Overexpression of Enterococcus faecalis elr operon protects from phagocytosis
- Naima G. Cortes-Perez1, 2, 5,
- Romain Dumoulin1, 2,
- Stéphane Gaubert1, 2,
- Caroline Lacoux1, 2,
- Francesca Bugli3,
- Rebeca Martin1, 2,
- Sophie Chat1, 2,
- Kevin Piquand1, 2,
- Thierry Meylheuc1, 2,
- Philippe Langella1, 2,
- Maurizio Sanguinetti3,
- Brunella Posteraro4,
- Lionel Rigottier-Gois1, 2 and
- Pascale Serror1, 2Email author
© Cortes-Perez et al.; licensee BioMed Central. 2015
Received: 14 January 2015
Accepted: 14 May 2015
Published: 25 May 2015
Mechanisms underlying the transition from commensalism to virulence in Enterococcus faecalis are not fully understood. We previously identified the enterococcal leucine-rich protein A (ElrA) as a virulence factor of E. faecalis. The elrA gene is part of an operon that comprises four other ORFs encoding putative surface proteins of unknown function.
In this work, we compared the susceptibility to phagocytosis of three E. faecalis strains, including a wild-type (WT), a ΔelrA strain, and a strain overexpressing the whole elr operon in order to understand the role of this operon in E. faecalis virulence. While both WT and ΔelrA strains were efficiently phagocytized by RAW 264.7 mouse macrophages, the elr operon-overexpressing strain showed a decreased capability to be internalized by the phagocytic cells. Consistently, the strain overexpressing elr operon was less adherent to macrophages than the WT strain, suggesting that overexpression of the elr operon could confer E. faecalis with additional anti-adhesion properties. In addition, increased virulence of the elr operon-overexpressing strain was shown in a mouse peritonitis model.
Altogether, our results indicate that overexpression of the elr operon facilitates the E. faecalis escape from host immune defenses.
As a natural inhabitant of the oral cavity, gastrointestinal tract, and female vaginal tract in humans, Enterococcus faecalis is normally considered a nonpathogenic microorganism. However, it is a common opportunistic pathogen in immunocompromised patients, causing nosocomial infections. While our current understanding of the mechanisms that lead to the lifestyle shift from commensalism to virulence in enterococci remains an emerging area of research, the pathogenesis of E. faecalis is clearly nonetheless a complex multifactorial process that currently remains poorly understood. In this regard, we have previously identified the enterococcal leucine-rich protein A (ElrA), a protein that possesses a leucine-rich repeat (LRR) domain and a carboxy-terminal WxL domain, which promotes non-covalent association to the bacterial surface . ElrA is encoded by the elr operon , which encodes two other WxL surface proteins, a small LPXTG-motif protein and a putative transmembrane protein proposed to form cell surface complexes [1–4]. Expression of the elr operon is under the control of the positive regulator elrR [4, 5]. The elrA gene is poorly expressed in vitro, but it can be induced by complex biological milieu such as serum or urine, which suggests the tightly regulated control of elrA expression in response to in vivo signals [5, 6]. Previously, we showed that inactivation of the elrA gene resulted in significantly reduced virulence in a mouse model of peritonitis . We also observed reduced secretion of interleukin-6 (IL-6, a pro-inflammatory cytokine) upon in vivo infection with the ΔelrA mutant strain and we hypothesized that ElrA may be involved in this modulation, by stimulating host immune cells to counteract E. faecalis infection .
Macrophages are potent antigen presenting cells that play a key role in initiating an immune response against invading bacteria. In turn, some pathogens have evolved strategies in order to circumvent macrophage functions . Previous studies have shown that E. faecalis can survive in peritoneal macrophages better than other non-pathogenic bacteria [8, 9]. In addition, it possesses mechanisms permitting escape from murine or human macrophages [8, 10, 11]. E. faecalis cell wall glycopolymers play a key role in the resistance to phagocytosis. In particular, capsular polysaccharide serotypes C and D contribute to complement evasion [12, 13] and rhamnopolysaccharide Epa protects from phagocytic killing [13, 14], most likely by preventing uptake by macrophages as we recently showed in zebrafish model .
In the present study, we sought to evaluate whether the expression of elrA alone or that of the entire elr operon most influences the capability of E. faecalis to be phagocytized by the RAW 264.7 mouse macrophages in vitro. To circumvent the aforementioned low level of elrA expression in vitro, a genetically modified E. faecalis strain harboring a constitutive promoter upstream of the elr operon (P+-elrA-E) was constructed. The ability of this elr-overexpressing strain to be internalized was compared with a wild-type strain of E. faecalis, and with different isogenic-elr mutant strains, obtained by genetic manipulation of the E. faecalis P+-elrA-E strain.
Results and discussion
Production of ElrA requires other gene(s) of the elr operon
Overexpression of elr operon impairs phagocytosis
Overexpression of elr operon modifies bacterial adhesion
Phagocytosis is initiated with the recognition of ligands on bacterial cell surfaces by receptors including scavenger receptors, glucan receptors, and integrins present on the membrane of macrophages, which leads to bacteria engulfment via an actin-dependent mechanism. To test whether the impairment of phagocytosis seen for the P+-elrA-E strain correlated with reduced adhesion of the bacterium to macrophage cells, we used cytochalasin D (CytD), which inhibits phagocytosis, but does not prevent the initial step of bacterial adhesion [17, 18]. Macrophages were therefore infected (as described above for the phagocytosis test) with either WT or P+-elrA-E strains in the presence or absence of CytD, and the percentages of GFP+ macrophages were measured by flow cytometry analysis (uninfected macrophages were used as negative control). Comparison of forward scatter (FSC) and side scatter (SSC) values from uninfected cells (CytD treated or untreated) confirmed that macrophages were not altered by CytD (Additional file 1: Figure S1). As shown in Fig. 3B, P+-elrA-E strain was 60 % less adherent to macrophages than the WT strain. These results are in agreement with scanning microscopy observations of infected macrophages, that showed a sharp contrast between adhesion of WT and P+-elrA-E strains (Fig. 3C).
Because proteins encoded by the elr operon demonstrate characteristics of surface proteins (WxL, and LPXTG motifs) and could form a surface complex, we hypothesized that overexpression of elr operon could result in the formation of surface structures, which in turn resulted in the inhibition of phagocytosis as observed in vitro. Analyses of bacterial strains using transmission electron microscopy (thin sections and negative staining) and scanning electron microscopy revealed no differences at the surface structure level between the WT and the elr operon-overexpressing strain (data not shown). This indicated that no major structural modification was detected under the tested conditions. Since high expression levels of surface proteins can modify physicochemical properties of bacterial cell surface such as charge or hydrophobicity [19, 20], we compared the affinity of bacterial cells of WT and P+-elrA-E strains to the solvents using a MATS test as described by Bellon-Fontaine et al. . Both strains exhibited similar affinity for the apolar solvents decane (~40 %) and hexadecane (~30 %) and for the acidic solvent chloroform (~70 %), indicating no major changes of the surface hydrophobicity upon elr overexpression. In turn, the affinity of the strain P+-elrA-E for the basic solvent ethyl acetate (~35 %) increased significantly compared to the WT (<1 %), indicating that expression of elr operon enhances the negative charge of the bacterial cells. Thus we hypothesize that poor adhesion of strain P+-elrA-E may result from repulsive forces between the negatively charged macrophage membrane and bacterial surface, which is loaded with Elr proteins.
Overexpression of elr operon increases E. faecalis virulence
Previous studies have shown that E. faecalis survives into peritoneal macrophages better than non-pathogenic bacteria . Since then, E. faecalis virulence factors able to interfere with uptake and survival in macrophages have been described . We previously linked the attenuated virulence of an E. faecalis strain deleted for elrA with decreased organ burden and survival in peritoneal macrophages . In this study, we show that overexpression of elr operon by E. faecalis confers resistance to phagocytosis by interfering with bacterial adhesion to macrophages. We also correlated E. faecalis avoidance of phagocytosis observed in vitro with increased virulence and dissemination in a mouse peritonitis model. These data contrast with our previous report that WT and ΔelrA strains were evenly phagocytosed . Nevertheless, these studies are difficult to compare since macrophage infections were performed differently (i.e. in vivo versus in vitro infection and duration of infection). Moreover, the expression level of Elr proteins in vivo is unknown. The tight control of expression of the elr operon suggests that the operon may be required in specific conditions that remain to be identified [4, 5]. The 162-fold increased level of erlA transcript in E. faecalis strain MMH594 grown in urine , supports that expression of Elr proteins may vary in response to host-derived cues. We assume that elr operon may enhance E. faecalis virulence by promoting initial dissemination in the host after escape of bacteria from phagocytosis, but also by contributing to E. faecalis survival within infected macrophages depending on the tissues or cell types encountered by E. faecalis. From this study we propose that high-level expression of elr operon may, in some circumstances, occur in vivo and promotes escape of E. faecalis from phagocytosis.
The present study also revealed that ElrA requires at least one other elr gene to be expressed at a detectable level and confirmed that elrA gene is cotranscribed with the other elr genes . The elr operon is a typical gene cluster of WxL surface proteins that associate non-covalently to the peptidoglycan of low-GC gram-positive bacteria. The operonic organization of the elr operon and the need of at least one other protein encoded by elr operon for ElrA production in vitro further support the hypothesis that cell-surface proteins, encoded by the elr operon, may participate in the formation of a multicomponent complex at the surface as it has been previously proposed [1, 2, 4]. Based on recent work by Galloway-Pena et al. who showed that WxL and DUF916 proteins interact in vitro , we believe that ElrA may be protected from degradation by interacting with at least another elr-encoded protein. If neither surface appendages nor modification could be observed upon overexpression of elr operon, other experiments are needed to establish if elr operon drives the formation of a surface complex in E. faecalis. Nevertheless, overexpression of Elr proteins seems to increase the negative charge of the bacterial surface, suggesting that E. faecalis evasion of phagocytosis by immune cells is driven by electrostatic repulsion. Even if elr overexpression emphasizes the steric or charge hindrance by Elr proteins in vitro, one cannot exclude that similar physicochemical changes occur in vivo in response to environmental cues , and confers to the E. faecalis cells anti-adhesion properties that promote escape from phagocytosis. These in vitro findings are reminiscent of the acidic LRR protein Slr from Streptococcus pyogenes that is involved in phagocytosis evasion , probably by enhancing the anti-adhesive properties of streptococcal cells. Another possibility would be that high level of Elr proteins sterically hinders E. faecalis-associated molecular patterns important for recognition by scavenger receptors. Altogether, this work shows that expression of elr operon contributes to the escape of E. faecalis from phagocytosis, promoting dissemination and enhancing virulence of the pathogen. Further investigations will focus on characterizing the precise role of each of the Elr proteins.
In summary, this work shows that high-level expression of elr operon by E. faecalis increases virulence and confers resistance to phagocytosis, probably through charge repulsion. Consistently, the strain expressing elr operon displays stabilization of ElrA, further supporting that Elr proteins form an extracellular protein complex as part of the virulence process. Structural and functional characterization of the Elr proteins will help to understand E. faecalis pathogenesis and provide clues on WxL- and associated proteins of low-GC Gram-positive bacteria.
All reagents were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise stated.
Bacterial strains and plasmids
Strains and plasmids used in this work
Designation relevant characteristics
Source or Reference
Fusr Rifr; plasmid-free wild-type strain
OG1RF P aphA3 ::elrA-E
OG1RF P aphA3 ::ΔelrA
OG1RF P aphA3 ::elrA-ΔelrB-E
OG1RF P aphA3 ::ΔelrA-E
supE hsdD5 thi (Δlac-proAB) F’ (traD36 proAB-lacZΔM15)
F− ompT gal dcm hsdSB(rB−mB−) λDE3
Ampr, Kanr, ori p15A
Ampr, ori colE1, T7 promoter, His-Tag coding sequence
Ampr, ori ColE1, linearized with 3’ T overhangs
Ermr, ori pWV01, repA(Ts)
pMV158 with the gene encoding the green fluorescent protein
pTCV-lac(P aphA3 )
Tetr, ori ColE1, ori pAMβ1, lacZ harboring P aphA3 promoter
Ermr, ori pWV01, repA(Ts), with elrA deletion
Ampr, pET2817 with 6x His::ElrA
Ampr, Kanr, ori p15A, ‘elrR-elrA’ region
Ampr, Kanr, ori p15A, ‘elrR-P aphA3 ::elrA’ region
Ermr, ori pWV01, repA(Ts), ‘elrR-P aphA3 ::elrA’ region
Ampr, ori colE1, with elrA-E deletion
Ampr, ori colE1, with elrB-E deletion
Ermr, ori pWV01, repA(Ts), with elrB-E deletion
Ampr, Kanr, ori p15A, with P aphA3 ::elrA-E deletion
Ermr, ori pWV01, repA(Ts), with P aphA3 ::elrA-E deletion
Ermr, ori pWV01, repA(Ts), with P aphA3 ::elrA deletion
Cell line and culture conditions
The RAW 264.7 mouse macrophage cell line (ATCC®−TIB-71) was maintained in DMEM supplemented with 10 % heat-inactivated fetal bovine serum (FBS) and 2 mM l-glutamine . For phagocytosis assays, cells were seeded at 0.5 × 106/well into 12-well tissue culture plates (TPP, Domique Dutscher, Brumath, France) and incubated overnight at 37 °C under 6 % CO2. For microscopy experiments, cells were cultured in tissue culture plates containing poly-L-lysine pretreated coverslips for microscopy or on Lab-tek chamber slides (Nunc, Domique Dutscher). Comparative analysis of phagocytosis using either heat-inactivated serum or serum-free media (Macrophage-SFM, GIBCO, Invitrogen) did not show differences (data not shown). Thus, for practical reasons we decided to use heat-inactivated serum in all experiments of this work.
Generation of anti-ElrA rat polyclonal antibodies
Primers used in this study
Source or Reference
Construction of mutant and over-expressing strains of E. faecalis
The E. faecalis elrA gene is part of a five-gene operon elrA (OG1RF_12055), elrB (OG1RF_12054), elrC (OG1RF_12053), elrD (OG1RF_12052), and elrE (OG1RF_12051) (Fig. 1), encoding putative surface proteins of unknown function. To circumvent the lack of ElrA production in vitro, we constructed a genetically modified E. faecalis strain harboring the constitutive promoter PaphA3 (hereafter named P+), instead of the native promoter, PelrA, upstream of the whole elr operon (i.e., elrA-E, Fig. 1). This genetically modified strain (called P+-elrA-E), was constructed by a double cross-over event using the pGhost9 plasmid . Briefly, two overlapping fragments were PCR-amplified from E. faecalis OG1RF chromosomal DNA with primers OEF343/OEF344 and OEF345/OEF346 (Table 2). The two PCR products were then fused by PCR using the external primers OEF344/OEF346, and the resulting product was cloned into purified XhoI-BamHI-digested pACYC177 vector, resulting in plasmid pVE14142. The PaphA3 promoter was PCR-amplified with primers Vlac1 and Vlac2 from pTCV-lac(PaphA3) plasmid . An EcoRI-BamHI fragment, containing the PaphA3 promoter, was then cloned into EcoRI-BglII-digested pVE14142 vector to obtain plasmid pVE14145. Then, a 2.3 kb XhoI-EaeI fragment from pVE14145 plasmid (containing the promoter and the targeted region) was cloned into pGhost9 vector to generate the final vector pVE14146. This plasmid was established in E. faecalis OG1RF strain and a markerless insertion of PaphA3 upstream of the elrA-E operon was performed as previously described . Correct integration of PaphA3 into the chromosomal locus was confirmed by sequencing. All the following mutant constructs were performed using P+−elrA-E strain as a recipient in order to have the same genetic background (Table 1). For the construction of a strain expressing only elrA under the control of PaphA3 promoter, a fused DNA fragment using primers OEF13/OEF595 and OEF596/OEF49 amplified from OG1RF strain DNA was cloned into pGEM-T easy vector (Promega) to generate pVE14179. A 4.5 kb PstI fragment was then cloned into PstI-digested pGhost9 to obtain plasmid pVE14450 and established in P+-elrA-E strain to obtain the P+-elrA-ΔelrB-E strain. For the construction of a strain expressing elr operon lacking elrA, a 6.6 kb Bst/ApeI fragment from pVE14009 was cloned into Bst/ApeI-digested pVE14146 vector, resulting in pVE14457. This plasmid was established in P+-elrA-E strain to obtain the P+-ΔelrA strain. To inactivate the whole elr operon (i.e., elrA-E), we first generated an in-frame deletion of the whole operon by PCR. For this, we used OEF15/OEF18 and OEF598/OEF49 primers described for the first PCR. The two PCR products were fused by PCR using external primers OEF49/OEF15, and the resulting product was cloned into pGEM-T, resulting in plasmid pVE14178. A BstAPI/AatII 890bp DNA fragment from pVE14178 was then cloned into pVE14145 to generate pVE14455. The final plasmid was generated by cloning a 4.5 kb XhoI DNA fragment from pVE14455 vector into XhoI-digested pGhost9 to obtain pVE14456. This plasmid was established in P+-elrA-E strain and the resulting strain was named P+-ΔelrA-E. All expected modifications or deletions were confirmed by sequencing.
Preparation of protein extracts, SDS gel electrophoresis, and immunoblot analysis
Total protein extraction from bacteria, SDS-PAGE, and Western blot immunodetection were carried out using standard methods (24) with some modifications. Strains were grown at 37 °C overnight and then diluted 100-fold and grown under the same conditions to an OD600~1. Protein crude extract was obtained by trichloroacetic acid (TCA) precipitation by mixing 800 μl of bacterial culture with 200 μl of ice cold TCA solution (100 % w/v). The protein pellet was then obtained by centrifugation and recovered directly into SDS sample buffer. Anti-ElrA antibody was used at a dilution of 1:500 for Western blot immunodetection.
RNA isolation and Northern blotting
Total RNA was extracted as previously described . Northern blots were performed on 40 μg of total RNA separated on a 0.9 % denaturing agarose gel as previously described . Specific oligonucleotides OEF9 and OEF212 were used to detect elrA transcripts. Oligonucleotides were labelled with [γ-32P]-ATP and T4 polynucleotide kinase (NEB Biolabs) according to the recommendations of the manufacturer (NEB Biolabs). Analysis was performed from RNA extracted from two independent experiments.
Phagocytosis assay with RAW macrophages
Fluorescent E. faecalis were grown on GM17 plates containing erythromycin (GM17-Ery), with a single colony subsequently being selected and grown overnight in GM17-Ery broth. A 100 μl aliquot was then transferred into 10 ml of fresh GM17-Ery and incubated until cultures reached an OD600 ~1. Bacteria were then pelleted by centrifugation, washed three times with PBS, and adjusted to a concentration of 1 × 109 CFU/ml in supplemented DMEM. The number of bacteria present in each suspension was confirmed by plating onto solid GM17-Ery.
For phagocytosis experiments, adherent RAW cells were infected with fluorescent E. faecalis at a multiplicity of infection (MOI) of 100:1 (bacterium/cell ratio). After 30 min of interaction, cells were washed twice with PBS, recovered with cell dissociation buffer (GIBCO, Invitrogen), washed again, and finally fixed in 3 % paraformaldehyde (PFA) solution. Fluorescence of RAW cells due to infecting bacteria was detected by a flow cytometer in the FL-1 channel. The phagocytosis index (PI) was calculated using the percent of fluorescent macrophages after E. faecalis wild-type (WT) strain infection and applying the following formula: PI = (percent of fluorescent macrophages after infection X 100 /percent of fluorescent macrophages after WT infection) [30, 31]. Results are expressed as the mean ± SEM from three independent experiments usually performed in duplicate or triplicate.
Bacterial adhesion assay
To separate adhesion from subsequent steps of phagocytosis, cells were pretreated 30 min with 1 μg/ml of cytochalasin D (CytD), an actin polymerization inhibitor, as described . A CytD (1000X) stock solution in DMSO was prepared according to manufacturer's recommendations and stored at −20 °C. DMEM supplemented medium (see above) was used to dilute stock solution. RAW cells were seeded at 1 × 106/well into 6-well tissue culture plates (TPP, Dominique Dutscher) and incubated O/N at 37 °C under 6 % CO2. Macrophages pre-treated with CytD were first washed twice with fresh medium and then infected at a MOI of 100:1, similar to phagocytosis analysis above; CytD-untreated and uninfected macrophages were used as negative controls. Fluorescence in RAW cells due to infecting bacteria was detected by flow cytometry. Adhesion Index (AI) = (percent of GFP+ macrophages pre-treated with CytD, after infection by the E. faecalis mutant strain X 100/percent of GFP+ macrophages pre-treated with CytD after WT infection).
Fluorescence and electron microscopy
Raw macrophages were seeded in 12-well cell culture plates on a glass slide and infected with GFP-labeled E. faecalis wild-type (WT) or P+-elrA-E strains at a MOI of 1:100, with uninfected macrophages serving as negative control. After 30 min of interaction, macrophages were washed twice with PBS, fixated and immunolabeled with Streptococcus group D antiserum (BD Diagnostics, Le Pont de Claix, France) as previously described . Fluorescence was examined using a Carl Zeiss microscope (Axiovert 200 M, in the ApoTome mode) at MIMA2 platform (INRA, Jouy en Josas). Images were processed with Axiovision version 4.6 (Carl Zeiss).
Imaging of bacterial-cells interaction was performed using a Hitachi S-4500 scanning electron microscope (SEM) at the MIMA2 imaging platform. Macrophages were seeded in 12-well cell culture plates and infected with either E. faecalis wild-type (WT) or P+-elrA-E strains at a MOI of 1:100, with uninfected macrophages serving as negative control. After 30 min of interaction, macrophages were washed twice with PBS, recovered with cell dissociation buffer (GIBCO, Invitrogen), washed again, and suspended in a fixative solution and treated as previously described .
Preparation of bacterial samples for transmission and scanning electron microscopy was performed as previously described [32, 33]. Thin-sections and negative-stains were observed with a Zeiss EM902 electron microscope operated at 80 kV (MIMA2 - UR 1196 Génomique et Physiologie de la Lactation, INRA, plateau de Microscopie Electronique, 78352 Jouy-en-Josas, France). Microphotographies were acquired using MegaView III CCD camera and analyzed with the ITEM software (Eloise SARL, Roissy CDG, France).
Microbial adhesion to solvents
Microbial adhesion to solvents (MATS) analysis was carried out as described previously by Bellon-Fontaine and collaborators . In brief, a single colony of each of the E. faecalis strains studied was subcultured four times in BHI and harvested at stationary phase. Bacterial cells were centrifuged at 5000 ×g for 8 min and washed twice in 0.15 M NaCl and re-suspended to a final OD400 ~0.8. Bacterial suspensions (2.4 ml) were vortexed for 1 min with 0.4 ml of highest purity grade chloroform (Sigma-Aldrich), hexadecane (Sigma-Aldrich), ethyl acetate (Merck), or decane (Merck). The emulsion was left to stand for 20 min to allow complete phase separation, and the OD400 of 1 ml from the aqueous phase was measured. Affinity of the cells for each solvent (% affinity) = ((ODf-ODi)/ ODi)x100 where OD i is the initial optical density of the bacterial suspension before mixing with the solvent, and OD f the final absorbance after mixing and phase separation. Analysis was performed twice in triplicate.
Mouse peritonitis model
The mouse experiments were approved by the Institutional Animal Use and Care Committee at the Università Cattolica del Sacro Cuore, Rome, Italy (permit number Z21, 1 November 2010), and authorized by the Italian Ministry of Health, according to the Legislative Decree 116/92, which implemented the European Directive 86/609/EEC on laboratory animal protection in Italy. Animal welfare was routinely checked by veterinarians of the Service for Animal Welfare.
Virulence of strains OG1RF, ΔelrA, and P+-elrA-E was tested as described previously . The inoculum size was confirmed by determining the number of CFU on brain heart infusion agar. Each inoculum was 10-fold diluted in 25 % sterile rat fecal extract prepared from a single batch as previously described . Groups of 10 ICR outbred mice (Harlan Italy Srl, San Pietro al Natisone, Italy) were challenged intraperitoneally with 1 ml of each bacterial inoculum, housed five per cage, and fed ad libitum. A control group of mice was injected with 25 % sterile rat fecal extract only. Survival was monitored every 3 to 6 h. In another set of experiments, groups of mice were killed 24 h postinfection, and livers and spleens were removed, weighed, homogenized, and serially diluted in saline solution for colony counts.
Statistics were performed using GraphPad Prism (Version 4.00 for Windows, GraphPad Software, San Diego California, USA). One-way analysis of variance (ANOVA) was followed by Dunnett's multiple-comparison test when comparing multiple groups for one factor. For animal experiments, survival estimates were constructed by the Kaplan-Meier method and compared by log rank analysis, and comparisons with P values of <0.05 were considered to be significant.
We thank A. Navickas and F. Wessner for technical support on MATS assays and RNA extractions, and P. Adenot and R. Fleurot of the Platform MIMA2 for access to the Apotome microscope. We also thank P. Lee, A. Gruss, D. Lereclus, C. Archambaud and S. Aymerich for critical revision of the manuscript. We are thankful to M. Mangan for careful reading of the manuscript for the English editing. This work was supported by the Institut National de la Recherche Agronomique. R.D. was supported by a fellowship from the Région Ile-de-France in the framework of the Dim MalinF.
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