Tissue-Specific Experimental Evolution Reveals Adaptive Trade-Offs in the Plant Vascular Pathogen Clavibacter michiganensis

Tissue-Specific Experimental Evolution Reveals Adaptive Trade-Offs in the Plant Vascular Pathogen Clavibacter michiganensis | 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 Tissue-Specific Experimental Evolution Reveals Adaptive Trade-Offs in the Plant Vascular Pathogen Clavibacter michiganensis Raj Kumar Verma , View ORCID Profile Veronica Roman-Reyna , Nathan Benmoche , Evanson Ngugi , Eduard Belausov , View ORCID Profile Maya Bar , View ORCID Profile Jonathan M. Jacobs , View ORCID Profile Doron Teper doi: https://doi.org/10.1101/2025.10.05.680514 Raj Kumar Verma 1 Dept. of Plant Pathology and Weed Research, Agricultural Research Organization - Volcani Institute , Rishon LeZion, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Veronica Roman-Reyna 2 Dept. Of Plant Pathology and Environmental Microbiology, Pennsylvania State University, University Park , PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Veronica Roman-Reyna Nathan Benmoche 1 Dept. of Plant Pathology and Weed Research, Agricultural Research Organization - Volcani Institute , Rishon LeZion, Israel 3 Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem , Rehovot, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Evanson Ngugi 1 Dept. of Plant Pathology and Weed Research, Agricultural Research Organization - Volcani Institute , Rishon LeZion, Israel 4 Dept. of Life Sciences, Ben-Gurion University of the Negev , Beer-Sheva, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Eduard Belausov 5 Dept. of Ornamental Plants and Agricultural Biotechnology, Agricultural Research Organization - Volcani Institute , Rishon LeZion, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Maya Bar 4 Dept. of Life Sciences, Ben-Gurion University of the Negev , Beer-Sheva, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maya Bar Jonathan M. Jacobs 6 Dept. of Plant Pathology, The Ohio State University , Columbus, OH, USA 7 Infectious Diseases Institute, The Ohio State University , Columbus, OH, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jonathan M. Jacobs Doron Teper 1 Dept. of Plant Pathology and Weed Research, Agricultural Research Organization - Volcani Institute , Rishon LeZion, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Doron Teper For correspondence: doront{at}volcani.agri.gov.il Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The plant pathogenic bacterium Clavibacter michiganensis (Cm) is a systemic vascular pathogen that colonizes both xylem vessels and the intracellular apoplast during different stages of infection. To identify traits and loci associated with adaptation to these distinct host microenvironments, we conducted tissue-specific experimental evolution. Twenty independent Cm lineages were repeatedly passaged in either tomato stems or leaves to promote adaptation to vascular or apoplastic lifestyles, respectively. After fifteen passages, adapted clones were characterized for virulence and virulence-related traits. These characterizations demonstrated clear differential associations of virulence-associated traits with the adapted tissue. The majority of vascular-adapted clones displayed enhanced surface attachment, reduced cellulase activity, reduced exopolysaccharide (EPS) production, and attenuated virulence on tomato compared to the parent clone. On the other hand, apoplast-adapted clones displayed reduced biofilm formation and enhanced EPS production while maintaining their virulence on tomato. Whole-genome sequencing of all adapted clones revealed candidate loci linked to tissue adaptation. Notably, six of ten vascular-adapted clones carried two independent mutations in CMM_1284 , a putative HipB/XRE-type transcriptional regulator. A CMM_1284 marker exchange mutant displayed phenotypes similar to vascular-adapted clones, suggesting a role for this regulator in vascular colonization. Together, these findings highlight the role of phenotypic plasticity in tissue adaptation of plant pathogens, showing that tissue-specific adaptation involves modulation of surface attachment, EPS production, and cell wall–degrading enzymes. They further reveal a regulated trade-off between vascular persistence, supported by strong surface attachment, and systemic virulence, which depends on bacterial dispersal and migration. Introduction Plant pathogenic microorganisms specialize in colonizing host tissues according to lifestyle: systemic pathogens spread through the vascular system, while non-systemic ones remain confined to apoplastic regions. These contrasting lifestyles shape disease etiology, epidemiology, and management. Non-systemic bacteria colonize the apoplast, inducing galls or necrotic lesions [ 1 – 3 ], spread locally by water splash, wounding, or insects, and rarely invade the vasculature [ 4 ]. Vascular pathogens specialize in xylem or phloem colonization. Phloem-associated pathogens are insect-transmitted, phloem-restricted, passively transported, and have reduced, low-GC genomes [ 5 ]. Xylem-associated pathogens show diverse transmission and tissue specificity: Xylella spp. are strictly xylem-limited, whereas vascular Xanthomonads, Ralstonia spp., Erwinia amylovora , and Clavibacter spp. also colonize apoplast tissues [ 6 , 7 ]. In E. amylovora , transitions between xylem and parenchyma are central: it enters the xylem from floral parenchyma, spreads systemically, and exits into bark parenchyma, causing tissue degradation and proliferation [ 8 ]. Mechanisms of tissue specificity and lifestyle transitions remain unclear; even related strains differ in tropism. In Xanthomonas , the glycosyl hydrolase gene cbsA distinguishes vascular from non-vascular lifestyles [ 9 ]. Xylem-associated bacteria produce biofilm-like matrices attaching to vessel walls, essential for systemic spread and implicated in drought-like symptoms by blocking water flow [ 10 ]. Mutational analyses show that balancing biofilm formation and dispersal is critical for systemic colonization [ 11 , 12 ]. Bacterial canker, caused by the Actinomycetota Clavibacter michiganensis (Cm), is one of the most destructive tomato diseases [ 13 ]. Symptoms include canker lesions on stems and branches, leaf wilting, necrosis, and ‘bird’s-eye’ spots on fruits [ 13 ]. Cm spreads by colonizing xylem vessels, causing vascular collapse through blockage of water-conducting elements [ 14 ]. The pathogen relies heavily on secreted hydrolases such as cellulases, xylanases, pectate lyases, and serine proteases, encoded mainly by the chp/tomA pathogenicity island (PAI) and plasmids pCM1 and pCM2 [ 15 , 16 ]. Variation in these regions strongly correlates with pathogenicity [ 17 – 19 ], and disruption of specific hydrolases such as celA , chpC , and pat-1 reduces virulence [ 20 – 23 ]. Microscopy has shown Cm forming biofilm-like aggregates attached to xylem walls [ 24 ], suggesting biofilms aid systemic colonization, though their composition, structure, and regulation are poorly understood. Only a few studies indirectly link biofilm formation to regulatory networks such as the stringent response and cell wall integrity [ 25 , 26 ]. Despite evidence for surface attachment in vitro and in planta , adhesion mechanisms remain undefined and no adhesion-associated macromolecules have been identified. Cm exopolysaccharides (EPS) were hypothesized to contribute to virulence but this hypothesize have yet to be tested by functional or genetic analyses [ 27 ]. The model strain NCPPB382 harbors at least four predicted EPS gene clusters [ 16 , 28 ], but their role in EPS production, xylem association, and virulence is unvalidated. Cm also colonizes the leaf apoplast, producing blister-like structures [ 15 , 29 ], though the contribution of this lifestyle to disease progression remains unclear. In this study, we employed a tissue-based experimental evolution approach to identify traits and genetic loci selected for adaptation to either the vascular or apoplastic lifestyle of Cm. Results Cm migrates from the xylem to apoplast tissue during infection Cm is traditionally classified as a xylem-inhabiting bacterium [ 7 ]. However, in contrast to xylem-restricted bacteria such as Xylella fastidiosa , Cm has also been reported to colonize intracellular apoplastic spaces within host tissues [ 7 , 24 , 30 ]. To assess Cm’s tissue localization, GFP-labeled Cm [ 24 ] was monitored for colonization throughout the plant following infection. First, we examined Cm colonization of the apoplast by syringe-infiltrating bacteria into tomato leaves ( Fig. 1A–C ). Cm caused localized water-soaked lesions that became necrotic within 5–10 days ( Fig. 1A ), accompanied by a ∼10,000-fold population increase by 3 days post-infiltration (dpi) ( Fig. 1B ). Confocal microscopy showed Cm cells in intracellular apoplastic spaces of leaf parenchyma ( Fig. 1C ), confirming its ability to thrive during apoplastic infection. Like many apoplastic pathogens, Cm-induced lesions stayed confined to infiltrated areas, and inoculated plants showed no wilt or canker symptoms typical of systemic infection. Thus, while Cm can act systemically, direct apoplastic introduction rarely leads to systemic spread. Download figure Open in new tab Fig. 1. In situ localization of Cm bacteria during infection. ( A – C ). Cultures (10 4 CFU/ml) of GFP-labeled Cm were infiltrated into six-leaf-stage tomato leaves using a needless syringe. ( A ) Representative leaf photographed 10 days post-infiltration (dpi). ( B ) Bacterial population at the infiltration site on days 0, 3, and 6 dpi. Data represent 26 biological repeats pooled from three independent experiments. ( C ) Infiltrated leaf tissues were visualized by confocal laser scanning microscopy using a GFP filter at 7 dpi. ( D , E ) Stem areas between the cotyledons of four-leaf-stage tomato plants were wound-inoculated using toothpicks soaked in 10 7 CFU/ml GFP-labeled Cm bacterial cultures. Bacteria were visualized by confocal laser scanning microscopy with a GFP filter. ( D ) In situ localization of Cm at 3 cm above the infection site, visualized in horizontal and vertical stem cross sections of Cm-inoculated plants at 7 dpi (prior to symptom development) and 14 dpi (when symptoms are apparent). ( E ) In situ localization of Cm in asymptomatic, wilted, and necrotic leaves at 14 dpi. The right and left panels show the two most common localization patterns at leaves during the described symptomatic stages. All depicted data represent at least 10 biological repeats conducted in at least two (C) or three (A, B, D, E) independent experiments. Next, we examined Cm localization during systemic infection by wound-inoculating stems with GFP-labeled Cm between the cotyledons. In situ monitoring was done 3 cm above the infection site at 7 dpi, before severe symptoms, and at 14 dpi, when ∼75% of leaflets were wilted and stem cankers were apparent. At 7 dpi, Cm was mainly in xylem vessels with little or no presence in pith parenchyma, consistent with its xylem-restricted lifestyle ( Fig. 1D , upper panels). By 14 dpi, bacteria had burst from the xylem into pith parenchyma intracellular spaces ( Fig. 1D , lower panels). In distal leaf tissues at 14 dpi, asymptomatic leaflets showed no bacteria or restriction to the main vein, wilted leaflets contained bacteria in vascular bundles or nearby tissues, and necrotic leaflets were heavily colonized in both vascular bundles and apoplastic parenchyma ( Fig. 1E ). Together, these findings indicate that Cm exits the vascular system into apoplastic parenchyma during late infection, and that xylem escape is key to its infection cycle. Experimental evolution of Cm under distinct vascular and apoplastic niches The xylem and apoplast microenvironments within a plant host differ greatly and pose unique challenges to invading pathogens [ 6 ]. Since Cm can thrive and transition between these habitats, it likely possesses traits supporting survival in each, along with phenotypic plasticity enabling the shift. We hypothesized that traits or loci associated with each environment could be identified by experimentally evolving Cm under repeated exposure to vascular or apoplastic conditions. To test this, we generated 20 parallel tissue-adapted Cm populations by repeated passaging in vascular or apoplastic niches. Ten clones were evolved in vascular tissue (S1–S10) and ten in the leaf apoplast (L1–L10). Experiments used Cm NCPPB382, hereafter referred to as Cm WT or parent clone. Vascular-adapted clones were passaged through distal stems: tomato stems were inoculated via toothpick puncture, and bacteria were re-isolated 14–21 days later from tissues 6 cm above the inoculation site. Isolates were pooled and used to inoculate new hosts for the next passage ( Fig. 2 ). Apoplast-adapted clones were passaged by infiltrating diluted Cm suspensions (10□ CFU/ml) into leaf apoplast with a needleless syringe. Bacteria were re-isolated 7–10 days later from the infiltration area, pooled, and used to inoculate new hosts ( Fig. 2 ). Parallel passaging continued for 15 cycles, after which one representative clone from each lineage was selected for phenotypic and genomic analyses. Download figure Open in new tab Fig. 2. Cartoon illustrating the experimental evolution procedure used in the study. Ten parallel Cm lines underwent repeated inoculation and re-isolation passages lasting 14–21 days, performed via stem (upper panel, vascular adaptation) and leaf (lower panel, apoplastic adaptation) inoculations. The cartoon was created with the assistance of cartoon photo editor ( https://play.google.com/store/apps/details?id=com.gamebrain.cartoon&hl=en ) and NIAID Visual & Medical Arts (bioart.niaid.nih.gov/bioart/386, bioart.niaid.nih.gov/bioart/506). Vascular-adapted clones exhibit reduced vascular and apoplastic virulence We investigated whether adaptation to specific plant tissues affected virulence and colonization in vascular and apoplastic tissues. After 15 passages, adapted clones and the parent Cm WT were inoculated into tomato stems or leaves, and plants were monitored for symptoms and bacterial growth. For vascular virulence, stems were toothpick-inoculated and wilt symptoms tracked for 21 days. Bacterial titers were also measured 1 and 10 cm above the inoculation site. While apoplast-adapted clones (L1–L10) caused wilt comparable to WT, eight vascular-adapted clones (S1, S2, S3, S4, S5, S6, S8, S9) showed reduced wilt ( Fig. 3A and 3B ), despite unchanged bacterial titers at 21 dpi ( Fig. 3C ). To test kinetic systemic spread, bacterial loads at 5 and 10 cm were measured at 7 and 14 dpi in three virulence attenuated vascular-adapted clones (S2, S4, S9). By 14 dpi, S2 and S4 colonization matched WT, while S4 showed a fivefold reduction 10 cm above the inoculation site ( Fig. 3D ), suggesting delayed systemic spread could explain its attenuated phenotype. For apoplast virulence, bacteria were syringe-infiltrated into leaves and symptoms scored for 10 days. Apoplast-adapted clones resembled WT (Fig. S1A, S1B). Four vascular-adapted clones (S2, S3, S4, S5) showed weaker or delayed symptoms, while S7 caused stronger, earlier lesions (Fig. S1A, S1B). However, all clones eventually reached similar apoplastic titers (Fig. S1C). Overall, vascular adaptation produced clones with attenuated virulence in both tissues, whereas apoplast adaptation preserved full virulence. Download figure Open in new tab Fig. 3. Vascular adaptations affect vascular virulence. Four-leaf stage tomato plants were wound-inoculated with the indicated vascular adapted clones (S1-S10), apoplast-adapted clones (L1-L10), and Cm382 (WT) at the stem areas between the cotyledons. (A) Representative plants (out of 14 repeats) were photographed at 21 dpi. ( B ) Wilt symptoms were scored by the percentage of wilted leaves according to the following scale: 0 – no wilting, 1 = 1-25%, 2= 25-50%, 3= 51-100%. The graph depicts the distribution of 14 repeats for each clone pooled from two experiments. “*” represent significant difference (chi-squared test, p-value < 0.05) compared to WT. ( C) Bacterial growth in stems at 1 and 10 cm above the sites of infections at 21 dpi. (D ) Bacterial growth of the indicated clones in stems at the site of infections, 5 cm above the site of infections, and 10 cm above the site infections was monitored at 7 and 14 dpi. ( C , D ) Data is depicted in a box plot graph (boxes represent 25%-75% of the data, line represents median) representing 10 repeats pooled from two experiments. “*” represent significant differences (U-Test, p-value < 0.05) compared to WT in the same sampling area at the same dpi. Vascular-adapted clones demonstrate reduced stem-to-leaf migration Most vascular-adapted clones colonized and migrated systemically through the stem similarly to the WT parent, suggesting that their reduced wilting symptoms are not solely due to limited stem colonization. Since our in situ localization experiments linked wilting to bacterial presence in leaves ( Fig. 1 ), we hypothesized that their attenuated virulence may reflect reduced migration from stem to leaves. To test this, tomato plants were stem-inoculated with Cm WT, vascular-adapted clones (S2, S4, S9), and apoplast-adapted clones (L2, L7, L9). Bacterial presence and populations were monitored in pooled terminal leaflets from the second to fourth true leaves at 7 and 14 dpi. Vascular-adapted clones showed markedly lower frequencies and titers compared to WT, whereas apoplast-adapted clones colonized leaves at equal or higher levels ( Fig. 4A and 4B ). At 7 dpi, over 50% of leaflets were colonized by WT and apoplast-adapted clones but only 20–30% by vascular-adapted clones. By 14 dpi, colonization in WT and apoplast-adapted clones exceeded 90%, while S2 and S4 reached ∼50% and S9 ∼75% ( Fig. 4A ). These results suggest vascular-adapted clones either migrate more slowly from stems or struggle to establish in leaves. To distinguish these, we directly infiltrated leaves with all seven representative clones and tracked colonization at 3, 6, and 9 dpi. No significant differences were detected ( Fig. 4C ), indicating reduced leaf colonization results from impaired stem-to-leaf migration rather than defective leaf colonization. We next asked if this defect reflected altered tissue localization. GFP-labeled S2 and S4 were compared with WT in distal stem and leaf tissues (Fig. S2). No clear differences in localization were observed, and patterns correlated with leaflet symptoms. Consistent with isolation data, bacteria were absent from many asymptomatic leaflets inoculated with S2 and S4, though this also occurred at lower frequency in WT. Download figure Open in new tab Fig. 4. Vascular and apoplastic adaptations affect stem-to-leaf migration. ( A , B ) Four-leaf stage tomato plants were wound-inoculated with the indicated vascular adapted clones (S2, S4, and S9), apoplast-adapted clones (L2, L7, and L9), and Cm382 (WT) at the stem areas between the cotyledons. Bacteria were isolated and quantified from pooled samples of terminal leaflets from the second, third, and fourth true leaves of each plant at 7 and 14 days post inoculation (dpi). Data represents at least 30 repeats pooled from five (for WT, S2, S4, S9) or three (for L2, L7, and L9) experimental repeats. ( A ) Stacked bar graphs represent presence/absence of bacteria in the sampled leaflets at 7 and 14 dpi. “*” indicates that data were significantly different compared to Cm WT (chi-squared test, p-value < 0.05) at the same dpi. ( B ) Bar graphs represents the averages and standard errors of leaf bacterial populations. “*” indicates that bacterial populations were significantly different compared to Cm WT (U-test, p-value < 0.05) at the same dpi. ( C ) Six-leaf stage tomato leaves were inoculated with the indicated clones through infiltration of bacterial cultures (10 4 CFU/ml) using a needless syringe. Bacterial growth in the infiltration sites at 0, 3, 6, and 9 dpi. Data represents 10 repeats for each clone pooled from two experiments. No significant differences (U-test) were observed between each of the clones to the WT. In summary, vascular-adapted clones show a pronounced defect in stem-to-leaf migration, likely underlying their attenuated virulence. Tissue-adapted clones demonstrate altered colony morphology, production of exopolysaccharides, and surface attachment Considering that vascular adaptation altered both vascular and non-vascular virulence, we hypothesized that traits linked to pathogenicity and xylem association were also modified. We therefore examined EPS production, surface attachment, exoenzyme activity, siderophore production, hypersensitive response (HR) elicitation in non-hosts, and expression of virulence-associated genes. Siderophore production and HR induction were unchanged in adapted clones relative to Cm WT (Fig. S3), but several traits shifted in a tissue-dependent manner. On rich or minimal media, eight of ten vascular-adapted clones (S1, S2, S3, S4, S5, S6, S8, S9) that also showed attenuated vascular virulence formed dry, condensed colonies, whereas apoplast-adapted clones resembled WT with wet, glossy colonies ( Fig. 5A ). This suggested reduced EPS secretion or altered composition. Quantification confirmed seven vascular-adapted clones produced less EPS ( Fig. 5B ), while six apoplast-adapted clones produced significantly more. To further investigate this pattern, we examined the transcriptional expression of genes predicted to be involved in EPS production [ 28 ] in representative vascular-adapted clones (S2, S4, and S9) with low EPS yields and observed significant reductions in specific target genes in some clones, but not others (Fig. S4). Since EPS production is traditionally associated with surface attachment and biofilm formation [ 31 , 32 ], we next conducted in vitro surface attachment assays, which also serve as a proxy for biofilm formation, on all adapted clones using a 24-well crystal violet staining assay ( Fig. 5C ). Unexpectedly, vascular-adapted clones with dry colonies and low EPS showed stronger attachment than WT, while apoplast-adapted clones, which produced more EPS, had weaker attachment ( Fig. 5C ). These results suggest that, unlike in other systems, EPS may reduce attachment and biofilm formation in Cm. Download figure Open in new tab Fig. 5. Vascular and apoplastic adaptations effect on EPS production and surface attachment. ( A , B ) Cm WT and the indicated vascular and apoplast adapted clones were spotted (OD600=1) on LB agar supplemented with 5% sucrose and incubated for four days. Plates were photographed at 5 dpi ( A ) and EPS was quantified in the resulting clones ( B ) using phenol–sulfuric acid hydrolysis assays following by colorimetric quantification and standardization to bacteria count. ( C) Bacteria were grown on LB media, standardized to OD600=0.5 and incubated in 24 well plates without shaking for 10 days. Supernatants were removed, wells were washed three times with distilled water, and attached bacteria were stained with crystal violet to quantify surface attachment followed by colorimetric quantification. (B , C) . Graphs represent one out of three experimental repeats each composed of three ( B ) or four ( C ) biological repeats showcasing similar results. “*” represent significant difference (U-test, p-value < 0.05) compared to Cm WT. Overall, EPS production and surface attachment appear shaped by tissue-specific selective pressures: vascular adaptation favored high attachment and low EPS, while apoplast adaptation favored low attachment and high EPS. Vascular adapted clones demonstrate reduced cellulase and amylase activity After establishing that EPS production and surface attachment are selected during tissue adaptation, we assessed exoenzyme activity and production using plate halo assays. Cm bacteria heavily depend on secreted hydrolases such as CAZymes and serine proteases to cause disease [ 15 ]. Therefore, we monitored plate exoenzyme activity by halo assays on multiple substrates and transcriptional expression of predicted virulence associated serine proteases and CAZymes. Halo assays revealed no tissue-adaptation bias in lipase and xylanase activity in the adapted clones (data not shown). However, a clear association was observed between tissue adaptation and amylase and cellulase activity, visualized using starch and CMC degradation, respectively ( Fig. 6A and 6B ). Specifically, all vascular-adapted clones demonstrated reduced amylase and cellulase activity, while no significant difference was observed in the apoplast-adapted clones compared to the WT ( Fig. 6A and 6B ). While amylase activity was not subjected to any study in Cm, previous studies reported that cellulase activity in Cm is mediated by the secreted endo-beta-1,4-glucanase CelA, and disruption of celA abolishes cellulase activity in CMC halo assays, leading to a significant reduction in virulence with minimal impact on in planta growth [ 20 , 33 ]. Therefore, we monitored the transcriptional expression of celA and the putative amylase encoding gene aglC in the representative vascular adapted clones S2, S4, and S9 by RT-qPCR, and identified that celA expression was reduced in all three clones while aglC expression was not significantly affected ( Fig. 6C ). To further confirm that celA expression is reduced in the vascular adapted clones, Cm WT, S2, S4, and S9 were introduced with a plasmid carrying transcriptional fusion of the celA or the gyrB (used as a control) promoter to a uidA ( GUS ) reporter gene and monitored for GUS activity. Supporting transcriptional data, celA promoter activity was significantly reduced in the vascular adapted clones compared to Cm WT while the gyrB promoter activity was not significantly altered ( Fig. 6D ). Next, we tested the transcriptional expression of additional CAzyme coding genes xysA and nagA , which did not change significantly compared to the Cm WT and three Chp/Pat-1 family serine proteases: chpC and chpE that were reduced in S2 and S4, and chpG that was unaltered (Fig. S4). These findings suggest that vascular-adaptation resulted in reduced exoenzyme activity and expression of specific secreted virulence factors. Download figure Open in new tab Fig. 6. Vascular-adapted clones demonstrate reduced amylase and cellulase activity. ( A , B ) Cm WT and the indicated vascular- and apoplast-adapted clones were spotted (OD600 = 1) onto M9 medium supplemented with 0.1% starch (A) or carboxymethyl cellulose (CMC) (B), and 0.01% sucrose, then incubated for seven (A) or four (B) days. Plates were stained with 0.1% iodine (A) or Congo red (B), photographed (upper panels), and halo diameters were measured with a ruler to assess starch or CMC degradation (lower panels). Graphs represent means ± SE of at least nine replicates pooled from two independent experiments. ( C ) mRNA transcript abundance of the putative amylase-encoding gene aglC (CMM_2797) and the cellulase-encoding gene celA (pCM1_0020) was quantified by RT-qPCR in the indicated Cm cultures after 24 h incubation in sucrose-supplemented M9 medium. gyrA (CMM_0007) mRNA levels were used for normalization. Graphs represent means ± SE of relative transcript abundance compared to Cm WT, based on six independent biological replicates pooled from two independent experiments. ( D ) GUS activity was measured in the indicated Cm cultures harboring plasmids carrying gyrB (CMM_0006) or celA promoter-GUS fusions after 24 h incubation in sucrose-supplemented M9 medium. Graphs represent means ± SE of relative GUS activity compared to Cm WT, based on nine independent biological replicates pooled from three independent experiments. ( A – D ) Asterisks (*) indicate statistically significant differences compared to Cm WT (U-test, p-value < 0.05). Overexpression of celA in vascular-adapted clones only partially restores virulence The cellulase CelA has been previously established as a key virulence factor in Cm that is essential for the development of wilting symptoms [ 20 , 33 ]. Hence, the attenuated virulence of the vascular-adapted clones may be a result of their reduced celA expression and, consequently, lower cellulase activity. To test this, we investigated whether ectopic expression of celA could restore virulence in these vascular-adapted clones. We introduced celA , fused to an HA epitope and driven by the groEL promoter from Microbacterium esteraromaticum (which we previously confirmed to be active in Cm, data not shown), into vascular-adapted clones S2, S4, and S9. CelA accumulation in the transformants was confirmed by restoration of cellulase activity to WT levels in plate assays and by Western blot analysis (Fig. S5A and S5B). Next, we inoculated tomato stems with Cm WT, the vascular-adapted clones, or the celA -expressing vascular-adapted clones and monitored wilting symptoms. Each CelA-expressing clone displayed increased virulence relative to its parental vascular-adapted clone but remained less virulent than Cm WT (Fig. S5C). This indicates that although restoring cellulase activity partially recovers virulence, it cannot fully compensate for the reduced virulence of the vascular-adapted clones. Genomic analyses of vascular- and apoplastic-adapted clones Vascular and apoplastic adaptation yielded clones with distinct phenotypes that strongly correlated to their adapted tissue. We hypothesized these changes were driven by genomic alterations and fixation of mutations underlying the observed traits. To test this, we sequenced genomes of all adapted clones used in phenotypic assays after 15 passages from each of the 20 lineages, as well as individual clones at passages 5 and 10 to assess mutation fixation over time. Three independent colonies of the parent strain Cm NCPPB382 were also sequenced for comparison. Sequencing was performed on an Illumina Nextseq2000 platform (150 bp paired-end reads) at the Applied Microbiology Services Laboratory, The Ohio State University. De novo assemblies were generated for all clones and compared with Cm NCPPB382 to detect large deletions and smaller variations via SNP and INDEL calling. Comparative analysis revealed no large deletions or plasmid loss. Mutations were limited to a small set of SNPs and INDELs (Table S1). Notably, despite major effects on cellulase activity, no mutations were detected in celA or its promoter in vascular-adapted clones. Tracking fixation patterns across passages 5, 10, and 15 showed some mutations arose early and persisted, while others appeared only transiently, indicating variable retention of diversity within lineages (Table S1). Most adapted clones harbored a few mutations in putative coding genes that resulted in gene disruption or shifts in amino acid composition (Tables 1 and S1). Some mutations appeared exclusively in tissue-specific groups. A G308→A substitution in CMM_2466 , encoding a putative restriction endonuclease, caused a G103→D change and was found in four apoplast-adapted clones (L1, L2, L3, L6) but absent in vascular clones ( Table 1 ). Conversely, six vascular-adapted clones carried two independent mutations in CMM_1284 , absent from apoplast clones: C58→T in S1, S2, S6 (R20C), and A110→G in S3, S4, S9 (H37R) ( Table 1 , Fig. S5A). CMM_1284 encodes a 107-aa HipB/XRE-type transcriptional regulator, with both substitutions located in its predicted helix-turn-helix DNA-binding domain (Fig. S6B). View this table: View inline View popup Table 1. Coding-sequence substitutions and short indels in vascular and apoplast-adapted clones These findings reveal specific mutations selected during vascular and apoplastic adaptation, providing a genetic basis for tissue-specific traits. CMM_1284 modulates virulence, EPS production, surface attachment, and cellulase activity Comparative genome analyses identified two independent mutations in the putative transcriptional regulator gene CMM_1284 across six vascular-adapted clones, all showing attenuated virulence, enhanced surface attachment, and reduced cellulase activity. These findings suggest CMM_1284 contributes to the vascular lifestyle by modulating these traits. To test this, we constructed a CMM_1284 marker exchange mutant (CmΩ1284; Fig. S6C and S6D) and examined its impact on traits altered in vascular-adapted clones. CmΩ1284 was first tested for virulence, systemic colonization, and stem-to-leaf migration in tomato. Like vascular-adapted clones, CmΩ1284-inoculated plants displayed significantly reduced wilting symptoms compared to WT ( Fig. 7A and 7B ), while systemic colonization in the stem was unaffected ( Fig. 7C ). In contrast, CmΩ1284 showed markedly reduced stem-to-leaf migration, with lower bacterial frequency and populations in the second to fourth leaves at 7 and 14 dpi ( Fig. 7D , and 7E). Finally, we assessed CmΩ1284 for in vitro virulence traits and found that it formed dry colonies, produced less EPS, exhibited stronger surface attachment, and showed reduced cellulase activity. These features closely resemble those of most vascular-adapted clones ( Fig. 7F–H ). Download figure Open in new tab Fig. 7. Characterization of the CMM_1284 marker exchange mutant. ( A – E ) Four-leaf stage tomato plants were wound-inoculated with Cm WT and the CMM_1284 marker exchange mutant (CmΩ1284) at the stem areas between the cotyledons. ( A ) Representative plants were photographed at 14 days post-inoculation (dpi). ( B ) Wilt symptoms were scored based on the percentage of wilted leaves using the following scale: 0 = no wilting, 1 = 1–25%, 2 = 25–50%, 3 = 51–100%. The stacked graph shows the distribution of 24 replicates for each clone pooled from three experiments at 14 dpi. ( C ) Bacterial growth of the indicated clones was monitored in the stem at the site of infection, 5 cm above the site of infection, and 10 cm above the site of infection at 7 and 14 dpi. Data are shown in a box plot, representing at least 19 replicates pooled from two experiments. ( D , E ) Bacteria were isolated and quantified from pooled terminal leaflet samples from the second, third, and fourth true leaves of each plant at 7 and 14 dpi. Data represent at least 30 replicates pooled from four (7 dpi) or three (14 dpi) experimental repeats. ( D ) Stacked bar graphs represent the presence/absence of bacteria in the sampled leaflets at 7 and 14 dpi. ( E ) Bar graphs show the averages and standard errors of bacterial populations in the leaves at 7 and 14 dpi. ( F ) Cm WT and CmΩ1284 were spotted (OD600 = 1) on LB agar supplemented with 5% sucrose and incubated for four days. Plates were photographed at 5 dpi (lower panel), and EPS production was quantified in the resulting clones (upper panel) using phenol– sulfuric acid hydrolysis assays followed by colorimetric quantification, standardized to bacterial count. ( G ) Cm WT and CmΩ1284 were grown on LB medium, standardized to OD600 = 0.5, and incubated in 24-well plates without shaking for 5 days. Supernatants were removed, wells were washed three times with distilled water, and attached bacteria were stained with crystal violet to quantify surface attachment (lower panel), followed by colorimetric quantification (upper panel). ( H ) Cm WT and CmΩ1284 were spotted (OD600 = 1) onto M9 medium supplemented with 0.1% carboxymethyl cellulose (CMC), and incubated for four days. Plates were stained with 0.1% Congo red, photographed (lower panel), and halo diameters were measured with a ruler to assess CMC degradation (upper panel). ( F , G , H ) Graphs represent means ± SE of at least ten biological replicates pooled from two experiments. ( B – H ) Asterisks (*) indicate that data were significantly different compared to Cm WT (U-test for C, E, F, G, and H; chi-squared test for B and D; p-value < 0.05). These results support CMM_1284 as a regulator of the vascular lifestyle and its associated traits. Media-adapted clones do not demonstrate directional phenotypic selection To confirm that tissue adaptation was due to host selective pressure rather than culture conditions, a control population of ten clones was generated by subculturing on LB agar for 15 passages. These clones (LB1–LB10) were analyzed for EPS production, cellulase activity, and virulence. Unlike tissue-adapted clones, which showed consistent shifts, media-adapted clones displayed scattered phenotypes: reduced EPS and cellulase activity in LB1 and LB3, complete cellulase loss in LB8, and altered virulence in LB9 and LB10 (Fig. S7). Thus, prolonged media culturing may cause mutations but not consistent, directional adaptation, likely reflecting clonal drift. Discussion Bacterial pathogens switch strategies within their host to complete the infection cycle, involving changes in behavior, target tissues, and movement. To adapt to different infection phases, they must overcome diverse challenges and exhibit phenotypic plasticity. We investigated this plasticity by subjecting Cm to tissue-specific experimental evolution, differentiating adaptation to vascular versus apoplastic lifestyles. Ten parallel clones in each condition diverged phenotypically: vascular-adapted clones showed reduced virulence, lower cellulase activity, decreased EPS production, and enhanced surface attachment, whereas apoplast-adapted clones retained virulence, increased EPS production, and reduced surface attachment. These results indicate that xylem and apoplast exert distinct selective pressures, driving directional selection of traits. During disease, Cm exits xylem vessels late in infection, associated with symptom onset. Xylem residence may represent a dormant stage enabling systemic spread without symptoms, while apoplastic colonization likely drives vascular collapse and tissue maceration, in a similar manner to previous reports on Erwinia amylovora [ 8 ]. The molecular signals governing this shift are not well defined. In other xylem pathogens such as E. amylovora and Ralstonia spp., shifts are linked to population sensing and host/environmental cues like metabolites, physiology, and temperature [ 34 , 35 ]. It is possible that during vascular adaptation in this study, we selected clones that linger longer in the dormant stage, resulting in attenuated virulence. Traits consistently observed in tissue-adapted clones suggest strong selection on EPS production and surface attachment, which appear negatively correlated in Cm. In the apoplast, higher EPS may protect against antimicrobial responses [ 36 – 38 ], while vascular adaptation favors surface attachment and biofilm formation for xylem colonization [ 10 ]. Repeated vascular exposure should thus select for strong attachment and dense biofilms, redundant in the apoplast. Notably, clones with higher surface attachment produced less EPS. Although EPS usually supports biofilms [ 31 ], composition and viscosity often matter more than quantity [ 32 ]. For instance, in Xylella fastidiosa , mutants lacking EPS-degrading enzymes overproduce EPS, leading to reduced surface attachment and impaired biofilm formation—similar to what we observed in apoplast-adapted clones [ 39 ]. EPS overproduction may inhibit cell-to-cell or surface attachment by masking cell wall-anchored proteins [ 40 ]. Notably, in many Gram-positive Firmicutes, surface attachment and biofilm formation are mediated by cell wall-bound proteins rather than EPS [ 40 – 42 ]. This suggests that EPS may not contribute significantly to biofilm formation in at least some Gram-positive species, contrasting with widely accepted Gram-negative-based models. This likely underlies attenuated virulence: vascular-adapted clones showed reduced stem-to-leaf migration, possibly due to impaired biofilm disassembly, and decreased vessel blockage, as EPS strongly contributes to wilting. Dynamic EPS modulation thus appears central to Cm colonization: low EPS supports local xylem establishment, while increased EPS promotes dispersal and systemic spread. These transitions are probably regulated by physiological and environmental cues. Another trait in vascular-adapted clones was reduced cellulase and amylase activity. Cellulase, linked to virulence through the secreted glucanase CelA, is essential for symptom development [ 20 , 33 ]. Cell wall–degrading enzymes can also affect EPS and biofilm disassembly [ 43 , 44 ], though not for CelA: celA mutants lack EPS phenotypes, overexpression does not restore vascular clone EPS traits, and natural isolates show no correlation between cellulase, morphology, or EPS. Therefore, CelA more likely modulates plant cell walls and xylem exit, promoting spread as in Pectobacterium [ 45 ]. Overexpressing celA only partially restored virulence in vascular clones, suggesting phenotypes depend more on EPS and surface attachment. Why cellulase was negatively selected remains unclear and warrants further study. Despite major phenotypic changes, adapted clones accumulated few SNPs/INDELs, with no large deletions or plasmid loss. Mutation frequencies align with previous studies [ 46 ], though stress may elevate rates via reactive oxygen species [ 47 ]. Importantly, neither celA , its promoter, pCM1, nor EPS-associated clusters were mutated, implying upstream regulatory changes. Indeed, six of ten vascular clones fixed mutations the in transcriptional regulator CMM_1284 , causing amino acid substitutions in its DNA-binding domain. A marker exchange mutant of CMM_1284 recapitulated vascular-clone phenotypes: enhanced attachment, reduced EPS and cellulase activity, and attenuated virulence, suggesting a role in vascular–apoplastic shifts. However, some clones lacked CMM_1284 changes yet showed similar phenotypes, and two vascular clones had no coding mutations at all, indicating regulatory, structural, or epigenetic mechanisms undetected by Illumina sequencing. In summary, experimental evolution identified tissue-specific traits in Cm. Biofilm formation and surface attachment were critical for vascular colonization but limited migration and virulence, suggesting that biofilm disassembly is essential for transitioning from a latent vascular phase to a virulent apoplastic phase. Materials and methods Bacterial Strains and Plant Material Cm and E. coli strains developed and used in this study are listed in Table S2. Cm strains were grown in Luria-Bertani (LB) medium at 28°C, supplemented with 100 µg/ml trimethoprim [ 48 ]. E. coli strains were grown in LB medium at 37°C. When required, media were supplemented with 25 µg/ml gentamicin, 75 µg/ml neomycin, 50 µg/ml kanamycin, or 100 µg/ml ampicillin. The plant cultivars used in this study were tomato ( Solanum lycopersicum cv. Moneymaker), eggplant ( Solanum melongena cv. Black Queen), and Nicotiana sylvestris . Plants were grown in a temperature-controlled glasshouse at 25°C under natural light conditions. Experimental Evolution Procedure Vascular, apoplastic, and media-adapted populations were generated by repeated passaging of Cm NCPPB382 in tomato stems, leaves, and LB medium. A full description is provided in Supplementary File 1. Briefly, for vascular passages, bacteria were toothpick-inoculated into stems of 4–6 leaf plants and re-isolated ∼6 cm above the inoculation site after 14–21 days. For apoplastic passages, diluted suspensions (10□ CFU/ml) were syringe-infiltrated into mature leaves and recovered from pooled foci after 7 days. Ten parallel lines per tissue type were passaged for 15 cycles, with populations stored in glycerol at –80 °C. Representative clones from passages 5, 10, and 15 were used for phenotyping and sequencing. Plant inoculations, disease severity assessments, apoplastic virulence, stem-to-leaf migration and quantification of stem/leaf bacterial populations The virulence assays were carried out using wound inoculation method similar to that described by [ 49 ], with some minor adjustments. A full description is provided in Supplementary File 1. Virulence assays were conducted using toothpick-mediated stem wound inoculation and needleless syringe infiltration of tomato leaves. Wilt symptoms in stems were scored at 14 or 21 dpi on a 0–3 scale based on the percentage of affected leaves. Leaf infiltration assays were evaluated at 10 dpi for chlorosis and necrosis on a 0–3 scale, and representative leaves were photographed. Bacterial populations were quantified from stem segments or leaf disks by plating serial dilutions, standardized to tissue weight or surface area. Hypersensitive response assays were performed in eggplant and Nicotiana sylvestris by syringe infiltration, with symptoms recorded at 36 h. Cloning and Bacterial Manipulation All plasmids and oligonucleotides produced and used in this study are listed in Tables S3 and S4, respectively. Detailed information regarding cloning techniques, and plasmids construction is provided in Supplementary File 1. The plasmid pMA-RQ:Cmp [ 18 ] served as the backbone for constructing marker exchange, overexpression, and reporter plasmids. A marker exchange construct (pCMAT:1284) targeting CMM_1284 was generated to obtain deletion mutants, which were confirmed by PCR. For overexpression, the backbone was modified with a promoter and a genomic integration site, producing derivatives such as pCMIARG, including a CelA-3×HA fusion confirmed by western blot. Reporter plasmids carrying celA and gyrB promoters fused to the uidA (GUS) gene were built in the Cm– E. coli shuttle vector pHN216 [ 50 ] and used for promoter activity assays. All constructs were introduced into Cm by electroporation, as described in [ 49 ]. EPS quantification, surface attachment, and plate halo assays Detailed descriptions of experimental procedures used for in vitro characterization is provided in Supplementary File 1. Briefly, EPS quantification was conducted on Cm bacteria grown in 5% supplanted LB plates, using the phenol-sulfuric acid estimation method [ 51 ]. Surface attachment/biofilm assays were performed using 24 well plates supplemented with Cm cultures previously grown on LB broth. Attachment was assessed and quantified after static incubation using crystal violet staining method as previously described [ 52 ] with modifications. Exoenzyme activity of Cm clones was tested by plate halo assays on media containing specific substrates for cellulase, amylase, xylanase, polygalacturonase, protease, or lipase, with halos visualized by staining and quantified by diameter measurements [ 53 ]. Siderophore production was assessed using the CAS agar assay [ 54 ] under iron-limiting conditions, with activity visualized as yellow/orange halos around colonies. In Situ Localization of Clavibacter michiganensis During Infection Using Confocal Laser Scanning Microscopy Cm clones NCPPB382, S2, and S4 were transformed with the E. coli – Clavibacter shuttle vector pK2-22 [ 24 ], which expresses EGFP under the control of the p CMP1 promoter. In situ localization of bacteria was monitored in infected plants using an Olympus IX81 confocal laser scanning microscope equipped with a GFP filter set. To visualize bacterial localization in stem tissues, ∼0.1□mm-thick horizontal and vertical cross-sections were collected approximately 3□cm above the inoculation site. For leaf tissues, ∼0.5□cm² sections were excised from areas exhibiting healthy, wilted, or necrotic phenotypes and directly mounted on microscope slides for imaging. Promoter Activity Assays and Gene Expression Analysis For promoter activity and gene expression assays, Cm WT, S2, S4, and S9 strains, either alone or carrying pHN:p celA : GUS or pHN: pgyrB : GUS , were grown overnight in LB, washed twice with distilled water, resuspended in M9 medium to induce virulence-associated gene expression [ 55 ], and incubated at 28 °C with rotation for 24 h. For promoter activity, OD 600 was measured, and 1.5 ml of each culture was lysed with 1 mg/ml lysozyme followed by sonication (SONIC-150W, MRC Labs). Lysates were centrifuged, and 80 µl of supernatant was mixed with 10 µl 10× Tris-based saline (pH 7.4) and 10 µl 10 mM PNPG. Reactions were incubated at 37 °C and monitored for yellow pigmentation, then stopped with 50 µl 1 M sodium bicarbonate. Absorbance at 405 nm was measured, and activity was calculated as OD 405 / (time × OD 600 ) and standardized to WT activity. For gene expression, total RNA was extracted from culture supernatants using the Hybrid-R Kit (GeneAll), reverse transcribed with the UltraScript™ cDNA Synthesis Kit (PCR Biosystems), and amplified using Fast SYBR qPCR Master Mix (Biogate) on a QuantStudio 3 system (Applied Biosystems) with gene-specific primers (Table S3). Expression was normalized to gyrA and calculated with the comparative Ct method [ 56 ]. Genome Sequencing, Assembly, and Comparative Genomic Analysis Three independent Cm NCPPB382 (WT) clones and tissue-adapted clones collected after 5, 10, and 15 passages were subjected to whole-genome sequencing. DNA was extracted from 10 ml overnight LB cultures using the Wizard Genomic DNA Purification Kit (Promega). Libraries were sequenced on an Illumina NextSeq2000 (150 bp paired-end) at the Applied Microbiology Services Laboratory (Ohio State University). Reads were cleaned with Trimmomatic [ 57 ] and assembled using Unicycler v0.5.0 and SPAdes v3.15.5 [ 58 , 59 ]; contigs <200 bp were removed. Genome completeness was assessed with BUSCO v5 (micrococcales_odb10) [ 60 ]. Genomes were deposited in the NCBI BioProject database under the ID PRJNA1333927. Genomic alterations were identified with Snippy ( https://github.com/tseemann/snippy ) and confirmed by BWA [ 61 ] against the NCPPB382 reference (GCA_000063485). Passage 10 of clone S6 and passage 5 of clone S10 were excluded due to poor sequencing quality. All INDELs and SNPs are listed in Table S1, excluding mutations found in any of the three resequenced WT clones. Ten representative ORF mutations were confirmed by Sanger sequencing (Table S1, *). The corresponding regions were PCR-amplified from Cm WT and adapted clones using specific primers (Table S4), sequenced (Hylabs Laboratories), and compared with assembled genomes. Acknowledgments This work was supported by the Israel Binational Science Foundation (BSF, grant no. 2021190 and 2023281, for D.T. and J.M.J). Funder Information Declared Israel Binational Science Foundation (BSF) , 2021190 , 2023281 References 1. ↵ Rico A , Preston GM . Pseudomonas syringae pv. tomato DC3000 Uses Constitutive and Apoplast-Induced Nutrient Assimilation Pathways to Catabolize Nutrients That Are Abundant in the Tomato Apoplast . Molecular Plant-Microbe Interactions® . 2008 ; 21 : 269 – 282 . doi: 10.1094/MPMI-21-2-0269 OpenUrl CrossRef PubMed Web of Science 2. Carlos Caicedo J , Villamizar S . Xanthomonas citri ssp. citri Pathogenicity, a Review. Citrus - Research , Development and Biotechnology. IntechOpen ; 2021 . doi: 10.5772/intechopen.97776 OpenUrl CrossRef 3. ↵ Dhaouadi S , A. H M , Rhouma A . The plant pathogen Rhodococcus fascians. History, disease symptomatology, host range, pathogenesis and plant–pathogen interaction . Annals of Applied Biology . 2020 ; 177 : 4 – 15 . doi: 10.1111/aab.12600 OpenUrl CrossRef 4. ↵ Beattie GA , Lindow SE . The Secret Life of Foliar Bacterial Pathogens on Leaves . Annu Rev Phytopathol . 1995 ; 33 : 145 – 172 . doi: 10.1146/annurev.py.33.090195.001045 OpenUrl CrossRef PubMed Web of Science 5. ↵ Bendix C , Lewis JD . The enemy within: phloem limited pathogens . Mol Plant Pathol . 2018 ; 19 : 238 – 254 . doi: 10.1111/mpp.12526 OpenUrl CrossRef PubMed 6. ↵ Yadeta KA , J. Thomma BPH . The xylem as battleground for plant hosts and vascular wilt pathogens . Front Plant Sci . 2013 ; 4 . doi: 10.3389/fpls.2013.00097 OpenUrl CrossRef PubMed 7. ↵ De La Fuente L , Merfa M V. , Cobine PA , Coleman JJ . Pathogen Adaptation to the Xylem Environment . Annu Rev Phytopathol . 2022 ; 60 : 163 – 186 . doi: 10.1146/annurev-phyto-021021-041716 OpenUrl CrossRef PubMed 8. ↵ Bogs J , Bruchmüller I , Erbar C , Geider K . Colonization of Host Plants by the Fire Blight Pathogen Erwinia amylovora Marked with Genes for Bioluminescence and Fluorescence . Phytopathology . 1998 ; 88 : 416 – 421 . doi: 10.1094/PHYTO.1998.88.5.416 OpenUrl CrossRef PubMed Web of Science 9. ↵ Gluck-Thaler E , Cerutti A , Perez-Quintero AL , Butchacas J , Roman-Reyna V , Madhavan VN , et al. Repeated gain and loss of a single gene modulates the evolution of vascular plant pathogen lifestyles . Sci Adv . 2020 ; 6 . doi: 10.1126/sciadv.abc4516 OpenUrl FREE Full Text 10. ↵ Mina IR , Jara NP , Criollo JE , Castillo JA . The critical role of biofilms in bacterial vascular plant pathogenesis . Plant Pathol . 2019 ; 68 : 1439 – 1447 . doi: 10.1111/ppa.13073 OpenUrl CrossRef 11. ↵ Rott P , Fleites L , Marlow G , Royer M , Gabriel DW . Identification of New Candidate Pathogenicity Factors in the Xylem-Invading Pathogen Xanthomonas albilineans by Transposon Mutagenesis . Molecular Plant-Microbe Interactions® . 2011 ; 24 : 594 – 605 . doi: 10.1094/MPMI-07-10-0156 OpenUrl CrossRef PubMed Web of Science 12. ↵ Burdman S , Bahar O , Parker JK , De La Fuente L . Involvement of Type IV Pili in Pathogenicity of Plant Pathogenic Bacteria . Genes (Basel ). 2011 ; 2 : 706 – 735 . doi: 10.3390/genes2040706 OpenUrl CrossRef 13. ↵ Osdaghi E , Abachi H , Jacques M. Clavibacter michiganensis Reframed: The Story of How the Genomics Era Made a New Face for an Old Enemy . Mol Plant Pathol . 2025 ; 26 . doi: 10.1111/mpp.70093 OpenUrl CrossRef 14. ↵ Park IW , Oh E-J , Hwang IS , Oh C-S . Reclassification and Pathogenesis of Clavibacter Species: A Model Pathogen for Studying the Molecular Interactions Between Gram-Positive Bacteria and Plants . Phytopathology . 2025 . doi: 10.1094/PHYTO-02-25-0084-IA OpenUrl CrossRef 15. ↵ Chalupowicz L , Barash I , Reuven M , Dror O , Sharabani G , Gartemann K-H , et al. Differential contribution of Clavibacter michiganensis subsp. michiganensis virulence factors to systemic and local infection in tomato . Mol Plant Pathol . 2016 . doi: 10.1111/mpp.12400 OpenUrl CrossRef PubMed 16. ↵ Gartemann K-H , Abt B , Bekel T , Burger A , Engemann J , Flul-gel M , et al. The Genome Sequence of the Tomato-Pathogenic Actinomycete Clavibacter michiganensis subsp. michiganensis NCPPB382 Reveals a Large Island Involved in Pathogenicity . J Bacteriol . 2008 ; 190 : 2138 – 2149 . doi: 10.1128/JB.01595-07 OpenUrl Abstract / FREE Full Text 17. ↵ Hwang IS , Nguyen TT , Oh E , Cho G , Kim JH , Kim K , et al. Two plasmid-borne virulence genomic islands of Clavibacter michiganensis are genetically diverse and determine the development of wilt symptoms in host plants . New Phytologist . 2025 . doi: 10.1111/nph.70329 OpenUrl CrossRef 18. ↵ Verma RK , Roman-Reyna V , Raanan H , Coaker G , Jacobs JM , Teper D . Allelic variations in the chpG effector gene within Clavibacter michiganensis populations determine pathogen host range . PLoS Pathog . 2024 ; 20 : e1012380 . doi: 10.1371/journal.ppat.1012380 OpenUrl CrossRef PubMed 19. ↵ Thapa SP , Pattathil S , Hahn MG , Jacques M-A , Gilbertson RL , Coaker G . Genomic Analysis of Clavibacter michiganensis Reveals Insight Into Virulence Strategies and Genetic Diversity of a Gram-Positive Bacterial Pathogen . Molecular Plant-Microbe Interactions® . 2017 ; 30 : 786 – 802 . doi: 10.1094/MPMI-06-17-0146-R OpenUrl CrossRef PubMed 20. ↵ Jahr H , Dreier J , Meletzus D , Bahro R , Eichenlaub R . The Endo-β-1,4-glucanase CelA of Clavibacter michiganensis subsp. michiganensis Is a Pathogenicity Determinant Required for Induction of Bacterial Wilt of Tomato . Molecular Plant-Microbe Interactions® . 2000 ; 13 : 703 – 714 . doi: 10.1094/MPMI.2000.13.7.703 OpenUrl CrossRef PubMed Web of Science 21. Stork I , Gartemann K-H , Burder A , Eichenlaub R . A family of serine proteases of Clavibacter michiganensis subsp. michiganensis : chpC plays a role in colonization of the host plant tomato . Mol Plant Pathol . 2008 ; 9 : 599 – 608 . doi: 10.1111/j.1364-3703.2008.00484.x OpenUrl CrossRef PubMed 22. Dreier J , Meletzus D , Eichenlaub R . Characterization of the Plasmid Encoded Virulence Region pat −1 of Phytopathogenic Clavibacter michiganensis subsp. michiganensis . Molecular Plant-Microbe Interactions® . 1997 ; 10 : 195 – 206 . doi: 10.1094/MPMI.1997.10.2.195 OpenUrl CrossRef PubMed Web of Science 23. ↵ Hwang IS , Oh E-J , Song E , Park IW , Lee Y , Sohn KH , et al. An Apoplastic Effector Pat-1Cm of the Gram-Positive Bacterium Clavibacter michiganensis Acts as Both a Pathogenicity Factor and an Immunity Elicitor in Plants . Front Plant Sci . 2022 ; 13 . doi: 10.3389/fpls.2022.888290 OpenUrl CrossRef 24. ↵ Chalupowicz L , Zellermann E-M , Fluegel M , Dror O , Eichenlaub R , Gartemann K-H , et al. Colonization and Movement of GFP-Labeled Clavibacter michiganensis subsp. michiganensis During Tomato Infection . Phytopathology . 2012 ; 102 : 23 – 31 . doi: 10.1094/PHYTO-05-11-0135 OpenUrl CrossRef PubMed 25. ↵ Bai K , Jiang N , Chen X , Xu X , Li J , Luo L . RNA-Seq Analysis Discovers the Critical Role of Rel in ppGpp Synthesis, Pathogenicity, and the VBNC State of Clavibacter michiganensis . Phytopathology . 2022 ; 112 : 1844 – 1858 . doi: 10.1094/PHYTO-01-22-0023-R OpenUrl CrossRef PubMed 26. ↵ Li Y , Chen X , Xu X , Yu C , Liu Y , Jiang N , et al. Deletion of pbpC Enhances Bacterial Pathogenicity on Tomato by Affecting Biofilm Formation, Exopolysaccharides Production, and Exoenzyme Activities in Clavibacter michiganensis . Int J Mol Sci . 2023 ; 24 : 5324 . doi: 10.3390/ijms24065324 OpenUrl CrossRef PubMed 27. ↵ Bermpohl A , Dreier J , Bahro R , Eichenlaub R . Exopolysaccharides in the pathogenic interaction of Clavibacter michiganensis subsp. michiganensis with tomato plants . Microbiol Res . 1996 ; 151 : 391 – 399 . doi: 10.1016/S0944-5013(96)80009-7 OpenUrl CrossRef Web of Science 28. ↵ Bentley SD , Corton C , Brown SE , Barron A , Clark L , Doggett J , et al. Genome of the Actinomycete Plant Pathogen Clavibacter michiganensis subsp. sepedonicus Suggests Recent Niche Adaptation . J Bacteriol . 2008 ; 190 : 2150 – 2160 . doi: 10.1128/JB.01598-07 OpenUrl Abstract / FREE Full Text 29. ↵ Hwang IS , Oh E-J , Oh C-S. A novel virulence gene, cviA1 of Clavibacter michiganensis for necrosis development in the Nicotiana benthamiana plant . Microbiol Res . 2024 ; 285 : 127743 . doi: 10.1016/j.micres.2024.127743 OpenUrl CrossRef 30. ↵ Tancos MA , Chalupowicz L , Barash I , Manulis-Sasson S , Smart CD . Tomato Fruit and Seed Colonization by Clavibacter michiganensis subsp. michiganensis through External and Internal Routes . Appl Environ Microbiol . 2013 ; 79 : 6948 – 6957 . doi: 10.1128/AEM.02495-13 OpenUrl Abstract / FREE Full Text 31. ↵ O’Toole G , Kaplan HB , Kolter R . Biofilm Formation as Microbial Development . Annu Rev Microbiol . 2000 ; 54 : 49 – 79 . doi: 10.1146/annurev.micro.54.1.49 OpenUrl CrossRef PubMed Web of Science 32. ↵ Ruhal R , Kataria R . Biofilm patterns in gram-positive and gram-negative bacteria . Microbiol Res . 2021 ; 251 : 126829 . doi: 10.1016/j.micres.2021.126829 OpenUrl CrossRef PubMed 33. ↵ Hwang IS , Oh E-J , Lee HB , Oh C-S . Functional Characterization of Two Cellulase Genes in the Gram-Positive Pathogenic Bacterium Clavibacter michiganensis for Wilting in Tomato . Molecular Plant-Microbe Interactions® . 2019 ; 32 : 491 – 501 . doi: 10.1094/MPMI-08-18-0227-R OpenUrl CrossRef 34. ↵ Kharadi RR , Schachterle JK , Yuan X , Castiblanco LF , Peng J , Slack SM , et al. Genetic Dissection of the Erwinia amylovora Disease Cycle . Annu Rev Phytopathol . 2021 ; 59 : 191 – 212 . doi: 10.1146/annurev-phyto-020620-095540 OpenUrl CrossRef PubMed 35. ↵ Planas-Marquès M , Kressin JP , Kashyap A , Panthee DR , Louws FJ , Coll NS , et al. Four bottlenecks restrict colonization and invasion by the pathogen Ralstonia solanacearum in resistant tomato . J Exp Bot . 2020 ; 71 : 2157 – 2171 . doi: 10.1093/jxb/erz562 OpenUrl CrossRef PubMed 36. ↵ Bianco MI , Toum L , Yaryura PM , Mielnichuk N , Gudesblat GE , Roeschlin R , et al. Xanthan Pyruvilation Is Essential for the Virulence of Xanthomonas campestris pv. campestris . Molecular Plant-Microbe Interactions® . 2016 ; 29 : 688 – 699 . doi: 10.1094/MPMI-06-16-0106-R OpenUrl CrossRef 37. Király Z , ElJZahaby HM , Klement Z . Role of Extracellular Polysaccharide (EPS) Slime of Plant Pathogenic Bacteria in Protecting Cells to Reactive Oxygen Species . Journal of Phytopathology . 1997 ; 145 : 59 – 68 . doi: 10.1111/j.1439-0434.1997.tb00365.x OpenUrl CrossRef 38. ↵ Du Y , Stegmann M , Misas Villamil JC . The apoplast as battleground for plant– microbe interactions . New Phytologist . 2016 ; 209 : 34 – 38 . doi: 10.1111/nph.13777 OpenUrl CrossRef PubMed 39. ↵ Chatterjee S , Newman KL , Lindow SE . Cell-to-Cell Signaling in Xylella fastidiosa Suppresses Movement and Xylem Vessel Colonization in Grape . Molecular Plant-Microbe Interactions® . 2008 ; 21 : 1309 – 1315 . doi: 10.1094/MPMI-21-10-1309 OpenUrl CrossRef PubMed Web of Science 40. ↵ Castro-Bravo N , Wells JM , Margolles A , Ruas-Madiedo P . Interactions of Surface Exopolysaccharides From Bifidobacterium and Lactobacillus Within the Intestinal Environment . Front Microbiol . 2018 ; 9 . doi: 10.3389/fmicb.2018.02426 OpenUrl CrossRef PubMed 41. Xu Z , Li Y , Xu A , Soteyome T , Yuan L , Ma Q , et al. Cell-wall-anchored proteins affect invasive host colonization and biofilm formation in Staphylococcus aureus . Microbiol Res . 2024 ; 285 : 127782 . doi: 10.1016/j.micres.2024.127782 OpenUrl CrossRef 42. ↵ Popowska M , Krawczyk-Balska A , Ostrowski R , Desvaux M . InlL from Listeria monocytogenes Is Involved in Biofilm Formation and Adhesion to Mucin . Front Microbiol . 2017 ; 8 . doi: 10.3389/fmicb.2017.00660 OpenUrl CrossRef PubMed 43. ↵ Dow JM , Crossman L , Findlay K , He Y-Q , Feng J-X , Tang J-L . Biofilm dispersal in Xanthomonas campestris is controlled by cell–cell signaling and is required for full virulence to plants . Proceedings of the National Academy of Sciences . 2003 ; 100 : 10995 – 11000 . doi: 10.1073/pnas.1833360100 OpenUrl Abstract / FREE Full Text 44. ↵ Li J , Zhang C , Hu X , Yoshida A , Osatomi K , Guo X , et al. Impact of different enzymes on biofilm formation and mussel settlement . Sci Rep . 2022 ; 12 : 4685 . doi: 10.1038/s41598-022-08530-4 OpenUrl CrossRef PubMed 45. ↵ Tsers I , Parfirova O , Moruzhenkova V , Petrova O , Gogoleva N , Vorob’ev V , et al. A Switch from Latent to Typical Infection during Pectobacterium atrosepticum— Tobacco Interactions: Predicted and True Molecular Players . Int J Mol Sci . 2023 ; 24 : 13283 . doi: 10.3390/ijms241713283 OpenUrl CrossRef PubMed 46. ↵ Wielgoss S , Barrick JE , Tenaillon O , Wiser MJ , Dittmar WJ , Cruveiller S , et al. Mutation rate dynamics in a bacterial population reflect tension between adaptation and genetic load . Proceedings of the National Academy of Sciences . 2013 ; 110 : 222 – 227 . doi: 10.1073/pnas.1219574110 OpenUrl Abstract / FREE Full Text 47. ↵ Qi W , Jonker MJ , de Leeuw W , Brul S , ter Kuile BH . Reactive oxygen species accelerate de novo acquisition of antibiotic resistance in E. coli. iScience . 2023 ; 26 : 108373 . doi: 10.1016/j.isci.2023.108373 OpenUrl CrossRef PubMed 48. ↵ Ftayeh RM , von Tiedemann A , Rudolph KWE . A New Selective Medium for Isolation of Clavibacter michiganensis subsp. michiganensis from Tomato Plants and Seed . Phytopathology . 2011 ; 101 : 1355 – 1364 . doi: 10.1094/PHYTO-02-11-0045 OpenUrl CrossRef PubMed 49. ↵ Verma RK , Teper D . Immune recognition of the secreted serine protease ChpG restricts the host range of Clavibacter michiganensis from eggplant varieties . Mol Plant Pathol . 2022 . doi: 10.1111/mpp.13215 OpenUrl CrossRef 50. ↵ Laine MJ , Nakhei H , Dreier J , Lehtilä K , Meletzus D , Eichenlaub R , et al. Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus with several cloning vectors . Appl Environ Microbiol . 1996 ; 62 : 1500 – 1506 . doi: 10.1128/AEM.62.5.1500-1506.1996 OpenUrl Abstract / FREE Full Text 51. ↵ DuBois Michel , Gilles KA , Hamilton JK , Rebers PA , Smith Fred . Colorimetric Method for Determination of Sugars and Related Substances . Anal Chem . 1956 ; 28 : 350 – 356 . doi: 10.1021/ac60111a017 OpenUrl CrossRef Web of Science 52. ↵ Merritt JH , Kadouri DE , O’Toole GA . Growing and Analyzing Static Biofilms . Curr Protoc Microbiol . 2006 ;00. doi: 10.1002/9780471729259.mc01b01s00 OpenUrl CrossRef PubMed 53. ↵ Hossain TJ , Chowdhury SI , Mozumder HA , Chowdhury MNA , Ali F , Rahman N , et al. Hydrolytic Exoenzymes Produced by Bacteria Isolated and Identified From the Gastrointestinal Tract of Bombay Duck . Front Microbiol . 2020 ; 11 . doi: 10.3389/fmicb.2020.02097 OpenUrl CrossRef 54. ↵ Louden BC , Haarmann D , Lynne AM . Use of Blue Agar CAS Assay for Siderophore Detection . J Microbiol Biol Educ . 2011 ; 12 : 51 – 53 . doi: 10.1128/jmbe.v12i1.249 OpenUrl CrossRef PubMed 55. ↵ Savidor A , Teper D , Gartemann KH , Eichenlaub R , Chalupowicz L , Manulis-Sasson S , et al. The Clavibacter michiganensis subsp. michiganensis -tomato interactome reveals the perception of pathogen by the host and suggests mechanisms of infection . J Proteome Res . 2012 ; 11 : 736 – 750 . OpenUrl PubMed 56. ↵ Pfaffl MW . A new mathematical model for relative quantification in real-time RT-PCR . Nucleic Acids Res . 2001 ; 29 : 45e – 445 . doi: 10.1093/nar/29.9.e45 OpenUrl CrossRef PubMed 57. ↵ Bolger AM , Lohse M , Usadel B . Trimmomatic: a flexible trimmer for Illumina sequence data . Bioinformatics . 2014 ; 30 : 2114 – 20 . doi: 10.1093/bioinformatics/btu170 OpenUrl CrossRef PubMed Web of Science 58. ↵ Bankevich A , Nurk S , Antipov D , Gurevich AA , Dvorkin M , Kulikov AS , et al. SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing . Journal of Computational Biology . 2012 ; 19 : 455 – 477 . doi: 10.1089/cmb.2012.0021 OpenUrl CrossRef PubMed 59. ↵ Wick RR , Judd LM , Gorrie CL , Holt KE . Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. Phillippy AM, editor . PLoS Comput Biol . 2017 ; 13 : e1005595 . doi: 10.1371/journal.pcbi.1005595 OpenUrl CrossRef PubMed 60. ↵ Manni M , Berkeley MR , Seppey M , Simão FA , Zdobnov EM . BUSCO Update: Novel and Streamlined Workflows along with Broader and Deeper Phylogenetic Coverage for Scoring of Eukaryotic , Prokaryotic, and Viral Genomes. Kelley J, editor. Mol Biol Evol . 2021 ; 38 : 4647 – 4654 . doi: 10.1093/molbev/msab199 OpenUrl CrossRef PubMed 61. ↵ Li H , Durbin R . Fast and accurate short read alignment with Burrows-Wheeler transform . Bioinformatics . 2009 ; 25 : 1754 – 60 . doi: 10.1093/bioinformatics/btp324 OpenUrl CrossRef PubMed Web of Science View the discussion thread. Back to top Previous Next Posted October 06, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Tissue-Specific Experimental Evolution Reveals Adaptive Trade-Offs in the Plant Vascular Pathogen Clavibacter michiganensis Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Tissue-Specific Experimental Evolution Reveals Adaptive Trade-Offs in the Plant Vascular Pathogen Clavibacter michiganensis Raj Kumar Verma , Veronica Roman-Reyna , Nathan Benmoche , Evanson Ngugi , Eduard Belausov , Maya Bar , Jonathan M. Jacobs , Doron Teper bioRxiv 2025.10.05.680514; doi: https://doi.org/10.1101/2025.10.05.680514 Share This Article: Copy Citation Tools Tissue-Specific Experimental Evolution Reveals Adaptive Trade-Offs in the Plant Vascular Pathogen Clavibacter michiganensis Raj Kumar Verma , Veronica Roman-Reyna , Nathan Benmoche , Evanson Ngugi , Eduard Belausov , Maya Bar , Jonathan M. Jacobs , Doron Teper bioRxiv 2025.10.05.680514; doi: https://doi.org/10.1101/2025.10.05.680514 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41911) Biophysics (21436) Cancer Biology (18578) Cell Biology (25482) Clinical Trials (138) Developmental Biology (13371) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15599) Genomics (22483) Immunology (17728) Microbiology (40364) Molecular Biology (17163) Neuroscience (88537) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15129) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)