Symbiosome functionality in Medicago truncatula nodules requires continuous clearing of pectins from the symbiosome space

Domain: biology
Published: 2025-12-13
Editorial outcome: minor_revision

Abstract
Central to the legume–rhizobium symbiosis is the formation of organelle-like symbiosomes where nitrogen-fixing bacteroids are enclosed by a host-derived symbiosome membrane. This creates the symbiosome space, which topologically resembles an apoplastic compartment within the cell. While the apoplast of plant cells is largely occupied by the cell wall, symbiosomes are devoid of cell wall polymers. Here, we describe a mechanism that functions to protect and maintain effective nitrogen fixation through the action of cell-wall-degrading enzymes that prevent accumulation of un-esterified pectin within symbiosomes. We identify two symbiotically-induced polygalacturonase (PG) genes in Medicago truncatula, SyPG1 and SyPG2, that are secreted into the symbiosome space. Silencing the expression of SyPG1/2 or editing SyPG1/2 via CRISPR-Cas9 both lead to nodule senescence and trigger excessive accumulation of un-esterified pectin in symbiosome containing cells. Additionally, we show that un-esterified pectins inhibit rhizobial growth both in vivo and in vitro. Together, our results provide evidence for a host-controlled cell wall clearance mechanism that is essential for symbiosome maintenance.

Introduction
Plant cells are surrounded by a cell wall (CW), that maintains cell shape and integrity. The composition of the CW not only differs between the plant lineages but also strongly depends on cell age, organ identity and extracellular stimuli1. The main components are cellulose, hemicelluloses, pectins, and structural proteins with pectins being highly abundant in young and more flexible cell walls. Pectins comprise a complex group of polysaccharides that includes homogalacturonan (HG), xylogalacturonan, apiogalacturonan, rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II), with HG being the most abundant and extensively studied form ref. 2. Pectin biosynthesis occurs in the Golgi apparatus, where it is produced in a highly methyl esterified form and subsequently secreted into the apoplast and integrated into the cell wall3. There, pectin methylesterases (PMEs) catalyze the removal of methyl groups from HG, generating regions of de-esterified pectin4,5. These de-esterified regions can either interact with calcium ions to form gel-like cross-linked networks or serve as substrates for pectin-degrading enzymes such as pectate lyase (PL) and polygalacturonase (PG)6. Through these enzymatic modifications, the structure and function of the cell wall are dynamically regulated during plant growth, development, and stress responses.

Serving as a physical barrier against pathogenic bacteria and fungi, micro-organisms have to actively overcome the cell wall to invade cells and tissues. To achieve this, they secrete a cocktail of cell wall degrading enzymes, including PLs, PGs, pectin lyases, hemicellulases and cellulases7. This situation may be inverted during symbiotic associations, such as the interaction between legumes and rhizobia when establishing the root nodule symbiosis. During this mutualistic bacteria-plant interaction, symbiotic rhizobia colonize legumes, and some other species mostly via root hairs, where the host initiates the formation of a membrane tunnel called the infection thread (IT)8. These invasively and polarly growing membrane structures progress transcellularly through the root cortex and branch in the developing nodule primordium, where bacteria are released into specialized cells8. The transcellular passage requires significant cell wall modifications at the cell-cell interface leading to the formation of the transcellular passage cleft9. Bacteria are released from ITs at specialized sites, called “infection droplets” that are devoid of a cell wall and may instead be stabilized by the symbiotic remorin protein SYMREM110. While rhizobia bud off from the droplet in a endocytosis-like process, they are and remain surrounded by a host derived membrane called the “symbiosome membrane” (SM). Depending on the plant species single or few bacteria are encapsulated in the SM where they differentiate into nitrogen-fixing bacteroids that deliver fixed nitrogen via the SM to the host cell. In return, the host provides photosynthetic sugars to the symbiont. Different to the above-described interactions, infected nodule cells are filled with thousands of independent symbiosomes that are entirely detached from the host membrane and are maintained as cell wall free compartments throughout their entire lifespan. Taking pectins as an example, it has been shown that methyl esterified pectins accumulate in the cell periphery of infected nodule cells but not within ITs, while de-methylesterified pectins were mainly found in the cell wall structure of ITs11. This finding was explained with the continuous activity of PMEs and the spatio-temporally restricted presence of the nodule pectate lyase NPL11. None of these pectin forms has, however, been detected in the symbiosome space, the area in between the SM and the bacterial surface, that is devoid of a plant cell wall11. So far, it has been hypothesized that upon bacterial release from cell wall-free infection droplets, symbiosomes remain devoid of cell wall components12,13. However, data from Pisum sativum suggest that RG-II accumulates to some extent in the symbiosome space of young symbiosomes with undifferentiated bacteroids, while it is depleted with increasing symbiosome age14. This could be explained in two different ways: First, pectin biosynthesis and secretion are entirely abolished in mature infected cells or second, that symbiosomes contain pectin-degrading enzymes that continuously remove pectins secreted into the symbiosome space. Here, we show evidence for the latter pathway and demonstrate that at least two polygalacturonases are required to continuously clear the symbiosome space from pectin accumulation.

Results
An nodule PME is enriched in the symbiosome space
Symbiosomes as the nitrogen-fixing organelles, lack a robust cell wall. In line with this, they neither accumulate pectins nor xyloglucans11. However, esterified pectins and xyloglucans do accumulate in the peripheral cell wall of infected cells11. This implies that although esterified pectin synthesis is persistent in these cells, they may be continuously cleared from the symbiosome space. This process would require the action of PLs or PGs as PME-driven de-esterification of secreted pectins is a pre-requisite of PL-/PG-mediated pectin degradation. Indeed, a symbiotic PME (SyPME) was reported to be induced by rhizobia inoculation and required for IT growth in Medicago truncatula (hereafter M. truncatula)11. This work also pointed to the presence of another PME (Medtr2g105560), which shows highest expression in nodules with a predominant enrichment in the nodule infection zone11. Thus, we named it nodule PME (nPME). To assess pectin dynamics in symbiosomes, we first monitored the presence of SyPME and nPME. While the previously described SyPME protein accumulated around ITs and in intercellular spaces in the infection zone of indeterminate M. truncatula nodules (Supplementary Fig. 1a–a”)11, nPME was strongly enriched in the symbiosome space at the interzone of M. truncatula nodules (Fig. 1a–d and Supplementary Fig. 2) but not in the cell periphery and ITs (Fig. 1e–e”). This indicates that active pectin de-esterification inside the symbiosome space is maintained and further supports the hypothesis that pectins are continuously cleared from this compartment.

Rhizobia-induced symbiotic PGs accumulate in the symbiosome space
To test the above hypothesis, we first confirmed that NPL does not accumulate around symbiosomes but rather localizes around nodular ITs and strongly accumulates at cell wall-less infection droplets as suggested earlier (Supplementary Fig. 1b–b”)11. As PGs provide an alternative path for pectin degradation, we selected candidate PGs based on their expression profiles using publicly available transcriptomic data15,16. Of the 80 PG genes annotated in the M. truncatula genome (Supplementary Fig. 3), expression profiles of 56 PGs could be retrieved from M. truncatula nodule transcriptomic data15,17. Based on these profiles, six PGs (Medtr4g094715, Medtr8g028725, Medtr6g028080, Medtr8g098840, Medtr2g032710, and Medtr7g078680) were initially selected as they were highly transcribed in nodules with their expression being restricted to either the infection zone or interzone of M. truncatula nodules (Supplementary Data 1 and Supplementary Fig. 4a). Among the six selected PGs, two of them are dependent on EFD (Supplementary Data 1 and Supplementary Fig. 4b), a key transcription factor that regulates rhizobial release and differentiation18. Thus, we re-named Medtr2g032710 and Medtr6g028080 as SyPG1 (Symbiotic PG1) and SyPG2, respectively.
To verify the expression patterns and to gain more cellular resolution, we generated promoter-GUS reporter constructs for SyPG1 and SyPG2 by using 2 kb upstream of their transcriptional start sites. Roots transformed with these reporter constructs were inoculated for two weeks with Sinorhizobium meliloti 2011 (hereafter S. meliloti 2011) before promoter activity was visualized based on β-glucuronidase (GUS) activity. Both SyPG1 and SyPG2 reporters showed activity in nodule primordia and within the vascular bundle of the root (Supplementary Fig. 5a, b), while staining was more restricted to the apical region (meristematic zone and infection zone) of mature nodules (Supplementary Fig. 5c, d). We additionally conducted in situ hybridization experiments to localize the respective mRNAs in nodule tissues. The transcripts of SyPG1 and SyPG2 both exhibited expression in nodule primordia (Supplementary Fig. 5e–l). Notably, the accumulation of SyPG1 and SyPG2 transcripts were predominantly detected within the interzone of mature nodules (Supplementary Fig. 5m–p). Given the dominant promoter activity in the meristematic and infection zone, it is hypothesized that the promoter sequence employed may be lacking in specific cis-elements necessary for the expression of those genes. Notably, it is also possible that the transcripts of those genes are either stabilized during cell differentiation and maturation in the nodule or that the corresponding mRNAs exhibit a certain extent of transcellular mobility.
To elucidate the subcellular patterning of the SyPG1 and SyPG2 proteins, we examined their localization in nodule cells by expressing SyPG1/2-GFP fusion proteins using a hairy root transformation system. For SyPG1, we observed distinct fluorescence along nodular ITs and in infection droplets, where bacteria are released into host cells (Supplementary Fig. 6a, b). By contrast, no SyPG2-GFP signal was detected around these sites (Supplementary Fig. 6c, d). Unexpectedly, we found comparably strong and symbiosome-confined accumulations of both SyPG1-GFP and SyPG2-GFP in infected cells of the nodule interzone (Fig. 2 and Supplementary Fig. 7), resembling the patterns observed for nPME (Fig. 1a–d). Thus, these data further strengthen the hypothesis that the symbiosome space is actively cleared from pectin material.

Knock-down the expression of SyPG1/2 lead to nodule senescence
To genetically assess whether SyPGs are important for nodule function, we cloned an RNA interference (RNAi) construct to simultaneously knock down the expression of both SyPG1 and SyPG2. Indeed, expression analysis of SyPG1 and SyPG2 transcripts in transgenic roots expressing the RNAi construct (SyPG1/2-RNAi) and being inoculated for 3 weeks with S. meliloti 2011 confirmed a significant reduction of both transcripts (Fig. 3a, b and Supplementary Fig. 8). The analysis of expression levels for the initially selected four other PGs indicated that only the transcripts of Medtr8g098840 were targeted by the SyPG1/2-RNAi construct. In contrast, transcript levels of the remaining three PGs exhibited a slight decrease, yet this reduction was not statistically significant (Supplementary Fig. 9). Phenotypically, no significant reduction in overall nodule number was observed at 3 wpi when comparing control plants carrying an empty vector (EV) with SyPG1/2 knockdown plants (Fig. 3c). We also checked IT growth at the 10 days post-inoculation, without any significant morphological differences being detected between EV and SyPG1/2 knockdown plants (Supplementary Fig. 10a, b). Further examination of the nodule structure revealed that the majority of nodules (19/22) from control plants exhibited a large nitrogen-fixing zone (FZ) with a limited senescence zone (SZ; Fig. 3d, e). In contrast, post-transcriptional gene silencing of SyPG1/2 resulted in nodules (18/25) with a drastically reduced nitrogen-FZ and an expanded SZ compared to the controls (Fig. 3f, g). Given that SyPG1 is also located at the ITs and infection droplets, we further examined ITs in the nodule infection zone of SyPG1/2 knockdown plants. However, no obvious morphological differences in the ITs between the control plants and the SyPG1/2 knockdown plants (Supplementary Fig. 10c–f).
To obtain more robust genetic evidence substantiating the function of SyPG1/2, we also utilized CRISPR/Cas9-mediated gene editing, specifically targeting SyPG1 and SyPG2 (SyPG1/2-Cas9). Sequencing analysis indicated that the roots transformed with SyPG1/2-Cas9 led to the premature termination of both SyPG1 and SyPG2 (Supplementary Fig. 11a). Subsequently, expression analysis suggested that it had no effect on the expression of other tested PG genes (Supplementary Fig. 11b–e). Phenotypic analysis of the nodules from SyPG1/2-Cas9 transformed plants revealed a similar phenotype, characterized by an enlarged SZ (Supplementary Fig. 11f–i). Those suggest that SyPG1/2 play a positive role in the maintenance of active symbiosome during symbiotic nitrogen fixation.

Un-esterified pectin is enriched around symbiosome in SyPG1/2-RNAi nodules
Given the role of PG in pectin degradation, we hypothesized that the down-regulation and/or loss of SyPG1 and SyPG2 in the symbiosome space may result in pectin accumulation within this compartment. To test this, we performed immunofluorescence assays using the LM19 antibody, which recognizes de-esterified pectins, the substrate of PGs. As shown before ref. 11, we confirmed that no reliable LM19 signal can be found in symbiosomes (Fig. 4a, b) of control nodules. By contrast, SyPG1/2-RNAi nodules strongly and significantly accumulated de-esterified pectins within infected cells (Fig. 4c–e). To gain resolution, we also applied transmission electron microscopy (TEM) to image the structure of symbiosomes and analysed LM19 signal deposition using an immuno-gold assay. Symbiosomes in infected cells of control (EV) nodules were highly organized with bacteroids displaying homogenous patterns (Fig. 4f, g). In contrast, symbiosomes in SyPG1/2 knock-down nodules were distorted with the SM only being loosely aligned with the bacteroids (Fig. 4h, i). Consistent with our immunofluorescence data, almost no gold particles were observed around the symbiosome in control nodules indicating the absence of de-methylesterified pectins in the symbiosome space (Fig. 4g). By contrast, a substantial accumulation of gold particles was found in the symbiosome spaces of nodules from SyPG1/2-RNAi transformed roots (Fig. 4i). In addition, we also detected methyl esterified pectin using the LM20 antibody, which showed no significant difference in the infected cells of control nodules and SyPG1/2-RNAi nodules (Supplementary Fig. 12). Performing these immunofluorescence assays on SyPG1/2-Cas9 transformed nodules, confirmed the same patterns that we observed on SyPG1/2-RNAi nodules (Supplementary Fig. 13). These data clearly indicate that the symbiosome space is continuously cleared from de-esterified pectins by the action of SyPG1/2.

Un-esterified pectin impairs the growth of rhizobia
To understand the positive function of SyPG1 and SyPG2 for symbiosome function, we assessed the direct impact of pectins on rhizobia. Polygalacturonic acids (PGAs) are un-esterified HGs that can be broken down into short-chain oligogalacturonic acids (OGs) in the presence of PGs. Thus, we investigated the effect of PGA on the growth of rhizobia. For this, we cultured S. meliloti 2011 in the presence of different concentrations of PGA and measured bacterial growth over a period of 10 h. Indeed, first inhibitory effects of PGA on bacterial growth were observed at a concentration of 0.5‰ with a strong inhibition occurring at 1‰ (Fig. 5a). These results were also recapitulated in bacterial growth assay on solid TY plates (Fig. 5b).
We then examined the effect of PGA on rhizobial cell integrity using a “live/dead” staining. In this assay, we visualized bacterial cells using SYTO9 and counterstained with propidium iodide (PI) at pH 5.0 that resembles the acidic environment of the symbiosome in M. truncatula nodules, which has been described to be between pH 4.5–5.019. While living cells are unable to take up PI, the stain is incorporated into dead cells turning them fluorescent. While most cells were only stained by SYTO9 in the control condition, PGA treatment of rhizobia resulted in high numbers of dead cells with more than 35% of the cells being stained by PI (magenta, Fig. 5c–e and Supplementary Fig. 14). This effect of PGA on rhizobia was also confirmed by using scanning electron microscopy (SEM). Here, significantly more rhizobia were damaged and showed ruffled surfaces after PGA treatment compared to the control (59/113 vs 3/107; Fig. 5f, g). In addition, we also investigated the effect of GalA2, a short-chain OG possibly produced during the degradation of PGA by PGs20, on rhizobial growth. Here, no effect of GalA2 (2‰) on rhizobial survival was observed (Supplementary Fig. 15a–d). Given the morphological and physiological differences between free-living bacteria and differentiated bacteroids within symbiosomes, we directly applied PGA and GalA2 onto nodule sections. As shown in Fig. 5h–k and Supplementary Fig. 15e–g, the activity of bacteroids experienced a significant reduction subsequent to PGA treatments, whereas it remained unaffected by GalA2 treatment. Moreover, we directly assessed the activity of bacteroids in nodules transformed with EV, SyPG1/2-RNAi, and SyPG1/2-Cas9. The results were in line with those of our in vitro assays, showing that the activity of bacteroids in SyPG1/2-RNAi and SyPG1/2-Cas9 transformed nodules was severely impaired (Fig. 5i–o and Supplementary Fig. 16). Altogether, those results indicate that un-esterified pectins have a negative effect on the activity of bacteria.

Methods
Constructs design
For subcellular localization: the coding sequence of nPME (Medtr2g105560), SyPG1 (Medtr2g032710) and SyPG2 (Medtr6g028080) were synthesized and then cloned into the GoldenGate EC50507 vector27, and Lotus ubiquitin (Ubi) promoter was used to drive the expression of those genes. For promoter activation: promoter sequences (2000 bp) of SyPG1 and SyPG2 were amplified by PCR from M. truncatula A17 genomic DNA and then ligated into the pCAMBIA1391 vector by Gibson assembly. For RNAi: the sequence was amplified and cloned into pZHY930 vector by Gibson assembly. For CRISPR gene editing, the amplified sequences were cloned into pGES402 vector via BsaI enzyme sites28. All primers used are listed in Supplementary Table 1.

Hairy root transformation
The constructs were transformed into the Agrobacterium rhizogenes (strR, CamR) ArQual strain and used for hairy root transformation as described previously29. In short, M. truncatula A17 seeds were surface-sterilized by sulfuric acid (10 min) and bleach solution (1.2% sodium hypochlorite, 0.1% sodium dodecyl sulfate; 60 s), with 5 times sterile water rinses between the two steps. Seeds were rinsed five additional times with sterile water before being plated on 1% agar plates and stratified at 4°C for 3 days. Subsequently, they were germinated overnight at 24°C in darkness and used for hairy root transformation. After transformation, the plants were kept in darkness at 24°C for 3 days and then in light at 24°C for 4 days (upper part of the plate only). Seedlings were then transferred to new Fahräeus medium plates (containing 0.5 mM NH4NO3) for an additional 10–14 days, and then positively transformed plants were identified with a portable fluorescent lamp (LUYOR-3415, American Router). The positive composite plants were then transferred to open pots (3:1 mix of vermiculite and perlite, 1 plant/pot) and grown in a greenhouse (12 h light/12 h dark, 25°C, light intensity 200 μmol/m2/s). Five days later, S. meliloti 2011 (OD600 = 0.03; 10 mL/plant) was used to inoculate the plants. Plants were harvested at 3 weeks post-inoculation for further analysis.

Technovit 8100 sectioning and Immunofluorescence
Nodules (3 wpi) were harvested and then immediately fixed in a PBS solution containing 4% paraformaldehyde (PFA) under vacuum for 15 min (twice) and stored at 4°C in the fixative solution overnight. They were then dehydrated in acetone/H2O graded series (50%, 70%, 80%, 90%, 100%, 1 h each at 4°C). Final dehydration in absolute acetone was repeated once, followed by incubation in acetone/Technovit infiltration solution (1:3, 2:2, 3:1, 60 min each at 4°C), and then the final infiltration in pure Technovit infiltration solution was carried out overnight at 4°C. Embedding of the samples was performed as directed by the manufacturer. Nodule sections (10 µm) for immunofluorescence labeling were obtained using a RM2155 microtome (Leica). The nodule sections were blocked with 3% BSA for 30 min at room temperature. Then the primary antibody (LM19 (2w00019S) and LM20 (2w00020s), from Abmart) was applied to the sections (1 to 50 diluted in PBS solution supplied with 3% BSA) and incubated for 1–2 h at room temperature. The sample was then washed at least 3 times (5 min each time) with PBS solution. The secondary antibody (conjugated with Alex Fluor 488 (A-11006), from Invitrogen) was then added diluted in PBS solution containing 3% BSA and then the sample was incubated for another 30 min to 1 h. The sample was then washed at least 3 times before images were taken. Note that after applying the secondary antibody all steps were performed in darkness. The samples were then imaged using a Leica SP8 confocal microscope.
For immuno-gold staining: the samples were embedded with LR White according to the protocol descript in Su et al.11. Goat-anti Rat IgG 10 nm gold conjugated polyclonal antibody (Sigma, G7035) was used as secondary antibody.

Technovit 7100 sections and GUS staining
The sample preparation followed same process described above for Technovit 8100, except that ethanol was used instead of acetone. The GUS staining was performed according to a previous study30. Briefly, tissue samples were collected in 5 mL tubes immersed in GUS staining solution, following vacuum twice for 10 min each time. The samples were incubated at 37°C for 4–8 h (in dark). After staining, the samples were cleared with 75% ethanol and stored in 75% ethanol for subsequent analysis. The stained nodules were embedded in Technovit 7100 for sectioning and visualization. All nodule sections were stained with 0.05% w/v Toluidine Blue or 0.1% w/v Ruthenium red prior to imaging. Images were taken with a ZEISS Axiolab 5 microscope.

In situ hybridization
Using specific primers (see Supplementary Table 1), the 254 bp sequence fragment of SyPG1 (corresponding to part of the coding sequence and the 3′ untranslated region) and the 251 bp coding sequence fragment of SyPG2 were amplified from cDNA using KOD polymerase (KFX-101, Stratagene, San Diego, CA). Subsequently, the amplified fragments were cloned into the pEASY®-Blunt3 vector (CB301-01, TransGen Biotech, San Diego, CA). Digoxigenin-labelled sense or antisense probes were synthesized using T7 or SP6 RNA polymerase. Medicago nodule samples were collected 7–10 days and 21 days post-inoculation. Following fixation in 4% PFA, the samples underwent paraffin embedding and sectioning at 8–10 µm thickness. After deparaffinization and dehydration, sections were subjected to hybridization staining according to previously described method31.

Localization analysis
14 days old nodules were embedded in 3% agarose and cut into 70 µm sections using a vibratome (VT1000S, Leica). FM4-64 (5 μg ml−1, from Sigma, F34653) was used to stain the membrane. The cell wall structure was stained with Calcofluor White M2R (0.1%, from Sigma, 910090). Images were acquired with a Leica SP8 confocal microscope with a 63×/1.2 (HC PL APO CS2) water immersion objective and an argon Light Laser (DAPI: 405 nm (ex)/415–465 nm; GFP: 488 nm (ex)/500–550 nm (em); mCherry: 561 nm (ex)/575–630 nm). All the image analyses and projections were performed using either the Fiji32 or Imaris software.

RNA extraction and quantitative real-time PCR
Total RNA was extracted using the Trizol reagent kit (Aidlab Biotechnologies Co., Ltd). The cDNA was synthesized using Hifair II 1st Strand cDNA Synthesis SuperMix (Yeasen Biotech, Shanghai, China). Quantitative real-time PCR (qRT-PCR) was accomplished using Hieff® qPCR SYBR Green Master Mix (No Rox) and CFX Connect real-time PCR detection system (Bio-Rad). The relative expression level was calculated by 2−ΔΔCt (ΔΔCt is the difference of ΔCt between the experimental group and the control group). PP2A was used as the reference gene33. All primers used are listed in Supplementary Table 1.

PGA treatment
For the PGA (Sigma, P3889) treatment S. meliloti 2011 were grown in TY liquid medium at 28°C to OD600 = 1.0, and then 1:100 added to the TY liquid medium containing different doses of PGA (0.1‰, 0.5‰, 1‰ and 2‰). The rhizobia were then incubated at 28°C and the OD600 was measured every 3 h with a spectrophotometer (Eppendorf BioPhotometer D30). For testing S. meliloti 2011 growth on PGA-containing solid plates the S. meliloti 2011 was cultured in TY liquid medium until it reached an OD600 = 1.0, then 10 μL of a dilution series was plated onto TY solid medium containing different concentrations of PGA and incubated at 28°C for 3–4 days. 2‰ PGA was used to treat the rhizobia for scanning electron microscopy analysis.

Scanning electron microscope analysis
Rhizobia were grown overnight in TY liquid medium, then diluted into fresh TY liquid medium with or without PGA (2‰, OD600 = 0.2) culture for 3 h. The rhizobia were then pelleted by centrifugation (1000 × g, 10 min) and washed with 0.01 M PBS buffer (pH 7.0). Immediately after washing, the rhizobia were placed in Formalin-Aceto-Alcohol (from Servicebio, G1103) fixative at 4°C overnight. The overnight fixed samples were dehydrated sequentially for 15–20 min in ethanol solutions of different concentrations (30%, 50%, 70%, 85%, 95%, and 100%), then the dehydrated samples were freeze-dried using a Labogene Scanvac CoolSafe 110-4. Finally, the samples were examined using a scanning electron microscope (FE-SEM, SU8010, HITACHI, Japan).

Live/dead staining
The rhizobial live/dead staining was carried out with solution containing SYTO9 (1:100; from Thermo Fisher, S34854) and PI (1:100, from Solarbio, C0080) in PBS (0.01 M, pH 7.0) for 1–5 min at room temperature. Regarding the nodule sections, the nodules (3 weeks post-inoculation) from the transformed roots of EV, SyPG1/2-RNAi, and SyPG1/2-Cas9 were embedded in 3% agarose. After that, vibratome sections with a thickness of 70 μm were obtained. Immediately, these sections were stained using live/dead staining for 5 min before undergoing microscopy imaging. For PGA and GalA2 treatments: The vibratome sections obtained from wild-type samples were immediately treated with PGA (2‰, from Sigma, P3889) and GalA2 (2, from Macklin, D981325) at room temperature for 3 h before performing live/dead staining for image acquisition. The results were observed using a Leica SP8 confocal microscope as described above.

Statistical analysis
The data were analysed using GraphPad Prism 10 built-in statistical tools.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript titled “Symbiosome functionality in Medicago truncatula nodules requires continuous clearing of pectins from the symbiosome space” by Chen et al. reports that SyPG1 and SyPG2 are localized around symbiosomes. The RNAi knockdown of SyPG1/2 results in early nodule senescence and an excessive accumulation of un-esterified pectin, which in turn inhibits rhizobia growth. While this study is intriguing, more robust results are needed to substantiate their conclusions and warrant publication.\n1. The genetic evidence presented is very weak, with only RNAi knockdown and a mild early senescence observed in the SyPG1/2 RNAi nodules, which does not fully support their findings.\n2. The accumulation of un-esterified pectin around the symbiosomes in the SyPG1/2 RNAi nodules led the authors to conduct an in vitro assay demonstrating that un-esterified pectin inhibits rhizobia growth. However, there is a significant difference between free-living rhizobia and those within symbiosomes. It is essential to have results from the SyPG1/2 mutants’ nodules to analyze rhizobia growth and activity.\n3. Both SyPG1 and SyPG2 are expressed at the apex of mature nodules, yet only the SyPG1/2 RNAi lines exhibit an early senescence phenotype, which should be observed in the nitrogen fixation zone. Additionally, SyPG1 is also localized to the infection threads (IT) and intercellular spaces (ID). This raises the question of whether SyPG1/2 is also necessary for IT formation and/or the release of rhizobia from ITs. To address this, a full analysis of the symbiotic phenotype in SyPG1/2 mutants is required.\n4. Given that MsPG3 has been reported in the interaction between M. sativa and rhizobia, but was not mentioned in this manuscript. Is MsPG3 an orthologue of SyPG1/2?", "reviewer_2": "The manuscript by Chen and colleagues, reports a key role for two polygalacturonase genes of Medicago truncatula in the maintenance of rhizobia as nitrogen-fixing organelles in root nodules. Silencing of SyPG1/2 caused the accumulation of un- esterified pectin in the symbiosome space and led to infected nodule cell senescence. Furthermore, they show that un- esterified pectin inhibits rhizobia growth in vitro, affecting rhizobial cell integrity.\nThe absence of pectins at the cell-wall free symbiosomes has been previously reported. Furthermore, it has been suggested that the plant delivers cell wall degrading enzymes, including enzymes for pectin turnover, to the symbiosomes to prevent the build up of cell wall material in these transient organelles (Gavrin et al., 2016). This work now add more mechanistic insight into this process by identifying two PG genes induced in nodules that contribute to the removal of un-esterified pectin. Furthermore, the data strongly indicate that symbiosome maintenance requires a continuous degradation of homogalacturones in the symbiosome space.\nOverall, the manuscript is clearly written and the data largely support the conclusions drawn. Below I have several suggestions:\nSurprisingly, the work by Gavrin et al. (2016) is not mentioned in the manuscript. It would be good to include this work in the introduction of the discussion section.\nThe role of pectin lyases and HGs in the processing of pectin can be clarified better. For example, HGs (first mentioned at line 81) are not properly introduced.\nAt line 85, a newly identified nodule PME is mentioned. It is not clear how this one was identified? Are these two genes the only PMEs in Medicago? It would be nice to add a phylogenetic analyses (both for PMEs and HGs).\nIt is not completely clear why the authors focussed on SyPG1 and SyPG2. From the presented public transcriptome data there are additional PGs that seem to be induced in nodules. Do different PGs have different biochemical activities? As additional PGs are highly expressed in the infection zone of the nodule, it raises the question whether these are also involved in the removal of HGs at the symbiosomes. Although it is mentioned that the SyPG1/2 RNAi construct also targets Medtr8g098840 in roots, it should also be tested whether in nodules other PGs might be affected by this construct.\nThe promoter GUS pattern for SyPG2 does not fit well with the expression profiles of these genes based on publically available RNAseq and gene chip data concerning its expression in the distal infection zone. This is not discussed. It would be good to confirm the expression domains using in situ hybridizations.\nAt line 185 it is concluded that “Medicago specifically evolved a subset of pectin degrading enzymes that allow spatially confined pectin processing without affecting the extracellular pectin accumulations”. I don’t see any evidence to support the claim that Medicago evolved a specific subset of genes for this purpose. A more evolutionary comparison would be needed to make this claim.\nFig. 1; which promoters are used to express the PME-GFP fusions?\nFig. 3; It would be good to add more detailed (confocal or plastic section) images of the infection zone to compare control vs SyPG1/2 RNAi nodules.\nFig. 4; in panel f LM19 on the y-axis is mentioned, but I guess it has to be LM20 according to the legend.", "reviewer_3": "The authors analyzed the formation of symbiosomes during the interaction between Medicago truncatula and S. meliloti. They notably identified a pectin methylesterase and two polygalacturonases present around the symbiosomes and they performed an RNAi SYPG1/PG2 line that presents nodules with senescence. Surprisingly, using immunofluorescence assays with antibodies directed against epitopes of unesterified pectins, they observed that in the case of the RNAi SYPG1/PG2 line, there are many unesterified pectins around the symbiosomes. They also showed that the growth of S. meliloti is inhibited by polygalacturonic acid (unesterified pectin). In conclusion all this work suggests that there is pectin remodeling within the symbiosomes in order to avoid the presence of unesterified pectins around symbiosomes. While the data are very interesting and surprising, there are several major issues with this manuscript that must still be addressed\nMy main concern is to understand where these pectins come from in these symbiosomes which are not supposed to have a wall if I understand correctly.\nIn Figure 6 the authors propose a model where they show that pectins are secreted at the level of symbiosomes. Their data does not allow this to be written. They see pectic epitopes recognized by the LM19 antibody (unesterified pectins) in the RNAi SyPG1/2 lines, but they should also write the fact that in Figure 4b’, they observe LM19 labeling around the cells, therefore in the wall? Can we imagine that the pectins come from endocytosis or even from the invagination of the plasma membrane at the time of the release of bacteria (and that the pectins are, for example, too methylesterified to be detected by LM20 and LM19 antibodies)? In this model they should also add their data about nPME? And SyPG1 is also present in symbiosome?\nThe confocal images showing nPME-GFP and SyPG-GFP are correct, but I think that to really understand what is happening at the level of the symbiosomes, we would need to obtain images with better resolution and in 3D (high resolution confocal image such as sSTED or STORM) in order to better understand where the enzymes are exactly in relation to the structures of the symbiosomes.\nSecondly, at this level, to understand the model evoked by the authors, images of the structure of symbiosomes in electron microscopy in the controls and the RNAIPG1/2 line with LM19 and LM20 labeling seem to me essential to conclude.\nThe discussion is far too short. The authors should discuss the arrival of pectins and at least how enzymes like nPME and PG could function around symbiosomes. How could nPME act on pectins at the level of symbiosomes with optimal activity at basic pH?\nOther comments :\nIn general, I find that the figures displaying confocal images are difficult to read for a non-expert. Would it be possible to add a diagram like in Figure 2 to understand which stage of development of the symbiosome corresponds to the image in Figure 4?\nFig 1 b Would it be possible to add an image representing the infected cell before zooming in on the symbiosome?\nIn Fig 2 H ; we're at the limit of the resolution and I can't really see SYPG1-GFP around all the bacteroids in the infection zone and at the level of the symbiosomes. 3D reconstructions and analyses using super-resolution microscopy such as STED or STORM will be useful..\nThe authors observed a more restricted GUS staining in the mature nodules ? Did they observe this restricted labeling using the SYPG1-GFP or SYPPG2-GFP in the mature nodules ? I can’t see this in the figure 2.\nAbout the chosen PG\n-SupplementaryTable 1 : Define what is A17 efd1\n-an alignment of the six PG sequences would be interesting to see the differences of sequences.\nFig3. Comment the figure 3E and G ? What do we observe using the ruthenium red staining ?\nFig4.\nexchange 4d’ and d images\nexchange 4e’ and e images\nIn SYPG1/2 RNAi nodules there is a strong labeling of LM19 around the symbiosome. I would like to get 3D labeling to really understand the spatial organisation of these LM19 signal and also noticed that LM19 strongly labels the cell wall of the infected cells (border of the cells).\nBy contrast there is a faint labeling of LM19 in control nodules. LM20 antibody was detected in the border of the cells and also in the symbiosome in the SYPG1/2 RNAi nodules. Is image 4e in the figure 4 a representative image because I observe more signal at the cell walls in the SYPG1/2 RNAi lines while the quantifications show similar results?\nFig. 5.\nThe authors tested the effects of PGA on the growth of the bacteria. PGA inhibits the growth in liquid and solid media. They then tested the effect of the tetragalacturonic acid and no effect was observed. Why the authors tested this oligosaccharide. They must first digest PGA in presence of SyPG1 for example and see what types of oligogalacturonides they will obtain. PG enzymes usually produces GalA, GalA2 and GalA3 products so at least the auhors need to treat bacteria with GalA and GalA2\nSupplementary Fig. 2.\nChange pother into other\nLine 184 change symbisome into symbiosome\nSupplementary Fig 2.\nI am not sure that the expression of other PGs is not impacted by this RNAi because only 5 values were obtained and there is a large variability in the control. This is why the sequence alignment of the PGs and the sequences used for RNAi would be important.\nDid the authors analyse several lines RNAiPG1/PG2 ?\nDefine S. from S. meliloti"}, "author_response": "Responses to the reviewers’ comments\nWe sincerely thank the reviewers for their thoughtful and constructive comments,\nwhich have substantially improved the quality of our manuscript. We have carefully\nconsidered each point raised and implemented the corresponding revisions. A detailed,\npoint-by-point response is provided below.\nIn summary, the principal revisions are as follows:\n1. The genetic evidence has been strengthened by validating all phenotypic\ncharacteristics (including nodule sections, pectin deposition, and bacterial activity)\nthrough gene editing in hairy roots;\n2. The quality of the images has been enhanced by increasing resolution and\ngenerating 3D reconstructions. In addition, Transmission Electron Microscopy (TEM)\nwas employed to examine symbiosomes and pectin deposition;\n3. Rhizobial activity has been directly assessed in SyPG1/2-RNAi and SyPG1/2-Cas9\nnodules. Furthermore, the effect of PGA (un-esterified pectin) on bacterial activity in\nvivo was evaluated by applying the treatment directly to nodule sections;\n4. The discussion section has been substantially extended and refined.\nREVIEWER COMMENTS\nReviewer #1:\nThe manuscript titled “Symbiosome functionality in Medicago truncatula nodules\nrequires continuous clearing of pectins from the symbiosome space” by Chen et al.\nreports that SyPG1 and SyPG2 are localized around symbiosomes. The RNAi\nknockdown of SyPG1/2 results in early nodule senescence and an excessive\naccumulation of un-esterified pectin, which in turn inhibits rhizobia growth. While\nthis study is intriguing, more robust results are needed to substantiate their\nconclusions and warrant publication.\nOur response: Thank you very much for the constructive and insightful comments,\nwhich have greatly improved the quality and clarity of our manuscript. We have\ncarefully considered each point raised, performed additional experiments, and revised\nthe manuscript accordingly.\nComment 1. The genetic evidence presented is very weak, with only RNAi\nknockdown and a mild early senescence observed in the SyPG1/2-RNAi nodules,\nwhich does not fully support their findings.\nOur response: We appreciate this important comment. To strengthen our genetic\nevidence, we generated CRISPR/Cas9-mediated knockout lines specifically targeting\nSyPG1 and SyPG2 (SyPG1/2-Cas9). The phenotypes observed in these edited nodules\nwere consistent with those from the SyPG1/2-RNAi lines, including early senescence\nand abnormal pectin deposition (Supplementary Fig. 11 and Supplementary Fig. 13).\nThese results validate the functional roles of SyPG1 and SyPG2 and provide stronger\ngenetic support for our conclusions. The new data have been incorporated into the\nrevised manuscript. We hope that all the new genetic evidences will address your\nconcerns.\nComment 2. The accumulation of un-esterified pectin around the symbiosomes in the\nSyPG1/2-RNAi nodules led the authors to conduct an in vitro assay demonstrating that\nun-esterified pectin inhibits rhizobia growth. However, there is a significant difference\nbetween free-living rhizobia and those within symbiosomes. It is essential to have\nresults from the SyPG1/2 mutants’ nodules to analyze rhizobia growth and activity.\nOur response: Thank you for this valuable suggestion. In response, we performed\nlive/dead staining on nodule sections from both SyPG1/2-RNAi and SyPG1/2-Cas9\ntransformed roots. These analyses revealed a significant reduction in bacteroid\nviability compared with control nodules (Fig. 5i–o and Supplementary Fig. 16).\nFurthermore, to directly test the effect of un-esterified pectin on bacteroid activity in\nvivo, we applied polygalacturonic acid (PGA) to wild-type (M. truncatula A17)\nnodule sections. This treatment also led to a pronounced decrease in bacteroid activity\n(Fig. 5h–k). Together, these results confirm that un-esterified pectin compromises\nrhizobial activity within nodules, supporting the physiological relevance of our earlier\nin vitro findings. We have included these results in the revised manuscript.\nComment 3. Both SyPG1 and SyPG2 are expressed at the apex of mature nodules, yet\nonly the SyPG1/2-RNAi lines exhibit an early senescence phenotype, which should be\nobserved in the nitrogen fixation zone. Additionally, SyPG1 is also localized to the\ninfection threads (IT) and intercellular spaces (ID). This raises the question of whether\nSyPG1/2 is also necessary for IT formation and/or the release of rhizobia from ITs. To\naddress this, a full analysis of the symbiotic phenotype in SyPG1/2 mutants is\nrequired.\nOur response: We appreciate this insightful comment. Our results show that although\nthe promoters of SyPG1 and SyPG2 are active in the apical region of mature nodules,\nthe encoded proteins exert their principal function within the symbiosome space\nlocated in the interzone, consistent with our in situ hybridization analyses\n(Supplementary Fig. 5e–l). We propose that the interzone is the site where pectin\nsecretion into the symbiosome space occurs; thus, loss of SyPG1/2 function leads to\npectin accumulation in this region, subsequently inducing premature senescence.\nTo address the infection phenotype, we examined IT morphology in SyPG1/2-RNAi\nnodules. No significant differences in IT development were observed compared with\nempty vector controls (Supplementary Fig. 10a–b). Although SyPG1 localizes to ITs\nand infection droplets (IDs), we did not detect obvious IT defects in the infection zone\nof SyPG1/2-RNAi nodules (Supplementary Fig. 10c–d). Enlarged IDs were\noccasionally observed, but rhizobial release proceeded normally. These results\nindicate that SyPG1/2 is not essential for IT growth or rhizobial release, but it cannot\nbe ruled out that SyPG1 may have redundant functions with other polygalacturonases\nlocalized to IT and ID. These observations and interpretations are now included in the\nrevised manuscript.\nComment 4. Given that MsPG3 has been reported in the interaction between M.\nsativa and rhizobia, but was not mentioned in this manuscript. Is MsPG3 an\northologue of SyPG1/2?\nOur response: We thank the reviewer for raising this question. Prior to selecting\nSyPG1/2 as candidate genes, we conducted a phylogenetic analysis including MsPG3.\nOur results indicate that MsPG3 and SyPG1/2 do not belong to the same evolutionary\nclade (Supplementary Fig. 3). Furthermore, transcriptomic data show that PGs\nclustering with MsPG3 are not expressed during rhizobial inoculation in M.\ntruncatula (see below). Taken together, these results demonstrate that SyPG1/2 are\nnot orthologues of MsPG3.\nFigure: Expression profiles of PGs clustered with MsPG3.\nReviewer #2:\nThe manuscript by Chen and colleagues, reports a key role for two polygalacturonase\ngenes of Medicago truncatula in the maintenance of rhizobia as nitrogen-fixing\norganelles in root nodules. Silencing of SyPG1/2 caused the accumulation of\nun-esterified pectin in the symbiosome space and led to infected nodule cell\nsenescence. Furthermore, they show that un-esterified pectin inhibits rhizobia growth\nin vitro, affecting rhizobial cell integrity. The absence of pectins at the cell-wall free\nsymbiosomes has been previously reported. Furthermore, it has been suggested that\nthe plant delivers cell wall degrading enzymes, including enzymes for pectin turnover,\nto the symbiosomes to prevent the build up of cell wall material in these transient\norganelles (Gavrin et al., 2016). This work now add more mechanistic insight into this\nprocess by identifying two PG genes induced in nodules that contribute to the removal\nof un-esterified pectin. Furthermore, the data strongly indicate that symbiosome\nmaintenance requires a continuous degradation of homogalacturones in the\nsymbiosome space. Overall, the manuscript is clearly written and the data largely\nsupport the conclusions drawn.\nOur response: We sincerely thank the reviewer for the positive assessment of our\nwork and for the constructive suggestions, which have helped us improve the clarity,\nrigor, and overall quality of the manuscript. We have carefully revised the text and\nincorporated additional supporting data in response to the comments, as detailed\nbelow.\nComment 1: Surprisingly, the work by Gavrin et al. (2016) is not mentioned in the\nmanuscript. It would be good to include this work in the introduction of the discussion\nsection.\nOur response: We thank the reviewer for this important suggestion. The study by\nGavrin et al. (2016) demonstrated that VAMP721 is required for transporting NPL to\ninfection droplets, where pectin degradation ensures rhizobial release1. This work\nprovides essential context for our findings. We have cited the study by Gavrin et al.\n(2016) in discussion sections of the revised manuscript1.\nComment 2: The role of pectin lyases and HGs in the processing of pectin can be\nclarified better. For example, HGs (first mentioned at line 81) are not properly\nintroduced.\nOur response: We appreciate this helpful comment. In the revised introduction, we\nhave expanded the description of pectin structure and clarified the roles of\nhomogalacturonans (HGs), pectin methylesterases (PMEs), polygalacturonases (PGs),\nand pectate lyases (PLs) in pectin processing.\nComment 3: At line 85, a newly identified nodule PME is mentioned. It is not clear\nhow this one was identified? Are these two genes the only PMEs in Medicago? It\nwould be nice to add a phylogenetic analyses (both for PMEs and HGs).\nOur response: Thank you for raising this point. The PME referred to was identified\nin our previous study based on strong rhizobia-induced expression. M. truncatula\ngenome encodes multiple PMEs, and a phylogenetic analysis of this gene family has\nbeen previously published2. In the revised manuscript, we provide details to clarify\nwhy this PME was chosen for functional characterization.\nWe agree that a phylogenetic analysis of PGs would be would be informative.\nAccordingly, we have performed this analysis and included the results in the revised\nmanuscript (Supplementary Fig. 3).\nComment 4: It is not completely clear why the authors focused on SyPG1 and SyPG2.\nFrom the presented public transcriptome data there are additional PGs that seem to be\ninduced in nodules. Do different PGs have different biochemical activities? As\nadditional PGs are highly expressed in the infection zone of the nodule, it raises the\nquestion whether these are also involved in the removal of HGs at the symbiosomes.\nAlthough it is mentioned that the SyPG1/2-RNAi construct also targets\nMedtr8g098840 in roots, it should also be tested whether in nodules other PGs might\nbe affected by this construct.\nOur response: We thank the reviewer for this insightful comment. We selected\nSyPG1 and SyPG2 based on their strong expression in the infection and interzone\nregions of nodules, and their regulation by EFD, a transcription factor essential for\nbacterial release and bacteroid differentiation. We have clarified this rationale in the\nrevised manuscript.\nAlthough multiple PGs are expressed in nodule and share the biochemical activity of\ndegrading un-esterified pectins, their distinct subcellular localizations likely confer\nspecialized functions. Our studies show that SyPG1 and SyPG2 specifically\naccumulate in the symbiosome space, supporting their direct role in\nhomogalacturonan (HG) removal at this site. While we cannot exclude the possibility\nthat other nodule-expressed PGs may also act in the symbiosome, the M. truncatula\ngenome encodes more than 80 PGs, making it impractical to assess the expression of\nall family members in SyPG1/2-RNAi roots. To validate the specific functions of\nSyPG1 and SyPG2, we generated CRISPR/Cas9 knockout constructs. These\nSyPG1/2-Cas9 lines recapitulated the RNAi phenotypes (Supplementary Fig. 11),\nthereby confirming the essential roles of SyPG1 and SyPG2 in symbiosome\nmaintenance. We believe that these new data and clarifications sufficiently address\nyour concerns.\nComment 5: The promoter GUS pattern for SyPG2 does not fit well with the\nexpression profiles of these genes based on publically available RNAseq and gene\nchip data concerning its expression in the distal infection zone. This is not discussed.\nIt would be good to confirm the expression domains using in situ hybridizations.\nOur response: We appreciate this important suggestion. We performed in situ\nhybridizations to validate the expression of SyPG1 and SyPG2. The results revealed\nthat both transcripts are predominantly localized to the nodule interzone of mature\nnodules, which contrasts with the earlier GUS staining pattern. Given the strong\npromoter activity (GUS) in the meristematic and infection zones, we propose that the\ntranscripts may either be stabilized during cell differentiation and maturation, or\nexhibit a certain degree of transcellular mobility. These new results and the\naccompanying discussion are now included in the revised manuscript (Supplementary\nFig. 5e–l).\nComment 6: At line 185 it is concluded that “Medicago specifically evolved a subset\nof pectin degrading enzymes that allow spatially confined pectin processing without\naffecting the extracellular pectin accumulations”. I don’t see any evidence to support\nthe claim that Medicago evolved a specific subset of genes for this purpose. A more\nevolutionary comparison would be needed to make this claim.\nOur response: We agree with the reviewer that our original statement was\ninappropriate. To avoids the over-interpretation, this claim has been removed in the\nrevised manuscript.\nComment 7: Fig. 1; which promoters are used to express the PME-GFP fusions?\nOur response: Thank you for pointing this out. The PME–GFP fusions were driven\nby the Lotus ubiquitin promoter, which has now been specified in the Methods section.\nWe also tested native promoters, which yielded similar localization patterns but with\nweaker fluorescence signals, so we used the ubiquitin promoter to obtain clearer\nimages.\nComment 8: Fig. 3; It would be good to add more detailed (confocal or plastic\nsection) images of the infection zone to compare control vs SyPG1/2-RNAi nodules.\nOur response: We thank the reviewer for this suggestion. We have revised all the\nimages to include images of the infection zone (Supplementary Fig. 10c-d), and we\nhave expanded the figure legend to provide more detailed descriptions.\nComment 9: Fig. 4; in panel of LM19 on the y-axis is mentioned, but I guess it has to\nbe LM20 according to the legend.\nOur response: We appreciate the reviewer’s careful reading. This was indeed an error,\nand we have corrected the label to “LM20” in the revised manuscript (Supplementary\nFig. 12).\nReviewer #3:\nThe authors analyzed the formation of symbiosomes during the interaction between\nMedicago truncatula and S. meliloti. They notably identified a pectin methylesterase\nand two polygalacturonases present around the symbiosomes and they performed an\nRNAi SyPG1/PG2 line that presents nodules with senescence. Surprisingly, using\nimmunofluorescence assays with antibodies directed against epitopes of unesterified\npectins, they observed that in the case of the RNAi SyPG1/PG2 line, there are many\nunesterified pectins around the symbiosomes. They also showed that the growth of S.\nmeliloti is inhibited by polygalacturonic acid (unesterified pectin). In conclusion all\nthis work suggests that there is pectin remodeling within the symbiosomes in order to\navoid the presence of unesterified pectins around symbiosomes. While the data are\nvery interesting and surprising, there are several major issues with this manuscript\nthat must still be addressed\nOur response: We are very grateful for the thorough and constructive evaluation.\nYour detailed comments and critical questions have greatly helped us strengthen the\nmanuscript. We have carefully revised the text, performed additional analyses,\nimproved the figures, and expanded the discussion accordingly. Below, we address\neach comment point-by-point.\nComment 1: My main concern is to understand where these pectins come from in\nthese symbiosomes which are not supposed to have a wall if I understand correctly. In\nFigure 6 the authors propose a model where they show that pectins are secreted at the\nlevel of symbiosomes. Their data does not allow this to be written. They see pectic\nepitopes recognized by the LM19 antibody (unesterified pectins) in the RNAi\nSyPG1/2 lines, but they should also write the fact that in Figure 4b’, they observe\nLM19 labeling around the cells, therefore in the wall? Can we imagine that the\npectins come from endocytosis or even from the invagination of the plasma\nmembrane at the time of the release of bacteria (and that the pectins are, for example,\ntoo methylesterified to be detected by LM20 and LM19 antibodies)? In this model\nthey should also add their data about nPME? And SyPG1 is also present in\nsymbiosome?\nOur response: We appreciate this important comment. Indeed, the LM19 signal\nobserved around cells in SyPG1/2-RNAi nodules corresponds to unesterified pectins in\nthe cell wall. However, this labeling is not consistently more intense than in control\nnodules. To address this, we have updated the figure with more representative images\n(Fig. 4a–d; Supplementary Fig. 13a–d).\nThe precise origin of pectins in the symbiosome space remains unclear. As the\nreviewer suggested, pectin endocytosis from the plasma membrane/cell wall is a\npossible source, consistent with the fundings of Gavrin et al. (2016), who reported\npectin-containing endosome-like structures in infected nodule cells. These vesicles\nmay originate from the plasma membrane/cell wall and subsequently be redirected to\nthe symbiosome. By contrast, delivery via plasma membrane invagination during\nrhizobial release seems less likely, since pectin is nearly undetectable in infection\ndroplets1.\nTo reflect these possibilities more accurately, we have revised our working model and\nexpanded the discussion to present a more balanced interpretation. We hope that these\nnew data and revised discussion will address your concerns.\nComment 3: The confocal images showing nPME-GFP and SyPG-GFP are correct,\nbut I think that to really understand what is happening at the level of the symbiosomes,\nwe would need to obtain images with better resolution and in 3D (high resolution\nconfocal image such as sSTED or STORM) in order to better understand where the\nenzymes are exactly in relation to the structures of the symbiosomes.\nOur response: Thank you for this valuable suggestion. We attempted super-resolution\nimaging using Nikon AXR NSPARC, but GFP signals bleached rapidly. Instead, we\noptimized confocal parameters with a Leica SP8 and generated 3D reconstructions\nusing Imaris (Fig. 1c; Fig. 2c, g). Additionally, co-staining with FM4-64 confirmed\nthat SyPG1/2–GFP signals localize to the symbiosome space (Supplementary Fig. 7).\nThese higher-quality images are included in the revised manuscript.\nComment 4: Secondly, at this level, to understand the model evoked by the authors,\nimages of the structure of symbiosomes in electron microscopy in the controls and the\nRNAi SyPG1/2 line with LM19 and LM20 labeling seem to me essential to conclude.\nOur response: Thank you for your valuable comments and suggestion. We fully\nagree. We have performed immuno-gold labeling on ultra-thin sections from control\nand SyPG1/2-RNAi nodules. These analyses confirmed structural differences in\nsymbiosomes and revealed a significant accumulation of LM19 epitopes in the\nSyPG1/2-RNAi lines (Fig. 4f–i). These new results strengthen our conclusions and are\nincluded in the revised manuscript.\nComment 5: The discussion is far too short. The authors should discuss the arrival of\npectins and at least how enzymes like and PG could function around symbiosomes.\nHow could nPME act on pectins at the level of symbiosomes with optimal activity at\nbasic pH?\nOur response: Thank you for this excellent suggestion. We have substantially\nexpanded the discussion to address possible mechanisms of pectin delivery to the\nsymbiosome space and how PGs and PMEs may function there. Notably, acidic pH\nstabilizes PME–PMEI complexes, inhibiting PME activity3. Thus, PME function in\nthe symbiosome may be facilitated by the absence of PMEI. Moreover, previous\nstudies have reported that PME retains partial activity under acidic conditions (pH ~5),\nwhich is compatible with the environment of the symbiosome3-5.\nOther comments:\nComment 1: In general, I find that the figures displaying confocal images are\ndifficult to read for a non-expert. Would it be possible to add a diagram like in Figure\n2 to understand which stage of development of the symbiosome corresponds to the\nimage in Figure 4?\nOur response: Thank you for the good suggestion. Diagrams and additional labels\nhave been included in the relevant figures to aid interpretation.\nComment 2: Fig 1b Would it be possible to add an image representing the infected\ncell before zooming in on the symbiosome?\nOur response: Thank you for your comments, we have added this context image\n(Supplementary Fig. 2).\nComment 3: In Fig 2H; we're at the limit of the resolution and I can't really see\nSyPG1-GFP around all the bacteroids in the infection zone and at the level of the\nsymbiosomes. 3D reconstructions and analyses using super-resolution microscopy\nsuch as STED or STORM will be useful..\nOur response: Thank you for your valuable comments and suggestion. As noted\nabove (Comment 3), we now provide 3D reconstructions using optimized confocal\nimaging (Fig. 2c, g).\nComment 4: The authors observed a more restricted GUS staining in the mature\nnodules? Did they observe this restricted labeling using the SyPG1-GFP or\nSPG2-GFP in the mature nodules ? I can’t see this in the figure 2.\nOur response: Yes. SyPG1/2–GFP localization is confined to the interzone region of\nmature nodules, with SyPG1 additionally detected in infection threads (IT) and\ninfection droplets (ID) within the infection zone. All images presented in the\nmanuscript were captured from these regions, and this has been clarified in the figure\nlegends in the revised manuscript.\nComment 5: About the chosen PG\n-SupplementaryTable 1: Define what is A17 efd1\n-an alignment of the six PG sequences would be interesting to see the differences of\nsequences.\nOur response: Thank you for your valuable comments and suggestion. We have\ndefined A17 and efd-1 in Supplementary Table 1 and added sequence alignments of\nthe six PGs (Supplementary Fig. 8).\nComment 6: Fig3. Comment the figure 3E and G? What do we observe using the\nruthenium red staining?\nOur response: Ruthenium red binds strongly to phospholipids and\nmucopolysaccharides; here, it was used to highlight cell borders.\nComment 7: Fig4.\nexchange 4d’ and d images; exchange 4e’ and e images\nOur response: Thank you for your careful review. The panels have been corrected\n(Supplementary Fig. 12).\nComment 8: In SYPG1/2 RNAi nodules there is a strong labeling of LM19 around the\nsymbiosome. I would like to get 3D labeling to really understand the spatial\norganisation of these LM19 signal and also noticed that LM19 strongly labels the cell\nwall of the infected cells (border of the cells).\nOur response: Thank you for this insightful comment. Due to limited antibody\npenetration in resin-embedded sections, 3D reconstruction of LM19 labeling is not\nfeasible. To address this, we performed immuno-gold labeling with ultra-thin sections,\nwhich confirmed LM19 accumulation at symbiosome peripheries in SyPG1/2-RNAi\nnodules (Fig. 4f–i). We did not observe a stronger LM19 signal labels the cell wall of\nthe infected cells in the SyPG1/2-RNAi lines compared to the control. Accordingly,\nmore representative images have been included in the revised manuscript (Fig. 4c–d\nand Supplementary Fig. 13a–d).\nComment 9: By contrast there is a faint labeling of LM19 in control nodules. LM20\nantibody was detected in the border of the cells and also in the symbiosome in the\nSYPG1/2 RNAi nodules. Is image 4e in the figure 4 a representative image because I\nobserve more signal at the cell walls in the SYPG1/2 RNAi lines while the\nquantifications show similar results?\nOur response: We appreciate your careful observation. Our quantification focused\nexclusively on intracellular signals in infected cells, excluding cell border labeling\nwhere methyl-esterified pectins naturally accumulate. We have revised the figure with\nmore representative images (Supplementary Figs. 12, 13f–i).\nComment 10: Fig. 5.\nThe authors tested the effects of PGA on the growth of the bacteria. PGA inhibits the\ngrowth in liquid and solid media. They then tested the effect of the tetragalacturonic\nacid and no effect was observed. Why the authors tested this oligosaccharide. They\nmust first digest PGA in presence of SyPG1 for example and see what types of\noligogalacturonides they will obtain. PG enzymes usually produces GalA, GalA2 and\nGalA3 products so at least the auhors need to treat bacteria with GalA and GalA2\nOur response: We thank the reviewer for this excellent suggestion. We have tested\nGalA2 and found that, unlike PGA, it does not impair bacterial activity in either in\nfree-living rhizobia or bacteroids (Supplementary Fig. 15). These results are presented\nin the revised manuscript.\nComment 11: Supplementary Fig. 2.\nChange pother into other\nOur response: Thank you for your careful review. This has been corrected.\nComment 12: Line 184 change symbisome into symbiosome\nOur response: Thank you for your careful review. This has been corrected.\nComment 13: Supplementary Fig 2.\nI am not sure that the expression of other PGs is not impacted by this RNAi because\nonly 5 values were obtained and there is a large variability in the control. This is why\nthe sequence alignment of the PGs and the sequences used for RNAi would be\nimportant.\nOur response: Thank you for your valuable comment. As noted above, we have\nincluded sequence alignments (Supplementary Fig. 8). To address concerns about\noff-target effects, we generated CRISPR/Cas9 lines specifically targeting SyPG1 and\nSyPG2. The observed phenotypes closely mirrored those of RNAi plants\n(Supplementary Fig. 11), confirming that the effects were due to loss of SyPG1/2\nrather than unintended silencing of other PGs.\nComment 15: Did the authors analyse several lines RNAi PG1/PG2?\nOur response: Thank you for raising this point. Yes. Multiple independent hairy root\ntransformation experiments were performed, and only roots with confirmed\nknockdown of both genes were analyzed. Furthermore, CRISPR/Cas9 knockout lines\nvalidated these results, strengthening our conclusions.\nComment 16: Define S. from S. meliloti\nOur response: Thank you for your careful review. This has been revised.\nWe thank the reviewers again for their thoughtful and constructive feedback. All\ncomments have been addressed, and the manuscript has been revised accordingly. We\nbelieve these changes have strengthened the study and hope the revised version meets\nwith the reviewers’ approval.\nReferences:\nrelease of bacteria in soybean nodules. New Phytol 210, 1011-1021 (2016).\nhttps://doi.org:10.1111/nph.13837\nroots is associated with locally confined cell wall modifications. Curr Biol 33,\n533-542 (2023). https://doi.org:10.1016/j.cub.2022.12.051\nthe pectin methylesterase-pectin methylesterase inhibitor complex. J Biol Chem 292,\n21538-21547 (2017). https://doi.org:10.1074/jbc.RA117.000197\npatterns for apple pectin methylesterase at pH 7.0 and 4.5. Carbohydr Res 327,\n385-393 (2000). https://doi.org:10.1016/s0008-6215(00)00070-7\ncell wall structure and hypocotyl elongation. Plant Cell Physiol 65, 301-318 (2024).\nhttps://doi.org:10.1093/pcp/pcad154"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript has satisfactorily addressed most of my previous questions. My remaining comments are minor, pertaining to a few small errors in the presentation and one conceptual point that may require further consideration.\nMajor Conceptual Point:\nThe study presents a compelling model where SyPG1/2 degrades pectins within the symbiosome space. This is justified as a mechanism to remove un-esterified pectin, which impairs bacterial growth, and potentially explains the absence of a cell wall around the symbiosome.\nThis model, however, raises an important mechanistic question:\nPGA degradation is generally, including to free-living bacteria, how do the bacteria survive within the infection threads (ITs)?\nThe ITs possess a cell wall that is \"reversed,\" meaning the matrix facing the bacteria would still contain pectins. A discussion of this apparent paradox would significantly strengthen the manuscript.\nMinor Corrections:\nPlease note the following small errors in the figures and text:\n• Figure 1a'': The white demarcation line is not visible. Please use a more contrasting color or a boldened line.\n• Figure 2 Legend (Line 541): The text references panels (b) and (f), but this seems to be (d) and (h). Please verify and correct the panel labels.\n• Figure 6:\no The meaning of the question mark (?) in the working model is unclear. Please add a detailed description of what it signifies in the figure legend.\no Furthermore, this working model should not only be cited but also be thoroughly described and interpreted in the main results or discussion text of the manuscript.", "reviewer_2": "The manuscript by Chen et al. identified an active role for two symbiotically induced polygalacturonase genes (SyPG1 and SyPG2) in the continuous degradation of un-esterified pectin from the symbiosome space in Medicago truncatula root nodules. Silencing or knock-out of these two genes led to the accumulation of unesterified pectin at the symbiosomes and led to early nodule senescence. This highlights the importance of continuous clearing of cell wall material from the symbiosome space to allow a successful symbiosis with rhizobium bacteria.\nThe revised manuscript has improved and largely addressed my previous concerns/comments.\nSeveral comments still remain:\nThe authors have now included in situ hybridization to determine the expression domains of SyPG1 and SyPG2. These show a predominant expression in the nodule interzone which is not in line with the promoter GUS analyses. Also publically available RNAseq data suggest predominant expression in the interzone/early mature zone (although this is not discussed). The discrepancy between the promoter-GUS analyses and the in situ hybridizations are now explained by either stabilization of transcripts or transcellular mobility. Although this cannot be ruled out, a more parsimonious explanation would be that the promoters used to drive the GUS constructs lack certain cis-regulatory elements to give the promoter expression pattern of these genes. This should be better discussed.\nThe images for the in situ hybridization are not very clear. More clearer pictures should be included.\nTo strengthen the specific role for SyPG1 and SyPG2 the authors now included CRISPR-Cas9 mutants. However, the details concerning these lines, how they were made (stable vs hairy root?, procedure?), how they were selected (which mutations occurred in the analysed roots/nodules, etc. .is lacking. More information needs to be added in the material and methods and results sections.\nFor Suppl fig 6 it should be indicated which promoters were used to drive the GFP constructs. Does this explain the difference in localization? If not, how to explain?\nIt would be good to provide more clearer images of the infection zone to support the claim that no effects on bacterial release or symbiosome differentiation were observed. Are there other PGs highly expressed in the infection zone that may contribute to the clearing of pectins from the symbiosomes in this part of the nodule?", "reviewer_3": "The changes made to the text by the authors are substantial and relevant. I now only have minor comments.\n1. The authors must update the article's abstract to indicate, on the one hand, that there are CRISPR mutants, and on the other hand, that bacterial activity is also reduced in vivo. I am not sure about keeping 'line 23 that are actively secreted into the symbiosome', as this is not demonstrated in the present work. It might be more appropriate to write 'actively expressed' instead or remove this part.\n2. Reference no. 1 is not the most appropriate for what the authors describe. It would be better to cite the article https://doi.org/10.1007/s00018-019-03388-8.\n3. Line 34 Callose should be removed from this sentence, as it is not a major component of the cell wall.\n4. Reference no. 4 add a more recent one such as https://doi.org/10.1016/j.ijbiomac.2023.125385\n5. Line 48 remove « these » in the sentence\n6. Line 50 add also the activity of pectin lyase.\n7. The authors generated CRISPR-Cas9 mutants, which significantly strengthens their data. They present the guides and the obtained sequences in Supplementary Figure 11. However, it would be helpful to add information on the resulting amino acid sequences (does it create a stop codon… ?) and to indicate which mutants were tested in symbiosis. Finally, I would have liked to see information on the expression of the PGs previously tested in the RNAi transformants.\n8. In sup Fig3, please highlight in a different color the four other PGs whose expression was tested in the RNAi transformants.\"\n9. In the Discussion section, around line 262, please also mention the generation of the CRISPR-Cas9 mutants.\n10. Mat met : add reference for PGA and GalA2 ?\n11. Mat met : add concentration of PBS line 391\n12. Define FAAfixative line 391"}, "author_response": "Responses to the reviewers’ comments\nWe are sincerely grateful to the reviewers for their valuable comments aimed at\nenhancing our manuscript. Below is the itemized responses to each point raised by the\nreviewers.\nReviewer #1\nGeneral: The revised manuscript has satisfactorily addressed most of my previous\nquestions. My remaining comments are minor, pertaining to a few small errors in the\npresentation and one conceptual point that may require further consideration.\nResponse: Thank you again for your valuable comments and suggestions on our\nmanuscript. We appreciate your constructive suggestions which helped us to improve\nthe quality of our manuscript.\nMajor Conceptual Point:\nComment 1: The study presents a compelling model where SyPG1/2 degrades pectins\nwithin the symbiosome space. This is justified as a mechanism to remove un-esterified\npectin, which impairs bacterial growth, and potentially explains the absence of a cell\nwall around the symbiosome. This model, however, raises an important mechanistic\nquestion: PGA degradation is generally, including to free-living bacteria, how do the\nbacteria survive within the infection threads (ITs)? The ITs possess a cell wall that is\n\"reversed,\" meaning the matrix facing the bacteria would still contain pectins. A\ndiscussion of this apparent paradox would significantly strengthen the manuscript.\nResponse: Thank you for your comment and for pointing this out. Indeed, it is\nintriguing that the bacteria within the IT can evade the influence of un-esterified pectin.\nOne possible explanation is that the bacteria inside the IT are embedded in the matrix,\nwhich potentially shields them from the un-esterified pectin. We note that PGA\ntreatment has a relatively limited effect on bacterial activity in IT (see Supplementary\nFigure 17), which supports our hypothesis. This result has been included in the revised\nmanuscript and is discussed therein (Line 282-287).\nMinor Corrections:\nPlease note the following small errors in the figures and text:\nComment 1: Figure 1a'': The white demarcation line is not visible. Please use a more\ncontrasting color or a boldened line.\nResponse: Thanks for your suggestion. In the revised manuscript, we have changed to\na more visible region for the label and employed a bolder line.\nComment 2: Figure 2 Legend (Line 541): The text references panels (b) and (f), but\nthis seems to be (d) and (h). Please verify and correct the panel labels.\nResponse: Thanks for your carefully reading. We have corrected those errors in the\nrevised manuscript.\nComment 3: Figure 6:\no The meaning of the question mark (?) in the working model is unclear. Please add a\ndetailed description of what it signifies in the figure legend.\no Furthermore, this working model should not only be cited but also be thoroughly\ndescribed and interpreted in the main results or discussion text of the manuscript.\nResponse: We sincerely appreciate your constructive suggestions. The question mark\n(?) in together with the dashed arrow indicate two potential pathways of pectin\ndeposition into symbiosome space. To enhance clarity, the “?” was removed from the\nfigure in the revised manuscript, and the gray dashed arrows and pink dashed arrows\nwere directly referenced in the figure legend. Furthermore, following your advice, a\ndescription of our working model has been included in the discussion section (Line\n262-267). Hope these modifications can address your concerns.\nReviewer #2:\nGeneral: The manuscript by Chen et al. identified an active role for two symbiotically\ninduced polygalacturonase genes (SyPG1 and SyPG2) in the continuous degradation of\nun-esterified pectin from the symbiosome space in Medicago truncatula root nodules.\nSilencing or knock-out of these two genes led to the accumulation of unesterified pectin\nat the symbiosomes and led to early nodule senescence. This highlights the importance\nof continuous clearing of cell wall material from the symbiosome space to allow a\nsuccessful symbiosis with rhizobium bacteria. The revised manuscript has improved\nand largely addressed my previous concerns/comments.\nResponse: Thank you again for your valuable comments and suggestions on our\nmanuscript, which helped us to improve the quality of our manuscript.\nComment 1: The authors have now included in situ hybridization to determine the\nexpression domains of SyPG1 and SyPG2. These show a predominant expression in the\nnodule interzone which is not in line with the promoter GUS analyses. Also publically\navailable RNAseq data suggest predominant expression in the interzone/early mature\nzone (although this is not discussed). The discrepancy between the promoter-GUS\nanalyses and the in situ hybridizations are now explained by either stabilization of\ntranscripts or transcellular mobility. Although this cannot be ruled out, a more\nparsimonious explanation would be that the promoters used to drive the GUS constructs\nlack certain cis-regulatory elements to give the promoter expression pattern of these\ngenes. This should be better discussed.\nResponse: Thank you for your comment and point this out. We agree with your point\nthat the promoter sequence we used may also lack certain cis-elements. Following your\nadvice, we have added this point in our revised manuscript (Line 141-147).\nComment 2: The images for the in situ hybridization are not very clear. More clearer\npictures should be included.\nResponse: Thank you for your comment. In the revised manuscript, we have replaced\nthe original in situ hybridization images (young nodule) with higher-resolution images\nto enhance the clarity of the results (Supplementary Figure 5).\nComment 3: To strengthen the specific role for SyPG1 and SyPG2 the authors now\nincluded CRISPR-Cas9 mutants. However, the details concerning these lines, how they\nwere made (stable vs hairy root?, procedure?), how they were selected (which\nmutations occurred in the analysed roots/nodules, etc. .is lacking. More information\nneeds to be added in the material and methods and results sections.\nResponse: We appreciate your suggestion. We carried out hairy root transformations\nusing the SyPG1/2-Cas9 construct, which is specifically designed to target SyPG1 and\nSyPG2. All the transformed roots (The construct employed in this study featured an\nmCherry marker (under a constitutive promoter) for selection purposes) were harvested,\nfollowed by DNA extraction and sequencing to elucidate the editing patterns of all the\ntransformed roots. Only nodules from the edited roots were employed for subsequent\nanalysis. Mainly two types of editing patterns identified are presented in\n(Supplementary Figure 11a). Regarding edited SyPG1, it encodes the N - terminal 68\namino acids, and for edited SyPG2, it encodes either 91 or 93 amino acids. All the\ninformation has been incorporated into the results (Line 186-189) and the figure legend\n(Supplementary Figure 11). Hope with these information can address your concerns.\nComment 4: For Suppl fig 6 it should be indicated which promoters were used to drive\nthe GFP constructs. Does this explain the difference in localization? If not, how to\nexplain?\nResponse: Thank you for your comment. In Supplementary Figure 6, the same\npromoter (LjUbi) was utilized to visualize the localization of SyPG1 and SyPG2. The\nresults suggest that the different localization is not due to the promoter employed. The\ndistinct localization patterns imply the potential existence of other regulatory\nmechanisms at the protein level, which recruit distinct PGs to different locations. This\nphenomenon was also observed in our previous study (Su et al., 2023), where SyPME\naccumulated at the site through which the infection thread passed, whereas NPL did not,\neven when the same promoter was utilized.\nComment 5: It would be good to provide more clearer images of the infection zone to\nsupport the claim that no effects on bacterial release or symbiosome differentiation were\nobserved. Are there other PGs highly expressed in the infection zone that may\ncontribute to the clearing of pectins from the symbiosomes in this part of the nodule?\nResponse: Thank you for your comment. In the revised manuscript, we have provided\nmore clearer Technovit 7100 images to show the nodule infection zone. Additionally,\nto further support our description, we provided nodule agarose sections stained with\nCalcofluor White to illustrate the infection threads and infection droplets in the\nnodules (nodule infection zone) harvested from the either empty vector or SyPG1/2 -\nRNAi transformed roots (Supplementary Figure 10).\nWe hypothesized that there exist other PG genes that are highly expressed in the nodule\ninfection zone and may also be located to the symbiosome space to perform the function\nof pectin clearance. Additionally, we also proposed that potentially, other members of\nthe PG family may be located at the infection droplet, functioning together with SyPG1\nto regulate bacterial release. This aspect has been incorporated into the discussion\nsection (Line 297-302) of the revised manuscript and also serves as a subject for our\nfuture research.\nReviewer #3:\nGeneral: The changes made to the text by the authors are substantial and relevant. I\nnow only have minor comments.\nResponse: Thank you again for your valuable comments on our manuscript, which\nhelped us to improve the quality of our manuscript.\nComment 1: The authors must update the article's abstract to indicate, on the one hand,\nthat there are CRISPR mutants, and on the other hand, that bacterial activity is also\nreduced in vivo. I am not sure about keeping 'line 23 that are actively secreted into the\nsymbiosome', as this is not demonstrated in the present work. It might be more\nappropriate to write 'actively expressed' instead or remove this part.\nResponse: Thank you for your suggestion. In accordance with your advice we have\nupdated our abstract. And to avoid over-interpretation, we have removed the “actively”\nfrom the sentence, only keep the “secreted into the symbiosome space” in the revised\nmanuscript.\nComment 2: Reference no. 1 is not the most appropriate for what the authors describe.\nIt would be better to cite the article https://doi.org/10.1007/s00018-019-03388-8.\nResponse: We express our sincere gratitude for your carefully reading and valuable\nsuggestion. In the revised manuscript, we have substituted the reference with the more\nappropriate one you recommended.\nComment 3: Line 34 Callose should be removed from this sentence, as it is not a major\ncomponent of the cell wall.\nResponse: Thank you for your comment. In the revised manuscript, we have removed\nthe “Callose” from the sentence.\nComment 4: Reference no. 4 add a more recent one such as\nhttps://doi.org/10.1016/j.ijbiomac.2023.125385\nResponse: Thank you for your comment. The reference you suggested has been added\nin the revised manuscript.\nComment 5: Line 48 remove « these » in the sentence\nResponse: Thank you for your suggestion, “these” has been removed.\nComment 6: Line 50 add also the activity of pectin lyase.\nResponse: Thank you for your suggestion, “pectin lyase” has been added in the\nsentence.\nComment 7: The authors generated CRISPR-Cas9 mutants, which significantly\nstrengthens their data. They present the guides and the obtained sequences in\nSupplementary Figure 11. However, it would be helpful to add information on the\nresulting amino acid sequences (does it create a stop codon… ?) and to indicate which\nmutants were tested in symbiosis. Finally, I would have liked to see information on the\nexpression of the PGs previously tested in the RNAi transformants.\nResponse: We appreciate your suggestion. We conducted hairy root transformations\nusing the SyPG1/2-Cas9 construct, which is designed to target SyPG1 and SyPG2. All\nthe transformed roots (The construct employed in this study featured an mCherry\nmarker (under a constitutive promoter) for selection purposes) were harvested,\nfollowed by DNA extraction and sequencing to elucidate the editing patterns of all the\ntransformed roots. Only nodules from the edited roots were employed for subsequent\nanalysis. Regarding the edited SyPG1, it encodes the N - terminal 68 amino acids. As\nfor the edited SyPG2, it can encode either the N - terminal 91 amino acids or the N -\nterminal 93 amino acids. All of this detailed information has been incorporated into the\nfigure legend (Supplementary Figure 11a).\nFurthermore, in reply to your inquiry, we investigated the expression of other PG genes\nin SyPG1/2-Cas9 transformed roots. The results shown that SyPG1/2-Cas9 transformed\nroots had no influence on the expression of other PG genes (Supplementary Figure\n11b-e). This result has been added in the revised manuscript.\nComment 8: In sup Fig3, please highlight in a different color the four other PGs whose\nexpression was tested in the RNAi transformants.\"\nResponse: Thank you for your suggestion, this has been done.\nComment 9: In the Discussion section, around line 262, please also mention the\ngeneration of the CRISPR-Cas9 mutants.\nResponse: Thank you for your suggestion. Indeed, we also examined pectin\ndistribution pattern in the SyPG1/2-Cas9 transformed nodules, this results also been\nmentioned in the sentence in the revised manuscript.\nComment 10: Mat met : add reference for PGA and GalA2 ?\nResponse: Thank you for your suggestion, the reference for PGA and GalA2 been\nadded.\nComment 11: Mat met : add concentration of PBS line 391\nResponse: Thank you for your suggestion. The concentration of the PBS solution used\nin this study has been clarified.\nComment 12: Define FAA fixative line 391\nResponse: Thank you for your suggestion. The FAA solution, which stands for\nformalin-acetic acid-alcohol fixative, has been clearly specified in the revised\nmanuscript.\nOnce again, we thank you for your valuable time and suggestions, which have helped\nus to improve the manuscript.\nReference\nSu, C. et al. Transcellular progression of infection threads in Medicago truncatula\nroots is associated with locally confined cell wall modifications. Curr Biol 33,\n533-542 (2023). https://doi.org:10.1016/j.cub.2022.12.051"}]

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