Zvyagilskaya 2021 ). In filamentous fungi, the PARP gene seems more generally present, as well as classic caspase-like protein cleavages by metacaspase ( Thrane, Kaufmann, Stumman, et al. 2004 ). Although baker’s yeast is an excellent model for basic metabolism, it seems to fall short of the multicellularity/nuclear strategizing seen in filamentous fungi that is important for conidia formation by emptying vegetative mycelium for production of conidia to spread to a more nutritious environment ( Thrane, Kaufmann, Stumman, et al. 2004 ; Semighini et al. 2005 ). Filamentous fungi are also known to have NLRs of types that should be able to sense bacterial and plant NSMPs. Some of these are known as Het proteins involved in heterogenous incompatibility proteins triggering local cell death in the model fungus Podospora anserina ( Leipe et al. 2004 ; Paoletti et al. 2007 ; Paoletti and Saupe 2009 ; Daskalov et al. 2012 ). Similarly and different from yeast fungal thalli (the multinucleate mycelium) can “migrate” from a hostile volume in the soil or search for a more “rewarding” volumes in soil by recycling nutrients from hostile soil volumes to support the migration through “barren” soil volumes ( Dowson et al. 1988 ; Rayner et al. 1995 ; Glass 2004 ; Fukasawa et al. 2020 ). In light of these capacities of filamentous fungi with a general high competence to thrive and grow in competition with bacteria and interacting with hyphae-invading bacteria, both pathogenic and beneficial to the fungus ( Ali et al. 2022 ), filamentous fungi should need signaling proteins like the well conserved MST3 kinase to relay the signals from NLR-receptors to innate immunity effectors like siderophore transporters to remove iron from their surroundings ( Ipcho et al. 2016 ). The lack of response to bacterial NSMPs for the ΔFgMST3 strains compared to the PH-1 background strain is in line with this and indicates that there must exist so far non-identified NLR proteins being exposed to endocytosed bacterial NSMP, recognizing them and triggering downstream responses, as the quick upregulation of the FgMirA ( Fig. 11 ) shows. Candidates for such NLRs can most probably be found among the approximately 100 identified F. graminearum STAND proteins ( Daskalov et al. 2012 ) that are implicated as possible bacterial NSMP receptors ( Paoletti et al. 2007 ; Paoletti and Saupe 2009 ). Bsr1/Irak4 kinases are short, while the MST3kinases are much longer, and their C-terminal part behind the N-terminal kinase domain contains an NLS sequence and a NES sequence, and between them a cleavage site for caspase that, when cleaved, further activates the kinase ( Lee et al. 2004 ). It is thus expected that the two fungal MST3-like kinases should have a domain structure like the human MST3-kinase if they have a similar and conserved function in innate immunity ( Fig. 2 , S1 , and Supplementary file SF1 ). On the other hand, the similarity should not necessarily be similar to S. cerevisiae since it has a very reduced genome and typically lacks genes for proteins important for multicellular organisms and for existing in multiple environmental settings ( Mamaev and Zvyagilskaya 2021 ). Thus, MoMST3 and FgMST3 could have a function as the mammalian MST3 gene in relaying signals from NLRs downstream to innate immunity, as the deletion of the FgMST3 affected the innate immunity effector reporter gene so that it does not respond in the mutants lacking FgMST3 ( Fig.11 ). MST3 gene deletions in both plant pathogenic fungi had negative effects on pathogenicity, most probably since the deletions affected conidia production with fewer conidia, changed the dynamics of conidia germination, and reduced colonization of the plant host tissues ( Fig. 7 - 8 ). Interestingly, both the MST3 mutants germinated faster than their respective background strains ( Fig. 5 ). We had deliberately selected the two fungal pathogens with different natural bacterial environments. F. graminearum has wet conidia and meets bacteria in the soil and rhizosphere/rhizoplane of plants, while M. oryzae mainly has dry melanized conidia and meets bacteria in the sunlit phylloplane of grass leaves. Because of these differences, the effect of MST3 deletions on genes downstream becomes detectable as differences in stress responses ( Fig. 6 , 9 , 10 ). Since F. graminearum lives in soil when not infecting a plant, it should be more dependent on its capacity to withstand biotic stresses in the soil and rhizosphere. M. oryzae should, on the other hand, be more dependent on a properly formed melanized cell wall and ROS defences since the environment on the leaves is very oxidative and prone to in ROS levels, also caused by intense sunlight. Inside the leaves, conditions are better controlled as long as there are nutrients available to run ROS defences. The largest difference we found in the change of sensitivity for the ΔMST3 strains in comparison with the background strains was in Zn 2+ resistance, where the ΔFgMST3 strains were more tolerant to Zn 2+ , although zinc can work as a fungicide towards F. graminearum ( Savi et al. 2015 ), and also very tolerant to the antibiotic gentamicin. ΔMoMST3 strains were less tolerant to both treatments compared to their respective background strains ( Fig. 9 - 10 ). The gentamicin treatments even stimulated the growth of F. graminearum, and the ΔFgMST3 strains were even more stimulated than the PH-1 strain, although we have at present no plausible explanation for that stimulation. Conclusion The detected reactions to Gram-negative bacterial NSMPs common in plant rhizospheres and the disappearance of these reactions when the MST3 gene is deleted indicate that FgMST must be relaying signals from a hitherto unknown fungal NLR-receptor of types postulated to be active, recognizing invading or interacting organisms, triggering fungal innate immunity that is downstream orchestrated by Mst3 proteins. The two identified MST3 genes have similar roles in both fungi and play a role in the normal functioning of plant infection processes of both these pathogens. The negative effect on pathogenicity of deleting the MST3 genes in both fungi might be caused by a lack of recognition of unknown plant NSMPs, which properly prepare the fungus for a successive plant invasion. Future work should try to identify NLR receptors as well as plant NSMPs to better understand how fungal non-self-recognition works and if it can be inhibited or manipulated, as well as teach us something about human innate immunity, since the fungal version seems a bit similar ( Ipcho et al. 2016 ). Materials and Methods Websites and programs that were used for the initial bioinformatics work We used NCBI for the download of protein sequences and for BLAST similarity searches, as and conserved domain descriptions. Multiple alignment of homologs was performed and visualized using the NCBI COBALT multiple alignment tool at the NCBI website. For phylogeny inferences of the found protein sequences, we used NGPhylogeny.fr ( Lemoine et al. 2019 ) ( https://ngphylogeny.fr/ ) using the ‘One click’ mode for tree building. ScreenCap 3 was used to predict putative caspase 3 cleavage sites ( https://scap.cbrc.pj.aist.go.jp/ScreenCap3/index.php ). Finally, the protein conserved domain structures were visualized using the free tool for Protein domain structure visualization: DOG 2.0 ( Yao and Xue 2009 ), available for download at http://dog.biocuckoo.org/ . The detailed step-by-step procedure of this bioinformatics work is available ( Supplemental File SF1) . Fungal strains, plant varieties, and plasmids The fungi we used are both well-studied model plant pathogenic Ascomycetes, Fusarium graminearum (teleomorph Gibberella zea ) wildtype strain PH-1, and Magnaporthe oryzae (formerly Magnaporthe grisea ) (Teleomorph Pyricularia oryzae wildtype strain of Guy11 with the gene MgKU80 required for non-homologous end joining inactivated, strain Ku80, to aid gene targeting ( Villalba et al. 2008 ). Information about genes and proteins for both strains can be found at NCBI ( F. graminearum NCBI:txid229533, M. oryzae NCBI:txid242507). Plants used were wheat ( Triticum aestivum ) variety Jimai 22, rice ( Oryzae sativa ) variety Co39, and barley ( Hordeum vulgare subsp. vulgare ) variety Xiyin No. 2. The plasmid used for knockout was PCMB containing Ampicillin resistance and HPH (Hygromycin resistance) for selection of putative successful knockouts. For complementations, we used plasmid PKNT-GFP containing Ampicillin resistance and G418 resistance for the selection of putative successful complementations. Gene knockout mutations and complementation of knockout strains with MST3-GFP constructs Targeted gene deletion and complementation were performed as described ( Chen et al., 2023 ). For respective ΔFgMST3 and ΔMoMST3 generation, ~800 bp 5’ and 3’ flanking regions were amplified and fused to a hygromycin resistance cassette (HYG), followed by polyethylene glycol (PEG)-mediated protoplast transformation. Primary transformants were selected on hygromycin (200 μg/mL) and validated by Southern blot. Complementation constructs (1.5 kb native promoter + GFP + FgMST3 or MoMST3) were cloned into pKNT and reintroduced into ΔFgMST3 or ΔMoMST3 . GFP fluorescence was confirmed via confocal microscopy (Nikon A1, Japan). Successful strain constructions were verified through PCR amplifying parts of flanking regions + knockout inserts and getting predicted band sizes, in combination with our own Gong/Olsson qPCR technique (GO-qPCR for short) to determine the HYG/Tubulin gene ratio in the mutants to detect possible ectopic integrations of the HYG gene used as a selection marker, using standard primers we have in the lab for these genes, amplifying only inside their exons. GO-qPCR is a faster and cheaper alternative to Southern blotting, developed for this study, that also quantifies the number of ectopic integrations in the genome. Using this method makes it less advantageous to use the KU80 strain to avoid multiple ectopic integrations through NHEJ, since screening for multiple integrations could simply be done as a first measure after transformation. This is feasible since only small amounts of gDNA are needed for qPCR, and could be done when colonies have first been obtained on the selective medium, using small samples taken directly from parts of colonies appearing on the hygromycin plates. Thus, subculturing/isolation needs only to be done from colonies showing one HYG gene integration in their genomes. G rowth of strains and stress treatments F. graminearum and M. oryzae mycelial disks were used as inoculum for CM, CMII, SYM, and MM agar plates that were incubated at 26 °C for 3 days (F. graminearum) or 7 days before the plates were photographed, and the colony diameters were measured, and relative inhibition rates were calculated ( Biregeya et al. 2022 ). For in vitro stress sensitivity assays of fungal strains were inoculated on CM solid media with one of the following additions 0.02% w/v calcofluor white (CFW), 1M NaCl, 1M KCl, 1mg/mL Congo Red (CR), 0.01% (w/v) sodium dodecyl sulfate (SDS), 0.03% w/v H2O2, or 1 M Sorbitol. Mycelial disks were used as an inoculum for the stress treatment plates and CM control plates, and they were incubated, photographed, and measured as before for the two pathogens, respectively. Conidia production and germination measurements Strains of F. graminearum grown on CM agar media were used as inoculum to shake cultures (180rpm) in CMC liquid medium and incubated at 25 °C days, and conidia were counted after 3, 6, and 9 days of incubation using a hemocytometer. Disks taken from CMII cultures of M. graminearum strains were used to inoculate rice bran plates and incubated inverted for 7 days before the aerial mycelium was scraped off into a clean disk and incubated at 26 °C under near ultraviolet illumination to promote conidia formation for 2 days. After incubation, the conidia were washed off and counted using a hemocytometer. For germination experiments, 10 μL conidia suspension 2*10 4 /mL pipetted to a hydrophobic microscope slide and incubated in high high-humidity chamber in the dark at 26 °C, and conidia germination, and appressorium formation ( M. oryzae ) ( Lee and Dean 1994 ), were investigated by microscopy after 2, 4, 6, and 8 hours. Pathogenicity of F. graminearum strains Wheat coleoptiles grown from alcohol sterilized seeds (cv. Jimai 22), on sterilized filter paper wetted with sterilized water, were used for testing. A F. graminearum conidia suspension containing 2 × 10 4 conidia/mL from a liquid CMC medium for 3-4 days was used as inoculum. The tip of the coleoptiles was cut and inoculated with 10 μL of conidia suspension, and the coleoptiles were placed in a tray covered with a plastic wrap. These trays with inoculated coleoptiles were then incubated at 26 °C with an 8/16 dark/light period for 3 days before lesion sizes were recorded. Wheat heads on field-grown winter wheat plants grown from seeds that were sown in mid-December were inoculated at the flowering stage in April-May the following year. Even-sized small disks cut from colonies of the F. graminearum strains were used for inoculation of the wheat heads. One disk was placed on the 4 th or 5 th floret from the bottom of each wheat head. Each head was covered with a plastic bag for 3 days, then after about 2 weeks, the wheat heads were cut off, photographed, and the levels of wheat head infection were estimated. Pathogenicity tests of M. oryzae strains Barley leaves (cv. Xiyin No. 2) with similar growth characteristics were selected from plants, 6-7 days old, and placed in a moist chamber made of a 15 cm Petri dish. A spore solution containing 2 × 10 4 conidia/mL was added to the barley leaves in spots of 15 μL/spot. The moist chamber containing the inoculated leaves was then incubated at 26 °C in the dark for 24 h, followed by 12/12 dark/light periods for 3-5 days before lesion sizes were recorded. Rice leaves from 4-week-old rice seedlings (cv. CO39) were spray-inoculated with 3 × 10 4 conidia/mL. Inoculated plants were maintained at 26°C (>90% humidity) for 5-7 days. Disease severity was scored on a 5-grade scale based on lesion size: grade 1 (0.4 mm) ( Chen et al. 2023 ). Rice plants (cv. CO39) were grown until the 3-leaf-1-heart stage, and leaves to be tested were excised for press-injured inoculation ( Takahashi et al. 1999 ). In short, 2 mm diameter press-injured spots were made using a pressing machine (Fujihara Co.), and 10 μL of a conidia suspension containing 5 × 10 5 per mL was added to each injured spot, and the spots were sealed with transparent tape. The leaves were then incubated at 26 °C with a 12/12 dark/light period for 10 days before lesion sizes were recorded. Confocal microscopy observations of localization in the strains complement strains expressing the FgMst3-GFP or MoMst3-GFP proteins Subcellular localization was analyzed using a Nikon A1 confocal microscope. EGFP-tagged strains were cultured on CM II, fixed in 4% paraformaldehyde, and imaged using blue light excitation (EGFP) (Argon laser 488. Excitation filter:470/40, Dichromatic mirror: 495LP, Barrier filter: 515/30 (the light imaged). Obtaining. E. coli OMVs containing bacterial NSMPs OMVs from overnight cultures of E.coli (strain DH5α) grown in 5ml Luria Broth LB at 37 °C were centrifuge-harvested at 10000 rpm, washed twice in sterile DD water, and finally resuspended in 1ml DD water. Then the cells were incubated for 1hour at 37 °C to release OMVs, which were finally harvested from the supernatant after centrifugation at 5000rpm to remove most cells. The supernatant with OMVs was sterile filtered through a 0.3 μm filter. This OMV suspension can be stored in the fridge at 4 °C until use. For treatments in the microscopy, the OMV suspension was diluted approximately 10 times, and the washed OMV suspension was added to washed and starved hyphae and incubated on the microscope slide for 1 or 2 test hours before the cover slip was put on and the slide was immediately investigated in the confocal microscope. For RT-qPCR, the OMV concentration used to expose the fungus was diluted 10 times before exposure, and the fungal mycelium was incubated for 1 or 2 h before immediate harvest of mRNA for RT-qPCR assessments. The procedure was as follows: The fungal hyphae to be treated with OMVs were produced in well-aerated liquid DFM medium cultures incubated for 3-4 days at 26 °C. After that incubation, the remaining medium and metabolites in the medium were removed by filtration, and the hyphae on the filter were transferred by shaking the filter in N-deficient DFM overnight. The mycelium was filter-washed again with DD water and finally transferred to a 50ml centrifuge tube containing 1mLsterile DD water, and 1 mL OMV suspension was added, exposing the fungus to OMVs containing bacterial NSMPs. The exposure of the starved fungal hyphae to OMVs from starved bacteria in DD-water mimics the starving conditions in the soil rhizosphere, where the fungus meets bacteria, takes up the few nutrients that are available, and interacts with the biotic and abiotic environment also through endocytosis. Author contributions SG First author, made MST3 mutants and experiments, including OMV extraction, and manuscript writing. XL, co-first author, made the reporter strain, tested the technique to extract OMVs, and tested whether RT-qPCR works with the reporter gene. SL refined the RT-qPCR technique for measuring reporter gene responses to OMVs so that it works reliably. SO Co First author and co-corresponding author, research idea, identification of reporter gene, OMV purification technique development, bioinformatics, manuscript writing, Supervision GL Co-supervision ZW Co-supervision YL, Corresponding author, provided funds and Co-Supervision. Supplementary figures Download figure Open in new tab Figure S1 Alignment of the MST3 like proteins in Figure 1 . From the alignment the kinase part of the protein aligns well for all proteins but absent from the IRAK4 and BSR1 proteins involved in relaying signals from TLR receptors. Download figure Open in new tab Figure S2. Deletion strategy for FgMST3 and MoMST3 Download figure Open in new tab Fig S3. Confirmation that the constructed strains have the right new genes in genomes and that there is no ectopic insertions of the gene used as selection marker. Download figure Open in new tab Figure S4. Confirmation that the constructed strains have the right new genes in their genomes and that there are no ectopic insertions of the gene used as a selection marker. Download figure Open in new tab Figure S5. RT-qPCR confirmation that there is no residual expression of the deleted genes in any of the mutants. Error bars indicate SEMs and *, **, ***, and **** symbolize P same <0.05, <0.01, <0.001 and <0.0001, respectively, for the null hypothesis that these measurements are the same as for the PH-1 or KU80, respectively. Download figure Open in new tab Figure S6. Subcellular localization. FgMst3-GFP localizes to the cytoplasm in hyphae (A) and conidia (B). Size bars = 5 μm (spore images), and 10 μm (hyphal images) Download figure Open in new tab Figure S7 Subcellular localization. MoMst3-GFP localizes to the cytoplasm in hyphae (A) and conidia (B). The central compartment of the conidium is heavily melanized and blocks the blue light needed to excite GFP but the signal appear clearly cytoplasmic as seen in the more enhanced image. Size bars = 5 μm (spore images), and 20 μm (hyphal images) Download figure Open in new tab Figure S8. Response of the reporter strain FgMirA-GFP by when treated with NSMPs from E. coli. The reporter strain was washed and starved in sterile water before treatment. Treatments: H 2 O washed sterile E.coli OMV suspension treatments for 1h, 5 replicate treatments E1-5. H 2 O-washed sterile E.coli OMV suspension treatments for 3h, 2 replicate treatments E31-2, seen in low magnification L. H 2 O control: H 2 O for 1 to 6 hours C1-C6. C2, C5 and C6 seen in in low magnification L. NOTE: The lookup table Fire was used to better visualize the differences of the responses to human eyes. Download figure Open in new tab Figure S9. Response of the reporter strain FgMirA-GFP by when treated with a plant NSMP. The reporter strain was washed and starved in sterile water before treatment. Treatments: Sterile sucrose solution (0.1 mg/L) treatments for 1h, 5 replicate treatments S1-5. Sterile sucrose solution (0.1 mg/L) treatments for 3h. Control: H 2 O for 1 to 6 hours C1-C6. C2, C5 and C6 seen in in low magnification L. NOTE: The lookup table Fire was used to better visualize the differences of the responses to human eyes. As a reference: The apoplast of plants contain 0.1-5 mM =0.1*360/1000 =0.036-0.18% W/V = 0.36-1.8g/L=360-1800mg/L. Thus 0.1mg/L is probably too low concentration of sucrose to for the fungus to grow on but could work as a plant NSMP signal to fast detect the presence of a plant when the fungus is on the rhizoplane since sucrose is virtually absent from soil due to its fast consumption by soil bacteria. Download figure Open in new tab Figure S10 RT-qPCR transcription test if the reporter gene FgmirA (FGSG_00539) upregulates in response to plant and bacterial NSMPs treatments for 1h. The responses, as Log2 gene transcription responses relative/water control, were used as the relative measurements. The responses were recorded after 1h of treatment. H2O = Autoclaved DD water, sterile filtered as a control treatment. Suc = Hyphae grown in liquid medium, washed, and then starved in water were exposed to 0.1 mg/L sucrose. Ecoli = E. coli OMVs, or just water as in Fig. S9 . The numbers above the bars are the probability for the null hypothesis (P same ) that the treatments are not different from the control. Acknowledgements Dr. Mengtian Pei is thanked for her help guiding Shanshan Gong in her laboratory work. 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Share Fungal Mst3-proteins are involved in fungal innate immunity needed for the recognition of bacteria surrounding the hyphae, as well as for plant pathogenicity Shanshan Gong , Xiaorong Lin , Shenghua Liu , Stefan Olsson , Guodong Lu , Zonghua Wang , Ya Li bioRxiv 2025.11.25.690610; doi: https://doi.org/10.1101/2025.11.25.690610 Share This Article: Copy Citation Tools Fungal Mst3-proteins are involved in fungal innate immunity needed for the recognition of bacteria surrounding the hyphae, as well as for plant pathogenicity Shanshan Gong , Xiaorong Lin , Shenghua Liu , Stefan Olsson , Guodong Lu , Zonghua Wang , Ya Li bioRxiv 2025.11.25.690610; doi: https://doi.org/10.1101/2025.11.25.690610 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 Immunology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17697) Bioengineering (13894) Bioinformatics (41951) Biophysics (21455) Cancer Biology (18593) Cell Biology (25509) Clinical Trials (138) Developmental Biology (13380) Ecology (19903) Epidemiology (2067) Evolutionary Biology (24322) Genetics (15611) Genomics (22509) Immunology (17737) Microbiology (40398) Molecular Biology (17183) Neuroscience (88619) Paleontology (667) Pathology (2833) Pharmacology and Toxicology (4825) Physiology (7644) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4296) Systems Biology (9825) Zoology (2271)