Jordan Vacheron

Guilhem Desbrosses

Sébastien Renoud

Rosa-Maria Padilla-Aguilar

Vincent Walker

Daniel Muller

Claire Prigent-Combaret
email: claire.prigent-combaret@univ-lyon1.fr

Rosa Padilla

Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays

Keywords: co-occurring plant-beneficial functions, maize, Arabidopsis, functional interactions, fluorescent Pseudomonas

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Plant growth-promoting rhizobacteria (PGPR) are root-colonizing bacteria able to provide growth and health benefits to plants through various mechanisms involving the expression of plant-beneficial functions (PBFs) in PGPR [START_REF] Bruto | Analysis of genes contributing to plant-beneficial functions in plant growth-promoting rhizobacteria and related Proteobacteria[END_REF][START_REF] Lemanceau | Let the core microbiota be functional[END_REF][START_REF] Richardson | Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth promotion by microorganisms[END_REF][START_REF] Vacheron | Plant growth-promoting rhizobacteria and root system functioning[END_REF]. For example, PGPR can modulate plant hormonal signalization by producing phytohormones such as auxins, cytokinins, gibberellins, nitric oxide (NO) [START_REF] Bashan | Chapter two -How the plant growth-promoting bacterium Azospirillum promotes plant growth-A critical assessment[END_REF], or decreasing the emission of ethylene [START_REF] Glick | A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria[END_REF][START_REF] Honma | Metabolism of 1-aminocyclopropane-1-carboxylic acid[END_REF]Vacheron et al. 2016a). PGPR may also help plant nutrition by solubilizing inorganic compounds such as phosphate, or other mineral elements [START_REF] Adesemoye | Plant-microbes interactions in enhanced fertilizer-use efficiency[END_REF][START_REF] Vessey | Plant growth promoting rhizobacteria as biofertilizers[END_REF]. Finally, some PGPR, in particular fluorescent pseudomonads, are producing antimicrobial compounds, such as 2,4diacetylphloroglucinol (DAPG), that protects plants against plant pathogen microorganisms [START_REF] Almario | Rhizosphere ecology and phytoprotection in soils naturally suppressive to Thielaviopsis black root rot of tobacco[END_REF][START_REF] Couillerot | Pseudomonas fluorescens and closely-related fluorescent pseudomonads as biocontrol agents of soil-borne phytopathogens[END_REF][START_REF] Lanteigne | Production of DAPG and HCN by Pseudomonas sp. LBUM300 contributes to the biological control of bacterial canker of tomato[END_REF]) and activates plant defenses [START_REF] Iavicoli | Induced systemic resistance in Arabidopsis thaliana in response to root inoculation with Pseudomonas fluorescens CHA0[END_REF]. Some of these antimicrobial compounds can also act as communication signal between bacteria [START_REF] Combes-Meynet | The Pseudomonas secondary metabolite 2,4-diacetylphloroglucinol is a signal inducing rhizoplane expression of Azospirillum genes involved in plant-growth promotion[END_REF].

The availability of an ever-increasing number of sequenced and annotated PGPR's genomes is allowing us to perform some comparative genomic analyses [START_REF] Flury | Insect pathogenicity in plant-beneficial pseudomonads: phylogenetic distribution and comparative genomics[END_REF][START_REF] Loper | Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions[END_REF]. The results of these analyses show the existence of combinations of PBF genes that are specific to some PGPR taxa [START_REF] Bruto | Analysis of genes contributing to plant-beneficial functions in plant growth-promoting rhizobacteria and related Proteobacteria[END_REF]. For example, in strains or species belonging to the Pseudomonas fluorescens group, the genes required for the synthesis of DAPG are present simultaneously with the ones required for cyanid hydrogen (HCN) synthesis [START_REF] Ramette | Prevalence of fluorescent pseudomonads producing antifungal phloroglucinols and/or hydrogen cyanide in soils naturally suppressive or conducive to tobacco black root rot[END_REF]. In Pseudomonas protegens, the production of pyoluteorin is co-occuring with the production of DAPG [START_REF] Ramette | Pseudomonas protegens sp. nov., widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin[END_REF]. The presence of cooccurring PBF genes in genomes suggests that these functions might be part of common bacterial regulatory or metabolic networks. Alternatively, [START_REF] Bashan | Chapter two -How the plant growth-promoting bacterium Azospirillum promotes plant growth-A critical assessment[END_REF] hypothesized that each PBF present in one PGPR's genome would improve plant growth either simultaneously or successively. In this additive hypothesis, the overall improvement of plant growth would result from the sum of effects of each co-occurring PBF. Although the relative contribution of several antimicrobial compounds to biocontrol performance has been well studied [START_REF] Calderón | The dar genes of Pseudomonas chlororaphis PCL1606 are crucial for biocontrol activity via production of the antifungal compound 2-hexyl, 5-propyl resorcinol[END_REF][START_REF] Iavicoli | Induced systemic resistance in Arabidopsis thaliana in response to root inoculation with Pseudomonas fluorescens CHA0[END_REF][START_REF] Romanowski | Circadian variation in Pseudomonas fluorescens (CHA0)-mediated paralysis of Caenorhabditis elegans[END_REF][START_REF] Voisard | Cyanide production by Pseudomonas fluorescens helps suppress black root rot of tobacco under gnotobiotic conditions[END_REF], the contribution of co-occurring PBFs in plant growth promotion is poorly documented. 4 Among fluorescent pseudomonads, the Pseudomonas kilonensis F113 strain (formerly Pseudomonas fluorescens; [START_REF] Almario | Distribution of 2, 4-Diacetylphloroglucinol biosynthetic genes among the Pseudomonas spp. reveals unexpected polyphyletism[END_REF] is a PGPR strain isolated from sugar beet rhizosphere [START_REF] Shanahan | Isolation of 2,4diacetylphloroglucinol from a fluorescent Pseudomonad and investigation of physiological parameters influencing its production[END_REF]). That strain is well known for its biocontrol activity against a broad range of plant pathogen microorganisms (Cronin et al. 1997a;Cronin et al. 1997b;[START_REF] Dunne | Combining proteolytic and phloroglucinol-producing bacteria for improved biocontrol of Pythium-mediated damping-off of sugar beet[END_REF]. Less known, but all the more important, F113 also promotes plant growth and development, particularly on maize (Vacheron et al. 2016a;[START_REF] Walker | Variation of secondary metabolite levels in maize seedling roots induced by inoculation with Azospirillum, Pseudomonas and Glomus consortium under field conditions[END_REF], probably because of its rhizosphere's competence or in other words, its ability to efficiently colonize plant roots and to compete with other rhizosphere competent bacteria [START_REF] Lugtenberg | What makes Pseudomonas bacteria rhizosphere competent?[END_REF]. The analysis of F113's genome sequence reveals the presence of multiple PBFs [START_REF] Redondo-Nieto | Genome sequence reveals that Pseudomonas fluorescens F113 possesses a large and diverse array of systems for rhizosphere function and host interaction[END_REF]. We can identify the phl genes, which enable the production of DAPG (phlD encodes a polyketide synthase that is a key determinant of the DAPG biosynthesis), and also gcd encoding a glucose dehydrogenase involved in phosphate solubilization [START_REF] Miller | Biochemical and genomic comparison of inorganic phosphate solubilization in Pseudomonas species[END_REF], acdS encoding the 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme [START_REF] Prigent-Combaret | Physical organization and phylogenetic analysis of acdR as leucine-responsive regulator of the 1-aminocyclopropane-1-carboxylate deaminase gene acdS in phytobeneficial Azospirillum lipoferum 4B and other Proteobacteria[END_REF]Vacheron et al. 2016a) and nirS encoding the nitrite reductase involved in NO production [START_REF] Choi | Assessing the impact of denitrifier-produced nitric oxide on other bacteria[END_REF]. Specific associations of co-occurring PBFs were frequently observed among Proteobacteria [START_REF] Bruto | Analysis of genes contributing to plant-beneficial functions in plant growth-promoting rhizobacteria and related Proteobacteria[END_REF], and may reflect the existence of functional interactions occurring between PBFs. In F113, the interaction between the 4 PBFs (i.e. DAPG production, ACC deamination, phosphate solubilization, and NO production) may happen at a regulatory or metabolic level and could result, when combined altogether, in enhanced plant growth. Following the additive hypothesis, we predict that the 4 co-occurring PBFs exert additive beneficial effects on the development of plant root system architecture (RSA) and on the overall growth of plants by P. kilonensis F113. To test this hypothesis, we used a genetic approach and constructed 5 mutant strains: 4 deleted for each PBFs, and on with deletion of the 4 PBFs. These 5 mutants were used to (i) analyze the functional relationships between F113 plant-beneficial activities and (ii) to determine if they are all equally contributing to the plant beneficial effects of F113 on Arabidopsis thaliana in in vitro conditions, and on maize (Zea mays L.) in soil pot experiment.

RESULTS

In vitro, most P. kilonensis F113's PBFs operate independently of each other

The existence of functional interactions between the 4 co-occurring PBFs of P. kilonensis F113 was first tested in vitro and in absence of plants. In the wild-type F113 strain (WT) and

Comment citer ce document : Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D., prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI :

5 in each various mutant strains (i.e. ∆acdS, ∆phlD, ∆nirS, ∆gcd and the quadruple mutant strain), we measured the activity of ACC deamination, phosphate solubilization, nitrite reduction and DAPG synthesis. As expected, the deamination of ACC into α-ketobutyrate was impaired in the ∆acdS and quadruple mutants (Fig. 1A). AcdS activity was weakly induced in ∆gcd and ∆nirS compared with F113 (1.7-fold and 1.3-fold respectively) and not significantly affected in the ∆phlD mutant (Fig. 1A). Moreover, the insoluble phosphate was no longer solubilized in the ∆gcd and quadruple mutants (Fig. 1B). Furthermore, the glucose dehydrogenase Gcd activity was weakly but significantly decreased (1.7-fold) in the ∆phlD mutant but remained identical to the one of F113 in ∆nirS and ∆acdS (Fig. 1B). The ∆nirS and the quadruple mutants were unable to reduce nitrite (and consequently to produce NO) (Fig. 1C). The NirS activity in the ∆acdS or ∆gcd or ∆phlD strain was identical to the one of the F113 (Fig. 1C). As expected, there was no longer DAPG synthesis in the ∆phlD and quadruple mutants (Fig. 1D). Interestingly, there was a strong accumulation of DAPG in the ∆gcd (82 times more than F113). By contrast the ∆acdS and ∆nirS produced as much DAPG as F113 (Fig. 1D). Thus, the main functional interaction that occurs in vitro between all the four different PBFs is between the cellular pathways containing the glucose dehydrogenase Gcd and the polyketide synthase PhlD that both are involved in the control of DAPG biosynthesis in P. kilonensis F113.

To further study the interaction between DAPG synthesis and the glucose dehydrogenase, the DAPG production and phosphate solubilization were compared between all double and triple deletion mutants (Fig. S1). On one hand, no phosphate solubilization was observed in all gcd double and triple mutants. It is also significantly reduced in the ∆phlD∆acdS (1.5-fold compared with F113), ∆phlD∆nirS (1.3-fold compared with F113), and ∆phlD∆acdS∆nirS (1.7-fold compared with F113) mutants but in similar way than in the single ∆phlD mutant (1.7-fold compared with F113). Thus, apart from the small positive impact of DAPG production on the solubilization of phosphate, there is no other function that interacts with the latter (Fig. S1). On the other hand, DAPG production is impaired in all phlD double and triple mutants but is unaffected in a ∆acdS∆nirS mutant (Fig. S1). Compared to the increase observed in a single gcd mutant (82 times higher than F113), DAPG production is much more increased in a ∆acdS∆gcd mutant (226 times higher than F113) but reduced in the ∆nirS∆gcd (30 times higher than F113) and ∆acdS∆gcd∆nirS mutants (26 times higher than F113)(Fig. S1). These results show that the cellular network involving the glucose dehydrogenase Gcd and the polyketide synthase PhlD is influenced negatively by acdS and positively by nirS (Fig. S1).

Deletion of PBF genes does not affect the fitness of P. kilonensis F113 in vitro but impair its rhizosphere soil competitiveness When a PGPR does not enhance plant growth, it can be due to either from the absence of a particular PBF in that strain, the poor expression of the PBF gene in the rhizosphere or to a reduced rhizosphere competence. To assess if any of the tested PBF can affect rhizosphere competence, we measured the growth of the WT strain and of every single and quadruple mutant strains in vitro and in vivo. In liquid culture, bacterial growth can be limited by the time required to divide (characterized by the growth rate) and the size of the cells population in the stationary phase (characterized by the final OD reached in the liquid culture).

The growth rate of the quadruple mutant is higher than the one of the WT or any given single mutant strain (Fig. 2, Table S1). The growth rates of all simple mutants are similar to the one of the WT (Fig. 2, Table S1). In terms of final OD reached in stationary phase, the quadruple and the ∆phlD mutants reached the highest optical density in the stationary growth phase compared to any other single mutants and to the WT strain (Fig. 2, Table S1). Since neither the ∆phlD nor the quadruple mutant do synthesize DAPG in vitro (Fig. 1D), these results suggest that the production of DAPG might become toxic to F113 cells at the stationary phase, in the tested conditions. To test that hypothesis, the growth of F113, ∆phlD and its complemented mutant was evaluated in presence or absence of DAPG (Fig. S2). First, the complemented ∆phlD mutant had a growth kinetic curve similar to that of F113 (Fig.

S2A

). Second, the addition of DAPG reduced significantly the optical density in the stationary growth stage of F113 (starting from 0.1 µg.ml -1 of DAPG) (Fig. S2B). Third, the growth of ∆phlD was slowed down in the exponential phase and reduced in the stationary growth by the addition of DAPG at 0.5 µg.ml -1 and 1 µg.ml -1 (Fig. S2C). Fourth, the addition of DAPG (at 0.5 µg.ml -1 and 1 µg.ml -1 ) slowed down the exponential growth stage of the complemented ∆phlD mutant, but the optical density reached in the stationary growth stage was quite similar to that of the complemented ∆phlD mutant and of F113 (Fig. S2D). Thus, when DAPG concentration reaches 0.5 µg.ml -1 , it negatively affects the growth of F113. Although the gcd mutant produced much more DAPG than the wild-type strain (and higher than 0.5 µg.ml -1 ), its growth is nevertheless not affected. One can suggest that non-target effects of the gcd mutation might result in protection of the bacterium against high DAPG concentration. In any 7 case, none of the simple and quadruple mutants have major deleterious effects on the F113

cell growth allowing us to analyze their role during the interaction of F113 with plant.

To monitor the contribution of PBF genes in the fitness of F113 in maize rhizosphere, F113 and mutants were inoculated on maize seeds grown in natural soil, in presence of the indigenous microbial communities. Same inoculation level of 5.10 6 bacteria per seed was used for each strain. F113 cell population was stable up to 8 dpi (days post-inoculation), but decreased at 20 dpi (Fig. 3). At 8 dpi, all mutant strains were found less abundant than F113 in the rhizosphere, the ∆phlD and quadruple mutants being the less numerous (Fig. 3). At 20 dpi, there was no significant difference in the rhizosphere colonization between F113 and the ∆phlD and ∆gcd mutants, whereas ∆acdS, ∆nirS and the quadruple mutants were less abundant than F113 (Fig. 3). Thus, all PBFs contribute to F113 rhizosphere competence but to various extends. In particular, phlD is relevant for root colonization up to 8 days and acdS and nirS genes are important for ensuring effective maize colonization over 20 days.

The acdS, nirS and gcd genes affect maize growth F113 and PBF mutants were inoculated on maize seeds grown in pot filled with soil. Twenty days after, shoot and root architecture traits were measured and the profiles obtained for each strain were compared using hierarchical clustering analyses (HCA). Compared to the noninoculated condition, F113 significantly enhanced root growth (with an increase of total root length of +50%, and of total root surface of +46%) and shoot growth (with an enhancement of the total leaf surface of +44%) (Tables S2,S3 and Fig. S3).

When comparing the inoculation effects of mutants to that of F113 on maize by HCA, two clusters were formed, grouping F113, ∆phlD and ∆gcd on one hand and ∆acdS, ∆nirS and the quadruple mutant in the other hand (Fig. 4). In the first cluster, all the plant parameters appear evenly affected during inoculation, giving rise to a shape like a circle similar to the one obtained with F113. It suggests that there are either no difference or minor differences between F113 and the ∆phlD and ∆gcd mutants. Consistent with that, there is no significant differences between F113 and ∆phlD for all plant parameters assessed (Table S2 and S3), while the inoculation with ∆gcd reduced significantly maize root length and surface with respectively 26% and 20% of decrease compared to F113 (Table S2).

In the second cluster, the plant parameters appear to be unevenly affected, giving rise to an unsymmetrical shape. The three strains belonging to that cluster (i.e. ∆acdS, ∆nirS and the quadruple mutant have a similar impact on maize development and growth (

). Contrasting with the strains of the first cluster, the inoculation of ∆acdS, ∆nirS and the quadruple mutant impacted significantly the root system architecture compared to F113 by reducing significantly the root length (-64%, -59% and -71%, respectively), the root surface (-50%, -50% and -57%, respectively), the number of root tips (respectively -62%; -45% and -71%) and the root fresh weight (respectively -31%, -38% and -42%) (Table S2). Thus, the RSA modifications triggered by F113 on maize were lost when ∆acdS, ∆nirS or the quadruple mutants were inoculated (Tables S2,S3 and Fig. S3).

In conclusion, F113's PBF genes and in particular acdS and nirS appear to alter mostly maize root parameters but not shoot parameters.

F113 PBFs differently affect A. thaliana root hair elongation and primary root growth in gnotobiotic agar conditions

To further investigate what is F113's PBF impact on root, we turned to in vitro experiments, as they allow a careful monitoring of F113's effects on root development or root hairs for example. Because maize is a large plant, with a root system that is likely to be too large to be evaluated in vitro, we turned instead to Arabidopsis, as its root development shares some similarities with the one of maize [START_REF] Atkinson | Branching out in roots: uncovering form, function, and regulation[END_REF][START_REF] Hochholdinger | Conserved and diverse mechanisms in root development[END_REF].

We first verified the effect of F113 inoculation and that of the various mutant strains on the Arabidopsis primary root growth. F113 inoculation inhibited significantly (2-fold) the primary root length of A. thaliana (Fig. 5A). When A. thaliana Col-0 was inoculated with ∆phlD, a smaller reduction of the primary root growth (1.5-fold) compared to F113 was observed. However, the difference with F113 was not significant, implying that DAPG production only weakly contributed to the primary root length inhibition triggered by the WT strain (Fig. 5A). Contrarily, primary root length inhibition was lost when ∆acdS, ∆gcd, ∆nirS or the quadruple mutants were inoculated (Fig. 5A). Therefore, acdS, gcd and nirS independently contribute to Arabidopsis primary root growth inhibition driven by F113 (Fig. 5A). Secondly, we investigated the effect of F113 and of the various mutant strains on the plant root hair length. F113 inoculation led to significantly longer root hairs (Fig. 5B).

Inoculation with the quadruple mutant generated the same plant phenotype than the noninoculated condition, suggesting that at least one of the deleted PBFs was responsible of enhanced root hair length (Fig. 5B). However, compared to F113 WT inoculated plants, the average root hair lengths were significantly reduced by 20% in ∆phlD, 14% in ∆acdS, 21% in ∆gcd inoculated plants, and up 41% in ∆nirS inoculated plants (Fig. 5B). Therefore, none of

Comment citer ce document : Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D., prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI :

9

F113's PBF functions is solely controlling root hair elongation. Because the activation of the plant ethylene signaling pathway results in root growth inhibition and root hair elongation [START_REF] Pitts | Auxin and ethylene promote root hair elongation in Arabidopsis[END_REF][START_REF] Swarup | Ethylene upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation[END_REF], and is also an indicator of the activation of a defense related mechanism [START_REF] Broekgaarden | Ethylene: traffic controller on hormonal crossroads to defense[END_REF], we looked at the effects of F113 and of the various mutants strains on the plant ethylene emission by photoacoustic infra-red spectrophotometry [START_REF] Cristescu | Current methods for detecting ethylene in plants[END_REF]. Plant ethylene emissions increased significantly when F113 (1.5 fold), and ∆acdS (2 fold) were inoculated comparatively to the noninoculated plant. The plant ethylene emission produced by ∆phlD, ∆gcD, ∆nirS and the quadruple mutant did not significantly differ from that of the non-inoculated condition (Fig.

6).

In conclusion, all F113's PBFs are contributing to the production of ethylene by

Arabidopsis, but in a distinct manner: acdS is reducing Arabidospis ethylene content whereas phlD, gcd and nirS are increasing it.

DISCUSSION

In this work, we evidence that interactions between PBFs might be different when bacteria are growing in vitro or in contact with a plant, where functional overlapping between PBFs may occur by affecting the same plant pathway. It highlights that inactivation of one PBF gene can affect the activity of other ones and that all PBFs harboured by PGPR have to be taken into account when analysing the plant response effects of PGPR inoculation.

Functional interactions between co-occurring PBFs in P. kilonensis F113

In vitro, the main functional interaction occurring in F113 cells between the four studied PBFs is between phosphate solubilization and biosynthesis of DAPG. We evidenced that the glucose dehydrogenase activity (Gcd) is repressing DAPG biosynthesis in F113 (Fig. 1D), confirming observations formerly made in P. protegens CHA0 (de Werra et al., 2009). In cells containing a functional Gcd, glucose-6P is transformed into gluconic acid, which is exported out of the cells, leading to medium acidification and phosphate solubilization (Fig.

S4A

) [START_REF] Stella | Gluconic acid production by bacteria to liberate phosphorus from insoluble phosphate complexes[END_REF]. When gcd is deleted, a higher quantity of glucose-6P could be provided to the glycolysis, resulting on the one hand in increased ATP production by the cell, and on the second hand in a higher amount of pyruvate that could then be incorporated to the tri-carboxylic acid cycle to produce higher amount of acetyl CoA, the precursor of DAPG [START_REF] Bangera | Identification and characterization of a gene cluster for synthesis of the polyketide antibiotic 2,4-diacetylphloroglucinol from Pseudomonas fluorescens Q2-87[END_REF][START_REF] Clifford | Phloroglucinol functions as an intracellular and intercellular chemical messenger influencing gene expression in Pseudomonas protegens[END_REF]) (Fig. S4B). Interestingly, a metabolic DAPG was shown to act as an intracellular messenger controlling transcription of different PBFs genes such as pyoluteorin production in P. protegens CHA0 and Pf-5 [START_REF] Clifford | Phloroglucinol functions as an intracellular and intercellular chemical messenger influencing gene expression in Pseudomonas protegens[END_REF][START_REF] De Werra | Plant-and microbederived compounds affect the expression of genes encoding antifungal compounds in a Pseudomonad with biocontrol activity[END_REF][START_REF] Kidarsa | Phloroglucinol mediates cross-talk between the pyoluteorin and 2,4-diacetylphloroglucinol biosynthetic pathways in Pseudomonas fluorescens Pf-5[END_REF][START_REF] Schnider-Keel | Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin[END_REF] or as an intercellular messenger inducing PBFS in other PGPR as auxin production [START_REF] Combes-Meynet | The Pseudomonas secondary metabolite 2,4-diacetylphloroglucinol is a signal inducing rhizoplane expression of Azospirillum genes involved in plant-growth promotion[END_REF]. Therefore, we hypothesize that the ∆gcd mutant strain could also show some specific modification at the transcriptional level of the expression of other PBFs encoding genes, resulting in potential functional compensation.

Distinct roles for F113 PBFs during colonization

The functional importance of F113's PBF functions and its competitiveness against the native soil populations of rhizobacteria was tested on maize plants growing in pot filled with nonsterile soil, a setup close to natural conditions. For this experiment, we selected maize as a model because it is frequently grown in fields where the soil we used has been collected

suggesting that this soil is enriched in a maize specific rhizosphere microbiota that is ideal to compare the rhizosphere competitiveness of a mutant vs a wild-type strain. In addition, maize plantlets have a large root system allowing an easy quantification of the colonization process.

Root secretion of organic substances is essential for the rhizosphere microbiota to thrive [START_REF] Haichar | Stable isotope probing of carbon flow in the plant holobiont[END_REF]. Since the root exudates are composed of a lot of organic molecules, which origin can be traced back to the photosynthetic activity of the plant, it is important that the two time points selected to sample the maize root rhizosphere will not differ in the physiological state of the plant. We sampled maize roots 8 days and 20 days after sowing. In both case, the maize seedlings are already performing photosynthesis and may release root exudate whose composition is not drastically dissimilar. Consistent with this hypothesis, the composition in secondary metabolites present in root exudates from 5 distinct maize inbred lines is less different between 8 and 10 days than it is between 3 and 5 days post-germination (Walker and Prigent-Combaret, unpublished results).

At 8 dpi, there are less ∆phlD mutant bacteria in the maize root rhizosphere than F113 while at 20 dpi, the ∆phlD mutant strain is equally abundant as the WT (Fig. 3). This suggests that DAPG is important at the beginning of the colonization process. DAPG is a well-known antimicrobial compounds secreted by F113 (Cronin et al. 1997). Its secretion could limit the 11 presence of competitor rhizobacteria or fungi in vivo, hence favoring an exclusive interaction between F113 and the host roots. Curiously, the ∆gcd mutant strain is equally abundant as the WT strain at both time points (Fig. 3). Yet, the ∆gcd mutant strain produces 3 times more DAPG in vitro than the WT strain (Fig. 1). Enhanced DAPG production may trigger some compensation mechanisms in the ∆gcd mutant strain, allowing it to colonize maize roots as efficiently as the wild-type strain.

At 20 dpi the colonization of the ∆nirS, ∆acdS and the quadruple mutant is less efficient than that of F113, ∆gcd, and ∆phlD (Fig. 3). AcdS + strains are catabolizing ACC exuded by plant roots in α-ketobutyric acid (carbon source) and NH 4 + (nitrogen source) [START_REF] Glick | A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria[END_REF]. acdS deletion can reduce the ability of F113 to fulfill its nutrient requirements in the rhizosphere, and thus its fitness and root colonization ability. NirS is allowing bacteria of the Pseudomonas genus, a strictly respiring genus, to respire NO 3 -and NO 2 -in the microaerophilic or anaerobic conditions that are frequently encountered in the rhizosphere [START_REF] Højberg | Oxygen-sensing reporter strain of Pseudomonas fluorescens for monitoring the distribution of low-oxygen habitats in soil[END_REF]). Thus, the loss of nitrogen respiring capacity may explain the lower root colonization ability of the ∆nirS mutant that has previously been observed [START_REF] Ghiglione | Relative involvement of nitrate and nitrite reduction in the competitiveness of Pseudomonas fluorescens in the rhizosphere of maize under non-limiting nitrate conditions[END_REF] as well as in our condition (Fig. 3). Since both NirS and AcdS are important for the colonization at 20 dpi, one could have expected that in the quadruple mutant strain, the effects of the two mutations would add up resulting in a strain with very poor ability to colonize at 20 dpi. Surprisingly, this is not the case (Fig. 3). This suggests the existence of potential epistatic interactions between the two genes nirS and acdS or with gcd and/or phlD that our in vitro analysis failed to unravel (Fig. 1). It also might be that plant exudates or microbe-microbe interactions in the rhizosphere result in activation of other genes that compensate for the lack of nirS and acdS.

The maize colonization process by F113 has a significant impact on the plant root and shoot growth.

To test if these different colonization patterns could affect maize growth in vivo, we measured 15 different physiological parameters (8 for shoots and 7 for roots; Table S2 andS3). To get a broad view of the effect of F113 on maize plants, we have used a hierarchical clustering analysis (HCA). Instead of focusing on each parameter, HCA takes into account parameter patterns that can be compared between the mutant and the wild-type strains (Fig. 4). The inoculation of maize with the ∆phlD or ∆gcd strains resulted in a pattern similar to the one of F113 (Fig. 4). Since phlD appears to play a role for maize root colonization at 8 dpi (Fig. 3), it suggests that F113 colonization of maize roots after 8 dpi is more important for the effects 12 on maize growth than up to 8 dpi. Consistent with that interpretation, the inoculation of maize roots with the ∆nirS or ∆acdS or the quadruple mutant strain (all these genes are important for the colonization at 20 dpi (Fig. 3)), is resulting in a pattern different from the one of the WT (Fig. 4).

Contrasting with F113, inoculation with ∆acdS or ∆nirS or the quadruple mutant resulted in a decrease in total root length, total root surface and number of root tips while at the same time, an increase in root diameter (Fig. 4, Table S2). This indicates that acdS and nirS, two genes that are important for the fitness of F113, are also important for the root growth promotion. How they could be beneficial to root maize growth remains unclear. For example, it could be a direct interference with the plant ethylene pathway for AcdS or with the plant NO pathways for NirS. At the level of the shoot, maize plants inoculated with ∆acdS or ∆nirS or the quadruple mutant show a weak decrease in leaf surfaces, as well as in chlorophyll content compared to F113 (Fig. 4; Table S3). While the leaf surface of inoculated maize plants by ∆acdS, ∆nirS or the quadruple mutant are slightly higher than in the noninoculated plants, the chlorophyll content is lower. As chlorophyll fluorescence is reduced in stressed plants [START_REF] Bolhàr-Nordenkampf | Chlorophyll fluorescence as a tool in photosynthesis research[END_REF], this result could suggest that maize plants inoculated with ∆acdS or ∆nirS or the quadruple mutant are stressed, whereas F113 may alleviate plant stress. Taken together, the PBF functions that contribute to the long-term fitness of F113 are also the ones that have the strongest effects on the growth of maize plants.

Relative contribution of PBFs on A. thaliana root architecture

To further study the contribution of the F113 various PBF's on the root development, we performed in vitro experiments with plants. We have used Arabidopsis as a model plant because it is small, it grows fast, it has a relatively simple root development program (tap root), and its root hairs are very easily observable. In addition, maize and Arabidopsis root development programs share some similarities [START_REF] Atkinson | Branching out in roots: uncovering form, function, and regulation[END_REF][START_REF] Hochholdinger | Conserved and diverse mechanisms in root development[END_REF]. Finally, F113, which has been initially isolated from sugar beet roots is known to colonize efficiently both monocotyledons such as wheat [START_REF] Couillerot | The role of the antimicrobial compound 2,4-diacetylphloroglucinol in the impact of biocontrol Pseudomonas fluorescens F113 on Azospirillum brasilense phytostimulators[END_REF][START_REF] De La Fuente | Host crop affects rhizosphere colonization and competitiveness of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens[END_REF]) and maize over more than 5 weeks [START_REF] Couillerot | Comparison of prominent Azospirillum strains in Azospirillum-Pseudomonas-Glomus consortia for promotion of maize growth[END_REF][START_REF] Von Felten | Quantification of Pseudomonas fluorescens strains F113, CHA0 and Pf153 in the rhizosphere of maize by strain-specific real-time PCR unaffected by the variability of DNA extraction efficiency[END_REF]) but also dicotyledonous crops such as alfalfa [START_REF] Sanchez-Contreras | Phenotypic selection and phase variation Occur during Alfalfa root colonization by Pseudomonas fluorescens F113[END_REF][START_REF] Villacieros | Colonization behaviour of Pseudomonas fluorescens and Sinorhizobium meliloti in the alfalfa (Medicago sativa) rhizosphere[END_REF] or tomato [START_REF] De Weert | Flagella-driven chemotaxis towards exudate components is an important trait for tomato root colonization by Pseudomonas fluorescens[END_REF].

In vitro F113 inoculation resulted in Arabidopsis primary root growth inhibition and strong promotion of root hair elongation (Fig. 5A andB). By contrast, the quadruple mutant showed no root architecture difference compared to the non-inoculated Arabidopsis, 13

Comment
suggesting that at least one F113 PBFs or a combination of PBFs may trigger root architecture modifications (Fig. 5, Fig. S3). Several plant hormones such as auxin (controlling cell division and elongation) [START_REF] Spaepen | Auxin and plant-microbe interactions[END_REF] or ethylene (controlling root cell elongation) [START_REF] Jung | Getting to the roots of it: genetic and hormonal control of root architecture[END_REF][START_REF] Street | Ethylene inhibits cell proliferation of the Arabidopsis root meristem[END_REF][START_REF] Swarup | Ethylene upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation[END_REF];) control primary root elongation. However, F113 does not produce auxinic compounds (Vacheron, Muller and Prigent-Combaret unpublished results) but instead is producing DAPG (Fig. 1).

One possibility is that DAPG could have an effect on the plant root system. Consistent with this, when tomato seedlings are growing in vitro and in presence of 10µM DAPG, their primary root growth is inhibited [START_REF] Brazelton | 2,4diacetylphloroglucinol alters plant root development[END_REF]). However, the ∆phlD mutant (none producing DAPG) remains able to inhibit Arabidopsis primary root elongation and the ∆gcd mutant (over-producing DAPG) does not show further reduced primary root length compared to F113 (Fig. 5). In addition, both the ∆phlD and ∆gcd mutants remain able to stimulate root hair length. These results imply that, aside from DAPG, other F113 PBFs are involved in the inhibition of the primary root elongation and stimulation of root hair length in A. thaliana.

Interestingly, the loss-of-function mutation in nirS affects the inhibition of primary root growth (Fig. 5A), and the root hair elongation (Fig. 5B) significantly. This could suggest that NO, produced by F113's NirS activity as evidenced by the use of the specific fluorescent probe diaminorhodamine-4M acetoxymethyl ester (Vacheron, Muller and Prigent-Combaret unpublished results), would act as an inhibitor of primary root growth and activator of root hair elongation. Consistent with this, NO is described as a positive regulator of root hair development [START_REF] Lombardo | Nitric oxide functions as a positive regulator of root hair development[END_REF][START_REF] Lombardo | Nitric oxide is essential for vesicle formation and trafficking in Arabidopsis root hair growth[END_REF]) and a negative regulator of primary root growth [START_REF] Fernández-Marcos | Nitric oxide causes root apical meristem defects and growth inhibition while reducing PIN-FORMED 1 Version postprint Comment citer ce document[END_REF].

In absence of PGPR, plant ethylene is known to inhibit primary root length and to activate root hairs elongation [START_REF] Cao | Differential ethylene sensitivity of epidermal cells is involved in the establishment of cell pattern in the Arabidopsis root[END_REF][START_REF] Pitts | Auxin and ethylene promote root hair elongation in Arabidopsis[END_REF]. F113 moderately increased plant ethylene emission, whereas all mutants but ∆acdS, shared similar plant-ethylene emission than non-inoculated condition (Fig. 6). Individually, each of the F113 PBFs contributes positively to ethylene production by the plant during the associative symbiosis with A. thaliana (Fig. 6). Contrasting with the other mutants, the ∆acdS strain colonization results in higher ethylene emission by the plant (Fig. 6). Hence, the AcdS activity negatively contributes to ethylene production. In our study, this mutant strain is the only one simple mutant unable to degrade in vitro the ethylene precursor ACC (Fig. 1A). Assuming that the mutant strain behaves similarly in vitro and in vivo, the lack of AcdS activity result in higher ethylene emission by the plant (Fig. 6). However, this increase is not observed with the production. Thus, it confirms that F113 colonization of the root system results in modulation of ethylene emission by the plant and that AcdS is not the only one F113's PBF contributing to this effect. However, it remains to be seen if this emission is greater in the root, in the shoot or both plant organs. It also confirms that AcdS activity is actively decreasing plant ethylene emission during the associative symbiosis. This result is consistent with a long-standing hypothesis on the function of AcdS during its interaction with plants ( [START_REF] Glick | A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria[END_REF]). Yet, this increased emission of ethylene in presence of the ∆acdS mutant strain does not result in a shorter primary and longer root hairs compared to the effect of the WT strain (Fig. 5A, andB). This absence of stronger root responses contrasts with previous observations suggesting that an absence of AcdS activity resulted in longer root hairs [START_REF] Galland | The ethylene pathway contributes to root hair elongation induced by the beneficial bacteria Phyllobacterium brassicacearum STM196[END_REF]. But

Phyllobacterium brassicacearum STM196, Rhizobium leguminosarum 128C53K and Pseudomonas sp. UW4, the strains used in that previous work, may not have the same combination of PBFs as the one of F113. Therefore, one should not rule out the possibility that this absence of stronger root responses with the ∆acdS strain is specific to F113 and its combination of PBFs. Another possibility that could explain the absence of stronger root responses, despite a significant increase in ethylene emission, is that F113 ∆acdS inoculated plants are becoming insensitive to the effect of high ethylene on root cells.

Taken together, F113 may trigger both ethylene-dependent and NO-dependent signalling pathways on Arabidospsis. Contrasting with the observations made on soil-pot grown maize, all F113's PBFs tested in vitro on Arabidopsis contribute to modification of the root architecture.

EXPERIMENTAL PROCEDURES Bacterial strains, plasmids and culture conditions

The bacterial strains and plasmids used in this study are listed in (Table S4). P. kilonensis F113 wild-type and its mutants were incubated at 28°C in King's B broth media [START_REF] Simon | The use of ampicillin in a simplified selective medium for the isolation of fluorescent pseudomonads[END_REF] supplemented when required with gentamycin (15 µg.ml -1 ) or kanamycin (Km; 50 µg.ml -1 ). Escherichia coli was incubated at 37°C in Luria Bertani broth [START_REF] Bertani | Studies on lyzogeny I[END_REF] supplemented with gentamycin (15 µg.ml -1 ) or ampicillin (75 µg.ml -1 ) and kanamycin (Km; 50 µg.ml -1 ) when necessary. In order to assess the effects of DAPG on the growth of F113

Comment citer ce document : Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D., prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI :

15 and its mutants, DAPG (Santa Cruz Biotechnology, USA) was added in King's B broth medium at 3 different concentrations (0.1 mg.ml -1 , 0.5 mg.ml -1 , and 1 mg.ml -1 ).

Growth kinetic assays

Bacterial growth was monitored during 24 h at 28°C by measuring optical density (OD) in a Bioscreen C analyzer (Labsystems, Finland) with one OD 600nm measured every 15 min.

Growth kinetic assays were performed three times independently with 5 technical repetitions.

Construction of deletion mutants and complementation of the phlD deletion mutant

To construct F113 PBF deletion mutants, about 700-pb fragments were amplified from regions upstream and downstream the PBF genes of interest using specific primers (Table S5)

and subcloned into pGEM ® -T easy (Promega, Madison, USA). The upstream and downstream DNA fragments were digested with AatII/MunI and SacII/SacI for ∆phlD and ∆gcd, AatII/PvuII and SacII/SacI for ∆acdS, PvuII/MunI and SacII/SacI for ∆nirS and then cloned one after another into pCM184, on both sides of the kanamycin resistance encoding gene [START_REF] Marx | Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria[END_REF]. All restriction enzymes were supplied by ThermoFisher Scientific (USA). All plasmid amplifications were done using E. coli JM109 chemically competent cells (Promega, Madison, USA). For validation of the correct insertion of upstream and downstream DNA fragments into pCM184, sequencing using primers S1F/S1R and S2F/S2R was performed. pCM184 harboring upstream and downstream regions of the plantbeneficial genes of interest were transformed into F113 by electroporation [START_REF] Højberg | Oxygen-sensing reporter strain of Pseudomonas fluorescens for monitoring the distribution of low-oxygen habitats in soil[END_REF]. Inactivation mutants resulting from a double recombination event of upstream and downstream regions were selected on King's B Km and screened for plant-beneficial gene deletion by PCR with specific primers. Then, the plasmid CM157 containing the cre-lox recombinase gene was introduced into F113 kanamycin resistant mutants in order to remove the antibiotic marker. To check the loss of the kanamycin cassette, mutant strains were screened for their inability to grow on King's B Km and by PCR using external primers. For complementation of the phlD deletion mutant, the coding region of the phlD gene of F113 was amplified by PCR using primers phlDCompF1 and phlDCompR1 and subcloned into pGEM-T easy. The phlD gene was extracted from pGEM-T easy using ApaI/SacI digestion and inserted into the multiple cloning site of vector pBBR1MCS-5, under control of the lac

Comment citer ce document : Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D., prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI :

16 promoter, resulting in pBBR1-MCS5:phlD. This plasmid was introduced into the ∆phlD mutant by electroporation [START_REF] Højberg | Oxygen-sensing reporter strain of Pseudomonas fluorescens for monitoring the distribution of low-oxygen habitats in soil[END_REF].

DAPG quantification by HPLC

DAPG production was measured by HPLC according to the method developed by [START_REF] Bonsall | Quantification of 2,4diacetylphloroglucinol produced by fluorescent Pseudomonas spp. in vitro and in the rhizosphere of wheat[END_REF]. Briefly, samples were analyzed with an Agilent 1200 series HPLC (Agilent Technologies, Santa Carla, USA) equipped with a degasser (G132A), a quaternary pump module (G1311A), an automatic sampler (G1329A) and a diode array detector (DAD G1315B). Separation of compounds was performed at room temperature with a C18 reversephase column. For each sample, 20 µl were injected and eluted at 1 ml.min -1 using a step-bystep gradient increasing acetonitrile (ACN) proportion in water: the gradient started at 40% of ACN over 4 min then rose from 40 to 64% in 7.5 min, then came to 75% at 16.5 min and ended at 100% at 18.5min (100% maintained for 3 min before decreasing to 40% for 5 min).

Chromatograms were recorded at 270nm (maximum of absorbance of DAPG). The chemstation Agilent software was used for integration of chromatograms, and quantitation of DAPG was done according to a reference curve with the chemical standard (Sigma-Aldrich).

This experiment was done independently three times.

ACC deaminase activity assay

The ACC deaminase activity of strains was performed as described by [START_REF] Blaha | Phylogeny of the 1aminocyclopropane-1-carboxylic acid deaminase-encoding gene acdS in phytobeneficial and pathogenic Proteobacteria and relation with strain biogeography[END_REF].

The method is based on the quantification of α-ketobutyrate produced by the deamination of ACC into α-ketobutyrate by the enzyme ACC deaminase. Total protein content in the assay was measured using the BCA Protein Assay Kit (Pierce, Rockford, IL). ACC deaminase activity was assessed three times for each strain. The data are expressed in nmol of ketobutyrate.h -1 .mg of protein -1 . This experiment was done independently three times.

Phosphate-solubilizing assay

The phosphate-solubilizing ability of strains was evaluated by measuring tricalcium 17 after 6 days of incubation at 28°C using ImageJ software. This experiment was done three times independently.

Denitrification activity measurement

The denitrification activity was estimated through the potential accumulation of N 2 O in the culture media, which is one step of the denitrification process. The accumulation of N 2 O was measured according to [START_REF] Bardon | Identification of B-type procyanidins in Fallopia spp. involved in biological denitrification inhibition[END_REF]. Briefly, 20 ml of King's B media supplemented with 20 mM of KNO 3 were placed in a plasma flask sealed by a rubber stopper. Anaerobic condition was obtained removing air and replaced it with He/C 2 H 2 mixture (90/10; v/v) in order to inhibit the reduction of N 2 O to N 2 and to allow the N 2 O accumulation. Each strain was inoculated at a final OD 600 of 0.1. N 2 O emissions were measured every 2h during 24h using gas chromatography coupled to a micro-katharometer detector (µGC-R3000, SRA instruments, Marcy l'Etoile, France). The assay was performed twice with two technical replicates. N 2 O emission was expressed as µg of N-N 2 O.h -1 .ml -1 of media. In addition, the ability of F113 to produce NO intracellularly was evaluated using the specific fluorescent probe diaminorhodamine-4M acetoxymethyl ester (Calbiochem), as previously described by [START_REF] Pothier | Promoter trap identification of wheat seed extract-induced genes in the plant-growthpromoting rhizobacterium Azospirillum brasilense Sp245[END_REF].

Bacterial inoculum preparation

The P. kilonensis F113 wild-type and related deletion mutants ∆phlD, ∆acdS, ∆gcd, ∆nirS and the quadruple mutant (∆phlD∆acdS∆gcd∆nirS) were incubated overnight at 28°C in King's B broth medium. Cell cultures were centrifuged at 3000 rpm. The pellet was washed with sterilized water and adjusted to 10 7 bacteria.ml -1 .

Bacterial inoculation effects on Arabidopsis root system architecture

The seeds of Arabidopsis thaliana Ecotype Columbia0 (Col-0) were prepared according to [START_REF] Mantelin | Nitrate-dependent control of root architecture and N nutrition are altered by a plant growth-promoting Phyllobacterium sp[END_REF]). After 7 days, plantlets were transferred to 50 ml of new plant agar medium containing F113 or mutant strains at 10 7 cells.ml -1 in square Petri dishes (120 x 120 mm). Inoculated plantlets were transferred to a phytotron set to the following parameters: 21°C/days; 17°C/night, 13h light periods, 9500 lux. Four Arabidopsis plantlets were disposed per Petri dish and 6 plates were prepared per condition. The 7 conditions tested were A. (1) F113 -control = α

(2) ((F113 -mutant) / α) x 100 = deleted gene relative contribution to F113 effect (%)

Arabidopsis ethylene emission

Measurements of in vivo ethylene emission by Arabidopsis plantlets were performed using infrared laser spectroscopy coupled to photo-acoustic detection [START_REF] Cristescu | Current methods for detecting ethylene in plants[END_REF]) (ETD-300, SensorSense, Nijmegen, Holland). Practically, we carefully poured 600 µl of sterile plant culture medium (as described above) inoculated or not with the various F113 strains at a final density of 2x10 7 cells.ml -1 in a 2 ml sterile Eppendorf tube. Once solidified, we carefully introduced two wild-type Arabidopsis plantlets aged of seven days and closed the lid. Lids were punched to allow a regular breathing of the plants. This system was then transferred to a phytotron (MLR-352-PE, Panasonic, Gunma, Japan) set to the following parameters: 21°C (day); 17°C (night), 13h light period, 9500 lux. No measure was made during the first 48 h to allow plant acclimation, and in order to recover stress associated to the transfer. Prior ethylene emission measurement, 200 µl of plant liquid medium was added in each tube in order to keep the head space humid, and therefore protecting the plant from the dry air coming from the compressed air bottle. Then, the lids of the tubes were removed and replaced by a rubber plug.

Needles were pushed through the plug to allow the headspace to be flushed to the ethylene detector by compressed air. Every 90 min, the ethylene content in the headspace was measured while the tubes remained in the phytotron. That measurement was performed during two to three consecutive days to collect enough data on ethylene content during the day. At the end of the experiment, plant fresh weight was measured to allow data normalization. Raw results were exported into a spreadsheet in which they were normalized and processed to obtain at least 10 different measures of ethylene content in presence of light in each condition.

Maize inoculation experiments in non-sterile soil

Comment citer ce document : Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D., prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI :

19

The semi-late maize hybrid PR37Y15 (Pioneer Semences, Aussonne, France) was used in this study. Maize seeds were surfaced disinfected. Briefly, seeds were soaked in sodium hypochlorite (3.4%) during one hour with agitation. Five washes were performed with sterile water. Four maize seeds were sown in non-sterile soil taken from La Côte Saint-André (LCSA) near Lyon (France; Vacheron et al. 2016a) and placed in 2dm 3 pots filled with 2.0kg of sieved soil (5mm) with a water content of 20% (v/v). The moisture was maintained at 20% during the experiment. Seeds (4 per pot) were inoculated with 100 µl of bacterial inoculum solution, corresponding to 10 6 bacteria. Ten pots per conditions were performed. Inoculum impact on plant growth was assessed at 20 dpi by characterizing maize root system architecture using WinRhizo (Reagent Instruments, Quebec City, Quebec, Canada). Leaf surface was measured with the WinFolia image analysis software (Regent Instruments, Quebec City, Quebec, Canada). Chlorophyll content was measured with a chlorophyll content meter (Hansatech instruments model CL-01, Amesbury, USA). Root and shoot fresh and dry biomasses were weighed. Ten plants were analyzed per conditions.

Monitoring the bacterial maize root colonization

The measurement of root colonization was assessed at 0, 8 and 20 dpi taking 10 seeds for each condition at each sampling time. Inoculated seeds or plants were harvested and disposed in a 50 ml Falcon tube and then soaked in liquid nitrogen. Seeds and root systems were lyophilized and then scratched in order to extract DNA from the seed surface and root adhering soil. DNA extractions from 500 mg of rhizosphere soils or seeds teguments were performed using a FastDNA™ SPIN Kit for Soil kit (MP Biomedicals, USA). DNA was also extracted from 1 ml of inoculum solution in order to ascertain the cell concentration of the inoculants. The seed and root bacterial colonization was assessed by real-time PCR as described by Von Felten et al. ( 2010). Real-time PCR were performed using the LightCycler® FastStart DNA Master SYBR® Green I kit and a LC-480 LightCycler (Roche Applied Science, Indianapolis, IN). 5 ng of extracted DNA were used to assess the abundance of seed and root colonization by the F113 parental and mutant strains using F113_1_for and F113_1_rev primers with a detection limit of 10 4 cells.seed -1 and 10 4 cells.g -1 of rhizosphere [START_REF] Von Felten | Quantification of Pseudomonas fluorescens strains F113, CHA0 and Pf153 in the rhizosphere of maize by strain-specific real-time PCR unaffected by the variability of DNA extraction efficiency[END_REF]. Real-time PCR quantification data were converted to gene copy number per gram of lyophilized root as described by [START_REF] Walker | Variation of secondary metabolite levels in maize seedling roots induced by inoculation with Azospirillum, Pseudomonas and Glomus consortium under field conditions[END_REF].

Statistical analysis

Comment citer ce document : Vacheron, J., Desbrosses, G., Renoud, S.

886

The data represent the means of five replicate cultures (standard errors were not presented but lower 887 than 10%). The experiment was done three times independently. The dotted line corresponds to the qPCR detection limit. NI: Non-inoculated condition. ‡ : PvuII restriction site ; *: size in F113 WT / size in the deleted mutant Kovach, M.E., Elzer, P.H., Hill, D.S. et al. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166: 175-176.

  Comment citer ce document : Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D., prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI : 10 interaction involving Gcd with another antimicrobial compound (i.e. pyoluteorin) has been evidenced in P. protegens CHA0 (de Werra et al. 2009).

  citer ce document : Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D., prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI :

  Comment citer ce document : Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D., prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI : 14 inoculation of the quadruple mutant, showing that the other PBFs activate plant ethylene

  phosphate solubilization halos on a National Botanical Research Institute's Phosphate (NBRIP) agar medium. Strains were cultured for 1 day at 28°C in King's B broth and then deposited 4 times (5 µl each) on NBRIP agar medium. Solubilization halos were measured Comment citer ce document : Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D., prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI :

  thaliana inoculated with F113, with the 4 simple mutants, with the quadruple mutant and the Comment citer ce document : Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D., prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI : 18 non-bacterized control condition. This experiment was made thrice independently. After 7 days, 3 plants per Petri dishes were scanned in order to analyze the primary root elongation and root hair length using ImageJ software (i.e. 18 analyzed plants per condition). The relative contribution of each tested PBF in the modulation of the Arabidopsis root architecture was calculated as follow:

Fig. 1 : 885 Fig. 2 :

 18852 FIG. LEGENDS

888Fig. 3 :

 3 Fig. 3: Comparison of maize root colonization levels between P. kilonensis F113 and its PBF

  892

Fig. 4 :Fig. 5 :Fig. 6 :

 456 Fig. 4: Comparison of the effects of P. kilonensis F113 PBF mutants on maize growth by

  Fig. 1 281x222mm (72 x 72 DPI)

  Fig. 2 132x91mm (300 x 300 DPI)

  Fig. 3 133x94mm (300 x 300 DPI)

  Fig. 4 112x83mm (300 x 300 DPI)

  Fig. 5 113x142mm (300 x 300 DPI)

  Fig. 6 118x81mm (300 x 300 DPI)

Fig. S2 :

 S2 Fig. S2: Effect of the addition of DAPG on growth curves of the P. fluorescens wild-type F113, the ∆phlD mutant, and the ∆phlD complemented mutant with restored DAPG production. Growth curves of F113 WT, ∆phlD and complemented ∆phlD (A). Effect of increasing DAPG concentrations on F113 WT (B), the ∆phlD mutant (C) and the complemented ∆phlD mutant (D). Comp.: Complemented. Results are presented as means of five replicate cultures (standard deviations were not presented but lower than 10%). The experiment was done three times independently.

Fig. S3 :

 S3 Fig. S3: Effects of P. fluorescens F113 and its PBF mutants on maize growth. For each plant parameters, ratio between data obtained with mutants and with F113 were calculated and expressed in percent. Root and shoot measured parameters are colored respectively in brown and green on the star plot. LS#1: surface of the leaf number 1 (same nomenclature for leaf 2 and 3); LSTot: Total leaf surface; Chl: chlorophyll content in leaf number 3 measured with a chlorophyll content meter (Hansatech instruments model CL-01; Dinç et al. 2012); FSM: Fresh shoot mass; DSM: Dry shoot mass. L: total root length, S: Total root surface; D: Root diameter; V: root volume; T: Root tips; FRM: Fresh root mass; DRM: Dry root mass. R/S ratio represents the ratio between the dry root biomass and the dry shoot biomass.Dinç, E., Ceppi, M.G., Tóth, S.Z., Bottka, S., Schansker, G. 2012. The chl a fluorescence intensity is remarkably insensitive to changes in the chlorophyll content of the leaf as long as the chl a/b ratio remains unaffected. Biochim. Biophys. Acta 1817:770-779.

Fig. S4 :

 S4 Fig. S4: Model presenting potential metabolic regulation involved in DAPG production in P. fluorescens F113. Glucose catabolism in F113 (A) and F113 gcd mutant (B). Enzymes involved in glucose metabolism are abbreviated using the same nomenclature as de Werra et al. (2009). The corresponding enzyme functions and F113 gene names are indicated in parenthesis: Gcd (glucose dehydrogenase -gcd); Glk (glucokinase -PSF113_1279), Gad (Gluconate dehydrogenase -PSF113_4842); Zwf (glucose-6-phosphate-1-dehydrogenase -PSF113_1290 and PSF113_2674); GnuK (gluconokinase -PSF113_1311); KguD (2-ketogluconate 6-phosphate reductase -kguK); KguK (2-ketogluconate kinase -kguK).

  for homologous recombination, Amp r , Km r , Tc r Marx and Lidstrom 2002 pCM157 Containing Cre recombinase activity, Amp r , Tc r Marx and Lidstrom 2002 pGEM-T easy:phlD phlD cloned into pGEM-T easy, Amp r This study pGEM-T easy:UPphlD Upstream sequence of phlD cloned into pGEM-T easy, Amp r This study pGEM-T easy:DWphlD Downstream sequence of phlD cloned into pGEM-T easy, Amp r This study pGEM-T easy:UPacdS Upstream sequence of acdS cloned into pGEM-T easy, Amp r This study pGEM-T easy:DWacdS Downstream sequence of acdS cloned into pGEM-T easy, Amp r This study pGEM-T easy:UPgcd Upstream sequence of gcd cloned into pGEM-T easy, Amp r This study pGEM-T easy:DWgcd Downstream sequence of gcd cloned into pGEM-T easy, Amp r This study pGEM-T easy:UPnirS Upstream sequence of nirS cloned into pGEM-T easy, Amp r This study pGEM-T easy:DWnirS Downstream sequence of nirS cloned into pGEM-T easy, Amp r This study pCM184:phlD Upstream and downstream sequence of phlD into pCM184, Amp r , Km r , Tc r This study pCM184:acdS Upstream and downstream sequence of acdS into pCM184, Amp r , Km r , Tc r This study pCM184:gcd Upstream and downstream sequence of gcd into pCM184, Amp r , Km r , Tc r This study pCM184:nirS Upstream and downstream sequence of nirS into pCM184, Amp r , Km r , Tc r This study pBBR1-MCS5:phlD phlD cloned into pBBR1-MCS5, Gm r This study a : Amp r Ampicillin resistant; Km r Kanamycin resistant; Tc r Tetracycline resistant, Gm r , Gentamycin resistant Kovach, M.E., Elzer, P.H., Hill, D.S. et al. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166: 175-176; Marx, C.J., Lidstrom, M.E. 2002. Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria. Biotechniques 33:1062-1067; Shanahan, P., O'Sullivan, D.J., Simpson, P., Glennon, J.D., O'Gara, F. 1992. Isolation of 2,4-diacetylphloroglucinol from a fluorescent Pseudomonad and investigation of physiological parameters influencing its production. Appl. Environ. Microbiol. 58:353-358. Comment citer ce document : Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D., prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI :

Table S2 and

	8
	Comment citer ce document :
	Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D.,
	prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of
	plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in
	Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI :

Table S1 : Slope comparison at the exponential growth stage and OD 600 at the stationary phase for F113 WT and its PBF mutants,

 S1

	Strains	Growth curve slopes at exponential growth stage	OD 600 at the stationary phase
	F113 WT	0.20 ± 0.02 b	1.29 ± 0.06 b
	∆phlD	0.20 ± 0.02 b	1.54 ± 0.05 a
	∆acdS	0.17 ± 0.04 b	1.29 ± 0.09 b
	∆gcd	0.18 ± 0.03 b	1.37 ± 0.08 b
	∆nirS	0.18 ± 0.03 b	1.35 ± 0.12 b
	Quadruple mutant	0.25 ± 0.02 a	1.52 ± 0.01 a
	Data were presented as mean ± standard deviation. Letters indicate statistical relations between
	strains for each tested PBF activities (ANOVA and Tukey's HSD test, P < 0.05)

Comment citer ce document : Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D., prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI :

Table S2 : Effect of P. fluorescens F113 and its related mutants on maize root architecture

 S2 Arbitrary Unit. Different letters correspond to significant differences for each plant parameter (ANOVA coupled with HSD's Tukey test P=0.05).

		Total root length (cm)	Total root surface (cm²)	Root diameter (mm)	Root total volume (cm 3 )	Number of root tips	Fresh root weight (g)	Dry root weight (g)
	F113 301.28 ± 61.68 a	70.96 ± 11.68 a	0.81 ± 0.05 b	1.35 ± 0.17 ab	2636.00 ± 755.92 a	1.39 ± 0.14 a	0.18 ± 0.04 a
	∆phlD 262.55 ± 43.59 a	68.59 ± 7.40 a	0.92 ± 0.09 ab	1.50 ± 0.13 a	2535.75 ± 551.14 a	1.44 ± 0.12 a	0.15 ± 0.01 ab
	∆acdS 108.83 ± 18.92 bc	35.47 ± 3.97 bc	1.12 ± 0.09 ab	0.96 ± 0.08 ab	995.29 ± 235.46 b	0.95 ± 0.09 b	0.13 ± 0.03 ab
	∆gcd 223.32 ± 61.34 b	56.39 ± 9.04 bc	0.93 ± 0.10 ab	1.23 ± 0.12 ab	2486.00 ± 592.98 a	1.24 ± 0.11 ab	0.14 ± 0.02 ab
	∆nirS 123.30 ± 27.81 bc	35.71 ± 5.44 bc	1.02 ± 0.08 ab	0.86 ± 0.10 b	1455.38 ± 400.02 b	0.86 ± 0.13 b	0.12 ± 0.03 b
	∆∆∆∆	86.85 ± 17.69 c	30.81 ± 4.35 c	1.26 ± 0.13 a	0.94 ± 0.16 ab	760.13 ± 163.47 b	0.80 ± 0.06 b	0.12 ± 0.02 b
	NI	201.23 ± 51.83 bc	48.66 ± 8.85 bc	0.90 ± 0.11 ab	1.01 ± 0.14 ab	1918.63 ± 506.78 a	1.14 ± 0.12 ab	0.14 ± 0.04 ab

* AU:

Comment citer ce document : Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D., prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI :

Table S4 : Bacterial strains and plasmids used in this study

 S4

	Bacterial strain or plasmid	Relevant Characteristics a	Source or reference
	Bacterial Strains
	Pseudomonas fluorescens
	F113	Wild-type, Phl + , acdS + , gcd + , nirS + HCN +	Shanahan et al. 1992
	∆phlD	F113 strain phlD gene deleted	This study
	∆acdS	F113 strain acdS gene deleted	This study
	∆gcd	F113 strain gcd gene deleted	This study
	∆nirS	F113 strain nirS gene deleted	This study
	∆phlD ∆acdS ∆gcd ∆nirS	F113 quadruple mutant	This study
	∆phlDComp	∆phlD with restored DAPG production capacity	This study
	Escherichia coli
	JM109	Host strain for the plasmids transformation	Promega

Table S5 : Primers used in this study

 S5

	Targeted regions		Names of primers	Sequences	Sizes of amplification	Reference
	Insertion verification into pGEMTeasy		M13 F M13 R	GTTTTCCCAGTCACGAC CAGGAAACAGCTATGAC	Variable according to the cloned gene	Promega
	Insertion verification into pBBR1-MCS5	pBBR1MCS5_F1 pBBR1MCS5_R1	TGCAAGGCGATTAAGTTGGGTA TATGACCATGATTACGCCAAGC	Variable according to the cloned gene	Kovach et al. 1995
	phlD gene amplification		phlDCompF1 phlDCompR1	ACTTTATTGGCTTCTCGCCG GGGTGTCGGTGTTTTATCGG	1104pb	This study This study
	pCM184 insertion site 1		S1F S1R	ACGGAAATGTTGAATACTCATACTC CGTTTCCCGTTGAATATGGC	Variable according to the cloned gene	This study This study
	pCM184 insertion site 2		S2F S2R	GCAGGTCCTAGATAACTTCG GGCTGGATCCTCTAGTGAG	Variable according to the cloned gene	This study This study
	Upstream region of phlD		phlDF1 phlDR1	TAGAGGTACCAGGCCACTAA † CAATTGTGACAGACCGATAAAACACCG	700pb	This study This study
	Downstream region of phlD		phlDF2 phlDR2	CATGACAAGTCCTCGGCGAGA GGGATTTTGCACATTACTGA	753pb	This study This study
	Upstream region of acdS		acdSF1 acdSR1	TAAGCGTAGAACTGCTTTTGG ‡ CAGCTGTAGAACGACAGTCGGAAGAT	773pb	This study This study
	Downstream region of acdS		acdSF2 acdSR2	ATTCAGTCGTGTCAGGTTTT CATGACTCTGCTCCTTGATAT	724pb	This study This study
	Upstream region of gcd		gcdF1 gcdR1	TTTCATCGATTTCGACTACG † CAATTGTAAGCCCAAGCTGCGCTC	793pb	This study This study
	Downstream region of gcd		gcdF2 gcdR2	CATAGTGTGTTTCTCCGTGAC CTTATCAATCCCCATGACTC	750pb	This study This study
	Upstream region of nirS		nirSF1 nirSR1	‡ CAGCTGCAGCAGGTGTTGCAGTTG † CAATTGAAGAACGTGATCAAGGACAAG	711pb	This study This study
	Downstream region of nirS		nirSF2 nirSR2	TTTTTTGTGGCTGATCAGCAT CTTCATCCCCTTGAGCAG	702pb	This study This study
	External primer		phlDExtf phlDExtr	ATTGAAGGAACAGTAGCCAG TGCAAAGGATATCGATGTGG	2378/1334pb *	This study This study
	External primer		acdSExtf acdSExtr	ACAGCGTGAGCATTAGACAA ACTCTTCAAGTTCACCGCAG	2896/1885pb	This study This study
	External primer		gcdExtf gcdExtr	CCATGAACCGTGAAGGTATT GTCAGTACCGTGAAGAAACT	4357/1949pb	This study This study
	External primer		nirSExtf nirSExtr	AGGAGAGGAAAGAACGGAT TTCAGCCAAAGTGGACATC	3550/1972pb	This study This study
	† : MunI (MfeI) restriction site ;

Molecular Plant-Microbe Interactions "First Look" paper • http://dx.doi.org/10.1094/MPMI-07-17-0185-R • posted 10/03/2017 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.

This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.

ACKNOWLEDGEMENTS

Jordan Vacheron was supported by a Ph.D grant from Academic Research Cluster 3 of Rhône-Alpes Region. We thank M. Ribierre for technical help. We thank Sara Puijalon and Vanessa Gardette from UMR CNRS 5023 for allowing us to use the Winfolia software. This work made use of the 'Serre' technical platform of FR41 at Université Lyon 1 and the AME plateform (UMR 5557) for denitrification assays. We also thank Delphine Aymoz for English proofreading of this paper. This study was supported by the ANR project SymbioMaize (ANR-12-JSV7-0014-01).

Version postprint

Comment citer ce document : Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D., prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI :

20

Regarding assays on each plant-beneficial function, data were log-transformed in order to respect normal distribution and variances homogeneity and a one-way ANalysis Of VAriance and Tukey's HSD tests were performed to detect significant differences between strains.

Arabidopsis root hair length data were compared using Kruskal-Wallis tests followed by posthoc non-parametric multiple comparisons. Arabidopsis primary root length data were square- UPGMA). All data were presented as mean ± standard error. All analyses were performed at P < 0.05, using R studio software (RStudio Team, 2015).

Version postprint

Comment citer ce document : Vacheron, J., Desbrosses, G., Renoud, S., Padilla-Aguilar, R.-M., Walker, V., Muller, D., prigent-combaret, c. (Auteur de correspondance) (2018). Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays. Molecular Plant-Microbe Interactions, 31 (2), 212-223. , DOI :