Plant Growth Benefits by the Coalition Function of Plant Microbiome

Plant Growth Benefits by the Coalition Function of Plant Microbiome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Plant Growth Benefits by the Coalition Function of Plant Microbiome Seon-Woo Lee, Sang-Moo Lee, Jungwook Park, Hyun-Hee Lee, Ju Hui Kim, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7560912/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Plant-associated microbial communities consist of plant holobiont and play an essential role in plant growth and development, yet their collective functions are not fully understood. Theoretically, microbiota can act as integrated consortia, conferring emergent properties beyond those of single species. Here, we show that the tomato rhizosphere microbiome, when stimulated by a Flavobacterium dauae , enhances plant growth by activating the phytosterol biosynthesis pathway in both the microbiota and the plant host, a function unattainable by individual microbial species. A reconstructed synthetic community, based on meta-transcriptome of plant microbiota, recapitulated this microbiome-driven activity upon stimulation by F. dauae . This synthetic community also restored the growth response in diverse sterol-deficient plant mutants. The microbial consortium exhibits multispecies biofilm formation and functional specialization among its members, constituting a microbial coalition that promotes plant growth. This study provides direct experimental evidence that plant microbiota function as a coordinated unit and orchestrate host plant development. We highlight this to be a plant holobiont function based on microbial community coalition in host plant. Biological sciences/Microbiology/Microbial communities/Microbiome Biological sciences/Microbiology/Microbial communities/Microbial ecology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Plant-associated microbiota influences host plant fitness, including growth, development, and stress tolerance, collectively contributing to the functional traits of the plant holobiont 1 – 7 . However, the functional coalition of microbial communities within the plant holobiont remains poorly characterized. Like other communities, microbial interactions are an important component for microbiota function in the plant rhizosphere 3 – 7 . Furthermore, metabolic interactions between living plant roots and microbiota, as well as microbial species, are key drivers of microbial cooperation in the rhizosphere 8 – 11 , while our understanding of the metabolic interactions is limited. Cooperative microbial interactions stabilize rhizosphere communities through metabolic exchanges among microbes and with the host 3,4,8–10,12−16 . Specific microbes may enhance phytohormone pathways, including auxin, gibberellin, salicylic acid, jasmonic acid, and abscisic acid, however, the effects of microbial community coalition on complex phytohormonal signaling in crop plants remain poorly characterized 16 , 17 – 21 . Negative interactions between microbial species, such as competition and antagonism, are generally predominant in natural microbiota 22 . Nevertheless, recent studies indicate that cooperative interactions of microbial species can elicit activities within the microbiota that support host plant performance, including plant growth promotion, stress tolerance, and disease control 16 , 23 , 24 . Compared with negative interactions, positive interactions such as cooperation typically requires close physical proximity and interdependence among species, resulting in the formation of microbial coalition in natural microbiota 15 , 25 – 28 . Once established, microbial coalition formed through cooperative interactions can enhance functional robustness against environmental perturbations and facilitate the evolution of complex traits within microbial communities 27 – 30 . In this context, multi-species biofilms not only enhance root colonization but also provide a cooperative state that stabilizes interactions among microbes and between microbes and the host plant 24 , 25 . However, how such community-level cooperation leading to microbiota coalition influences host plant performance remains poorly characterized. Plants produce a diverse array of phytosterols, such as campesterol, sitosterol, and stigmasterol 31 , 32 . Phytosterols function not only as precursors of plant growth hormones like brassinosteroids but also as key regulators of plant responses to biotic and abiotic stresses 31 , 32 . Notably, fine-tuning of sterol composition can modulate plant responses to diverse microbes, particularly phytopathogens 32 – 36 . During plant–pathogen interactions, the equilibrium of phytosterols in the plasma membrane can shift 32 – 34 . These alterations affect plasma membrane composition and permeability 31 , 32 , 34 , 35 . However, whether and how beneficial plant-associated microbiota collectively influence host sterol-related processes through cooperative interactions remains unclear 32 . Here we employed multi-omics approaches and a synthetic community (SynCom) approach to tomato rhizosphere to investigate how rhizosphere microbiota functions as a unified cooperative population. SynComs have been developed as a reductionist approach to dissect the mechanisms underlying microbiota function 10 , 37 , 38 . Typically, the design of SynCom primarily rely on the quantitative dynamics of microbial communities to achieve targeted agricultural traits 10 , 38 , 39 . Based on gene expression patterns in both rhizosphere microbiota and host plant, we hypothesized that the rhizosphere microbiota occupies an ecological niche as a functionally integrated consortium, i.e. microbiota coalition, through cooperative interactions. To test this, we used cooperative rhizosphere microbiota stimulated by Flavobacterium dauae 40 and tomato plant. Upon stimulation, the phytosterol biosynthesis pathway was activated in both the rhizosphere microbiota and tomato plant, indicating coordinated interactions that contribute to plant growth. We further demonstrated that SynCom recapitulates the activity of the native rhizosphere microbiota through multi-species biofilm formation and functional specialization among SynCom members. Results Microbial stimulator for plant growth promotion with native soil microbiota. Previously, we identified an auxotrophic F. dauae TCH3-2 from the tomato rhizosphere, which exhibited the strongest plant growth-promoting activity among 32 Flavobacterium isolates 7 , 40 . Compared to other Flavobacterium species genomes, TCH3-2 genome is highly reduced (2.9 Mb), lacking most growth factor biosynthesis genes and indicating strict auxotrophy (Supplementary Fig. 1, Supplementary Table 1). Unique genes present in TCH3-2 genome included those for transposases, Type IX secretion, proline iminopeptidase, N-acetylglutamate synthase, DoxX-like proteins, and the flavanone-binding protein YndB (Supplementary Tables 2 and 3). The treatment of TCH3-2 significantly increased the fresh weight, dry weight, height, stem width, the number of flowers and fruits in tomato plants grown in upland soil compared to control (Fig. 1 a,b and Extended Data Fig. 1 and Supplementary Fig. 2). However, the plant growth-promoting (PGP) activity by TCH3-2 treatment was completely diminished in the autoclaved upland soil (Fig. 1 b and Supplementary Fig. 2). The PGP activity by TCH3-2 treatment was not specific to soil type or tomato cultivar (Supplementary Figs. 3 and 4). Co-treatment of TCH3-2 with the microbial fraction of upland soil (UpMF), independent of soil particles, promoted tomato growth compared to individual treatments and the control (Fig. 1 c,d). In addition, another closely-related Flavobacterium strains did not promote tomato growth with the UpMF (Supplementary Fig. 5). In particular, the upland soil-derived strain F. daejeonense RCH33 7 , which possesses PGP potential through auxin production, failed to promote tomato growth in the presence of the UpMF (Supplementary Fig. 5a,b). These results indicated a unique cooperative interaction between TCH3-2 and the indigenous soil microbiota for PGP activity. Microbiome alteration by the stimulatory Flavobacterium dauae TCH3-2 Next, we analyzed the impact of TCH3-2 on tomato root microbiota using 16S rRNA gene amplicon sequencing (Supplementary Table 4 and Supplementary Fig. 6). TCH3-2 treatment altered bacterial relative abundance of diverse bacterial taxa in both rhizosphere and root endosphere compared to controls (Supplementary Fig. 7). Although TCH3-2 did not affect the richness and evenness of microbiota (Supplementary Fig. 8), microbiota comparison (β-diversity) revealed a significant shift in the rhizosphere bacterial community ( P < 0.01) (Extended Data Fig. 2 ). TCH3-2 enriched a more diverse set of OTUs from various classes both in rhizosphere and in endosphere (Supplementary Figs. 9 and 10, Supplementary Table 5–8). Additionally, the abundance of TCH3-2 were significantly higher in both rhizosphere and root endosphere (Supplementary Fig. 11). These results indicated that the colonization of auxotrophic TCH3-2 alters the microbiota structure in tomato rhizosphere and root endosphere. Rhizosphere microbiota genes stimulated by F. dauae TCH3-2 To investigate the role of TCH3-2 in the rhizosphere microbiota, we analyzed the metatranscriptome of the rhizosphere soil (Supplementary Fig. 12). RNA-seq and de novo assembly produced 1,050,679 contigs, with 40.7% bacterial origin, yielding 223,840 unigenes. DEGseq analysis identified 4,699 upregulated and 4,363 downregulated genes in the TCH3-2 treatment (Supplementary Figs. 13 and 14 and Supplementary Table 9–12). WGCNA analysis identified 16 co-expressed modules, ranging in scales from 152 KOs (darkred) to 1,631 KOs (brown) (Extended Data Fig. 3 and Supplementary Table 13). The blue and greenyellow modules showed the strongest positive (595 KOs) and negative (303 KOs) correlations with TCH3-2 treatment, respectively (Fig. 2 a, Extended Data Fig. 3 c and Supplementary Figs. 14 and 15). GO annotation showed strong enrichment of biosynthetic and metabolic processes, including sterol, thiamine diphosphate, ubiquinone-6, methionine and carbon/nitrogen metabolism, in blue module (Fig. 2 b, Extended Data Fig. 3 d and Supplementary Tables 14 and 15). KEGG enrichment analysis supported these findings, showing that 375 KOs in the blue module were involved in 228 KEGG pathways and 205 modules (Supplementary Tables 16 and 17), with significant enrichment ( p < 0.01) in pathways overlapping key GO terms. Those include sucrose and starch metabolism, oxidative phosphorylation, glycolysis/gluconeogenesis, sterol and steroid biosynthesis, arginine metabolism, and methionine biosynthesis (Supplementary Tables 14 and 16). Expression of rhizosphere microbiota genes involved in steroid biosynthesis We further focused on steroid biosynthesis in blue module, as it was enriched in both GO (GO:0016126) and KEGG (ko00100) terms (Supplementary Tables 14 and 16). All DEGs involved—7-dehydrocholesterol reductase, squalene monooxygenase, farnesyl-diphosphate farnesyltransferase, sterol 14-demethylase, delta24-sterol reductase, and methylsterol monooxygenase—showed high gene significance (> 0.5) with the trait (Fig. 2 c and Supplementary Table 18), and the module also contained 39 genes for biofilm-related processes (Supplementary Table 19). A total of 63 KOs were taken as potential hubs in the blue module, based on high module membership and gene significance (> 0.8) with statistical significance ( P < 0.05). The hub network contained meaningful features connected to hubs, including 66 up-regulated DEGs, 15 biological functions, and 301 interactions (Fig. 2 d). K00213 (7-dehydrocholesterol reductase) in the steroid biosynthesis pathway showed strong interactions (0.27–0.38 TOM) with many upregulated DEGs, including other steroid-related genes (Fig. 2 e). Notably, the upregulated steroid biosynthesis–related DEGs were originated from a taxonomically diverse set of bacteria, including class Actinomycetes, Bacilli, Alphaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, Flavobacteriia, Chlamydiia, Gemmatimonadia, and Planctomycetia (Fig. 2 c-e and Supplementary Table 18). These results indicated that the functional roles of TCH3-2 in rhizosphere microbiota are relevant to stimulate steroid-related pathway of the microbiota. Differential expression of tomato genes under rhizosphere microbiota To assess the impact of TCH3-2 and upland microbiota on tomato, we analyzed tomato root transcriptomes under TCH3-2 and upland soil microbiota using RNA-seq (Extended Data Fig. 4 , Supplementary Fig. 16 and Supplementary Table 20). A total of 1,251 DEGs were identified with 566 up-regulated and 685 down-regulated genes in TCH3-2 treatment (Extended Data Fig. 4 a). GO enrichment analysis of the up-regulated DEGs highlighted hormone-related categories, including gibberellin (GA) and brassinosteroid (BR) (Supplementary Table 21). KEGG analysis revealed that TCH3-2 significantly enriched 12 KEGG pathways, with steroid biosynthesis showing the strongest enrichment, together with terpenoid, zeatin, and hormone signaling (Extended Data Fig. 4 b). Notably, steroid biosynthesis genes were strongly activated among most DEGs involved in converting the precursor farnesyl diphosphate to phytosterols like campesterol and sitosterol, except for SMT1/2 and CYP710A genes (Extended Data Fig. 4 c). These results indicated that the TCH3-2 and native soil microbiota influence the steroid synthesis pathway in tomato plants. Recapitulation of native rhizosphere microbiota activity by synthetic microbial community We investigated whether a purpose-built synthetic microbial community (SynCom) could recapitulate the cooperative plant growth effects of native microbiota by targeting the sterol biosynthesis pathway. From 479 upland soil-derived isolates and type strains, our multi-omics data identified seven sterol-related strains for SynCom assembly: Preistia megaterium UR39 ( Pm ), Novosphingobium guangzhouense UR4 ( Ng ), Gordonia polyisoprenivorans UT158 ( Gp ), Dyella japonica FT133 ( Dj ) were used. In case of Fluviicola taffensis RW262 ( Ft ), Ketobacter alkanivorans KCTC52659 ( Ka ) and Archangium gephyra ATCC25201 ( Ag ), we used the type strains (Fig. 3 a,b and Supplementary Table 22). These bacterial strains harbor diverse sterol biosynthesis-related genes or its homologous genes from Arabidopsis or tomato within their genomes (Fig. 3 b and Supplementary Table 23). Especially, Ka encoded the most sterol biosynthesis homologs (8 genes), whereas TCH3-2 lacked them. All SynCom members and TCH3-2 did not exhibit antagonistic interactions with each other in vitro ; instead, specific combinations of SynCom and TCH3-2 showed positive interactions (Extended Data Fig. 5 ), such as TCH3-2 promoting the growth of Dj and Ka , and SynCom members like Ft , Ng , Dj , and Gp enhancing the growth of other SynCom strains or TCH3-2. The SynCom combined with TCH3-2 (ST) exhibited the highest PGP activity in both soil and hydroponic system, outperforming mixtures with other Flavobacterium strains (Fig. 3 c,d and Supplementary Figs. 17 and 18). However, the individual SynCom members did not show the PGP activity (Supplementary Fig. 19a). In addition, drop-out experiments identified Ka and Ag as essential, with Pm , Gp , and Dj also contributing (Fig. 3 e). However, neither the single treatment of Ka nor the combination treatment of Ka and TCH3-2 promoted the growth of tomato plant (Supplementary Fig. 19b). The ST treatment consistently promoted hypocotyl growth in tomato at 7-, 10-, and 14- days post transplantation, resulting in a progressively greater difference compared with individual treatment (T, S) or loss of Ka (ST-K) treatments, indicative of a cumulative growth-promoting effect (Fig. 3 f and Supplementary Fig. 20). These results showed that the unique cooperation between the upland-mimicking SynCom and TCH3-2 promotes plant growth. To verify whether the combination of SynCom and TCH3-2 induce the activation of sterol synthesis pathway in SynCom, we examined the expression pattern of fdtt1 gene, sqle gene, smo2 gene in Pm , Ft , and Ag under hydroponic condition, respectively (Fig. 3 g). Compared with the S treatment, the fdtt1 gene in Pm , the sqle gene in Ft , and the smo2 gene in Ag were each upregulated by at least two-fold in the ST treatment at 30 min and 5 days after transplantation, respectively (Fig. 3 g). However, lack of Ka in ST (ST-K) down-regulated the expression level of the three genes under hydroponic condition. These results suggested that the cooperation between the SynCom and TCH3-2 elicits sterol-related process in SynCom members. Activation of phytosterol-related pathway in host plant by SynCom with TCH3-2 To determine whether PGP activity of ST involves sterol pathways 41 , we analyzed the expression of BR receptor and sterol biosynthesis genes in tomato root tissues (Fig. 4 a-c, Extended Data Fig. 6 , and Supplementary Table 24). First, ST significantly up-regulated BR receptors SlBRI1 and SlBAK1 42 , compared with the control and individual treatments (Fig. 4 a,b and Extended Data Fig. 6 ). ST also activated 13 phytosterol and BR biosynthesis genes 41 including SlFDFT1 , SlSQLE , SlCPI1 , SlCYP51 , SlHYD2 , SlSMO2 , DHCR7 , SlDWF1 , CYP710A11 , CYP90B3 , SlCPD , SlDET2 , and CYP85A1 (Fig. 4 c and Extended Data Fig. 6 ). The expression pattern of tomato genes under ST treatment match that under natural soil microbiota treated with TCH3-2 (Extended Data Fig. 4 c) except for CYP710A11 31 . ST activated CYP710A11 , encoding sterol C-22 desaturase for sitosterol-stigmasterol conversion in tomato, and its Arabidopsis orthologs CYP710A1 / 2 , which also function as BR C-24 desaturase 43 , while the gene CYP710A11 was not induced under natural microbiota with TCH3-2. ST did not induce the auxin marker SlIAA8 44 but activated SlGID1a , which is suppressed by exogenous gibberellin 45 (Fig. 4 d), indicating that the interaction of SynCom and TCH3-2 specifically activate the sterol-related pathway in tomato plant. To functionally validate SynCom activity, we assessed the effects of ST treatment in a sterol-deficient tomato and Arabidopsis mutants with defects in sterol biosynthesis or signaling (Supplementary Fig. 21, and Fig. 5 ). First, we used the two tomato cultivars including the cultivar Moneymaker and a BR-deficient cultivar Micro-Tom. ST promoted Moneymaker growth but not BR-deficient cultivar Micro-Tom carrying a mutation of CYP85A1 , catalyzing the C-6 oxidation of 6-deoxocastasterone to castasterone, which is active form of brassinosteroid in tomato 46 (Supplementary Fig. 21). This is because the bacterial members of ST lacked homologs of the plant CYP85A1 gene defective in Micro-Tom (Fig. 3 b). We further assessed the PGP activity of ST in sterol- and BR-related Arabidopsis mutants: dwarf1 ( dwf1 ), defective in both BR C24 reductase and sterol C24 reductase activity 47 ; cyp710a1 and cyp710a2 43 , impaired in sterol C-22 desaturase activity for sitosterol-stigmasterol conversion; and the BR-insensitive mutant brassinosteroid insensitive1-301 ( bri1-301 ) 48 , defective in BR signal perception 49 (Fig. 5 ). Interestingly, the complete combination ( i.e. ST) enhanced shoot growth and hypocotyl elongation in wild-type Col-0 and sterol-related mutants ( dwf1 , cyp710a1 , cyp710a2 ) under light and dark conditions, compared to control and its incomplete combination ( i.e. S, T, or ST-K), indicating restoration of sterol- and BR-deficient phenotypes (Fig. 5 a-h). However, ST failed to rescue the growth of bri1-301 (Fig. 5 a-d), indicating its effects act via sterol/BR biosynthesis rather than BR signaling pathway. Notably, the absence of Ka abolished the restore the Arabidopsis BR-related mutants’ phenotype, highlighting its essential role (Fig. 5 ). These results suggest that SynCom with TCH3-2 restores sterol-deficient phenotypes by enhancing sterol/BR biosynthesis. Rhizosphere competency and multispecies biofilm formation by cooperative interaction between SynCom and TCH3-2 TCH3-2 induced biofilm-related gene expression in rhizosphere microbiota (Supplementary Table 19), suggesting cooperative biofilm formation with SynCom and TCH3-2. Individual inoculation of SynCom and TCH3-2 or pairwise combinations of SynCom members with TCH3-2 failed to form biofilms, while ST markedly enhanced biofilm formation in TRM medium (Supplementary Fig. 22). By contrast, SynCom with other Flavobacterium species did not induce biofilm formation (Supplementary Fig. 23). In presence of root exudate, ST significantly enhanced biofilm formation but each SynCom member failed to induce substantial biofilm formation (Fig. 6 a, Supplementary Fig. 24). Notably, lack of specific taxon including Ka , Pm , Ng , and Ag reduced the biofilm formation of ST, highlighting the requirement of both TCH3-2 and key SynCom members (Fig. 6 b). Enhanced biofilm formation by ST significantly increased the total sessile bacterial population on plant roots (Fig. 6 c). While the abundance of TCH3-2 within biofilms declined in the absence of the SynCom, it was stabilized under the ST treatment, and most SynCom members—particularly Ka , Ag , Pm , and Ft —showed significant increases in their root-associated populations (Fig. 6 d, Supplementary Fig. 25, and Supplementary Table 25). In contrast, Gp , Dj , and Ng exhibited only modest and non-significant changes. Notably, total planktonic cell density did not differ significantly among the S, ST, and ST-K treatments (Fig. 6 e), indicating that ST did not promote overall bacterial growth. However, removal of Ka , which impaired biofilm formation, led to a significant reduction in the planktonic populations of Ft , Gp , Ag , and Dj (Fig. 6 f), suggesting destabilization of community structure in the absence of cooperative interactions. Together, these results demonstrate that cooperative interactions in ST support a stable, biofilm-associated microbial community on plant roots. Specialized functional division among SynCom members Building on the finding that TCH3-2 triggers cooperative traits including biofilm formation, root colonization, microbial interaction and PGP activity. We hypothesized that TCH3-2 acts as “a community conductor”, coordinating the SynCom function. Hierarchical clustering of SynCom traits in the presence of TCH3-2 revealed three key factors—(i) the number of sterol-specific genes, (ii) the ability to promote biofilm formation, and (iii) root colonization efficiency—were significantly associated with plant growth promoting (PGP) activity with 95% confidence (Fig. 7 a,b). Principal coordinates analysis (PCoA) further grouped SynCom members into three functional ranks (Fig. 7 c): Rank 1 ( Ka , Ag , and Pm ) specialized in core PGP traits with sterol genes; Rank 2 ( Ft , Ng , and Dj ) supported Rank 1 and Rank 3; Rank 3 ( Gp ) primarily promoted growth of the auxotrophic TCH3-2 (Fig. 7 c). The minimized SynCom composed of Rank 1 and 2 members retained PGP activity, comparable to that observed with the ST treatment, and a cumulative effect was observed only when at least TCH3-2, Ka , Ag , and Ft were present (Supplementary Fig. 26). These results indicate that the functional specialization of SynCom members is orchestrated by TCH3-2, enabling microbiota coalition that collectively promote plant growth. Discussion This study provides the new experimental insight into how microbiota coalition can have beneficial interaction with plant host. This supports the growing concept of plant holobiont. Central to this system is F. dauae TCH3-2, a beneficiary auxotrophic bacterium with streamlined-genome, which depends on metabolic support from neighboring microbes and plant-derived compounds 16 , 30 , 40 , 50 . Although TCH3-2 is a metabolic beneficiary but somehow may acts as “community conductor”, recruiting cooperative partners and modulating community-level coalition function. TCH3-2 functions as a founder cell that initiates multispecies biofilm formation, serving as a driving force for cooperative microbial interactions in the rhizosphere by enhancing colonization (Fig. 6 ) 23 , 24 , 51 – 53 . TCH3-2-initiated multispecies biofilms may contribute to the establishment of a cooperative microbial environment via complex metabolic exchange at the root surface 54 – 56 . Theoretical models predict that communities dominated by competitive interactions are relatively stable, whereas cooperative communities are intrinsically less stable in natural environments due to strong interdependence and the need for close physical proximity 15 , 16 , 26 – 28 , 57 , 58 . Consistent with this framework, loss of keystone taxa in ST perturbed multiple functions linked to plant fitness, indicating cooperation with strong interdependency (Figs. 3 – 6 ). Notably, F. dauae TCH3-2, a beneficiary auxotroph, may act as a community organizer that reinforces interspecies dependency. Once formed, cooperative interactions may enhance metabolic efficiency and enable functional outputs that are not attainable by individual species alone 15 , 16 , 26 – 28 . Our result further suggest that, in the rhizosphere, the multispecies biofilm formed by ST on the root surface provides physical proximity and a spatially structured niche that stabilizes these cooperative interactions, enhances root colonization, and buffers the community against environmental fluctuations 23 , 24 , 59 . Together, these findings support the view that interaction structure rather than microbial diversity plays a central role in determining microbiome function, and that multispecies biofilm–mediated cooperation in ST can stabilize otherwise fragile interactions and confer plant-beneficial effects. Metatranscriptome and host transcriptome analyses revealed that co-inoculation with TCH3-2 and UpMF activated multiple pathways (Fig. 2 ; Extended Data Fig. 4 ). By contrast, a metatranscriptome-based SynCom combined with TCH3-2 selectively recapitulated the cooperative growth-promoting function of UpMF by consistently activating sterol biosynthesis and sterol-responsive gene expression, whereas auxin- and gibberellin-related changes did not align with growth phenotypes or mutant responses (Figs. 4 and 5 ). These results indicate that sterol-related processes constitute a core component of the cooperative traits between UpMF and TCH3-2. ST activated both sterol biosynthesis and responsive genes in tomato plant and SynCom itself (Figs. 3 g, 4 b, and 4 c). Notably, ST activated the entire phytosterol biosynthesis pathway in tomato, from squalene to brassinolide via the campesterol branch or to stigmasterol via the sitosterol branch 41 , 60 . Especially, ST treatment can recover or mitigate dwarf phenotype in BR-deficient dwf1 but not BR-insensitive mutant bri1-301 (Fig. 5 ), indicating ST treatment can enhance plant growth via activation of BR biosynthesis pathway 47 – 49 . Furthermore, ST treatment rescued the growth of cyp710a1 , and cyp710a2 mutants—defective in sitosterol branch, respectively—but only partially restored growth in dwf1 mutants, which lack both campesterol and sitosterol branches 22 , 41 – 43 , 47 . ST treatment might promotes plant growth by increasing some specific intermediates for phytosterol biosynthesis or sterol level like BR or by optimizing the overall sterol balance between campesterol, sitosterol, or stigmasterol in plant 23 – 25 . Although ST-mediated plant growth promotion was linked to sterol-associated processes, we were unable to consistently detect sterol production directly from ST cultures (data not shown). Several factors may account for this limitation. First, strong cooperative interactions among ST members likely occur within spatially confined multispecies biofilms (Fig. 6 ), where metabolic exchange is localized and sterol levels may fall below detection limits. Second, the growth-promoting activity of ST appears to depend on sustained interactions between living plants and the community; cultivation of planktonic communities alone reduced community stability and led to loss or decline of taxa critical for cooperative function (Figs. 3 f, 6 and Supplementary Fig. 20). Third, during plant–microbe interactions, distinguishing plant from microbially derived sterols remains technically challenging, as sterol localization and temporal accumulation are difficult to resolve with conventional assays 23 . Non-invasive in situ mass spectrometry ( e.g. , liquid microjunction surface-sampling probe–mass spectrometry) may enable detection of low-abundance sterols directly from intact biofilms and rhizosphere microenvironments 61 . Our hierarchical model illustrates that microbial members are functionally differentiated under the influence of TCH3-2: Rank 1 interact closely with TCH3-2 and actively colonize the rhizosphere as key taxa contributing to integrated PGP activity via sterol-related unique genes; Rank 2 supports the growth of other groups; Rank 3 primarily support the beneficiary TCH3-2 (Fig. 7 g). This functional specialization, potentially division of labor, and microbial coalition may enhance phytosterol-associated benefits for the host 62 – 66 . Transcriptomic changes in both microbes and the plant, along with Arabidopsis mutant assays, potentially suggest cooperative production of sterol-like compounds (Figs. 2 , 4 , 5 and Extended Data Fig. 4 ). While simple sterols like lanosterol or zymosterol 60 , 67 – 69 occur in some prokaryotes, complex phytosterols remain unique to plants, as key biosynthetic genes are absent in individual bacterial species. This study illuminates the biosynthetic potential of phytosterols by microbiota coalition in a unique niche of host plant. The microbiota may benefit from the fast-growing plant host, gaining access to greater resources and space that enhance its stability and proliferation in the root environment. Further metabolomic analysis is required to identify specific metabolites derived from SynCom stimulated by TCH3-2. It is also essential to characterize specific factors involved in the community stimulating role of F. dauae TCH3-2. Taken together, this study suggests that auxotrophic F. dauae TCH3-2 orchestrates a functionally stratified rhizosphere microbiota, where distinct groups contribute cooperatively to coalition function of microbiota and plant benefit. This may resemble division of labor in microbiota seen in multicellular systems 70 . The division of labor may reduce metabolic burdens of individual community members while enhancing collective function 71 . Rather than acting independently, these microbes interact within a structured community, allowing the microbiota as a whole to exert functions that are not readily achieved by individual taxa alone. Structured as multispecies biofilms initiated by founder cells, such microbiota exhibit emergent properties like functional specialization and host responsiveness, reflecting a potential holobiont-level adaptation 24 , 72 . Microbiota coalitions may vary across ecosystems, depending on the common good between the microbiota conductor and its cooperative partners. While the underlying mechanisms remain to be clarified, our study provides the initial insight on microbiota functional coalition at community-level to expand the functional repertoire of plant-associated microbiota beyond what has been observed in single-strain or limited-consortium studies. By moving beyond reductionist approaches, this work provides a conceptual framework for understanding how cooperative microbial interactions can support plant functions and inform the engineering of resilient plant–microbe systems. Methods Methods, including statements of data availability and any associated accession codes and references, are available in the online version of the paper. Declarations Competing interests: The authors declare no competing financial interests. Author Contributions: SWL and YSS designed the project. SML, JHK, HS, EK, PAL, JUS, SEK, and SYC performed most of plant experiments with microbiota and SynCom. EK, SML, and KC performed microbiome analysis. JP, HHL, HJ, and GH conducted rhizosphere metatranscriptome and tomato transcriptome analysis. SEJ, AAU and GTK conducted Arabidopsis mutant analysis. JHK, HS and SML conducted microbial gene expression and plant gene expression analysis. SML, JHK, HS and KN conducted co-culture experiment and biofilm assay. SML, KN, and JHK performed qPCR for root colonization. JC performed pangenome analysis. SML, JP, HHL, JHK, and SWL wrote a draft. GTK, YSS and SWL edited manuscript. All of the authors read and approved the manuscript before submission. Acknowledgements: We thank Paul Schulze-Lefert at Max Planck Institute for Plant Breeding Research (Cologne, Germany) for his suggestions and for critically reading the manuscript. We also thank Hyoung Ju Lee and Kwang Yeol Baek at Dong-A University for technical assistance on plant experiment. This research was supported by the National Research Foundation of Korea (NRF) grant (No. RS-2020-NR049596 to SWL), Biomaterials Specialized Graduate Program funded by the Korea government (MOE, MCEE) and Research program for Agriculture Science and Technology Development (No. RS-2025-02653099 to SWL and YSS) and Next-Generation BioGreen 21 program to SWL and YSS (PJ01313101 and PJ01313102) through Rural Development Administration, Republic of Korea. 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Nat Commun 7:10508 Joshi RV, Gunawan C, Mann R (2021) We are one: multispecies metabolism of a biofilm consortium and their treatment strategies. Front Microbiol 12:635432 Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTableNatureComm.xlsx Supplementary Tables SupplementaryFigureNatComm.docx ExtendedDataFigureLegends.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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08:43:50","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":192575,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7560912/v1/fc02098e7961c6760d129851.html"},{"id":100560887,"identity":"edc0d93e-36ef-4897-91a4-b6aa7766a24b","added_by":"auto","created_at":"2026-01-19 08:43:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":452349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlant growth promotion of tomato plants (cv. Hawaii 7996) treated with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFlavobacterium dauae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e TCH3-2 and indigenous upland soil microbiota. a,\u003c/strong\u003e Plant growth promotion by TCH3-2 in non-autoclaved upland soil. This photo was taken at 5 weeks post transplantation. \u003cstrong\u003eb,\u003c/strong\u003e The comparison of plant growth promotion (PGP) by TCH3-2 in tomato plants grown at non-autoclaved and autoclaved upland soil. The fresh weight and height of plants were measured at 3 weeks post transplantation. The experiment was conducted in triplicate with 15 plants per treatment and repeated three times with similar results. Significant difference was noticed by two-sample \u003cem\u003eT-\u003c/em\u003etest (***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). Control, sterilized distilled water negative control; TCH3-2, treatment of TCH3-2 suspension. \u003cstrong\u003ec,d,\u003c/strong\u003eThe combination of TCH3-2 and upland soil microbial fraction (UpMF) promoted tomato growth compared to (\u003cstrong\u003ec\u003c/strong\u003e) the single TCH3-2 treatment (\u003cem\u003en\u003c/em\u003e=10 plants, triplicated) and (\u003cstrong\u003ed\u003c/strong\u003e) the UpMF treatment (\u003cem\u003en\u003c/em\u003e=10 plants, repeated four times). Different letters on the box plot in Fig. 1c represent significant difference among means of each treatment by one-way univariate analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) \u003cem\u003epost hoc\u003c/em\u003e test (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). Significant difference was noticed by two-sample \u003cem\u003eT\u003c/em\u003e-test in Fig. 1d (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Diamonds and bolded lines of the boxplot are the average and median of indicated values, respectively. MES, 2.5 mM MES buffer; MES+TCH3-2, treatment of TCH3-2 suspended in 2.5 mM MES buffer; UpMF, UpMF fractionated using a 2.5 mM MES buffer; UpMF+TCH3-2, co-treatment of UpMF and TCH3-2.\u003c/p\u003e","description":"","filename":"MainFigure11.png","url":"https://assets-eu.researchsquare.com/files/rs-7560912/v1/d16acc27c888a6f1fc676a00.png"},{"id":100560856,"identity":"b78ae7ff-2195-4501-9b63-e574eb81ddc3","added_by":"auto","created_at":"2026-01-19 08:43:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1206002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe metatranscriptome analysis of TCH3-2 treated tomato rhizosphere\u003c/strong\u003e \u003cstrong\u003emicrobiota. a,\u003c/strong\u003e Co-expressed modules, highly correlated with the TCH3-2 treatment condition, are depicted in a heatmap. Cell colors and numeric values indicate the correlation coefficients (red, positive; green, negative) and KO counts, respectively. The modules with the strongest connection to the trait are highlighted with a white check mark. The right panel shows module eigengenes for each module in each treatment. High levels of module eigengene reflect high expression pattern of KOs within that module. \u003cstrong\u003eb,\u003c/strong\u003e The GO functional enrichment analysis in the blue modules. The most significantly enriched GO terms belonging to the category biological process with the \u003cem\u003eP\u003c/em\u003e-value \u0026lt;0.01 visualized as a bar graph. An x-axis shows the GO terms and a y-axis indicates the −log\u003csub\u003e10\u003c/sub\u003e\u003cem\u003eP\u003c/em\u003e-value. \u003cstrong\u003ec,\u003c/strong\u003e Phytosterol biosynthesis pathway identified in the blue module. The schematic molecular interaction/reaction network diagram shows that KOs in the blue module are mapped onto the KEGG steroid biosynthesis pathway (ko00100). Two node types are illustrated: rectangular, KO mapped in blue module, and circular, chemical compound. The scale indicates changes in the gene significance, which are represented by a yellow–red color. Bacterial classes identified to carry the genes in each corresponding rectangular node were indicated in the pathway based on metatranscriptome data. \u003cstrong\u003ed,e, \u003c/strong\u003eVisualization of intramodular hub network with biological systems.\u003c/p\u003e","description":"","filename":"MainFigure21.png","url":"https://assets-eu.researchsquare.com/files/rs-7560912/v1/671ce3f6c9eec37aff63eb24.png"},{"id":100594690,"identity":"5e9edcc2-72fe-4f10-8bcb-1c763f679c59","added_by":"auto","created_at":"2026-01-19 13:43:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":431997,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction of upland soil-mimicking SynCom for plant growth promoting activity \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein planta\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a,\u003c/strong\u003e The biosynthesis pathway of phytosterols. \u003cstrong\u003eb,\u003c/strong\u003e The SynCom members predicted to carry the various of genes related to sterol biosynthesis. K, F, P, N, G, A, and D, \u003cem\u003eK. alkanivorans\u003c/em\u003e, \u003cem\u003eF. taffensis\u003c/em\u003e, \u003cem\u003eP. megaterium\u003c/em\u003e, \u003cem\u003eN. guangzhouense\u003c/em\u003e, \u003cem\u003eG. polyisoprenivorans\u003c/em\u003e, \u003cem\u003eA. gephyra\u003c/em\u003e, and \u003cem\u003eD. japonica\u003c/em\u003e, respectively; blue box, the sterol biosynthesis genes identified from SynCom members; orange to yellow box, the putative genes with similarities to the indicated sterol-producing genes. 1, farnesyl-diphosphate farnesyltransferase (KEGG accession: K00801); 2, squalene monooxygenase (K00511); 3, cycloartenol synthase (K01853); 4, SAM-dependent methyltransferase (K00559); 5, 4,4-dimethylsterol C-4alpha-methyl-monooxygenase (K14423); 6\u0026amp;12, 3-beta hydroxysteroid dehydrogenase (K23558); 7, lanosterol synthase (K06045/K01852); 8, Cycloeucalenol cycloisomerase (K08246); 9, sterol 14alpha-demethylase\u0026nbsp;(K05917); 10, 24-methylenesterol C-methyltransferase (K08242); 11, 4alpha-monomethylsterol monooxygenase (K14424); 13, Delta7-sterol 5-desaturase (K00227); 14, Isoprenylcysteine carboxyl methyltransferase (K00213); 15, Delta24-sterol reductase (K09828); 16, sterol 22-desaturase (K09832). (\u003cstrong\u003ec-e\u003c/strong\u003e) The PGP activity of combination of SynCom and TCH3-2. \u003cstrong\u003ec,\u003c/strong\u003e The fresh weight of tomato plant were measured at 5 weeks post transplantation in autoclaved nursery soil. The experiment was conducted in triplicate with 15 plants per treatment and repeated three times with similar results. \u003cstrong\u003ed,\u003c/strong\u003e The hypocotyl length of tomato seedlings were measured at 2 weeks post transplantation under hydroponic condition. The experiment was conducted in triplicate with 12 plants per treatment (\u003cem\u003en \u003c/em\u003e= 36). \u003cstrong\u003ee,\u003c/strong\u003e The drop-out experiment to define keystone taxa in SynCom and TCH3-2. The hypocotyl length of tomato seedlings was measured at 2 weeks post transplantation under hydroponic condition. The experiment was conducted in triplicate with 8 plants per treatment (\u003cem\u003en\u003c/em\u003e=24). Different letters on the box plot represent significant difference among means of each treatment by ANOVA and Tukey’s HSD \u003cem\u003epost hoc\u003c/em\u003e test (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05). \u003cstrong\u003ef,\u003c/strong\u003e Time-course analysis of hypocotyl growth under different treatments. Data are presented as mean ± standard error (SE). Lines connect mean values across time points for each treatment, and shaded areas indicate SE. \u003cstrong\u003eg,\u003c/strong\u003e The expression patterns of sterol synthesis-related genes in SynCom members under hydroponic condition at 30 mins and 5 days post transplantation. \u003cem\u003ePm\u003c/em\u003e_\u003cem\u003efdt1\u003c/em\u003e, farnesyl-diphosphate farnesyltransferase gene in \u003cem\u003eP. megaterium\u003c/em\u003e; \u003cem\u003eFt\u003c/em\u003e_\u003cem\u003esqle\u003c/em\u003e, squalene monooxygenase gene in \u003cem\u003eF. taffensis\u003c/em\u003e; \u003cem\u003eAg\u003c/em\u003e_\u003cem\u003esmo2 \u003c/em\u003e4alpha-monomethylsterol monooxygenase gene in \u003cem\u003eA. gephyra\u003c/em\u003e. Different letters on the box plot indicate statistically significant differences among treatment based on Welch’s \u003cem\u003eT\u003c/em\u003e-test (\u003cem\u003en\u003c/em\u003e = 3; \u003csup\u003e*\u003c/sup\u003e, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; \u003csup\u003e**,\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; \u003csup\u003e***\u003c/sup\u003e, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001). C, control, (c) SDW treatment, (d-g) 2.5 mM MES buffer; T, the treatment of TCH3-2 suspension; S, the treatment of SynCom mixture; ST, the co-treatment of SynCom and TCH3-2; ST-K, ST-F, ST-P, ST-N, ST-G, ST-A and ST-D, the suspension of ST mixtures lacking one strain each, including \u003cem\u003eK. alkanivorans\u003c/em\u003e,\u003cem\u003e F. taffensis\u003c/em\u003e, \u003cem\u003eP. megaterium\u003c/em\u003e, \u003cem\u003eN. guangzhouense\u003c/em\u003e, \u003cem\u003eG. polyisoprenivorans\u003c/em\u003e, \u003cem\u003eA. gephyra\u003c/em\u003e, and \u003cem\u003eD. japonica\u003c/em\u003e, respectively.\u003c/p\u003e","description":"","filename":"MainFigure31.png","url":"https://assets-eu.researchsquare.com/files/rs-7560912/v1/52063e3337ff80c55b43a688.png"},{"id":100560898,"identity":"1d364720-d584-47be-9af6-176b6456baa8","added_by":"auto","created_at":"2026-01-19 08:43:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":293228,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynCom with TCH3-2 activates the BR-signaling and biosynthesis pathway in tomato plants. a,\u003c/strong\u003e The sterol biosynthesis pathway in tomato plant. \u003cstrong\u003eb,c,\u003c/strong\u003e The combination treatment of TCH3-2 and SynCom exhibited a statistically significant increase in the expression of genes involved in (b) BR signaling and (c) phytosterol biosynthesis pathways compared to the control at 3 weeks post-treatment (Extended Data Fig. 6 for statistical comparison). \u003cem\u003eSlFDFT1, \u003c/em\u003efarnesyl-diphosphate farnesyltransferase;\u003cem\u003e SlSQLE, \u003c/em\u003esqualene monooxygenase; \u003cem\u003eSlCPI1, \u003c/em\u003ecycloeucalenol cycloisomerase; \u003cem\u003eSlCYP51,\u003c/em\u003e sterol 14alpha-demethylase; \u003cem\u003eSlHYD2, \u003c/em\u003eDelta14-sterol reductase; \u003cem\u003eSlSMO2, \u003c/em\u003eplant 4 alpha-monomethylsterol monooxygenase; \u003cem\u003eDHCR7, \u003c/em\u003e7-dehydrocholesterol reductase, \u003cem\u003eSlDWF1, \u003c/em\u003edelta24-sterol reductase; \u003cem\u003eCYP710A11, \u003c/em\u003esterol 22-desaturase; \u003cem\u003eCYP90B3, \u003c/em\u003esteroid 22S-hydroxylase, \u003cem\u003eSlCPD, \u003c/em\u003ecytochrome P450 monooxygenase; \u003cem\u003eSlDET2, \u003c/em\u003esteroid 5 alpha reductase; \u003cem\u003eCYP85A1, \u003c/em\u003ebrassinosteroid C-6 Oxidase; C, control; T, TCH3-2; S, SynCom; ST, SynCom + TCH3-2. \u003cstrong\u003ed,\u003c/strong\u003e The expression patterns of auxin marker gene \u003cem\u003eSlIAA8\u003c/em\u003e and gibberellin marker gene \u003cem\u003eSlGID1a\u003c/em\u003e in ST treated tomato roots. \u003cem\u003eSlIAA8\u003c/em\u003e, indole-3-acetic acid (IAA)/Aux gene; \u003cem\u003eSl\u003c/em\u003eGID1a, \u003cem\u003eGIBBERELLIN INSENSITIVE DWARF1 A\u003c/em\u003e gene. Different letters on the box plot represent significant difference among means of each treatment by ANOVA and Tukey’s HSD \u003cem\u003epost hoc\u003c/em\u003e test (\u003cem\u003en\u003c/em\u003e = 3 samples, triplicated).\u003c/p\u003e","description":"","filename":"MainFigure41.png","url":"https://assets-eu.researchsquare.com/files/rs-7560912/v1/f8d6ad3ff282ce16d11466ad.png"},{"id":100560971,"identity":"49cb51b8-c641-4424-9296-82221db009d8","added_by":"auto","created_at":"2026-01-19 08:43:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":897538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlant growth promotion of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e wild type and sterol-related mutants\u003c/strong\u003e \u003cstrong\u003eby SynCom and TCH3-2 treatment.\u003c/strong\u003e \u003cstrong\u003ea,b\u003c/strong\u003e The plant growth by SynCom and TCH3-2 in \u003cem\u003eArabidopsis\u003c/em\u003e wild type and BR-related mutants under the light condition. The experiment was conducted in triplicate (\u003cem\u003en\u003c/em\u003e= 8).\u003cstrong\u003e c,d,\u003c/strong\u003e The hypocotyl growth by SynCom and TCH3-2 in \u003cem\u003eArabidopsis\u003c/em\u003e wild type and BR-related mutants under the dark condition. The experiment was conducted in triplicate (\u003cem\u003en\u003c/em\u003e= 8). \u003cstrong\u003ee,f,\u003c/strong\u003e The plant growth by SynCom and TCH3-2 in \u003cem\u003eArabidopsis\u003c/em\u003e wild type and stigmasterol and dolichosterone-deficient mutants under the light condition. The experiment was conducted in triplicate (\u003cem\u003en\u003c/em\u003e= 6). \u003cstrong\u003eg,h,\u003c/strong\u003e The hypocotyl growth by SynCom and TCH3-2 in \u003cem\u003eArabidopsis\u003c/em\u003e wild type and stigmasterol and dolichosterone-deficient mutants under the dark condition. The experiment was conducted in triplicate (\u003cem\u003en\u003c/em\u003e = 6). Statistical analysis were conducted separately for each \u003cem\u003eArabidopsis\u003c/em\u003e genotype. Different letters on the box plot represent significant difference among means of each treatment by ANOVA and Tukey’s HSD \u003cem\u003epost hoc\u003c/em\u003e test (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05). C, control; T, TCH3-2; S, SynCom; ST, SynCom + TCH3-2; ST-K, ST without \u003cem\u003eK. alkanivorans\u003c/em\u003e.; Col-0, Arabidopsis wild type Colombia-0; \u003cem\u003edwf1,\u003c/em\u003e knockout mutant of \u003cem\u003eDWARF 1 (DWF1)\u003c/em\u003e gene encoding a sterol C-24 reductase (SALK_006932); \u003cem\u003ebri1-301\u003c/em\u003e, knockout mutant of \u003cem\u003eBRASSINOSTEROID INSENSITIVE1 \u003c/em\u003e(\u003cem\u003eBRI1\u003c/em\u003e) gene; \u003cem\u003ecyp710a1\u003c/em\u003e (SALK_014626)\u003cem\u003e \u003c/em\u003eand\u003cem\u003e cyp710a2 \u003c/em\u003e(SALK_001175); knockout mutant of CYP710a encoding a sterol C-22 desaturase.\u003c/p\u003e","description":"","filename":"MainFigure51.png","url":"https://assets-eu.researchsquare.com/files/rs-7560912/v1/3cb7466a63a891eb60505cf0.png"},{"id":100560920,"identity":"a40acaf2-80b8-4c2c-8d60-31fddc4e909d","added_by":"auto","created_at":"2026-01-19 08:43:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":65496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCooperative biofilm formation between TCH3-2 and SynCom stabilizes rhizosphere microbial communities. a,\u003c/strong\u003e Biofilm formation by SynCom and TCH3-2 combination in TRM medium supplemented with root exudate \u003cstrong\u003eb,\u003c/strong\u003e Dropping out of SynCom member reduces the multispecies biofilm formation in TRM medium supplemented with root exudate. The biofilm assay was in triplicates with the 6 wells per treatment. Different letters on the box plot represent significant difference among means of each treatment by ANOVA and Tukey’s HSD \u003cem\u003epost hoc\u003c/em\u003e test (\u003cem\u003en\u003c/em\u003e = 18 samples). \u003cstrong\u003ec,\u003c/strong\u003e Total sessile bacterial population in the multispecies biofilms. Different letters on the box plot represent significant difference among means of each treatment by ANOVA and Tukey’s HSD \u003cem\u003epost hoc\u003c/em\u003e test (\u003cem\u003en\u003c/em\u003e = 3, triplicated). \u003cstrong\u003ed,\u003c/strong\u003e Sessile bacterial population dynamics in multispecies biofilm attached on tomato root surface. The experiment was conducted in triplicate (\u003cem\u003en \u003c/em\u003e= 9)\u003cem\u003e.\u003c/em\u003e Significant difference was noticed by two-sample \u003cem\u003eT\u003c/em\u003e-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt;0 .01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). C, control (TRM broth with sterilized tomato root exudates) treatment; T, the treatment of TCH3-2 suspension; S, the treatment of SynCom mixture; ST, the co-treatment of of SynCom and TCH3-2; ST-K, ST-F, ST-P, ST-N, ST-G, ST-A and ST-D, the suspension of ST mixtures lacking one strain each, including \u003cem\u003eK. alkanivorans \u003c/em\u003e(\u003cem\u003eKa\u003c/em\u003e),\u003cem\u003e F. taffensis \u003c/em\u003e(\u003cem\u003eFt\u003c/em\u003e), \u003cem\u003eP. megaterium \u003c/em\u003e(\u003cem\u003ePm\u003c/em\u003e), \u003cem\u003eN. guangzhouense\u003c/em\u003e (\u003cem\u003eNg\u003c/em\u003e), \u003cem\u003eG. polyisoprenivorans \u003c/em\u003e(\u003cem\u003eGp\u003c/em\u003e), \u003cem\u003eA. gephyra \u003c/em\u003e(\u003cem\u003eAg\u003c/em\u003e), and \u003cem\u003eD. japonica \u003c/em\u003e(\u003cem\u003eDj\u003c/em\u003e), respectively. \u003cstrong\u003ee, \u003c/strong\u003ePlanktonic cell density (OD\u003csub\u003e600\u003c/sub\u003e) of TCH3-2 and SynCom culture solution at 2 days after inoculation using TRM medium. Different letters on the box plot represent significant difference among means of each treatment by ANOVA and Tukey’s HSD \u003cem\u003epost hoc\u003c/em\u003e test (\u003cem\u003en\u003c/em\u003e = 2 per treatment, triplicated). \u003cstrong\u003ef, \u003c/strong\u003ePlanktonic bacterial population dynamics in ST-K culture solution at 0 and 2 days after inoculation using TRM medium. Different letters on the box plot represent significant difference among means of each treatment by ANOVA and Tukey’s HSD \u003cem\u003epost hoc\u003c/em\u003e test (Day 0, \u003cem\u003en\u003c/em\u003e = 8; Day 2, \u003cem\u003en\u003c/em\u003e = 12; triplicated).\u003c/p\u003e","description":"","filename":"MainFigure61.png","url":"https://assets-eu.researchsquare.com/files/rs-7560912/v1/0815ba4c6ca3c3c4a5630b0b.png"},{"id":100560822,"identity":"bbcdd006-0734-4c7d-925d-b87dd13fe87e","added_by":"auto","created_at":"2026-01-19 08:43:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":103629,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional characterization of each SynCom member in rhizosphere for plant growth promotion. a,\u003c/strong\u003e Hierarchical clustering heatmap based on seven functional traits across seven SynCom members. Trait values include number of sterol-related unique genes (Sterol genes), contribution to plant growth promotion (PGP activity), contribution to biofilm formation (Biofilm formation), contribution to root colonization (Root colonization), support to TCH3-2 growth (TCH3-2 growth), and contribution to other SynCom member growth (SynCom growth). Euclidean distance and Ward's minimum variance method (ward.D2) were used to construct the dendrogram. Color intensity reflects the magnitude of trait values, and numbers in cells indicate exact values. \u003cstrong\u003eb,\u003c/strong\u003eDendrogram showing trait-based clustering of bacterial species using Euclidean distance and Ward.D2 linkage (Bootstrap value: 1,000). The x-axis represents the degree of Euclidean dissimilarity between clusters. This analysis highlights distinct functional groupings based on combined trait profiles. au, Approximately unbiased \u003cem\u003eP\u003c/em\u003e-value; bp, Bootstrap probability. \u003cstrong\u003ec,\u003c/strong\u003e Principal coordinates analysis (PCoA) of bacterial strains based on six functional traits using Euclidean dissimilarity. Arrows show trait influence on the ordination analyzed by \u003cem\u003eenvfit\u003c/em\u003e, with length proportional to the coefficient of determination (\u003cem\u003er²\u003c/em\u003e). \u003cem\u003eKa, K. alkanivorans\u003c/em\u003e; \u003cem\u003eAg, A. gephyra\u003c/em\u003e;\u003cem\u003e Pm, P. megaterium\u003c/em\u003e;\u003cem\u003eFt, F. taffensis\u003c/em\u003e; \u003cem\u003eNg\u003c/em\u003e, \u003cem\u003eN. guangzhouense\u003c/em\u003e;\u003cem\u003e Dj\u003c/em\u003e,\u003cem\u003eD. japonica\u003c/em\u003e; \u003cem\u003eGp\u003c/em\u003e, \u003cem\u003eG. polyisoprenivorans\u003c/em\u003e. Colors indicate functional rank: red, rank 1; blue, rank 2; green, rank 3. \u003cstrong\u003ed.\u003c/strong\u003e A proposed model for TCH3-2-mediated microbiota coalition in the rhizosphere. Auxotrophic TCH3-2 initiates microbial cooperation via early colonization and biofilm formation, inducing functional specialization among rhizosphere microbes. Rank 1 bacteria closely interact with TCH3-2, promote its biofilm, and contribute plant growth promotion via unique sterol-related genes. Rank 2 acts as a mediator supporting Rank 1 and Rank 3, while Rank 3 specializes in supporting TCH3-2 growth. This community-level cooperation represents microbiota coalition and may induce sterol precursor production or modulate plant phytosterol/BR pathways, enhancing plant growth.\u003c/p\u003e","description":"","filename":"MainFigure71.png","url":"https://assets-eu.researchsquare.com/files/rs-7560912/v1/3fd6945c639039fe1374c4f3.png"},{"id":100597260,"identity":"af8bbf7c-ec3d-4219-9b3a-f4d81fd44295","added_by":"auto","created_at":"2026-01-19 14:16:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4288582,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7560912/v1/f417fbf4-65ea-4085-9ce6-3d12194cfdff.pdf"},{"id":100561023,"identity":"66da067a-14b7-436e-8bb5-f5a8422699ef","added_by":"auto","created_at":"2026-01-19 08:43:55","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2256812,"visible":true,"origin":"","legend":"Supplementary Tables","description":"","filename":"SupplementaryTableNatureComm.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7560912/v1/036ccf48f4e0f5a31e6f395d.xlsx"},{"id":100560876,"identity":"f9331669-dd68-4eb6-9c4f-51c8d0d17d7f","added_by":"auto","created_at":"2026-01-19 08:43:52","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6013550,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureNatComm.docx","url":"https://assets-eu.researchsquare.com/files/rs-7560912/v1/5d1ad14c47ec212bfcafb5d3.docx"},{"id":100560621,"identity":"0000fab4-3cd9-4a83-8d7f-c3398d1491e5","added_by":"auto","created_at":"2026-01-19 08:43:49","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18792,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFigureLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-7560912/v1/9b0b127f13dc80d1ea5f4db3.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Plant Growth Benefits by the Coalition Function of Plant Microbiome","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant-associated microbiota influences host plant fitness, including growth, development, and stress tolerance, collectively contributing to the functional traits of the plant holobiont\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, the functional coalition of microbial communities within the plant holobiont remains poorly characterized. Like other communities, microbial interactions are an important component for microbiota function in the plant rhizosphere\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Furthermore, metabolic interactions between living plant roots and microbiota, as well as microbial species, are key drivers of microbial cooperation in the rhizosphere\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, while our understanding of the metabolic interactions is limited. Cooperative microbial interactions stabilize rhizosphere communities through metabolic exchanges among microbes and with the host\u003csup\u003e3,4,8\u0026ndash;10,12\u0026minus;16\u003c/sup\u003e. Specific microbes may enhance phytohormone pathways, including auxin, gibberellin, salicylic acid, jasmonic acid, and abscisic acid, however, the effects of microbial community coalition on complex phytohormonal signaling in crop plants remain poorly characterized\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNegative interactions between microbial species, such as competition and antagonism, are generally predominant in natural microbiota\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Nevertheless, recent studies indicate that cooperative interactions of microbial species can elicit activities within the microbiota that support host plant performance, including plant growth promotion, stress tolerance, and disease control\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Compared with negative interactions, positive interactions such as cooperation typically requires close physical proximity and interdependence among species, resulting in the formation of microbial coalition in natural microbiota\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Once established, microbial coalition formed through cooperative interactions can enhance functional robustness against environmental perturbations and facilitate the evolution of complex traits within microbial communities\u003csup\u003e\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In this context, multi-species biofilms not only enhance root colonization but also provide a cooperative state that stabilizes interactions among microbes and between microbes and the host plant\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, how such community-level cooperation leading to microbiota coalition influences host plant performance remains poorly characterized.\u003c/p\u003e \u003cp\u003ePlants produce a diverse array of phytosterols, such as campesterol, sitosterol, and stigmasterol\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Phytosterols function not only as precursors of plant growth hormones like brassinosteroids but also as key regulators of plant responses to biotic and abiotic stresses\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Notably, fine-tuning of sterol composition can modulate plant responses to diverse microbes, particularly phytopathogens\u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34 CR35\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. During plant\u0026ndash;pathogen interactions, the equilibrium of phytosterols in the plasma membrane can shift\u003csup\u003e\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. These alterations affect plasma membrane composition and permeability\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. However, whether and how beneficial plant-associated microbiota collectively influence host sterol-related processes through cooperative interactions remains unclear\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere we employed multi-omics approaches and a synthetic community (SynCom) approach to tomato rhizosphere to investigate how rhizosphere microbiota functions as a unified cooperative population. SynComs have been developed as a reductionist approach to dissect the mechanisms underlying microbiota function\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Typically, the design of SynCom primarily rely on the quantitative dynamics of microbial communities to achieve targeted agricultural traits\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Based on gene expression patterns in both rhizosphere microbiota and host plant, we hypothesized that the rhizosphere microbiota occupies an ecological niche as a functionally integrated consortium, \u003cem\u003ei.e.\u003c/em\u003e microbiota coalition, through cooperative interactions. To test this, we used cooperative rhizosphere microbiota stimulated by \u003cem\u003eFlavobacterium dauae\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and tomato plant. Upon stimulation, the phytosterol biosynthesis pathway was activated in both the rhizosphere microbiota and tomato plant, indicating coordinated interactions that contribute to plant growth. We further demonstrated that SynCom recapitulates the activity of the native rhizosphere microbiota through multi-species biofilm formation and functional specialization among SynCom members.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eMicrobial stimulator for plant growth promotion with native soil microbiota.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePreviously, we identified an auxotrophic \u003cem\u003eF. dauae\u003c/em\u003e TCH3-2 from the tomato rhizosphere, which exhibited the strongest plant growth-promoting activity among 32 \u003cem\u003eFlavobacterium\u003c/em\u003e isolates\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Compared to other \u003cem\u003eFlavobacterium\u003c/em\u003e species genomes, TCH3-2 genome is highly reduced (2.9 Mb), lacking most growth factor biosynthesis genes and indicating strict auxotrophy (Supplementary Fig.\u0026nbsp;1, Supplementary Table\u0026nbsp;1). Unique genes present in TCH3-2 genome included those for transposases, Type IX secretion, proline iminopeptidase, N-acetylglutamate synthase, DoxX-like proteins, and the flavanone-binding protein YndB (Supplementary Tables\u0026nbsp;2 and 3). The treatment of TCH3-2 significantly increased the fresh weight, dry weight, height, stem width, the number of flowers and fruits in tomato plants grown in upland soil compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea,b and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Fig.\u0026nbsp;2). However, the plant growth-promoting (PGP) activity by TCH3-2 treatment was completely diminished in the autoclaved upland soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;2). The PGP activity by TCH3-2 treatment was not specific to soil type or tomato cultivar (Supplementary Figs.\u0026nbsp;3 and 4). Co-treatment of TCH3-2 with the microbial fraction of upland soil (UpMF), independent of soil particles, promoted tomato growth compared to individual treatments and the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec,d). In addition, another closely-related \u003cem\u003eFlavobacterium\u003c/em\u003e strains did not promote tomato growth with the UpMF (Supplementary Fig.\u0026nbsp;5). In particular, the upland soil-derived strain \u003cem\u003eF. daejeonense\u003c/em\u003e RCH33\u003csup\u003e7\u003c/sup\u003e, which possesses PGP potential through auxin production, failed to promote tomato growth in the presence of the UpMF (Supplementary Fig.\u0026nbsp;5a,b). These results indicated a unique cooperative interaction between TCH3-2 and the indigenous soil microbiota for PGP activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMicrobiome alteration by the stimulatory\u003c/b\u003e \u003cb\u003eFlavobacterium dauae\u003c/b\u003e \u003cb\u003eTCH3-2\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNext, we analyzed the impact of TCH3-2 on tomato root microbiota using 16S rRNA gene amplicon sequencing (Supplementary Table\u0026nbsp;4 and Supplementary Fig.\u0026nbsp;6). TCH3-2 treatment altered bacterial relative abundance of diverse bacterial taxa in both rhizosphere and root endosphere compared to controls (Supplementary Fig.\u0026nbsp;7). Although TCH3-2 did not affect the richness and evenness of microbiota (Supplementary Fig.\u0026nbsp;8), microbiota comparison (β-diversity) revealed a significant shift in the rhizosphere bacterial community (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). TCH3-2 enriched a more diverse set of OTUs from various classes both in rhizosphere and in endosphere (Supplementary Figs.\u0026nbsp;9 and 10, Supplementary Table\u0026nbsp;5\u0026ndash;8). Additionally, the abundance of TCH3-2 were significantly higher in both rhizosphere and root endosphere (Supplementary Fig.\u0026nbsp;11). These results indicated that the colonization of auxotrophic TCH3-2 alters the microbiota structure in tomato rhizosphere and root endosphere.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRhizosphere microbiota genes stimulated by\u003c/b\u003e \u003cb\u003eF. dauae\u003c/b\u003e \u003cb\u003eTCH3-2\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the role of TCH3-2 in the rhizosphere microbiota, we analyzed the metatranscriptome of the rhizosphere soil (Supplementary Fig.\u0026nbsp;12). RNA-seq and \u003cem\u003ede novo\u003c/em\u003e assembly produced 1,050,679 contigs, with 40.7% bacterial origin, yielding 223,840 unigenes. DEGseq analysis identified 4,699 upregulated and 4,363 downregulated genes in the TCH3-2 treatment (Supplementary Figs.\u0026nbsp;13 and 14 and Supplementary Table\u0026nbsp;9\u0026ndash;12). WGCNA analysis identified 16 co-expressed modules, ranging in scales from 152 KOs (darkred) to 1,631 KOs (brown) (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Supplementary Table\u0026nbsp;13). The blue and greenyellow modules showed the strongest positive (595 KOs) and negative (303 KOs) correlations with TCH3-2 treatment, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and Supplementary Figs.\u0026nbsp;14 and 15). GO annotation showed strong enrichment of biosynthetic and metabolic processes, including sterol, thiamine diphosphate, ubiquinone-6, methionine and carbon/nitrogen metabolism, in blue module (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and Supplementary Tables\u0026nbsp;14 and 15). KEGG enrichment analysis supported these findings, showing that 375 KOs in the blue module were involved in 228 KEGG pathways and 205 modules (Supplementary Tables\u0026nbsp;16 and 17), with significant enrichment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in pathways overlapping key GO terms. Those include sucrose and starch metabolism, oxidative phosphorylation, glycolysis/gluconeogenesis, sterol and steroid biosynthesis, arginine metabolism, and methionine biosynthesis (Supplementary Tables\u0026nbsp;14 and 16).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExpression of rhizosphere microbiota genes involved in steroid biosynthesis\u003c/h2\u003e \u003cp\u003eWe further focused on steroid biosynthesis in blue module, as it was enriched in both GO (GO:0016126) and KEGG (ko00100) terms (Supplementary Tables\u0026nbsp;14 and 16). All DEGs involved\u0026mdash;7-dehydrocholesterol reductase, squalene monooxygenase, farnesyl-diphosphate farnesyltransferase, sterol 14-demethylase, delta24-sterol reductase, and methylsterol monooxygenase\u0026mdash;showed high gene significance (\u0026gt;\u0026thinsp;0.5) with the trait (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and Supplementary Table\u0026nbsp;18), and the module also contained 39 genes for biofilm-related processes (Supplementary Table\u0026nbsp;19). A total of 63 KOs were taken as potential hubs in the blue module, based on high module membership and gene significance (\u0026gt;\u0026thinsp;0.8) with statistical significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The hub network contained meaningful features connected to hubs, including 66 up-regulated DEGs, 15 biological functions, and 301 interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). K00213 (7-dehydrocholesterol reductase) in the steroid biosynthesis pathway showed strong interactions (0.27\u0026ndash;0.38 TOM) with many upregulated DEGs, including other steroid-related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Notably, the upregulated steroid biosynthesis\u0026ndash;related DEGs were originated from a taxonomically diverse set of bacteria, including class Actinomycetes, Bacilli, Alphaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, Flavobacteriia, Chlamydiia, Gemmatimonadia, and Planctomycetia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-e and Supplementary Table\u0026nbsp;18). These results indicated that the functional roles of TCH3-2 in rhizosphere microbiota are relevant to stimulate steroid-related pathway of the microbiota.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDifferential expression of tomato genes under rhizosphere microbiota\u003c/h3\u003e\n\u003cp\u003eTo assess the impact of TCH3-2 and upland microbiota on tomato, we analyzed tomato root transcriptomes under TCH3-2 and upland soil microbiota using RNA-seq (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Fig.\u0026nbsp;16 and Supplementary Table\u0026nbsp;20). A total of 1,251 DEGs were identified with 566 up-regulated and 685 down-regulated genes in TCH3-2 treatment (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). GO enrichment analysis of the up-regulated DEGs highlighted hormone-related categories, including gibberellin (GA) and brassinosteroid (BR) (Supplementary Table\u0026nbsp;21). KEGG analysis revealed that TCH3-2 significantly enriched 12 KEGG pathways, with steroid biosynthesis showing the strongest enrichment, together with terpenoid, zeatin, and hormone signaling (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Notably, steroid biosynthesis genes were strongly activated among most DEGs involved in converting the precursor farnesyl diphosphate to phytosterols like campesterol and sitosterol, except for \u003cem\u003eSMT1/2\u003c/em\u003e and \u003cem\u003eCYP710A\u003c/em\u003e genes (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). These results indicated that the TCH3-2 and native soil microbiota influence the steroid synthesis pathway in tomato plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eRecapitulation of native rhizosphere microbiota activity by synthetic microbial community\u003c/h3\u003e\n\u003cp\u003eWe investigated whether a purpose-built synthetic microbial community (SynCom) could recapitulate the cooperative plant growth effects of native microbiota by targeting the sterol biosynthesis pathway. From 479 upland soil-derived isolates and type strains, our multi-omics data identified seven sterol-related strains for SynCom assembly: \u003cem\u003ePreistia megaterium\u003c/em\u003e UR39 (\u003cem\u003ePm\u003c/em\u003e), \u003cem\u003eNovosphingobium guangzhouense\u003c/em\u003e UR4 (\u003cem\u003eNg\u003c/em\u003e), \u003cem\u003eGordonia polyisoprenivorans\u003c/em\u003e UT158 (\u003cem\u003eGp\u003c/em\u003e), \u003cem\u003eDyella japonica\u003c/em\u003e FT133 (\u003cem\u003eDj\u003c/em\u003e) were used. In case of \u003cem\u003eFluviicola taffensis\u003c/em\u003e RW262 (\u003cem\u003eFt\u003c/em\u003e), \u003cem\u003eKetobacter alkanivorans\u003c/em\u003e KCTC52659 (\u003cem\u003eKa\u003c/em\u003e) and \u003cem\u003eArchangium gephyra\u003c/em\u003e ATCC25201 (\u003cem\u003eAg\u003c/em\u003e), we used the type strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b and Supplementary Table\u0026nbsp;22). These bacterial strains harbor diverse sterol biosynthesis-related genes or its homologous genes from \u003cem\u003eArabidopsis\u003c/em\u003e or tomato within their genomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and Supplementary Table\u0026nbsp;23). Especially, \u003cem\u003eKa\u003c/em\u003e encoded the most sterol biosynthesis homologs (8 genes), whereas TCH3-2 lacked them. All SynCom members and TCH3-2 did not exhibit antagonistic interactions with each other \u003cem\u003ein vitro\u003c/em\u003e; instead, specific combinations of SynCom and TCH3-2 showed positive interactions (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), such as TCH3-2 promoting the growth of \u003cem\u003eDj\u003c/em\u003e and \u003cem\u003eKa\u003c/em\u003e, and SynCom members like \u003cem\u003eFt\u003c/em\u003e, \u003cem\u003eNg\u003c/em\u003e, \u003cem\u003eDj\u003c/em\u003e, and \u003cem\u003eGp\u003c/em\u003e enhancing the growth of other SynCom strains or TCH3-2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe SynCom combined with TCH3-2 (ST) exhibited the highest PGP activity in both soil and hydroponic system, outperforming mixtures with other \u003cem\u003eFlavobacterium\u003c/em\u003e strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec,d and Supplementary Figs.\u0026nbsp;17 and 18). However, the individual SynCom members did not show the PGP activity (Supplementary Fig.\u0026nbsp;19a). In addition, drop-out experiments identified \u003cem\u003eKa\u003c/em\u003e and \u003cem\u003eAg\u003c/em\u003e as essential, with \u003cem\u003ePm\u003c/em\u003e, \u003cem\u003eGp\u003c/em\u003e, and \u003cem\u003eDj\u003c/em\u003e also contributing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). However, neither the single treatment of \u003cem\u003eKa\u003c/em\u003e nor the combination treatment of \u003cem\u003eKa\u003c/em\u003e and TCH3-2 promoted the growth of tomato plant (Supplementary Fig.\u0026nbsp;19b). The ST treatment consistently promoted hypocotyl growth in tomato at 7-, 10-, and 14- days post transplantation, resulting in a progressively greater difference compared with individual treatment (T, S) or loss of \u003cem\u003eKa\u003c/em\u003e (ST-K) treatments, indicative of a cumulative growth-promoting effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef and Supplementary Fig.\u0026nbsp;20). These results showed that the unique cooperation between the upland-mimicking SynCom and TCH3-2 promotes plant growth.\u003c/p\u003e \u003cp\u003eTo verify whether the combination of SynCom and TCH3-2 induce the activation of sterol synthesis pathway in SynCom, we examined the expression pattern of \u003cem\u003efdtt1\u003c/em\u003egene, \u003cem\u003esqle\u003c/em\u003e gene, \u003cem\u003esmo2\u003c/em\u003e gene in \u003cem\u003ePm\u003c/em\u003e, \u003cem\u003eFt\u003c/em\u003e, and \u003cem\u003eAg\u003c/em\u003e under hydroponic condition, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). Compared with the S treatment, the \u003cem\u003efdtt1\u003c/em\u003e gene in \u003cem\u003ePm\u003c/em\u003e, the \u003cem\u003esqle\u003c/em\u003e gene in \u003cem\u003eFt\u003c/em\u003e, and the \u003cem\u003esmo2\u003c/em\u003e gene in \u003cem\u003eAg\u003c/em\u003e were each upregulated by at least two-fold in the ST treatment at 30 min and 5 days after transplantation, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). However, lack of \u003cem\u003eKa\u003c/em\u003e in ST (ST-K) down-regulated the expression level of the three genes under hydroponic condition. These results suggested that the cooperation between the SynCom and TCH3-2 elicits sterol-related process in SynCom members.\u003c/p\u003e\n\u003ch3\u003eActivation of phytosterol-related pathway in host plant by SynCom with TCH3-2\u003c/h3\u003e\n\u003cp\u003eTo determine whether PGP activity of ST involves sterol pathways\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, we analyzed the expression of BR receptor and sterol biosynthesis genes in tomato root tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, and Supplementary Table\u0026nbsp;24). First, ST significantly up-regulated BR receptors \u003cem\u003eSlBRI1\u003c/em\u003e and \u003cem\u003eSlBAK1\u003c/em\u003e\u003csup\u003e42\u003c/sup\u003e, compared with the control and individual treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). ST also activated 13 phytosterol and BR biosynthesis genes\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e including \u003cem\u003eSlFDFT1\u003c/em\u003e, \u003cem\u003eSlSQLE\u003c/em\u003e, \u003cem\u003eSlCPI1\u003c/em\u003e, \u003cem\u003eSlCYP51\u003c/em\u003e, \u003cem\u003eSlHYD2\u003c/em\u003e, \u003cem\u003eSlSMO2\u003c/em\u003e, \u003cem\u003eDHCR7\u003c/em\u003e, \u003cem\u003eSlDWF1\u003c/em\u003e, \u003cem\u003eCYP710A11\u003c/em\u003e, \u003cem\u003eCYP90B3\u003c/em\u003e, \u003cem\u003eSlCPD\u003c/em\u003e, \u003cem\u003eSlDET2\u003c/em\u003e, and \u003cem\u003eCYP85A1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The expression pattern of tomato genes under ST treatment match that under natural soil microbiota treated with TCH3-2 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) except for \u003cem\u003eCYP710A11\u003c/em\u003e\u003csup\u003e31\u003c/sup\u003e. ST activated \u003cem\u003eCYP710A11\u003c/em\u003e, encoding sterol C-22 desaturase for sitosterol-stigmasterol conversion in tomato, and its \u003cem\u003eArabidopsis\u003c/em\u003e orthologs \u003cem\u003eCYP710A1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e, which also function as BR C-24 desaturase\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, while the gene \u003cem\u003eCYP710A11\u003c/em\u003e was not induced under natural microbiota with TCH3-2. ST did not induce the auxin marker \u003cem\u003eSlIAA8\u003c/em\u003e\u003csup\u003e44\u003c/sup\u003e but activated \u003cem\u003eSlGID1a\u003c/em\u003e, which is suppressed by exogenous gibberellin\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), indicating that the interaction of SynCom and TCH3-2 specifically activate the sterol-related pathway in tomato plant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo functionally validate SynCom activity, we assessed the effects of ST treatment in a sterol-deficient tomato and \u003cem\u003eArabidopsis\u003c/em\u003e mutants with defects in sterol biosynthesis or signaling (Supplementary Fig.\u0026nbsp;21, and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). First, we used the two tomato cultivars including the cultivar Moneymaker and a BR-deficient cultivar Micro-Tom. ST promoted Moneymaker growth but not BR-deficient cultivar Micro-Tom carrying a mutation of \u003cem\u003eCYP85A1\u003c/em\u003e, catalyzing the C-6 oxidation of 6-deoxocastasterone to castasterone, which is active form of brassinosteroid in tomato\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e(Supplementary Fig.\u0026nbsp;21). This is because the bacterial members of ST lacked homologs of the plant \u003cem\u003eCYP85A1\u003c/em\u003e gene defective in Micro-Tom (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). We further assessed the PGP activity of ST in sterol- and BR-related \u003cem\u003eArabidopsis\u003c/em\u003e mutants: \u003cem\u003edwarf1\u003c/em\u003e (\u003cem\u003edwf1\u003c/em\u003e), defective in both BR C24 reductase and sterol C24 reductase activity\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e; \u003cem\u003ecyp710a1\u003c/em\u003e and \u003cem\u003ecyp710a2\u003c/em\u003e\u003csup\u003e43\u003c/sup\u003e, impaired in sterol C-22 desaturase activity for sitosterol-stigmasterol conversion; and the BR-insensitive mutant \u003cem\u003ebrassinosteroid insensitive1-301\u003c/em\u003e (\u003cem\u003ebri1-301\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, defective in BR signal perception\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Interestingly, the complete combination (\u003cem\u003ei.e.\u003c/em\u003e ST) enhanced shoot growth and hypocotyl elongation in wild-type Col-0 and sterol-related mutants (\u003cem\u003edwf1\u003c/em\u003e, \u003cem\u003ecyp710a1\u003c/em\u003e, \u003cem\u003ecyp710a2\u003c/em\u003e) under light and dark conditions, compared to control and its incomplete combination (\u003cem\u003ei.e.\u003c/em\u003e S, T, or ST-K), indicating restoration of sterol- and BR-deficient phenotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-h). However, ST failed to rescue the growth of \u003cem\u003ebri1-301\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d), indicating its effects act via sterol/BR biosynthesis rather than BR signaling pathway. Notably, the absence of \u003cem\u003eKa\u003c/em\u003e abolished the restore the \u003cem\u003eArabidopsis\u003c/em\u003e BR-related mutants\u0026rsquo; phenotype, highlighting its essential role (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results suggest that SynCom with TCH3-2 restores sterol-deficient phenotypes by enhancing sterol/BR biosynthesis.\u003c/p\u003e\n\u003ch3\u003eRhizosphere competency and multispecies biofilm formation by cooperative interaction between SynCom and TCH3-2\u003c/h3\u003e\n\u003cp\u003eTCH3-2 induced biofilm-related gene expression in rhizosphere microbiota (Supplementary Table\u0026nbsp;19), suggesting cooperative biofilm formation with SynCom and TCH3-2. Individual inoculation of SynCom and TCH3-2 or pairwise combinations of SynCom members with TCH3-2 failed to form biofilms, while ST markedly enhanced biofilm formation in TRM medium (Supplementary Fig.\u0026nbsp;22). By contrast, SynCom with other \u003cem\u003eFlavobacterium\u003c/em\u003e species did not induce biofilm formation (Supplementary Fig.\u0026nbsp;23). In presence of root exudate, ST significantly enhanced biofilm formation but each SynCom member failed to induce substantial biofilm formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, Supplementary Fig.\u0026nbsp;24). Notably, lack of specific taxon including \u003cem\u003eKa\u003c/em\u003e, \u003cem\u003ePm\u003c/em\u003e, \u003cem\u003eNg\u003c/em\u003e, and \u003cem\u003eAg\u003c/em\u003e reduced the biofilm formation of ST, highlighting the requirement of both TCH3-2 and key SynCom members (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eEnhanced biofilm formation by ST significantly increased the total sessile bacterial population on plant roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). While the abundance of TCH3-2 within biofilms declined in the absence of the SynCom, it was stabilized under the ST treatment, and most SynCom members\u0026mdash;particularly \u003cem\u003eKa\u003c/em\u003e, \u003cem\u003eAg\u003c/em\u003e, \u003cem\u003ePm\u003c/em\u003e, and \u003cem\u003eFt\u003c/em\u003e\u0026mdash;showed significant increases in their root-associated populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, Supplementary Fig.\u0026nbsp;25, and Supplementary Table\u0026nbsp;25). In contrast, \u003cem\u003eGp\u003c/em\u003e, \u003cem\u003eDj\u003c/em\u003e, and \u003cem\u003eNg\u003c/em\u003e exhibited only modest and non-significant changes. Notably, total planktonic cell density did not differ significantly among the S, ST, and ST-K treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee), indicating that ST did not promote overall bacterial growth. However, removal of \u003cem\u003eKa\u003c/em\u003e, which impaired biofilm formation, led to a significant reduction in the planktonic populations of \u003cem\u003eFt\u003c/em\u003e, \u003cem\u003eGp\u003c/em\u003e, \u003cem\u003eAg\u003c/em\u003e, and \u003cem\u003eDj\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef), suggesting destabilization of community structure in the absence of cooperative interactions. Together, these results demonstrate that cooperative interactions in ST support a stable, biofilm-associated microbial community on plant roots.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSpecialized functional division among SynCom members\u003c/h2\u003e \u003cp\u003eBuilding on the finding that TCH3-2 triggers cooperative traits including biofilm formation, root colonization, microbial interaction and PGP activity. We hypothesized that TCH3-2 acts as \u0026ldquo;a community conductor\u0026rdquo;, coordinating the SynCom function. Hierarchical clustering of SynCom traits in the presence of TCH3-2 revealed three key factors\u0026mdash;(i) the number of sterol-specific genes, (ii) the ability to promote biofilm formation, and (iii) root colonization efficiency\u0026mdash;were significantly associated with plant growth promoting (PGP) activity with 95% confidence (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea,b). Principal coordinates analysis (PCoA) further grouped SynCom members into three functional ranks (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec): Rank 1 (\u003cem\u003eKa\u003c/em\u003e, \u003cem\u003eAg\u003c/em\u003e, and \u003cem\u003ePm\u003c/em\u003e) specialized in core PGP traits with sterol genes; Rank 2 (\u003cem\u003eFt\u003c/em\u003e, \u003cem\u003eNg\u003c/em\u003e, and \u003cem\u003eDj\u003c/em\u003e) supported Rank 1 and Rank 3; Rank 3 (\u003cem\u003eGp\u003c/em\u003e) primarily promoted growth of the auxotrophic TCH3-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). The minimized SynCom composed of Rank 1 and 2 members retained PGP activity, comparable to that observed with the ST treatment, and a cumulative effect was observed only when at least TCH3-2, \u003cem\u003eKa\u003c/em\u003e, \u003cem\u003eAg\u003c/em\u003e, and \u003cem\u003eFt\u003c/em\u003e were present (Supplementary Fig.\u0026nbsp;26). These results indicate that the functional specialization of SynCom members is orchestrated by TCH3-2, enabling microbiota coalition that collectively promote plant growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides the new experimental insight into how microbiota coalition can have beneficial interaction with plant host. This supports the growing concept of plant holobiont. Central to this system is \u003cem\u003eF. dauae\u003c/em\u003e TCH3-2, a beneficiary auxotrophic bacterium with streamlined-genome, which depends on metabolic support from neighboring microbes and plant-derived compounds\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Although TCH3-2 is a metabolic beneficiary but somehow may acts as \u0026ldquo;community conductor\u0026rdquo;, recruiting cooperative partners and modulating community-level coalition function. TCH3-2 functions as a founder cell that initiates multispecies biofilm formation, serving as a driving force for cooperative microbial interactions in the rhizosphere by enhancing colonization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. TCH3-2-initiated multispecies biofilms may contribute to the establishment of a cooperative microbial environment via complex metabolic exchange at the root surface\u003csup\u003e\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTheoretical models predict that communities dominated by competitive interactions are relatively stable, whereas cooperative communities are intrinsically less stable in natural environments due to strong interdependence and the need for close physical proximity\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Consistent with this framework, loss of keystone taxa in ST perturbed multiple functions linked to plant fitness, indicating cooperation with strong interdependency (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Notably, \u003cem\u003eF. dauae\u003c/em\u003e TCH3-2, a beneficiary auxotroph, may act as a community organizer that reinforces interspecies dependency. Once formed, cooperative interactions may enhance metabolic efficiency and enable functional outputs that are not attainable by individual species alone\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Our result further suggest that, in the rhizosphere, the multispecies biofilm formed by ST on the root surface provides physical proximity and a spatially structured niche that stabilizes these cooperative interactions, enhances root colonization, and buffers the community against environmental fluctuations\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Together, these findings support the view that interaction structure rather than microbial diversity plays a central role in determining microbiome function, and that multispecies biofilm\u0026ndash;mediated cooperation in ST can stabilize otherwise fragile interactions and confer plant-beneficial effects.\u003c/p\u003e \u003cp\u003eMetatranscriptome and host transcriptome analyses revealed that co-inoculation with TCH3-2 and UpMF activated multiple pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). By contrast, a metatranscriptome-based SynCom combined with TCH3-2 selectively recapitulated the cooperative growth-promoting function of UpMF by consistently activating sterol biosynthesis and sterol-responsive gene expression, whereas auxin- and gibberellin-related changes did not align with growth phenotypes or mutant responses (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results indicate that sterol-related processes constitute a core component of the cooperative traits between UpMF and TCH3-2. ST activated both sterol biosynthesis and responsive genes in tomato plant and SynCom itself (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Notably, ST activated the entire phytosterol biosynthesis pathway in tomato, from squalene to brassinolide via the campesterol branch or to stigmasterol via the sitosterol branch\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Especially, ST treatment can recover or mitigate dwarf phenotype in BR-deficient \u003cem\u003edwf1\u003c/em\u003e but not BR-insensitive mutant \u003cem\u003ebri1-301\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), indicating ST treatment can enhance plant growth via activation of BR biosynthesis pathway\u003csup\u003e\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Furthermore, ST treatment rescued the growth of \u003cem\u003ecyp710a1\u003c/em\u003e, and \u003cem\u003ecyp710a2\u003c/em\u003e mutants\u0026mdash;defective in sitosterol branch, respectively\u0026mdash;but only partially restored growth in \u003cem\u003edwf1\u003c/em\u003e mutants, which lack both campesterol and sitosterol branches\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. ST treatment might promotes plant growth by increasing some specific intermediates for phytosterol biosynthesis or sterol level like BR or by optimizing the overall sterol balance between campesterol, sitosterol, or stigmasterol in plant\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough ST-mediated plant growth promotion was linked to sterol-associated processes, we were unable to consistently detect sterol production directly from ST cultures (data not shown). Several factors may account for this limitation. First, strong cooperative interactions among ST members likely occur within spatially confined multispecies biofilms (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), where metabolic exchange is localized and sterol levels may fall below detection limits. Second, the growth-promoting activity of ST appears to depend on sustained interactions between living plants and the community; cultivation of planktonic communities alone reduced community stability and led to loss or decline of taxa critical for cooperative function (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Supplementary Fig.\u0026nbsp;20). Third, during plant\u0026ndash;microbe interactions, distinguishing plant from microbially derived sterols remains technically challenging, as sterol localization and temporal accumulation are difficult to resolve with conventional assays\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Non-invasive \u003cem\u003ein situ\u003c/em\u003e mass spectrometry (\u003cem\u003ee.g.\u003c/em\u003e, liquid microjunction surface-sampling probe\u0026ndash;mass spectrometry) may enable detection of low-abundance sterols directly from intact biofilms and rhizosphere microenvironments\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur hierarchical model illustrates that microbial members are functionally differentiated under the influence of TCH3-2: Rank 1 interact closely with TCH3-2 and actively colonize the rhizosphere as key taxa contributing to integrated PGP activity via sterol-related unique genes; Rank 2 supports the growth of other groups; Rank 3 primarily support the beneficiary TCH3-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). This functional specialization, potentially division of labor, and microbial coalition may enhance phytosterol-associated benefits for the host\u003csup\u003e\u003cspan additionalcitationids=\"CR63 CR64 CR65\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Transcriptomic changes in both microbes and the plant, along with \u003cem\u003eArabidopsis\u003c/em\u003e mutant assays, potentially suggest cooperative production of sterol-like compounds (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). While simple sterols like lanosterol or zymosterol\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan additionalcitationids=\"CR68\" citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e occur in some prokaryotes, complex phytosterols remain unique to plants, as key biosynthetic genes are absent in individual bacterial species. This study illuminates the biosynthetic potential of phytosterols by microbiota coalition in a unique niche of host plant. The microbiota may benefit from the fast-growing plant host, gaining access to greater resources and space that enhance its stability and proliferation in the root environment. Further metabolomic analysis is required to identify specific metabolites derived from SynCom stimulated by TCH3-2. It is also essential to characterize specific factors involved in the community stimulating role of \u003cem\u003eF. dauae\u003c/em\u003e TCH3-2.\u003c/p\u003e \u003cp\u003eTaken together, this study suggests that auxotrophic \u003cem\u003eF. dauae\u003c/em\u003e TCH3-2 orchestrates a functionally stratified rhizosphere microbiota, where distinct groups contribute cooperatively to coalition function of microbiota and plant benefit. This may resemble division of labor in microbiota seen in multicellular systems\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. The division of labor may reduce metabolic burdens of individual community members while enhancing collective function\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Rather than acting independently, these microbes interact within a structured community, allowing the microbiota as a whole to exert functions that are not readily achieved by individual taxa alone. Structured as multispecies biofilms initiated by founder cells, such microbiota exhibit emergent properties like functional specialization and host responsiveness, reflecting a potential holobiont-level adaptation\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Microbiota coalitions may vary across ecosystems, depending on the common good between the microbiota conductor and its cooperative partners. While the underlying mechanisms remain to be clarified, our study provides the initial insight on microbiota functional coalition at community-level to expand the functional repertoire of plant-associated microbiota beyond what has been observed in single-strain or limited-consortium studies. By moving beyond reductionist approaches, this work provides a conceptual framework for understanding how cooperative microbial interactions can support plant functions and inform the engineering of resilient plant\u0026ndash;microbe systems.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eMethods, including statements of data availability and any associated accession codes and references, are available in the online version of the paper.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e \u003cp\u003eSWL and YSS designed the project. SML, JHK, HS, EK, PAL, JUS, SEK, and SYC performed most of plant experiments with microbiota and SynCom. EK, SML, and KC performed microbiome analysis. JP, HHL, HJ, and GH conducted rhizosphere metatranscriptome and tomato transcriptome analysis. SEJ, AAU and GTK conducted \u003cem\u003eArabidopsis\u003c/em\u003e mutant analysis. JHK, HS and SML conducted microbial gene expression and plant gene expression analysis. SML, JHK, HS and KN conducted co-culture experiment and biofilm assay. SML, KN, and JHK performed qPCR for root colonization. JC performed pangenome analysis. SML, JP, HHL, JHK, and SWL wrote a draft. GTK, YSS and SWL edited manuscript. All of the authors read and approved the manuscript before submission.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eWe thank Paul Schulze-Lefert at Max Planck Institute for Plant Breeding Research (Cologne, Germany) for his suggestions and for critically reading the manuscript. We also thank Hyoung Ju Lee and Kwang Yeol Baek at Dong-A University for technical assistance on plant experiment. This research was supported by the National Research Foundation of Korea (NRF) grant (No. RS-2020-NR049596 to SWL), Biomaterials Specialized Graduate Program funded by the Korea government (MOE, MCEE) and Research program for Agriculture Science and Technology Development (No. RS-2025-02653099 to SWL and YSS) and Next-Generation BioGreen 21 program to SWL and YSS (PJ01313101 and PJ01313102) through Rural Development Administration, Republic of Korea.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMargulis L, Fester R (1991) Symbiosis as a source of evolutionary innovation: speciation and morphogenesis. MIT Press\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVandenkoornhuyse P, Quaiser A, Duhamel M, Le Van A, Dufresne A (2015) The importance of the microbiome of the plant holobiont. 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Front Microbiol 12:635432\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7560912/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7560912/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant-associated microbial communities consist of plant holobiont and play an essential role in plant growth and development, yet their collective functions are not fully understood. Theoretically, microbiota can act as integrated consortia, conferring emergent properties beyond those of single species. Here, we show that the tomato rhizosphere microbiome, when stimulated by a \u003cem\u003eFlavobacterium dauae\u003c/em\u003e, enhances plant growth by activating the phytosterol biosynthesis pathway in both the microbiota and the plant host, a function unattainable by individual microbial species. A reconstructed synthetic community, based on meta-transcriptome of plant microbiota, recapitulated this microbiome-driven activity upon stimulation by \u003cem\u003eF. dauae\u003c/em\u003e. This synthetic community also restored the growth response in diverse sterol-deficient plant mutants. The microbial consortium exhibits multispecies biofilm formation and functional specialization among its members, constituting a microbial coalition that promotes plant growth. This study provides direct experimental evidence that plant microbiota function as a coordinated unit and orchestrate host plant development. We highlight this to be a plant holobiont function based on microbial community coalition in host plant.\u003c/p\u003e","manuscriptTitle":"Plant Growth Benefits by the Coalition Function of Plant Microbiome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 08:27:44","doi":"10.21203/rs.3.rs-7560912/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6c128dae-cc5e-49a4-b6a1-97bb41b5cc4c","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61242333,"name":"Biological sciences/Microbiology/Microbial communities/Microbiome"},{"id":61242334,"name":"Biological sciences/Microbiology/Microbial communities/Microbial ecology"}],"tags":[],"updatedAt":"2026-04-13T10:04:40+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-19 08:27:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7560912","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7560912","identity":"rs-7560912","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}