# Pectic homogalacturonan sensed by Bacillus acts as host associated cue to promote establishment and persistence in the rhizosphere

Farah Boubsi, Grégory Hoff, Anthony Arguelles Arias, Sébastien Steels, Sofija Andrić, Adrien Anckaert, Romain Roulard, Augustin Rigolet, Olivier van Wuytswinkel, Marc Ongena

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## To cite this version:

Farah Boubsi, Grégory Hoff, Anthony Arguelles Arias, Sébastien Steels, Sofija Andrić, et al., Pectic homogalacturonan sensed by Bacillus acts as host associated cue to promote establishment and persistence in the rhizosphere. iScience, 2023, pp.107925. 10.1016/j.isci.2023.107925. hal-04213115

HAL Id: hal-04213115

https://u-picardie.hal.science/hal-04213115v1

Submitted on 21 Sep 2023

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Pectic homogalacturonan sensed by *Bacillus* acts as host associated cue to promote establishment and persistence in the rhizosphere

Farah Boubsi, Grégory Hoff, Anthony Arguelles Arias, Sébastien Steels, Sofija Andrić, Adrien Anckaert, Romain Roulard, Augustin Rigolet, Olivier van Wuytswinkel, Marc Ongena

PII: S2589-0042(23)02002-3

DOI: https://doi.org/10.1016/j.isci.2023.107925

Reference: ISCI 107925

To appear in: *ISCIENCE*

Received Date: 7 April 2023

Revised Date: 19 July 2023

Accepted Date: 12 September 2023

Please cite this article as: Boubsi, F., Hoff, G., Arias, A.A., Steels, S., Andrić, S., Anckaert, A., Roulard, R., Rigolet, A., van Wuytswinkel, O., Ongena, M., Pectic homogalacturonan sensed by *Bacillus* acts as host associated cue to promote establishment and persistence in the rhizosphere, *ISCIENCE* (2023), doi: https://doi.org/10.1016/j.isci.2023.107925.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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B. velezensis
GA1

Establishment
and
peristence

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Pectin backbone
(HG)

HG / High DP OGs

Short DP OGs

Signal ✓

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Signal

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Biofilm
formation

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Sporulation

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Bioactive
secondary
metabolites
1 **Pectic homogalacturonan sensed by *Bacillus* acts as host associated cue to**
2 **promote establishment and persistence in the rhizosphere**

3

4 Farah Boubsi¹*, Grégory Hoff¹,², Anthony Arguelles Arias¹, Sébastien Steels¹, Sofija Andrić¹, Adrien

5 Anckaert¹, Romain Roulard³, Augustin Rigolet¹, Olivier van Wuytswinkel³, and Marc Ongena¹,⁴*

6

7 ¹Microbial Processes and Interactions, TERRA Teaching and Research Center, University of Liège - Gembloux Agro-Bio Tech,

8 Gembloux, 5030, Belgium

9 ²Present address: MYCOPHYTO, Grasse, 06130, France

10 ³UMRT INRAe 1158 Plant Biology and Innovation, University of Picardie Jules Verne, UFR des Sciences, Amiens, 80039,

11 France

12

13 ⁴Lead contact

14 *Correspondence : farah.boubsi@uliege.be, marc.ongena@uliege.be

15

16 **Abstract**

17

18 *Bacillus velezensis* isolates are among the most promising plant-associated beneficial bacteria used as
19 biocontrol agents. However, various aspects of the chemical communication between the plant and
20 these beneficials, determining root colonization ability, remain poorly described. Here we investigated
21 the molecular basis of such interkingdom interaction occurring upon contact between *Bacillus
22 velezensis* and its host via the sensing of pectin backbone homogalacturonan (HG). We showed that *B.
23 velezensis* stimulates key developmental traits via a dynamic process involving two conserved
24 pectinolytic enzymes. This response integrates transcriptional changes leading to the switch from
25 planktonic to sessile cells, a strong increase in biofilm formation and an accelerated sporulation
26 dynamics while conserving the potential to efficiently produce specialized secondary metabolites. As
27 a whole, we anticipate that this response of *Bacillus* to cell wall-derived host cues contributes to its
28 establishment and persistence in the competitive rhizosphere niche and *ipso facto* to its activity as
29 biocontrol agent.

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33 **Key Words:** *Bacillus velezensis*, pectin, homogalacturonan, biofilm, sporulation, bioactive
34 secondary metabolites, plant-bacteria interactions, root colonization

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## 42 Introduction

The rhizosphere is viewed as the narrow interface between plant roots and bulk soil directly influenced by root exudates¹. These root-secreted compounds represent the major nutrient source for carbon-starved soil dwelling microbes and contribute to create a densely populated but spatially limited niche characterized by intense interspecies interactions being neutral, cooperative or antagonistic²⁻⁴. Root exudates thus shape the assembly of microbial communities associated with roots according to the potential of individuals to adapt to the host and to face such highly competitive context. This results in dynamic and complex rhizosphere microbiomes that comprise commensal, pathogenic but also beneficial microorganisms including bacterial species that have evolved as true mutualists establishing intricate interactions with the plant. These bacteria use root exuded chemicals to fuel their catabolism and sustain growth while in turn, they provide beneficial services to their host by promoting growth and/or by augmenting resistance to biotic and abiotic stresses⁵⁻⁷. These beneficial rhizobacteria are usually characterized by efficient root colonization ability and stress tolerance⁸,⁹. Moreover, they possess a higher number of genes involved in chemotaxis, motility and biofilm formation compared with bacteria found in the bulk soil¹⁰,¹¹.

Species of the Bacillaceae belonging to the *Bacillus subtilis* complex are common inhabitants of the rhizosphere and are among the most studied plant-beneficial bacteria (PBB), especially for their positive effects on plant health and growth¹²,¹³. It includes *Bacillus velezensis*, one of the most promising rhizobacterial species to be used as biological control agent¹⁴⁻¹⁷. This biocontrol activity mainly relies on i) the ability to compete for space and nutrients with the other inhabitants of the rhizosphere, ii) to develop strong direct antagonism towards pathogenic (micro)organisms and iii) to stimulate host immunity leading to induced systemic resistance (ISR)¹²,¹⁸. The expression of these traits correlates with the potential of *B. velezensis* to produce a wide range of chemically diverse and functionally specialized secondary metabolites (SMs) acting as signals, siderophores, antimicrobials or immunity elicitors¹⁹,²⁰. From an ecological perspective, this arsenal is also considered as an adaptive trait to improve establishment and persistence in the competitive rhizosphere niche.

A few recent reports have described how soil bacilli and other plant beneficial species may modulate the expression of developmental traits and adapt the production of SMs upon facing microbial competitors in interspecies interactions²¹⁻²⁵. However, the molecular dialogue occurring with the host plant at the root level also represents a key component of the multitrophic rhizospheric interactions that may influence the behaviour of these beneficials. Certain root-exuded compounds, essentially amino acids, organic acids and to a lesser extent sugars are perceived as host-derived signals by rhizobacteria which in response, move towards the host roots *via* chemotaxis²⁶⁻²⁹. This promotes bacterial cell attachment to the root surface to initiate root colonization by forming biofilms, which are highly-organized bacterial community assembly comprising vegetative cells and spores enclosed in a self-produced matrix²⁵,³⁰. Root exudates have also been shown to modulate SMs production, among which surfactin³¹,³², and the expression of genes involved in bacillibactin, bacilysin, bacilaene, difficidin, macrolactin, surfactin and fengycin synthesis³³,³⁴ in diverse *B. velezensis* strains.
Another facet of the plant-rhizobacteria interaction, occurring upon direct contact with the host plant, involves the perception of plant cell wall polymers (CWPs). We and others previously showed that the production of the multifunctional lipopeptide surfactin is specifically stimulated upon sensing pectin<sup>35,36</sup>, and more precisely pectin backbone<sup>32</sup>, playing a key role in the first steps of root colonization. Moreover, pectin but also arabinogalactan and xylan were shown to promote biofilm formation in several bacilli species<sup>36-38</sup> through the induction of the expression of biofilm matrix-related genes<sup>36,37</sup>. In *B. subtilis*, the galactose contained in these polymers is used as a substrate to synthesize matrix exopolysaccharides<sup>37,39</sup>. However, the phenotypic and metabolite responses of beneficial bacilli to CWPs perception still remain largely uncharacterized.

In this study, we investigated the mechanistic underlying the impact of pectin, as first CWP potentially sensed by *B. velezensis* upon contact with root tissues<sup>40</sup>, on the expression of key developmental traits sustaining efficient colonization and on SMs production. We showed that the pectin backbone homogalacturonan (HG) acts as host-associated cue that triggers early biofilm formation, fast sporulation and enhanced production of some specialized secondary metabolites. Our data indicated that such HG sensing contributes to the establishment and persistence of *B. velezensis* cell populations in the rhizosphere via a dynamic process involving bacterial pectinolytic enzymes readily active in planta.

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## 103 Results

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## 105 Conserved polygalacturonate lyases contribute to root colonization by *B. velezensis*

In this work, the genetically amenable plant-associated strain GA1 (GenBank: CP046386) was used as representative of the *B. velezensis* species<sup>41</sup>. We previously showed that GA1 possesses two genes encoding polygalacturonate lyases (*pelA*, GL331_08735; *pelB*, GL331_04125) that are conserved with a high nucleotide identity in most strains of the *B. velezensis* species which belong to the functional group *amyloliquefaciens* within the *B. subtilis* complex<sup>32</sup>. These lyases act as pectin degrading enzymes by generating oligogalacturonides from pectin backbone (homogalacturonan), constituting up to 65% of total pectin encountered in the plant primary cell wall<sup>40,42</sup>. Comparative genomic analysis of the enzymatic content of pectin remodelling and degrading enzymes in other soil-dwelling bacilli reveals that PelA and PelB are the only enzymes potentially involved in pectin degradation/remodelling that are produced by *B. velezensis* (Table1, TableS1). On the one hand, it highlights a highly reduced pectinase arsenal in GA1 compared to other saprophytic soil species such as *B. subtilis*, *B. paralicheniformis* or *B. licheniformis* (Table1, TableS1). On the other hand, none of these enzymes are synthesized by ubiquitous species such as *B. mycoides*, *B. simplex*, *B. thuringiensis* or *B. weihenstephanensis* (Table1). We assume that such a limited potential to synthesize pectin-degrading enzymes in *B. velezensis* might be linked to its typical plant-associated lifestyle<sup>43</sup>. In terms of phylogenetic relationship between pectinases of the PL1 and PL3 families found in these soil-dwelling bacilli, the maximum likelihood tree shows low similarity between PelA and PelB of GA1 with pectinases of the other *Bacillus* species (Fig1A,
TableS2). Moreover, PelA and PelB of *B. velezensis* are separated in two different clusters, suggesting notable differences in their structure and function.

In the plant cell wall, pectin backbone is mainly low methylesterified, but it can also be found in its highly methylesterified form⁴⁰. Therefore, we wanted to assess whether PelA and PelB may display some substrate specificity regarding the level of methylation of the HG polymer, allowing to distinguish between pectin and pectate lyase activities⁴². To that end, we generated and cultivated the two GA1 mutants ΔpeI/A and ΔpeI/B in a medium containing the specific sugars, organic acids and amino acids found in root exudates of Solanaceae³¹ (root exudates mimicking medium). Pel enzymes are readily produced in this medium and we measured the activity of the corresponding cell-free supernatants on commercially available homogalacturonan polymer of low (≤ 5%, HG) and high (≥ 85%, HGHM) methylesterification degree. Our data reveal that PelB is moderately but exclusively active on HGHM (Fig1B) while PelA is highly and preferentially active on HG (Fig1C), suggesting that PelA is a pectate lyase and PelB is a pectin lyase. In silico analysis of structural features in the protein sequences of PelA and PelB strongly suggests that the low activity of PelB compared to PelA might be due to the absence of disordered domain(s) (TableS3), responsible of structural plasticity and proper exposition of the catalytic site for optimal functioning of the enzyme⁴⁴,⁴⁵. Therefore, PelA and PelB thus display complementary lyase activities on HG. In addition, the double mutant ΔpeI/AΔpeI/B is fully impaired in its potential to breakdown HG, providing functional evidence in support to genomic data that these two Pels represent the only enzymes involved in HG degradation in GA1 (Fig1D).

As mutualistic rhizobacteria, one of the key traits of *B. velezensis* isolates relies on their potential to efficiently colonize host roots. We previously showed that GA1 tends to form biofilm structures when colonizing tomato plantlets roots⁴¹. As they represent unique functional pectin-degrading enzymes that have been conserved in the species, we postulated that PelA/B lyases may play some role in the rhizosphere fitness of the bacterium. Monitoring of bacterial population dynamics on tomato roots on solidified Hoagland medium reveals that the mutant ΔpeI/AΔpeI/B impaired in lyases production is also significantly affected in its colonization potential compared to the wild type strain (Fig2A). In addition, ΔpeI/AΔpeI/B is strongly outcompeted by GA1 upon co-inoculation at the same initial cell concentration, further indicating some reduced root fitness related to the loss of PelA/B synthesis (Fig2B). In support to the role of these enzymes in the *Bacillus*-plant interaction *in vivo*, we detected substantial amounts of oligogalacturonides released from native pectin extracted from tomato roots (PEC) after treatment with a PelA/B-enriched extract (Fig2C). This indicates that these enzymes may effectively alter the pectin polymer naturally found in the plant cell wall.

Based on all these data, we assumed that PelA/B are readily produced by the bacterium quite early upon root colonization. In liquid cultures, pectin degrading activity is detected as soon as GA1 cells enter into the exponential growth phase (Fig2D), suggesting that quorum sensing might regulate their expression. This population-dependent process plays a key role in various developmental traits driving the fate of cell communities including biofilm formation and *in*
extenso root colonization<sup>46,47</sup>. RT-qPCR measurements of the expression levels of *pelA* and *pelB* in a *ΔcomA* mutant unable to synthesize the major regulator of quorum sensing in bacilli show that *pelA* is not expressed anymore while the expression of *pelB* is markedly repressed compared to GA1 (Fig2E). This indicates that these enzymes also differ in their regulation in addition to differences in their activities.

Therefore, GA1 may somehow degrade to a limited extent pectin backbone during root colonization via its Pel enzymes, without impairing cell wall integrity since we never observed any adverse effect on plant health and growth caused by this beneficial bacterium. We thus hypothesized that besides root exudates, the bacterium may use oligomers of galacturonic acid (GA) generated from HG degradation as an additional carbon source to sustain growth and thereby, favour root colonization. However, according to KEGG database<sup>48</sup>, the pentose and glucuronate interconversion pathway necessary for the assimilation of oligomers of GA is incomplete in *B. velezensis* (Fig2F). Intriguingly, GA1 possesses the complete pathway to metabolize and use GA monomers as carbon source but is unable to generate GA monomers from HG since it does not synthesize polygalacturonases (EC.3.2.1.15) and the other required isomerases. We confirmed this experimentally since GA1 and the mutant *ΔpelAΔpelB* display a reduced but similar growth rate in minimal medium supplemented with HG while it is significantly higher in presence of GA (Fig2G). Taking all these results together, the ability to degrade HG confers to GA1 an advantage for its establishment on roots at the early stages of colonization but without using HG degradation products as an additional carbon source.

## Homogalacturonan sensing stimulates early biofilm formation in *B. velezensis*

The root colonization potential of *B. velezensis* and related species such as *B. subtilis* correlates positively with their ability to form robust biofilm *in vitro* and *in vivo*<sup>49–52</sup>. Therefore, we next hypothesized that enhanced fitness of GA1 during root colonization may be due to an improved potential to form biofilm in response to HG or HG degradation products. Based on measurements of biofilm pellicles at the air-liquid interface, we observed an enhanced biofilm formation in presence of HG for both GA1 and the mutant *ΔpelAΔpelB* compared to unsupplemented control conditions (Fig3A). Furthermore, *ΔpelAΔpelB* responds optimally to HG of high degree of polymerization (DP) by increasing biofilm formation but only moderately to oligogalacturonides with intermediate DP (OGA). However, it does not respond to low-DP oligomers (OGB) or GA monomer (Fig3B, FigS1A for DP distributions of OGA and OGB). This indicates that HG of high DP is responsible for triggering biofilm formation in GA1. To further investigate the dynamics of this phenomenon, we observed the biofilm pellicle formation by microscopy at early stages. In presence of HG, bacterial cells start to agglomerate within a few hours and form long chains reducing their motility (VideoS1A), while individual cells remain highly motile in control conditions 7h post inoculation (VideoS1B). This earlier shift from motile to sessile behaviour upon HG supplementation correlates with a global repression of genes related to flagellar protein synthesis as revealed by genome-wide RNAseq analysis of the transcriptional reprogramming induced in GA1 within the first 8 hours in presence of pectin backbone (Fig3C).
In bacilli, biofilm initiation and maturation is related to the production of an extracellular matrix mainly composed of exopolysaccharides (EPS), cohesion proteins (TasA) and extracellular DNA (eDNA), which is essential to hold the bacterial community together within the biofilm structure²⁵. Measurements of the expression level of *epsB* and *tasA* by RT-qPCR on GA1 cells harvested from biofilm at the early stages of formation reveal an overexpression of both genes in the medium supplemented with HG compared to control conditions (Fig3D). Our RNAseq data also confirmed that all the genes constituting the operons responsible of EPS and cohesion protein synthesis are up-regulated in presence of HG (FigS2A). Interestingly, among genes of the *epsA-O* operon, the protein encoded by *epsE*, in addition of being a key protein involved in EPS synthesis, has been shown to be involved in motility cessation by acting like a clutch on the flagellar motor switch protein FliG⁵³,⁵⁴.

On the other hand, it has been shown that the lipopeptide surfactin acts as signal triggering the expression of *eps* and *tasA* genes⁵⁵,⁵⁶ and in a previous study, we showed that the production of this compound by GA1 is stimulated in response to HG in the early exponential growth phase³². Therefore, we wanted to evaluate whether this early surfactin production in presence of HG could have an impact on the enhanced biofilm formation observed here. Colorimetric quantification of biofilm formation by crystal violet staining shows that the mutant ΔsrfAA impaired in surfactin synthesis is strongly affected in its potential to produce biofilm compared with GA1, but still displays a high responsiveness to HG regarding both pellicle formation (FigS2B) and biofilm gene expression (FigS2C). This indicates that surfactin synthesis stimulation by HG does not markedly contribute to the strongly enhanced biofilm phenotype triggered by the polymer in GA1.

Besides EPS and cohesion proteins, eDNA is also required at early stage of biofilm formation and contributes to its 3D architecture⁵⁷. Relative eDNA quantification assay shows a significantly higher eDNA content in pellicles formed in presence of HG compared with the control medium after 24h (Fig3E), which is concomitant with a higher number of cells embedded in the biofilm pellicle (Inset Fig3E). However, no significant differences were observed in terms of total number of cells in the wells (i.e. planktonic cells and cells embedded in the biofilm) in both conditions (approximately 4.5x10⁸ CFU/ml at 24h after inoculation), indicating that the accumulation of eDNA is not due to cell lysis but rather to an active secretion as reported for *B. subtilis*⁵⁸.

Accordingly, both macroscopic observations and colorimetric quantification of biofilm show that GA1 pellicle starts to form earlier in presence of HG and gives rise to a thicker biofilm after 24h compared with control conditions (Fig3F-G). A similar increase in biofilm formation was also measured upon addition of natural pectin extracted from tomato plantlets roots (PEC) at the same concentration (Fig3G). In addition, we observed a significant stimulation of biofilm formation upon addition of HG at concentrations as low as 0.03 g/l (Fig3H), suggesting the involvement of a quite sensitive perception system of the polymer in GA1. This would also discard the possibility that HG acts indirectly by creating some osmotic stress if added at sufficient concentration to the medium, which is supported by the fact that the chemical
polymer PEG, added at the same concentration, does not trigger any response in GA1 (FigS1B). That said, HG of high polymerization degree with low level of methylesterification can form egg-box structures stabilized by calcium bridges⁵⁹ that might favour cell cohesion and contribute to biofilm architecture via a biologically-unrelated process as it has been shown for cellulose in E. coli biofilms⁶⁰. Nevertheless, involvement of such a physico-chemical structuring effect can be ruled out since a similar increase in biofilm pellicle formation is observed upon addition of highly-methylated HG, unable to form such supra-molecular conformation (Fig3I). Collectively, these data strongly suggest that pectin perception underpins a signalling process through which bacterial cells actively detect homogalacturonan fragments with a minimal length for triggering biofilm establishment.

## Homogalacturonan sensing enhances the sporulation dynamics in B. velezensis

It is commonly assumed that during biofilm formation, *Bacillus* enters into a process of cell differentiation, leading to the coexistence of vegetative cells, endospores and spores into the same multicellular structure⁶¹. Within the population colonizing tomato roots, we observed that a majority of cells tends to rapidly differentiate into spores, representing more than half of the total population after 3 days and around 90% after 6 days (Fig4A). This prompted us to also investigate the possible impact of HG recognition on sporulation as key developmental trait. We set up a method to monitor the sporulation dynamics of GA1 cells by flow cytometry using Redox Sensor Green dye (RSG) as a marker of cellular activity and cell size as proxies (See STAR method and FigS3A). A strong increase in spore population is observed upon supplementation of liquid cultures of GA1 with both PEC or HG compared with the control medium after 48h (Fig4B). We next investigated the dynamics of spore formation in GA1 population between 24h and 48h, this time frame corresponding to the end of the exponential growth phase/beginning of the stationary phase in our culture conditions. We observed that sporulation is triggered earlier and faster in GA1 cells upon HG sensing, allowing the biomass to remain constant while it sharply decreases in control conditions (Fig4C). As observed for biofilms, the response of the bacterium regarding sporulation in presence of HG is DP-dependant. Undegraded HG and OG~A~ induce a strong increase in spore population in the Δ*pelAΔpelB* mutant while OG~B~ and GA monomer display a limited or no effect respectively compared with the control conditions (Fig4D).

The initiation of sporulation in bacilli is regulated by the master transcription factor Spo0A, depending on its abundance and phosphorylation state⁶². The active form Spo0A-P is indeed responsible of the direct regulation of hundreds of genes involved in the sporulation mechanism. It includes repression of the global transition state regulator AbrB, known to influence the expression of a large number of genes at late growth phase⁶³,⁶⁴. We therefore anticipated that differences in expression of both *spo0A* and *abrB* genes should be induced in response to HG before the release of the first mature spores observed after 30h. Accordingly, RT-qPCR measurements show a significant increase in *spo0A* expression and a concomitant repression of *abrB* in presence of HG in cells collected after 24h (Fig4E). The strong repression
of *abrB* in presence of HG thus reflects a higher cellular concentration of Spo0A-P, necessary to initiate the sporulation mechanism.

Next, we wanted to verify if enhanced sporulation was also triggered by HG in biofilm pellicles of GA1. We analysed by flow cytometry the population diversity of cells embedded in biofilm pellicles of GA1 and we observed that cells also sporulate faster upon HG supplementation, with a 8.5 fold increase of mature spore population in the 48h-pellicle compared with the control medium. As a consequence, the bacterial population in the mature biofilm in presence of HG is almost exclusively constituted of spores after 120h (Fig4F). Still, such a higher sporulation dynamics could be an indirect effect of HG rather than a direct effect of its perception since it has been shown that the sporulation rate of *B. subtilis* NCIB 3610 increases when the bacterium produces exopolysaccharides⁶⁵. However, sporulation is also significantly increased in the non-EPS producing mutant Δ*epsA-O* upon HG supplementation (Fig4G), confirming that HG perception has a direct effect on the sporulation mechanism.

**Homogalacturonan sensing impacts the production of bioactive secondary metabolites in B. velezensis**

We also investigated the possible modulation of SMs production by *B. velezensis* in response to HG since these small-size chemicals contribute to the rhizosphere fitness of the bacterium and play key roles in its biocontrol activity¹⁴,¹⁵,⁶⁶. We first performed an untargeted profiling of metabolites secreted by planktonic GA1 cells in liquid cultures supplemented or not with HG. Cell-free extracts collected at stationary growth phase were analysed by UPLC-qTOF-MS and data processing with MZmine²⁶⁷ allowed us to detect features and the corresponding compounds differentially produced upon HG sensing. Beside unknown hits, we could identify some non-ribosomal lipopeptides (LPs) and polyketide (PKs)-type metabolites with production modulated in response to HG but in a specific way depending on the family (Fig5A). The production of the surfactin- and iturin-type LPs and to a lower extent, the PKs bacillaene and difficidin, are significantly increased in presence of HG while fengycins and macrolactins were not or negatively impacted respectively (Fig5A-Fig5A4). However, due to specific transcriptional regulations, the kinetic of synthesis of these metabolites differs depending on the growth phase, which means that the amplitude and timing of the stimulating effect of HG can vary according to the molecule and sampling time. Thus, we decided to refine the time course analysis focusing on the LPs as key compounds widely produced by several plant-beneficial *Bacillus* species⁶⁸. We observed a pronounced early stimulation of surfactins production (Fig5B left) as previously described³², which correlates with an enhanced expression of the *srfAA* gene, one of the genes encoding for the surfactins synthetase (Inset Fig5B left). However, our data also reveal a clear stimulation of iturins and fengycins production upon HG supplementation (Fig5B middle and right), which is concomitant with a higher expression of the related biosynthesis genes *ituC* and *fenC* (Inset Fig5B middle and right). This phenomenon is independent of pectin degradation products since the stimulation
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is also observed in cultures of ΔpeI/ΔpeI/B cells upon HG supplementation as shown for iturins
production (FigS4B).

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We also wondered whether the acceleration of sporulation dynamics in GA1 upon HG perception has an effect on the potential of the bacterium to efficiently synthesize these bioactive SMs when it forms thick spore-enriched biofilms. UPLC-qTOF-MS profiling of LPs produced by mature biofilm of GA1 with and without HG added to the medium reveal similar amounts of iturins, fengycins and surfactins in both conditions (Fig5C). Therefore, the faster increase in spore population in biofilm triggered upon HG perception does not impair the ability of the bacterial community to efficiently synthesize these SMs. This can be explained considering the higher production rate of iturins, surfactins and fengycins by active cells in the population even if the relative proportion of these vegetative cells is lower in presence of HG.

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Discussion

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According to our current knowledge, the molecular dialogue between beneficial rhizobacteria and their host plant is mainly mediated by small-size diffusible compounds³²,⁶⁹,⁷⁰. However, signaling that may occur upon direct contact remains relatively unknown. In this work, we demonstrate that pectin backbone homogalacturonan (HG) is perceived as a host cue by the beneficial species *B. velezensis*, which in turn, readily stimulates key developmental traits such as biofilm formation, sporulation, but also production of various bioactive secondary metabolites with specific functions. Our data come in support to the few studies mainly performed on *B. subtilis* reporting that some plant cell wall polysaccharides may act as trigger of biofilm formation³²,³⁶⁻³⁹. However, we provide a more comprehensive picture of the multifaceted response mounted by the typical plant-associated *B. velezensis* aiming at increasing its fitness and ensuring its persistence in its natural competitive ecological niche.

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We found that the potential of *B. velezensis* to perceive and respond to HG is associated with its ability to secrete two pectin/pectate lyases, constituting its very limited arsenal of pectin remodeling and degrading enzymes (Table1-TableS1). These Pels contribute to bacterial fitness since depleted mutants are significantly affected in root colonization (Fig2A-B). *B. velezensis* can feed on galacturonic acid, the constitutive monomer of HG, only if it is already available in the rhizosphere due to its incomplete pathway to assimilate HG degradation products (Fig2F-G), in contrast with microbial plant pathogens generating and consuming monomers from HG directly encountered in the plant cell wall⁷¹,⁷². From an ecological point of view, the presence of such enzymes in the genome of beneficials raises questions. Indeed, the ability to synthesize active plant cell wall degrading enzymes, which are very abundant in phytopathogenic microorganisms, is considered as one of the main traits responsible of their virulence⁷²⁻⁷⁴. Therefore, it is tempting to speculate that such mutualistic bacteria living in intricate association with roots might have co-evolved with their host, leading to a decrease of their arsenal of pectin degrading enzymes to be tolerated by the plant. We assume that the role of Pels in colonization of plant roots by *B. velezensis* relies on the fact that their activity
may either facilitate accessibility to the HG backbone for the bacterium without being detri-
mental to the host plant and release some high-DP OGs from the complex polymer, to which
the bacterium is able to respond. A reduced arsenal of pectin degrading enzymes, with mod-
erate activity, could also be used to weaken cell-to-cell adhesion by gently degrading pectin
of the middle lamella to colonize intercellular spaces in root tissues as it has been shown for
*Enterobacter* sp. SA187⁷⁵.

The ability of bacteria to form robust biofilms in their natural ecological niche is con-
sidered as an essential adaptive trait. In the case of rhizobacteria, it facilitates attachment to
the root and efficient colonization, which is required to exert their beneficial effects on the
host plant<sup>25,30,51,76,77</sup>. Here we showed that, in *B. velezensis*, this process is favoured by the
ability of the bacterium to sense HG and high-DP OGs (<strong>Fig3B</strong>). Early biofilm pellicle formation
triggered by HG (<strong>Fig3F</strong>) correlates with transcriptional activation of genes playing key roles in
biofilm initiation (i.e. EPS and cohesion proteins synthesis) and concomitant repression of
genes involved in motility (<strong>Fig3C-D</strong>), further illustrating the trade-off and incompatibility of
both processes as reported in *B. subtilis*<sup>78</sup>. Moreover, we detected a higher eDNA content in
the biofilm pellicle upon HG sensing (<strong>Fig3E</strong>), which can contribute to biofilm robustness since
eDNA is known to interact with EPS to promote cell adhesion<sup>57</sup>. Besides ensuring cell cohesion
and adhesion to surfaces, which are two traits governing biofilm stability<sup>79</sup>, EPS and the hy-
drophobin layer of the biofilm also act as a shield to protect the cell community within the
biofilm from external stresses and against toxins and/or infiltration by competitors<sup>25,80,81</sup>. This
confers an advantage to *B. velezensis* in terms of ecological fitness in the highly competitive
rhizosphere niche where microbial warfare dominates.

Cell differentiation is inherent to biofilm formation and guarantees the survival of
the community and proliferation of the species in response to adverse abiotic conditions or
microbial competitors⁸²⁻⁸⁵. Biofilm formation and sporulation can be seen as interlinked pro-
cesses since both are controlled by the same global regulators⁶²,⁸⁶,⁸⁷. Interestingly, our data
show that the sporulation dynamics of GA1 cells is highly accelerated upon HG sensing both
in biofilms and in cells in planktonic cultures (**Fig4B**, **Fig4F**), independently of EPS synthesis
(**Fig4G**) indicating that it results directly from HG perception inducing transcriptional changes
in key regulators genes of sporulation such as *spoOA* and *abrB* (**Fig4E**). Via an enhanced spor-
ulation, GA1 also avoids the strong decrease in population observed at the end of the expo-
nential growth phase in liquid cultures (**Fig4C**). Bacilli are known to exhibit a social behaviour
called cannibalism when they face nutrient starvation during which a portion of the popula-
tion feed itself on the other to survive⁸⁸⁻⁹¹. Therefore, by enhancing sporulation dynamics
upon HG perception, GA1 might highjack this phenomenon and allow a greater persistence of
cells on plant roots. A prompt formation of spore-enriched biofilms should globally favour the
establishment of robust populations on roots.

The secretion of SMs is also one of the factors contributing to the modulation of the
fitness of the bacteria in the highly competitive rhizosphere and *B. velezensis* is one of the
most prolific species regarding this aspect¹⁴⁻¹⁷. In this work, we observed that HG sensing also
results in modulation of the secondary metabolome of GA1 (*Fig5A-B*,*Fig5A-C*). As recently
reported, it includes an increased synthesis of the cyclic LPs surfactins, favouring biofilm
formation and motility and priming the plant immune system leading to systemic
reinforcement of the host against phytopathogens³²,⁹². Iturins and fengycins are other
lipopeptides boosted upon HG perception, mainly described for their strong antifungal activity
against a wide range of phytopathogens¹⁵,¹⁹ but also reported as elicitors of plant systemic
resistance⁹³⁻⁹⁶. PKs synthesis of the bacillaene and difficidin families, with broad-spectrum
antibiotic activities against gram-negative bacteria⁹⁷⁻¹⁰¹, is also enhanced in response to HG.
Enhanced production of these SMs is thus of clear benefit for plant health and biocontrol.
However, in the rhizosphere as in any other populated environment, microbes dwell in a
context of competitive interactions where antibiotics are raised as weapons for chemical
warfare¹²,⁶⁸. The changes occurring in the secondary metabolome described here may result
in an enhanced global antimicrobial potential of *B. velezensis* as we reported recently upon
sensing bacterial competitors in interspecies interactions²¹. Importantly, our data also show
that efficient production of these bioactive metabolites by *B. velezensis* is still occuring in
spore-enriched biofilms (*Fig5C*) and therefore in multicellular communities developing on
roots of its host on which the bacteria tend to rapidly differentiate into spores (*Fig4A*).

That said, the molecular basis underpinning HG perception by bacilli cells is still
unclear. Our data show that the bacterial response is not indirectly triggered by some physico-
chemical cues such as structuring effect or osmotic stress that may have been caused by the
addition of the polymer (Fig3I, FigS1B). Moreover, *B. velezensis* responds to relatively low
concentrations of HG (Fig3H) but also does not react to short-DP OGs and GA monomer,
suggesting some sensitive recognition system triggered selectively by high DP HG of a minimal
length. Therefore, we assume that polymer sensing may rely on a specific receptor-based
recognition process as it has been reported for *Sphingomonas spp*¹⁰², but further investigation
is required to identify such putative receptor. The membrane-anchored histidine kinases
(KinA-E) are good candidates since in *B. subtilis*, they are activated in response to a range of
environmental cues including potassium leakage induced by surfactin²², plant
polysaccharides³⁷ and potentially oxygen, light and redox potential¹⁰³,¹⁰⁴ which results in
phosphorylation of the master regulator Spo0A acting on both biofilm formation and
sporulation, according to the level of its phosphorylation state⁶²,⁸⁵. However, whether such
Kin system is involved or not in the perception of HG by *B. velezensis* remains to be
established.

In conclusion, perception of HG as host-associated cue underpins a new aspect of
interkingdom molecular interactions between plants and their bacterial associates such as *B. velezensis*. Using HG as signal allows *Bacillus* to eavesdrop its host, establish and proliferate in
the rhizosphere. Improved fitness in terms of root colonization and persistence means a
higher niche competition and establishment of threshold populations necessary to produce,
in biologically relevant amounts, its unique arsenal of antimicrobials and plant immunity elic-
itors, all of them contributing to a better protection of the plant against pathogens and *in fine*
to biocontrol. So beyond the interest in microbial chemical ecology in the broad sense, this
442 knowledge is essential for optimizing the use of *B. velezensis* as environmentally robust inoculant in sustainable crop production.
443

444

## 445 Limitations of the study

446

447 In this study, we highlight the importance of HG as major constituent of pectin in the context of plant-bacteria interactions. However, the mechanism underlying the perception of this polymer as molecular pattern by *B. velezensis* remains to be determined, including the identification of potential specific receptor at the bacterial cell surface. Moreover, it could be relevant to study if this ability to perceive and respond to such plant cell wall polymer is also found in other bacterial species and genera commonly used as PGPR. Biocontrol assays would also be interesting to assess if the stimulation of lipopeptide production by *B. velezensis* upon HG supplementation may lead to higher protection of the host plant against the attack of phytopathogens. Finally, it would be relevant to extend the scope of this study and further investigate the impact of the application of exogenous HG at the plant microbiome level.
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457

## 458 Author contributions

459

460 F.B and M.O conceived, designed and coordinated the project. F.B, G.H, A.A.A, S.S, S.A, A.A, R.R, A.R and O.VW generated materials, performed experiments, and/or analysed data. F.B and M.O wrote the manuscript. O.VW, A.A and G.H revised the manuscript. O.VW and G.H were involved in the discussion of the work.
461
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463

464

## 465 Main figure titles and legends

466

467 **Figure 1.** General characterization of *B. velezensis* pectin degrading enzymes PelA and PelB. (A) Phylogenetic relationship of HG degrading enzymes between PelA and PelB of *B. velezensis* GA1 and archetype strains of other soil-dwelling bacilli. (B and C) Time-course measurement of pectin degradation activity of cell-free culture supernatants of (B) *B. velezensis* GA1 ΔpelA and (C) ΔpelB mutants on HG (light gray bars) and HGHM (dark grey bars) (Mean±SD, n=3). (D) Kinetics of oligogalacturonides released over time from HG by 24h-cell-free culture supernatants of GA1 (black line) and the mutant ΔpelAΔpelB (light grey line). See also Table S2 and S3.
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472

473 **Figure 2.** Conserved polygalacturonate lyases contribute to root colonization by *B. velezensis*. (A and B) Time-course measurement of (A) GA1 (white bars) and ΔpelAΔpelB (grey bars) populations on tomato plantlets roots and (B) upon co-inoculation (50:50) on tomato plantlets roots (Mean±SD, n=3 for (A) and n=7 for (B), t-test; ns, non-significative; *, P<0.05; ****, P<0.0001). (C) Schematic representation of pectin degradation products generated by Pels enzymes from pectin extracted from tomato plant roots (PEC, Created with biorender.com). Column chart represents simple sugar composition (expressed as molar ratio percentages) of PEC fraction. Line graph represents the kinetics of oligogalacturonides released over time from PEC fraction by cell-free culture supernatants of GA1. (D) Time-course measurement of pectin degrading activity of cell-free culture supernatants of GA1 (bars, left axis) regarding bacterial growth (line, right axis) (Mean±SD, n=3). (E) Expression pattern of pelA (up) and pelB (down) in GA1 (white bars) and ΔcomA mutant (grey bars) at early growth stages (Mean±SD, n=3, t-test; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). (F) Simplified pathway of HG degradation and assimilation (pentose and glucuronate interconversion, adapted from KEGG pathway database<sup>48</sup>). Enzymes encoded and not encoded in GA1 genome are marked in green and red respectively. (G) Growth profile of GA1 and ΔpelAΔpelB mutant on minimal medium supplemented with 0.2% (w/v) HG (black and grey line respectively) or GA (green and red lines respectively) (mean±SD, n=3). Growth rate in each condition is indicated next to the corresponding curve and was determined by calculating the slope of the linear part of ln(OD<sub>600</sub>).
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487

488 **Figure 3.** Homogalacturonan sensing stimulates early biofilm formation in *B. velezensis*. (A and B) Assessment of biofilm formation at the air-liquid interface by (A) GA1 and ΔpelAΔpelB in the control medium (white bars) and upon supplementation
489
with 0.1% (m/v) HG (grey bars) in a 96-well microplate by crystal violet staining and (B) by GA1 upon supplementation with 0.1% (m/v) HG of different DP (HG, mean DP≈170³²; OGₐ, mean DP=13; OGᵇ, mean DP=4; GA, DP=1) (Mean±SD, n=8, Tukey's multiple comparisons, α=0.05). (C) Heatmap representing differential expression of genes involved in motility in GA1 upon supplementation with 0.1% (m/v) HG normalized to the control medium after 2.5, 5 and 8h of culture. Green and red shadings represent respectively higher and lower relative expression level compared with the control medium. (D) Expression pattern of epsB (left) and tasA (right) genes in GA1 in the control medium (white bars) and upon supplementation with 0.1% (m/v) HG (grey bars) at early biofilm formation stages (Mean±SD, n=3, t-test; **, P<0.01; ***, P<0.001). (E) Time-course measurement of eDNA content in biofilm pellicles of GA1 formed at the air-liquid interface in the control medium (black line) and upon supplementation with 0.1% (m/v) HG (grey line) (Mean±SD, n=8, t-test; ****, P<0.0001). Inset: Time-course measurement of the number of cells embedded into the biofilm pellicle in the control medium (white bars) and upon supplementation with 0.1% (m/v) HG (Mean±SD, n=3, t-test; *, P<0.05; **, P<0.01). (F) Macroscopic observation of the kinetic of biofilm formation at the air-liquid interface by GA1 in the control medium and upon supplementation with 0.1% (m/v) HG. (G) Assessment of biofilm formation (as described in panel A and B) by GA1 in the control medium and upon supplementation with 0.1% (m/v) pectin fraction extracted from tomato plant roots (PEC) and HG (Mean±SD, n=8, Tukey's multiple comparisons, α=0.05). (H) Assessment of biofilm formation (as described in panel A and B) by GA1 upon supplementation of HG at different concentrations (Mean±SD, n=8, Tukey's multiple comparisons, α=0.05). (I) Assessment of biofilm formation (as described in panel A and B) by GA1 in the control medium and upon supplementation with 0.1% (m/v) HG and HGHM (Mean±SD, n=8, Tukey's multiple comparisons, α=0.05). See also Figure S1 and S2.

**Figure 4. Homogalacturonan sensing enhances the sporulation dynamics in B. velezensis.** (A) Time-course measurement of GA1 population (vegetative cells and spores) by CFU counting upon root colonization of tomato plantlets roots (Mean±SD, n=6). (B) Assessment of spores percentages by flow cytometry (see STAR method) in planktonic cultures of GA1 after 48h upon supplementation with 0.1% (m/v) PEC or HG (Mean±SD, n=3, Tukey's multiple comparisons, α=0.05). (C) Time-course measurement of sporulation dynamics over time of GA1 (left axis) in control medium (light grey bars) and upon supplementation with 0.1% (m/v) HG (dark grey bars) regarding bacterial OD₆₀₀ (right axis) in the control medium (red dots) and upon supplementation with 0.1% (m/v) HG (green dots) (Mean±SD, n=3). (D) Assessment of spores percentages (as described in Panel B) in planktonic cultures of GA1 after 48h upon supplementation with 0.1% (m/v) HG of different DP (HG, mean DP≈170³²; OGₐ, mean DP=13; OGᵇ, mean DP=4; GA, DP=1) (Mean±SD, n=3, Tukey's multiple comparisons, α=0.05). (E) Relative gene expression of spo0A and abrB in GA1 cells in the control medium (white bars) and upon supplementation with 0.1% (m/v) HG (grey bars) in planktonic cultures of 24h (Mean±SD, n=3, t-test; *, P<0.05; **, P<0.01). (F) Population diversity in biofilm pellicles formed at the air-liquid interface determined by flow cytometry after RSG staining (see STAR method) in the control medium and upon supplementation with 0.1% (m/v) HG. Gate P1, P2, P3, P4 stand for highly metabolically active vegetative cells, low metabolically active vegetative cells, spore-forming cells and mature spores respectively. (G) Assessment of spores percentages (as described in panel B) in planktonic cultures of GA1 and the mutant ΔepsA-O after 48h upon supplementation with 0.1% (m/v) HG (Mean±SD, n=3, t-test; ns, ****, P<0.0001). See also Figure S3.

**Figure 5. Homogalacturonan sensing impacts the production of bioactive secondary metabolites in B. velezensis.** (A) Impact of HG supplementation on the secondary metabolome of GA1 at late exponential/stationary phase. Each dot represents a feature detected corresponding to a specific compound (molecular ion with exact mass in UPLC-MSMS). The variety of dots in each family of metabolites reflects the natural co-production of structural variants differing in the length and isomery of the fatty acid chain and in the position of some amino acid residues in the peptide moiety. Data are expressed as peak area fold change per OD₆₀₀, compared to control culture (Mean±SD, n=3). (B) Relative production of surfactins, iturins and fengycins (from left to right) in planktonic cultures of GA1 in the control medium (white bars) and upon supplementation with 0.1% (m/v) HG (grey bars) (Mean±SD, n=3, t-test; ns, non-significative; *, P<0.05; **, P<0.01; ***, P<0.001). Inset: Expression pattern of srfAA, ituC and fenC genes (from left to right) in planktonic cultures of GA1 in the control medium (white bars) and upon supplementation with 0.1% (m/v) HG (grey bars) (Mean±SD, n=3, t-test; ns, non-significative; **, P<0.01; ***, P<0.001). (C) UPLC-MS total ion chromatogram (TIC) illustrating the relative abundance of lipopeptides surfactins, iturins and fengycins and their corresponding structure synthesized by GA1 cells forming biofilm at the air liquid interface in the control medium (red) and upon supplementation with 0.1% (m/v) HG (black) after 72h. The different peaks eluted for each lipopeptide family correspond to structural variants and homologs as described in panel A. See also Figure S4.

## 539 Main tables and legends

**Table 1.** Content of enzymes involved in pectin degradation (polygalacturonases, (PG); rhamnogalacturonan hydrolases, (RGH); polygalacturonate lyases, (PL)) and pectin remodelling (pectin methylesterases, (PME) and pectin acetylesterases, (PAE)) in archetype strains of soil-dwelling bacilli. Enzymes are grouped by family following the classification established in the CAZy database¹⁰⁵. * means that one or several protein sequences are referred as domain of unknown function (DUF), unknown protein, hypothetical protein or putative protein. See also Table S1.
<table><thead><tr><th rowspan="2"></th><th colspan="3">Pectin degradation</th><th colspan="3">Pectin remodelling</th></tr><tr><th>PG</th><th>RGH</th><th>PL</th><th>PME</th><th>PAE</th><th>TOTAL</th></tr></thead><tbody><tr><td><b>B. altitudinis CHB19</b></td><td>1</td><td>1</td><td>2*</td><td>1</td><td>1</td><td><b>6</b></td></tr><tr><td><b>B. atrophaeus GQJK17</b></td><td>0</td><td>2</td><td>6*</td><td>0</td><td>3</td><td><b>11</b></td></tr><tr><td><b>B. clausii DSM8716</b></td><td>0</td><td>2</td><td>4*</td><td>0</td><td>0</td><td><b>6</b></td></tr><tr><td><b>B. licheniformis ATCC-14180</b></td><td>1</td><td>2</td><td>8*</td><td>1</td><td>4</td><td><b>16</b></td></tr><tr><td><b>B. mycoides KBAB4</b></td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td><b>0</b></td></tr><tr><td><b>B. paralicheniformis CBMAI1303</b></td><td>1</td><td>2</td><td>8*</td><td>1</td><td>4</td><td><b>16</b></td></tr><tr><td><b>B. pumilus SAFR032</b></td><td>1</td><td>1</td><td>2*</td><td>1</td><td>1</td><td><b>6</b></td></tr><tr><td><b>B. simplex SH-B26</b></td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td><b>0</b></td></tr><tr><td><b>B. subtilis 168</b></td><td>0</td><td>2</td><td>7*</td><td>0</td><td>3</td><td><b>12</b></td></tr><tr><td><b>B. thuringiensis CT-43</b></td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td><b>0</b></td></tr><tr><td><b>B. velezensis GA1/FZB42</b></td><td>0</td><td>0</td><td>3*</td><td>0</td><td>0</td><td><b>3</b></td></tr><tr><td><b>B. weihenstephanensis WSBC 10204</b></td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td><b>0</b></td></tr></tbody></table>

561

562

563 STAR methods

564 Resource availability

565 Lead contact

566 Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Marc Ongena (marc.ongena@uliege.be).

568

569 Materials availability

570

571 Bacterial strains and mutants generated in this study are available upon request from Marc Ongena (marc.ongena@uliege.be). This study did not generate new unique plasmids or reagents.

574

575 Data and code availability

576

• Data

577 The RNA-seq data sets (generated by Hoff et al.³²) exploited for this study are deposited at https://www.ebi.ac.uk/ena/ under the project reference PRJEB39762.

579
580 • *Code*

581 This paper does not report original code.

582 • Any other item

583

584 Any additional information regarding the data reported in this study is available from

585 the lead contact upon request.

586

587 **Experimental model and subject details**

588

589 *Bacterial strains and construction of B. velezensis GA1 knockout mutants*

590 All the bacterial strains used in this study are listed in **TableS4**. Knockout mutant strains of B.

591 *velezensis* GA1 were constructed by gene replacement by homologous recombination. Briefly,

592 a cassette containing a chloramphenicol or phleomycin resistance gene flanked by 1 kb of the

593 upstream and downstream region of the targeted gene (see **TableS5** for primers) was

594 constructed by PCR overlap following the method developed by Bryksin and Matsumura¹⁰⁶.

595 This recombinant cassette was introduced into *B. velezensis* GA1 following a slightly modified

596 protocol developed by Jarmer *et al*.¹⁰⁷ to induce natural competence *via* nitrogen limitation.

597 Briefly, one colony of GA1 was inoculated in LB medium during 6h at 37°C under shaking

598 (160rpm). Then, 1mg of the recombinant cassette was added to the GA1 washed cell

599 suspension adjusted to an OD₆₀₀ of 0.01 in MMG medium at pH 7 (19g/l K₂HPO₄, 6g/l KH₂PO₄,

600 1g/l Na₃citrate, 0.2g/l MgSO₄.7H₂O, 2g/l Na₂SO₄, 50mM FeCl₃ [sterilized by filtration at

601 0.22µm], 2mM MnSO₄ [sterilized by filtration at 0.22 µm], 8g/l glucose, and 2g/l L-glutamic

602 acid). Incubation was performed at 37°C under shaking (160rpm) during 24h. Cells were plated

603 on LB medium solidified with agar (14g/l) supplemented with 5µg/ml chloramphenicol or

604 4µg/ml phleomycin to only select cells having integrated the recombinant cassette by double

605 crossing over. Gene deletions were confirmed by PCR analysis with the corresponding specific

606 upstream forward and downstream reverse primers.

607 *Culture media and growth conditions*

608 Bacterial cultures were performed at 26°C under shaking (160rpm) in root exudates mimicking

609 medium diluted to the half (REM)³¹ at pH 7: 1g/l glucose, 1.7g/l fructose, 0.2g/l maltose, 0.3g/l

610 ribose, 2g/l citrate, 2g/l oxalate, 1.5g/l succinate, 0.5g/l malate, 0.5g/l fumarate, 0.34g/l

611 KH₂PO₄, 10.5g/l MOPS, 0.25g/l MgSO₄ 7H₂O, 0.25g/l KCl, 0.5g/l yeast extract, 0.5g/l Casamino

612 acids, 1g/l (NH₄)₂SO₄, 600µg/l Fe₂(SO₄)₃, 200µg/l MnSO₄, 800µg/l CuSO₄, 2mg/l Na₂MoO₄.

613 Following the experiment, PEC, HG(HM) (Elicityl), oligogalacturonides (OGs) or galacturonic

614 acid (GA, Sigma) were normalized in terms of weight and were added at a final concentration

615 of 0.1% (m/v) in the REM.

616 To assess the use of pectin backbone as a carbon source, cultures were performed in modified

617 M9 medium (M9 stock solution adjusted to pH7: 42.5g/l Na₂HPO₄.2H₂O, 15g/l KH₂PO₄, 5g/l

618 NH₄Cl, 2.5g/l NaCl; trace elements solution: 0.1g/l MnCl₂.4H₂O, 0.17g/l ZnCl₂, 0.043g/l

619 CuCl₂.2H₂O, 0.06g/l CoCl₂.6H₂O, 0.06g/l Na₂MoO₄.2H₂O; Calcium chloride solution: 14.7g/l
CaCl₂·2H₂O; Magnesium sulfate solution: 246g/l MgSO₄·7H₂O; Iron chloride solution: 13.5g/l
FeCl₃·6H₂O, 21g/l citric acid·H₂O). All the solutions were filter sterilized (0.22µm CA
membrane). M9 medium was reconstituted by mixing 200ml M9 stock solution, 1ml calcium
chloride solution, 10ml trace elements solution, 1ml magnesium sulfate solution, 1ml iron
chloride solution, 0.1% (m/v) glycerol and the volume was adjusted to 1l with ultrapure water.
The carbon sources tested were normalized in terms of weight and were added to the medium
at a final concentration of 0.2% (w/v).

For planktonic and biofilm cultures, the cells of an overnight preculture of the strain of interest were washed twice with sterile phosphate-buffered saline (PBS, 8g/l NaCl, 0.2g/l KCl, 1.44g/l Na₂HPO₄, 0.24g/l KH₂PO₄; pH 7.0), before being inoculated in the liquid medium at a final OD₆₀₀ of 0.02 (planktonic cultures) or 0.1 (biofilm cultures).

## Plant growth conditions

For plant root colonization assays, tomato seeds were sterilized in a 70% (v/v) ethanol solution for 2min, transferred in a 20% (v/v) bleach solution under shaking during 20min, and rinsed three times with sterile water. Sterilized tomato seeds were pre-germinated during 4 days in square petri dishes containing Hoagland medium solidified with agar (14g/l) at 22°C under a 16 h/8 h day/night cycle.

To extract pectin from tomato plant roots, tomato seeds were sterilized as described above. Then, seeds were transferred into seedholders filled with Hoagland medium with 0.7% (m/v) agar and grown during 4 weeks under a 16h/8h day/night cycle in hydroponic systems (Araponics, Belgium) filled with a nutritive solution (500µl/l FloraBloom, FloraMicro and FloraGro (General Hydroponics Europe)).

642

## Method details

### Phylogenetic and sequence identity analysis

All the analysis were carried out on MEGA11 software¹⁰⁸. Phylogenetic analysis was performed on a total of 20 pectin/pectate lyase sequences (classified in PL1 and PL3 family following the CAZy Database¹⁰⁵) of soil-dwelling bacilli species retrieved from NCBI protein database. To construct the maximum likelihood tree, protein sequences were aligned by MUSCLE alignment and phylogenetic evolution was inferred using the “Maximum likelihood” method and “Whelan And Goldman” model (WAG) with a discrete Gamma distribution (+G). The best-fit substitution model was selected among all the substitution models implemented in MEGA11 by choosing the one yielding to the lowest BIC score for the dataset analyzed.

### Pectin degradation activity measurement

Pectin degradation activity of cell-free culture supernatants (CFCS) of *B. velezensis* GA1 or GA1 knockout mutants was measured by spectrophotometry. Accumulation of unsaturated oligogalacturonates generated by the enzymes via the mechanism of β-elimination was evaluated by measuring the increase of absorbance at 232nm. Briefly, CFCS of the different strains were obtained by centrifugation (6000rpm, 10min) of the samples from the planktonic
bacterial cultures in REM at different time points before being sterilized on a 0.22µm PTFE filter. Then, 25µl of the CFCS were added to 100µl of HG or HGHM (2.5g/l) dissolved in 50mM Tris-HCl buffer at pH 8. The reaction mixture was then incubated at 30°C under shaking and the increase of absorbance was measured at 232nm in a multiplate reader (Tecan SPARK, Männedorf, Switzerland). The molar extinction coefficient for unsaturated oligogalacturonates at 232nm is 4600 M⁻¹ cm⁻¹ ¹⁰⁹.

### root colonization assay

Root colonization assays were performed on tomato plantlets under gnotobiotic conditions. 4 days-old tomato plantlets roots were inoculated with the bacterial strain of interest in the square petri dishes of solidified Hoagland medium in which the seeds were pre-germinated. To do so, cells from an overnight preculture in REM were washed twice with sterile PBS and OD₆₀₀ was adjusted to 0.5. For the co-inoculation assay, 500µl of GA1 and ΔpeIΔpeI_B cell suspensions, both at OD₆₀₀ adjusted to 0.5, were mixed together in an Eppendorf and vortexed. Then, 5µl of the bacterial suspensions were deposited on the root top. To quantify the bacterial colonization in-planta overtime, the roots were separated from the aerial part of the plant after 1, 3 and 7 days of colonization. The roots were placed in tubes containing a solution of PBS supplemented with 0.1% (m/v) Tween20 and vortexed vigorously to tear off the bacterial cells from the roots. The total colony-forming units (CFUs) were determined by plating serial dilutions of the bacterial solution on solid LB medium (for GA1 cells) or solid LB medium supplemented with 5µg/ml chloramphenicol (for ΔpeIΔpeI_B mutant cells) and incubated overnight at 30°C before proceeding to plate counting. For spores counting, the same solution containing the bacteria was heated at 85°C during 20min to kill vegetative cells before proceeding to the plating.

### Extraction of pectin from tomato roots

Roots of 4 weeks-old tomato plants were harvested and lyophilized before being grinded with a Retsh MM400 mixer mill. The extraction of root cell wall was performed following an adapted protocol from Carpita¹¹⁰ and Silva et al¹¹¹. Briefly, 200mg of cell wall powder were resuspended into 40ml of ethanol 80% (v/v) and incubated at 90°C during 20min. After centrifugation (10,000g, 5min), supernatant was discarded and the same procedure described above was repeated 3 times on the pellet. Then, the pellet was washed with 20ml of ultrapure water and after centrifugation (10,000g, 5min), the pellet was resuspended in 10ml of acetone. The solution was centrifuged (10,000g, 5min) and the pellet was lyophilized to obtain the alcoholic residue (AR) for fractionation. The AR was resuspended in 20ml of ultrapure water and incubated at 80°C during 1h. After centrifugation (10,000g, 5min), the pellet was resuspended in 100ml of ammonium oxalate 1% (v/v) and incubated at 80°C during 2h. After centrifugation (10,000g, 5min), the supernatant was recovered, dialyzed on a 3.5kD MWCO Spectra/Por membrane (Spectrum Laboratories Inc.) and lyophilized to obtain the PEC fraction.

### Simple sugar analysis of pectin fraction

To hydrolyse simple sugars from PEC fraction, 1ml of 2M trifluoroacetic acid (TFA) were added to 2mg of lyophilized pectin fraction and incubated at 121°C during 90min. Then, TFA was
evaporated under N₂ flow and the residue was resuspended in 1ml ultrapure water and filtered on a 0.2µm filter. Before analysis, the solution was diluted to 1mg/ml in ultrapure water. Neutral and acidic monosaccharide composition was determined by high-performance anion exchange chromatography (ICS3000 system) coupled with a pulsed amperometric detector (HPAEC-PAD) (Dionex, Thermo Scientific) equipped with a CarboPac PA-1 column (4mm × 250mm) and a CarboPac PA1 guard column (ID 4mm × 50mm). The injection volume was 25µl and elution was performed at 30°C with a constant flow rate of 1ml/min in gradient mode. For neutral sugar quantification, the mobile phases were ultrapure H₂O (A), 160 mM NaOH (B) and 200 mM NaOH (C). The elution profiles were as follows: 0–25 min 90% A and 10% B, 25–26 min 0–100% C, 26–35 min 100% C, 25–36 min 100–0% C, 36–50 min 90% A and 10% B. For uronic acids quantification, the mobile phases were 0.16 M NaOH (A) and 0.6 M NaOAc in 0.16 M NaOH (B). The elution profiles were as follows: 0–5 min 100% A, 5–35 min 0–100% B, 35–40 min 100% B, 40–42 min 100–0% B and finally column re-equilibration by 100% A from 42 to 50 min. Monosaccharides arabinose, fucose, galactose, glucose, rhamnose, xylose, galacturonic acid and glucuronic acid (Sigma-Aldrich) were used as standards for quantification.

### 716 RNA extraction and RT-qPCR analysis

RNA extractions were performed with the NucleoSpin RNA Kit (Macherey Nagel, Germany), following the manufacturer's protocol for Gram positive bacteria. The amount of RNAs extracted and their purity were assessed with the UV-vis spectrophotometer NanoDrop 2000 (Thermo scientific). RT-qPCR was performed in a ABI StepOne™ qPCR apparatus (Applied Biosystems) using the kit Luna® Universal One-Step RT-qPCR Kit (New England Biolabs, Ipswich, MA, United States). Reactions were carried out in a 20µl volume containing 10µL of Luna Universal One Step Reaction Mix, 1µL of Luna WarmStart® RT Enzyme Mix, 0.8µl of each primer (10µM) and 100ng of RNA under the following thermal cycling conditions: a 10min reverse transcription step at 55°C, a 1min initial denaturation step at 95°C and 40 cycles of 10s at 95°C and 30s at 60°C. Melting curves were also realized from 65 to 95°C with an increase rate of 0.5°C/5s to evaluate the specificity of the amplified products. The transcription level of genes of interest was analysed using the mathematical model proposed by Pfaff¹¹². The *gyrA* gene encoding the DNA gyrase subunit A was used as housekeeping gene to normalise the expression level of the gene of interest.

### 731 Biofilm formation assay

Quantification of total biofilm was performed by crystal violet staining. Briefly, the strain of interest was inoculated in a 96-wells microplate containing 200µl of REM supplemented or not with 0.1% (m/v) HG, HGHM, OGs or GA. The plate was incubated at 30°C during 24h without shaking. Medium and planktonic cells were discarded, and wells were washed with PBS. The biofilm pellicle was stained with 0.1% (v/v) crystal violet during 10min and then wells were washed three times with PBS. The stained biofilm was dissolved with 30% (v/v) acetic acid. Absorbance was measured at 595nm.

739

740
741 *Generation of short DP OGs*

742 OGs of short polymerization degree (OG<sub>B</sub>) were generated from the reaction of concentrated
743 24h-CFCS of *B. velezensis* GA1 Δsfp unable to synthesize lipopeptides, on HG as substrate.
744 Briefly, the CFCS was obtained as described in the section “Pectin degradation activity
745 measurement”. The CFCS was then concentrated 50x on a Vivaspin20 centrifugal concentrator
746 with a polyethylene sulfone membrane of 10,000Da MWCO (Sartorius). The enzymatic
747 reaction was performed at 30°C by mixing 25% (v/v) of concentrated supernatant with a
748 solution of 0.5% (m/v) HG resuspended in 50mM Tris-HCl buffer at pH 8. Absorbance at 232nm
749 was followed until the enzymatic reaction reached a plateau. Then, short DP OGs were
750 separated from larger molecules according to the protocol of Voxeur et al<sup>113</sup> via ethanol
751 precipitation. The supernatant was then recovered and lyophilized.

752 *Analysis of OGs profile*

753 Relative quantification and polymerization degree of OGs were evaluated by UPLC-qTOF MS.
754 For each sample, a volume of 10µl was injected in an Agilent 1290 Infinity II apparatus coupled
755 with a diode array detector and a mass detector (Jet Stream ESI-Q-TOF 6530) with the
756 parameters set up as follows: capillary voltage: 3.5kV; nebulizer pressure: 35psi; drying gas:
757 8l/min; drying gas temperature: 300°C; flow rate of sheath gas: 11l/min; sheath gas
758 temperature: 350°C; fragmentor voltage: 175V; skimmer voltage: 65V; octopole RF: 750V.
759 Accurate mass spectra were recorded in negative mode in the range of m/z = 50-1700. OGs
760 were separated on a Luna HILIC column (3mm x 150mm x 5µm, Phenomenex, Torrance,
761 California, USA) using a gradient of water (solvent A) and 90% acetonitrile (solvent B) that
762 were both acidified with 0.1% (v/v) formic acid and supplemented with 15mM ammonium
763 formate as mobile phase, with a constant flow rate of 0.5ml/min and a temperature of 40°C.
764 For the separation of OGs, initial gradient was decreased from 100% B to 85% B in 4min before
765 decreasing to 0% B in 16min. Solvent B was kept at 0% during 10min before going back to
766 100% B. Polymerization degree of the detected OGs was determined based on accurate mass
767 measurement and relative quantification was based on peak area of the extracted ion
768 chromatogram generated by MassHunter worksation v10.0.

769 *Extracellular DNA (eDNA) detection in biofilm pellicles*

770 Biofilms of GA1 were grown in a 96-wells black microplate containing 200µl of REM
771 supplemented or not with 0.1%(m/v) HG and incubated at 30°C without shaking. eDNA
772 present in the biofilm pellicles formed at the air-liquid interface was measured using the kit
773 QuantiFluor dsDNA system (Promega, Madison, WI, USA) according to manufacturer's
774 protocol. Briefly, medium and planktonic cells were discarded, and wells were washed with
775 PBS in order to only keep the biofilm pellicles. Then, 200µl of QuantiFluor® dsDNA Dye working
776 solution (1:400 QuantiFluor® dsDNA Dye in 1X TE buffer) were dispensed into each well and
777 homogenized with the pellicles by pipetting up and down. Microplate was incubated in the
778 dark at room temperature during 5min and the fluorescence intensity (504nm<sub>Ex</sub>/531nm<sub>Em</sub>)
779 was measured using a microplate reader (Tecan SPARK, Männedorf, Switzerland).

780
781 *Cell counting in biofilm pellicles*

782 To evaluate the bacterial population embedded in the biofilm pellicles, GA1 cells were
783 inoculated in a 24-wells microplate containing 2ml of REM supplemented or not with 0.1%
784 (w/v) HG and incubated at 30°C without shaking. The biofilm pellicle formed at the air-liquid
785 interface was then collected, resuspended into 2ml PBS and mildly sonicated (Power 60%, 2
786 cycles, 1min) on a Sonoplus apparatus HD 2070 (Bandelin). Several dilutions were plated on
787 solid LB medium and incubated at 30°C overnight before counting.

788 *Stereomicroscopy acquisitions*

789 Pictures of biofilm pellicles of GA1 formed at the air-liquid interface were taken with a Nikon
790 SMZ1270 stereomicroscope (Nikon, Japan) equipped with a Nikon DS-Qi2 monochrome
791 microscope camera and a DS-F 1x F-mount adapter. Pictures were captured in the bright field
792 channel with an ED Plan Apo 1x/WF objective and a 0.63x magnification of with an exposure
793 time of 40ms. Additionally, pictures of biofilm pellicles at 6h were captured with a 3x
794 magnification and an exposure time of 10ms to get a better view of the structures in
795 formation. Acquisitions were then processed with NIS-Element AR software (Nikon, Japan).

796 *Microscopy acquisitions*

797 Microscopy acquisitions were performed using a Nikon Ti2-E inverted microscope (Nikon, 798 Japan) equipped with a ×20/0.45 NA S Plan Fluor objective lens (Nikon, Switzerland) and a 799 Nikon DS-Qi2 monochrome microscope camera. Images and videos taken in the bright field 800 channel were acquired using a Ti2 Illuminator-DIA and an exposure time of 20ms. GA1 cells 801 strained with RedoxSenor Green (RSG) (BacLight vitality kit, Thermofisher) were visualized by 802 conventional epifluorescence microscopy with a ×100/1 oil NA S Plan Fluor objective lens 803 (Nikon, Switzerland). A lumencor sola illuminator (Lumencor, USA) was used as the source of 804 excitation with an exposure time of 500ms and the GFP-B HC Bright-Line Basic Filter was used. 805 Acquisitions were then processed with NIS-Element AR software (Nikon, Japan).

806 *Flow cytometry analysis*

807 Cell activity of GA1 cells was evaluated by RSG staining. For the experiments conducted under
808 biofilm conditions, the pellicles formed at the air-liquid interface were collected, resuspended
809 into PBS and mildly sonicated as described in the section "Cell counting in biofilm pellicles",
810 before staining. To stain GA1 cells, 1µl of RSG 10x diluted were added to 1ml of cell culture
811 appropriately diluted in filtered PBS and incubated in the dark during 10min. RSG fluorescence
812 (FL1-490nmex/520nmem) and cell size (FSC-H) were measured by flow cytometry using a BD
813 Accuri™ C6 appartus (BD Biosciences) under the following conditions: Medium fluidics
814 (35µl/min); 20,000 events recorded; FSC-H threshold 20,000.

815

816 *Analysis of BSMs*

817 The detection of *B. velezensis* metabolites was performed by UPLC-qTOF MS. Before analysis, 818 samples from planktonic cell cultures were filtered on a 0.22µm PTFE filter. For the analysis of 819 metabolites of biofilm-forming cells, the content of each well of the microplate was mildly
sonicated as described in the section “Cell counting in biofilm pellicles”, before filtration. For each sample, a volume of 5µl was injected in an Agilent 1290 Infinity II appartus coupled with a diode array detector and mass detector (Jet Stream ESI-Q-TOF 6530) with the parameters set up as described in the section “Analysis of OGs profile”. Accurate mass spectra were recorded in positive mode in the range of m/z =100–1700. Metabolites were separated on a C18 Acquity UPLC BEH column (2.1 × 50mm × 1.7µm; Waters, milford, MA, USA) using a gradient of water (solvent A) and acetonitrile (solvent B) both acidified with 0.1% formic acid as a mobile phase with a constant flow rate 0.6ml/min and a temperature of 40°C. The gradient was set up to start at 10% B and kept in this condition during 1min before raising to 100% B in 10 min. Solvent B was kept at 100% for 3.5 min before going back to the initial ratio. MassHunter v10.0 workstation and the open-source software MzMine2⁶⁷ were used for data collection and analysis.

RNA-seq data analysis

833

The analysis of RNAseq data used in this study has been described in details previously³². In the present study, we focused especially on genes involved in motility and EPS and cohesion protein synthesis. Briefly, raw RNA-seq reads were trimmed with Trimmomatic v0.39¹¹⁴ and quality control was assessed using FastQC v0.11.8 (Babraham Bioinformatics). The trimmed reads were then aligned to the genome of B. velezensis GA1 (GenBank: CP046386) using BWA-MEM v0.7.17¹¹⁵. Read counts were calculated using the python-based tool HTSeq v0.9¹¹⁶ and the tables of fragments per kilobase of transcript per million mapped reads (FPKM) were generated using the Cufflinks function cuffnorm¹¹⁷. Genes with read counts <25 were excluded. Differential expression analysis was conducted using DESeq2 pipeline¹¹⁸, considering the following parameters: P-value<0.05 and fold change ≥1.5. Heatmaps were performed on GraphPad Prism 9.

845

Quantification and Statistical Analyses

All statistical analyses were performed on GraphPad Prism 9. Results are expressed as mean ± SD (standard deviation). Analysis of variance (ANOVA) was performed on each data set and statistical differences between means were evaluated by two-tailed Student's t-test or Tukey's multiple comparisons (α=0.05). The number of biological replicates used for each experiment and P-values are indicated in the legends of the corresponding figures.

852

Video files

VideoS1A. Microscopic observation of early biofilm formation (at 7 hours post-inoculation) of B. velezensis GA1 in microplate in the control medium. Related to Figure 3.

VideoS1B. Microscopic observation of early biofilm formation (at 7 hours post-inoculation) of B. velezensis GA1 in microplate upon supplementation with 0.1% (m/v) HG. Related to Figure 3.

858

859

860

861
862 Acknowledgments

863

This work was supported by the PDR research project ID 26084552 from the F.R.S.-FNRS (National fund for Scientific Research in Belgium) and by the EOS project ID 30650620 from the FWO/F.R.S.-FNRS. F.B., A.A. and A.R. are recipient of a F.R.I.A. fellowship (F.R.S.-FNRS) and MO is Research Director at the F.R.S.-FNRS. We gratefully acknowledge Emmanuel Petit for providing the oligogalacturonides of high polymerization degree and Catherine Helmus for technical help.

869

870 Declaration of interests

872

The authors declare no competing interests.

874

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1208
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<figure><img src="image_47.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_48.png" /></figure>
Highlights

* *B. velezensis* (*B.v*) secretes two unique enzymes degrading homogalacturonan (HG)

* These pectinases contribute to bacterial fitness and root colonization

* HG stimulates biofilm, sporulation and secondary metabolite production in *B.v*

* These traits contribute to establishment and persistence of B.v in the rhizosphere

<figure><img src="image_49.png" /></figure>
KEY RESOURCES TABLE

The table highlights the reagents, genetically modified organisms and strains, cell lines, software, instrumentation, and source data **essential** to reproduce results presented in the manuscript. Depending on the nature of the study, this may include standard laboratory materials (i.e., food chow for metabolism studies, support material for catalysis studies), but the table is **not** meant to be a comprehensive list of all materials and resources used (e.g., essential chemicals such as standard solvents, SDS, sucrose, or standard culture media do not need to be listed in the table). **Items in the table must also be reported in the method details section within the context of their use.** To maximize readability, the number of **oligonucleotides and RNA sequences** that may be listed in the table is restricted to no more than 10 each. If there are more than 10 oligonucleotides or RNA sequences to report, please provide this information as a supplementary document and reference the file (e.g., See Table S1 for XX) in the key resources table.

**Please note that ALL references cited in the key resources table must be included in the main references list.** Please report the information as follows:

* **REAGENT or RESOURCE:** Provide the full descriptive name of the item so that it can be identified and linked with its description in the manuscript (e.g., provide version number for software, host source for antibody, strain name). In the experimental models section (applicable only to experimental life science studies), please include all models used in the paper and describe each line/strain as: model organism: name used for strain/line in paper: genotype. (i.e., Mouse: OXTR<sup>fl/fl</sup>: B6.129(SJL)-Oxtr<sup>tm1.1Wsy/J</sup>). In the biological samples section (applicable only to experimental life science studies), please list all samples obtained from commercial sources or biological repositories. Please note that software mentioned in the methods details or data and code availability section needs to also be included in the table. See the sample tables at the end of this document for examples of how to report reagents.

* **SOURCE:** Report the company, manufacturer, or individual that provided the item or where the item can be obtained (e.g., stock center or repository). For materials distributed by Addgene, please cite the article describing the plasmid and include "Addgene" as part of the identifier. If an item is from another lab, please include the name of the principal investigator and a citation if it has been previously published. If the material is being reported for the first time in the current paper, please indicate as "this paper." For software, please provide the company name if it is commercially available or cite the paper in which it has been initially described.

* **IDENTIFIER:** Include catalog numbers (entered in the column as "Cat#" followed by the number, e.g., Cat#3879S). Where available, please include unique entities such as **RRIDs**, Model Organism Database numbers, accession numbers, and PDB, CAS, or CCDC IDs. For antibodies, if applicable and available, please also include the lot number or clone identity. For software or data resources, please include the URL where the resource can be downloaded. Please ensure accuracy of the identifiers, as they are essential for generation of hyperlinks to external sources when available. Please see the Elsevier **list of data repositories** with automated bidirectional linking for details. When listing more than one identifier for the same item, use semicolons to separate them (e.g., Cat#3879S; RRID: AB_2255011). If an identifier is not available, please enter "N/A" in the column.

* **A NOTE ABOUT RRIDs:** We highly recommend using RRIDs as the identifier (in particular for antibodies and organisms but also for software tools and databases). For more details on how to obtain or generate an RRID for existing or newly generated resources, please visit the **Rll** or **search for RRIDs**.

Please use the empty table that follows to organize the information in the sections defined by the subheading, skipping sections not relevant to your study. Please do not add subheadings. To add a row,
place the cursor at the end of the row above where you would like to add the row, just outside the right border of the table. Then press the ENTER key to add the row. Please delete empty rows. Each entry must be on a separate row; do not list multiple items in a single table cell. Please see the sample tables at the end of this document for relevant examples in the life and physical sciences of how reagents and instrumentation should be cited.

TABLE FOR AUTHOR TO COMPLETE

Please upload the completed table as a separate document. **Please do not add subheadings to the key resources table.** If you wish to make an entry that does not fall into one of the subheadings below, please contact your handling editor. **Any subheadings not relevant to your study can be skipped.** (NOTE: References within the KRT should be in numbered style rather than Harvard.)

Key resources table

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td><strong>Antibodies</strong></td><td></td><td></td></tr><tr><td>N/A</td><td></td><td></td></tr><tr><td><strong>Bacterial and virus strains</strong></td><td></td><td></td></tr><tr><td>N/A</td><td></td><td></td></tr><tr><td><strong>Biological samples</strong></td><td></td><td></td></tr><tr><td>N/A</td><td></td><td></td></tr><tr><td><strong>Chemicals, peptides, and recombinant proteins</strong></td><td></td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>Cat#M1254</td></tr><tr><td>Bacto™ Casamino acids</td><td>ThermoFisher Scientific</td><td>Cat#223050</td></tr><tr><td>Bacto™ Yeast extract</td><td>ThermoFisher Scientific</td><td>Cat#212750</td></tr><tr><td>HG: Galacturonan polysaccharides LM</td><td>Elicityl</td><td>Cat#GAT100</td></tr><tr><td>HGHM: Galacturonan polysaccharides HM</td><td>Elicityl</td><td>Cat#GAT101</td></tr><tr><td>GA: Galacturonic acid</td><td>Sigma</td><td>Cat#93478</td></tr><tr><td>OGA</td><td>Pr. Emmanuel Petit (UPJV, France)</td><td>N/A</td></tr><tr><td>OGB</td><td>This study</td><td>N/A</td></tr><tr><td>PEC</td><td>This study</td><td>N/A</td></tr><tr><td>Chloramphenicol</td><td>AppliChem</td><td>Cat#A1806</td></tr><tr><td>Phleomycin</td><td>InvivoGen</td><td>Cat#ant-ph-1</td></tr><tr><td>Tris-HCl</td><td>Sigma</td><td>Cat#T3253</td></tr><tr><td>Tween20</td><td>Sigma</td><td>Cat#P9416</td></tr><tr><td>TFA: Trifluoroacetic acid</td><td>Sigma</td><td>Cat#302031</td></tr><tr><td>Crystal Violet solution</td><td>Sigma</td><td>Cat#V5265</td></tr><tr><td>FloraBloom</td><td>General Hydroponics Europe (GHE)</td><td>N/A</td></tr><tr><td>FloraMicro</td><td>General Hydroponics Europe (GHE)</td><td>N/A</td></tr><tr><td>FloraGro</td><td>General Hydroponics Europe (GHE)</td><td>N/A</td></tr></tbody></table>
<table><thead><tr><th colspan="3">Critical commercial assays</th></tr></thead><tbody><tr><td>NucleoSpin RNA Kit</td><td>Macherey Nagel</td><td>Cat#740955.250</td></tr><tr><td>Luna® Universal One-Step RT-qPCR Kit</td><td>New England Biolabs</td><td>Cat#E3005L</td></tr><tr><td>QuantiFluor dsDNA system</td><td>Promega</td><td>Cat#E2670</td></tr><tr><td>BacLight™ RedoxSensor™ Green vitality kit</td><td>ThermoFisher Scientific</td><td>Cat#B34954</td></tr><tr><th colspan="3">Deposited data</th></tr><tr><td>RNAseq data exploited in this paper</td><td>Hoff et al., 2021</td><td>https://www.ebi.ac.uk/ena/<br>Project reference:<br>PRJEB39762</td></tr><tr><th colspan="3">Experimental models: Cell lines</th></tr><tr><td colspan="3">N/A</td></tr><tr><th colspan="3">Experimental models: Organisms/strains</th></tr><tr><td>Bacterial strains (see TableS4)</td><td></td><td></td></tr><tr><td>Tomato: Solanum Lycopersicum L.</td><td>Sluis Garden</td><td>Cat#SL0765</td></tr><tr><th colspan="3">Oligonucleotides</th></tr><tr><td>Oligonucleotides (see TableS5)</td><td>This study</td><td>N/A</td></tr><tr><th colspan="3">Recombinant DNA</th></tr><tr><td colspan="3">N/A</td></tr><tr><th colspan="3">Software and algorithms</th></tr><tr><td>MEGA11</td><td>Tamura et al., 2021</td><td>https://www.megasof<br>tware.net/</td></tr><tr><td>MassHunter v10.0</td><td>Agilent</td><td>https://www.agilent.com/en/product/software-informatics/mass-spectrometry-software</td></tr><tr><td>MzMine2</td><td>Pluskal et al., 2010</td><td>http://mzmine.github.io/features.html</td></tr><tr><td>Trimmomatic v0.39</td><td>Bolger et al., 2014</td><td>http://www.usadellab.org/cms/?page=trimmaticy</td></tr><tr><td>FastQC v0.11.8</td><td>Babraham Bioinformatics</td><td>https://www.bioinformatics.babraham.ac.uk/projects/fastqc/</td></tr><tr><td>BWA-MEM v0.7.17</td><td>Li and Durbin., (2009)</td><td>https://maq.sourceforge.net/</td></tr><tr><td>HTSeq v0.9</td><td>Anders et al., 2015</td><td>https://htseq.readthe/docs.io/en/release_0.9.1/history.html#version-0-9-0</td></tr><tr><td>DESeq2</td><td>Love et al., 2014</td><td>https://bioconductor.org/packages/releas/e/bioc/html/DESeq2.html</td></tr></tbody></table>
<table><tr><td>NIS-Element AR software</td><td>Nikon</td><td>https://www.microscope.healthcare.nikon.com/products/software/nis-elements/nis-elements-advanced-research</td></tr><tr><td>GraphPad Prism 9</td><td>GraphPad Software</td><td>https://www graphpad.com/features</td></tr><tr><td colspan="3"><strong>Other</strong></td></tr><tr><td>SPARK multiplate reader</td><td>Tecan</td><td>https://lifesciences.tecan.com/multimode-plate-reader</td></tr><tr><td>Mixer Mill MM400</td><td>Retsch</td><td>https://www.retsch.com/products/milling/ball-mills/mixer-mill-mm-400/</td></tr><tr><td>Hydroponic systems</td><td>Araponics</td><td>Cat#K72L</td></tr><tr><td>Dionex™ ICS-3000 Ion Chromatography System</td><td>Conquer scientific</td><td>https://conquerscientific.com/product/dionex-ics-3000-ion-chromatography-system/</td></tr><tr><td>Dionex™CarboPac™ PA-1 column (4mm x 250mm)</td><td>ThermoFisher Scientific</td><td>Cat#035391</td></tr><tr><td>NanoDrop 2000</td><td>ThermoFisher Scientific</td><td>Cat#ND-2000</td></tr><tr><td>StepOne™ Real-Time PCR system</td><td>ThermoFisher Scientific</td><td>Cat#4376357</td></tr><tr><td>3.5kD MWCO Spectra/Por dialysis membrane</td><td>VWR</td><td>Cat#25219-085</td></tr><tr><td>Vivaspin20 centrifugal concentrator polyethylene sulfone 10,000Da MWCO</td><td>Sartorius</td><td>Cat#VS2001</td></tr><tr><td>UHPLC Agilent 1290 Infinity II</td><td>Agilent</td><td>https://www.agilent.com/en/product/liquid-chromatography/hplc-systems/analytical-hplc-systems/1290-infinity-ii-lc-system</td></tr><tr><td>Luna® HILIC 200 Å column (150 x 3 mm x 5 µm)</td><td>Phenomenex</td><td>Cat#00F-4450-Y0</td></tr><tr><td>Acquity UPLC BEH C18 column (2.1 x 50mm x 1.7µm)</td><td>Waters</td><td>Cat#186002350</td></tr><tr><td>6530 Q-TOF mass spectrometer</td><td>Agilent</td><td>Cat#G6530AA</td></tr><tr><td>Sonoplus HD 2070 ultrasonic homogenizer</td><td>Bandelin</td><td>https://profilab24.com/en/laboratory/ultrasonic/bandelin-sonopuls-hd-2070-homogeniser</td></tr></table>
<table><tr><td>BD Accuri™ C6 Plus Flow cytometer</td><td>BD Biosciences</td><td>https://www.bdbiosciences.com/en-us/products/instruments/flow-cytometers/research-cell-analyzers/bd-accuri-c6-plus</td></tr><tr><td>Nikon SMZ1270 stereomicroscope</td><td>Nikon</td><td>https://www.microscope.healthcare.nikon.com/products/stereo-microscopes-macroscopes/smz1270-smz1270i</td></tr><tr><td>Nikon Ti2-E inverted microscope</td><td>Nikon</td><td>https://www.microscope.healthcare.nikon.com/products/inverted-microscopes/eclipse-ti2-series</td></tr></table>