Fabricio D Cassan

Anahí Coniglio

Edgar Amavizca

Guillermo Maroniche

Eric Cascales

Yoav Bashan

Luz E De-Bashan

SI: Multifaceted Plant-Microbe Interactions The Azospirillum brasilense Type VI secretion system promotes cell aggregation, biocontrol protection against phytopathogens and attachment to the microalgae Chlorella sorokiniana

Keywords: Attachment, aggregation, Plant Growth Promoting Bacteria (PGPB), Azospirillum, bacteria-microalgae interaction, Chlorella, Type VI secretion system (T6SS)

The plant-growth promoting bacterium Azospirillum brasilense is able to associate with the microalgae Chlorella sorokiniana. Attachment of A. brasilense increases the metabolic performances of the microalgae. Recent genome analyses have revealed that the A. brasilense Az39 genome contains two complete sets of genes encoding Type VI secretion systems (T6SS), including the T6SS1 that is induced by the indole-3-acetic acid (IAA) phytohormone. The T6SS is a multiprotein machine, widespread in gram-negative bacteria, that delivers protein effectors in both prokaryotic and eukaryotic cells. Here we show that the A. brasilense T6SS is required for Chlorella-Azospirillum synthetic mutualism. Our data demonstrate that the T6SS is an important determinant to promote production of carbohydrates and photosynthetic pigments by the microalgae. We further show that this is likely due to the role of the T6SS during the attachment stage and for the production of IAA phytohormones. Finally, we demonstrate that the A. brasilense T6SS provides antagonistic activities against a number of plant pathogens such as Agrobacterium, Pectobacterium, Dickeya and Ralstonia species in vitro, suggesting that, in addition to promoting growth, A. brasilense might confer T6SS-dependent bio-control protection to microalgae and plants against bacterial pathogens.

Significance

Azospirillum brasilense, a plant growth promoting bacterium, increases the metabolic performances of plants and microalgae. Here we show that the A. brasilense type VI secretion system (T6SS) is an important determinant of Azospirillum attachment to the microalgae Chlorella sorokiniana, and hence that a mutant defective in T6SS decreases metabolite production in the microalga. In addition, we show that the T6SS confers antibacterial activity against competitor 4 bacterial cells, including phytopathogens in vitro, suggesting that the T6SS is also an important determinant for bioprotection. This study therefore demonstrates that a secretion system, involved in the secretion of bacterial effectors and toxins, participates in processes that are beneficial to plant and microalgae cells.

Introduction

Azospirillum spp. is one of the best-characterized genera of plant growth-promoting bacteria (PGPB) and can colonize more than 130 plant species in 37 families [START_REF] Pereg | Assessment of affinity and specificity of Azospirillum for plants[END_REF]. The association of Azospirillum spp. with these plants significantly improves growth, development, and in many cases, yield under field conditions [START_REF] Cassan | Everything you must know about Azospirillum and its impact on science, research, agriculture and beyond[END_REF]. There is no single mechanism involved in promoting plant growth with Azospirillum, but rather a combination of mechanisms in different cases of inoculation. These mechanisms work together or in tandem, and the phenomenon is commonly known as "multiple mechanism theory" [START_REF] Bashan | How the plant growth-promoting bacterium Azospirillum promotes plant growth -a critical assessment[END_REF]. The production of indole-3-acetic acid (IAA) by Azospirillum spp. is one of the main mechanisms proposed for the effect of this bacterial species on plant growth [START_REF] Bashan | How the plant growth-promoting bacterium Azospirillum promotes plant growth -a critical assessment[END_REF].

The A. brasilense Az39 used in this study was isolated in 1982 from surface-sterilized wheat seedlings in Marcos Juarez, Argentina. This strain was selected for inoculant formulation, based on its ability to increase crop yields of maize and wheat under agronomic conditions [START_REF] Díaz-Zorita | Field performance of a liquid formulation of Azospirillum brasilense on dryland wheat productivity[END_REF]. Currently, A. brasilense Az39 is one of the most used strains for crops such as maize and sorghum in Argentina and South America [START_REF] Cassan | Everything you must know about Azospirillum and its impact on science, research, agriculture and beyond[END_REF].

For decades, Azospirillum spp. has been known to associate to roots of higher plants [START_REF] Bashan | How the plant growth-promoting bacterium Azospirillum promotes plant growth -a critical assessment[END_REF], such as cereals, tomato, pepper, cotton, and soybean, and with the microalgae Chlorella sorokiniana [START_REF] Levanony | Ultrastructural localization and identification of Azospirillum brasilense Cd on and within wheat root by immuno-gold labeling[END_REF][START_REF] Bashan | Root surface colonization of non-cereal crop plants by pleomorphic Azospirillum brasilense Cd[END_REF][START_REF] Assmus | Situ localization of Azospirillum brasilense in the rhizosphere of wheat with fluorescently labeled, rRNA-targeted oligonucleotide probes and scanning confocal laser microscopy[END_REF]. The first step of the association corresponds to attachment to the root, a critical step because all azospirilla are highly motile bacteria in vitro [START_REF] Bashan | Root-to-root travel of the beneficial bacterium Azospirillum brasilense[END_REF][START_REF] Alexandre | Energy taxis is the dominant behavior in Azospirillum brasilense[END_REF], in soil [START_REF] Bashan | Horizontal and vertical movement of Azospirillum brasilense Cd in the soil and along the rhizosphere of wheat and weeds in controlled and field environments[END_REF], and in water [START_REF] Puente | Root surface colonization of black mangrove seedlings by Azospirillum halofraeference and Azospirillum brasilense in seawater[END_REF]. In addition to motility, root colonization also requires the production of surface polysaccharides [START_REF] Jofre | Disruption of dTDP-rhamnose biosynthesis modifies lipopolysaccharide core, exopolysaccharide production, and root colonization in Azospirillum brasilense[END_REF]. After attachment, azospirilla colonize the roots. Host colonization usually requires the delivery of effector proteins by dedicated secretion systems. Up to eleven secretion systems (Types I-XI) have been described so far in gram-negative bacteria [START_REF] Costa | Secretion systems in Gram-negative bacteria: structural and mechanistic insights[END_REF]. Genome analyses show that A. brasilense strains are lacking Type III and Type IV secretion systems.

Transcriptomics and bioinformatics analyses show that A. brasilense strains Sp245 and Az39 contain gene clusters encoding putative Type VI secretion systems (T6SSs) [START_REF] Van Puyvelde | Transcriptome analysis of the rhizosphere bacterium Azospirillum brasilense reveals an extensive auxin response[END_REF][START_REF] Rivera | Complete genome sequence of the model rhizosphere strain Azospirillum brasilense Az39, successfully applied in agriculture[END_REF]Fig. 1; Table S1). The T6SS is a widespread, multi-protein apparatus, found in gram-negative bacteria [START_REF] Bingle | Type VI secretion: a beginner's guide[END_REF][START_REF] Cascales | The type VI secretion toolkit[END_REF]. The T6SS is not only distributed in human, animal, and plant pathogenic bacteria where it is involved in virulence and pathogenesis, but also found in commensal and symbiotic PGPB [START_REF] Bingle | Type VI secretion: a beginner's guide[END_REF][START_REF] Russell | Type VI secretion system effectors: poisons with a purpose[END_REF]. In these bacteria, the T6SS participates to intermicrobial rivalry in bacterial and fungal competition, biofilm formation, and establishment of symbiosis [START_REF] Jiménez-Guerrero | Bacterial protein secretion systems. Implications in beneficial associations with plants[END_REF][START_REF] Durand | Tle, and beyond: the versatile arsenal of Type VI secretion effectors[END_REF][START_REF] Russell | Type VI secretion system effectors: poisons with a purpose[END_REF][START_REF] Ryu | Against friend and foe: Type 6 effectors in plant-associated bacteria[END_REF][START_REF] Bernal | The Pseudomonas putida T6SS is a plant warden against phytopathogens[END_REF][START_REF] Trunk | A new front in microbial warfare -delivery of antifungal effectors by the type VI secretion system[END_REF]Gallegos-Monterossa and Coulthurst, 2021). At the molecular level, the T6SS acts like a cellular "biological syringe": it assembles a bacteriophage tail-like structure anchored to the cell envelope by a membrane complex [START_REF] Zoued | Architecture and assembly of the Type VI secretion system[END_REF][START_REF] Coulthurst | The Type VI secretion system: a versatile bacterial weapon[END_REF][START_REF] Cherrak | Structure and activity of the type VI secretion system[END_REF]. The tail-like structure consists of an inner tube made of hexamers of the Hcp protein capped by a puncturing VgrG-PAAR complex and wrapped in the TssBC contractile sheath [START_REF] Zoued | Architecture and assembly of the Type VI secretion system[END_REF][START_REF] Cherrak | Structure and activity of the type VI secretion system[END_REF][START_REF] Basler | Type VI secretion system: secretion by a contractile nanomachine[END_REF]. Effector toxins are loaded onto the VgrG o PAAR proteins or within the Hcp tube [START_REF] Hernandez | Type VI secretion system effector proteins: Effective weapons for bacterial competitiveness[END_REF]Jurenas and Journet, 2021). Contraction of the sheath propels the inner tube towards the target cell [START_REF] Basler | Type VI secretion system: secretion by a contractile nanomachine[END_REF][START_REF] Brackmann | Using force to punch holes: mechanics of contractile nanomachines[END_REF]. Penetration of the tube into the target cell allows delivery of the effectors [START_REF] Hernandez | Type VI secretion system effector proteins: Effective weapons for bacterial competitiveness[END_REF]Jurenas and Journet, 2021). While the mechanisms of action and the role of T6SS in animal and plant pathogens have been well described in recent years, it has been less studied in plant symbiotic and associative bacteria [START_REF] Jiménez-Guerrero | Bacterial protein secretion systems. Implications in beneficial associations with plants[END_REF][START_REF] Ryu | Against friend and foe: Type 6 effectors in plant-associated bacteria[END_REF][START_REF] Wu | The Agrobacterium type VI secretion system: a contractile nanomachine for interbacterial competition[END_REF][START_REF] Wu | Plant-pathogenic Agrobacterium tumefaciens strains have diverse type VI effector-immunity pairs and vary in in-planta competitiveness[END_REF][START_REF] Bernal | Type VI secretion systems in plant-associated bacteria[END_REF][START_REF] Borrero De Acuña | Plant holobiont interactions mediated by the type VI secretion system and the membrane vesicles: promising tools for a greener agriculture[END_REF]. In Rhizobium leguminosarum, the T6SS affects the attachment, nodulation, and symbiosis processes [START_REF] Roest | A Rhizobium leguminosarum biovar trifolii locus not localized on the sym plasmid hinders effective nodulation on plants of the pea cross-inoculation group[END_REF][START_REF] Bladergroen | Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion[END_REF].

In Pseudomonas fluorescens, the T6SS is involved in bacterial competition, motility, biofilm formation, and is critical to persist in the rhizosphere microbiome [START_REF] Decoin | A type VI secretion system is involved in Pseudomonas fluorescens bacterial competition[END_REF]2015;[START_REF] Gallique | Contribution of the Pseudomonas fluorescens MFE01 type VI secretion system to biofilm formation[END_REF][START_REF] Durán | Pseudomonas fluorescens F113 type VI secretion systems mediate bacterial killing and adaption to the rhizosphere microbiome[END_REF]. P. putida produces three T6SSs, including K1-T6SS that confers antibacterial activity against numerous bacterial phytopathogens, and hence can be used as a biocontrol agent against plant pathogens [START_REF] Bernal | The Pseudomonas putida T6SS is a plant warden against phytopathogens[END_REF]. Finally, Agrobacterium tumefaciens was shown to deploy a T6SS to destroy competitors and to increase colonization in planta [START_REF] Ma | Agrobacterium tumefaciens deploys a superfamily of type VI secretion DNase effectors as weapons for interbacterial competition in planta[END_REF][START_REF] Wu | The Agrobacterium type VI secretion system: a contractile nanomachine for interbacterial competition[END_REF][START_REF] Wu | Plant-pathogenic Agrobacterium tumefaciens strains have diverse type VI effector-immunity pairs and vary in in-planta competitiveness[END_REF]. A. brasilense Az39 contains two gene clusters encoding T6SSs. A transcriptomic study of A. brasilense Sp245 showed that the T6SS1 gene cluster is induced by indole-3-acetic acid (IAA) [START_REF] Van Puyvelde | Transcriptome analysis of the rhizosphere bacterium Azospirillum brasilense reveals an extensive auxin response[END_REF]. The activation of T6SS1 by a phytohormone in A. brasilense prompted us to investigate the role of this T6SS in interbacterial competition and its interaction with microalgal cells.

The freshwater unicellular microalgae Chlorella spp. can associate with several bacterial species [START_REF] Imase | Construction of an artificial symbiotic community using a Chlorella-symbiont association as a model[END_REF], including Azospirillum spp. [START_REF] Hernandez | Growth promotion of the freshwater microalga Chlorella vulgaris by the nitrogen-fixing, plant growth-promoting bacterium Bacillus pumilus from arid zone soils[END_REF][START_REF] De-Bashan | Interaction of Azospirillum spp. with microalgae; a basic eukaryotic-prokaryotic model and its biotechnological applications[END_REF]. The Chlorella-Azospirillum association is an established model system of synthetic mutualism to study cellular mechanisms during plant-bacteria interaction (de- Bashan and Bashan, 2008;[START_REF] De-Bashan | Establishment of stable synthetic mutualism without coevolution between microalgae and bacteria demonstrated by mutual transfer of metabolites (NanoSIMS isotopic imaging) and persistent physical association (Fluorescent in situ hybridization)[END_REF]. This association significantly promotes the production of carbohydrates, fatty acids, lipids, and pigments of the microalgae, both autotrophically and heterotrophically (de- [START_REF] De-Bashan | Increased pigment and lipid content, lipid variety, and cell and population size of the microalgae Chlorella spp. when co-immobilized in alginate beads with the microalgae-growthpromoting bacterium Azospirillum brasilenses[END_REF][START_REF] Bashan | Increase in auxiliary photoprotective photosynthetic pigments in wheat seedlings induced by Azospirillum brasilense[END_REF]Choix et al., 2012a, b;[START_REF] Leyva | Accumulation of fatty acids in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoA carboxylase, temperature, and co-immobilization with Azospirillum brasilense[END_REF].

Previously, we tested the attachment and mutual aggregation between the microalga Chlorella sorokiniana and Azospirillum brasilense immobilized in alginate beads to facilitate the initial stage of formation of this synthetic association (de- Bashan and Bashan, 2008;[START_REF] De-Bashan | Interaction of Azospirillum spp. with microalgae; a basic eukaryotic-prokaryotic model and its biotechnological applications[END_REF][START_REF] De-Bashan | Establishment of stable synthetic mutualism without coevolution between microalgae and bacteria demonstrated by mutual transfer of metabolites (NanoSIMS isotopic imaging) and persistent physical association (Fluorescent in situ hybridization)[END_REF]. Attachment of Azospirillum brasilense to Chlorella spp. cells involve fibrillar connections [START_REF] Lebsky | Ultrastructure of interaction in alginate beads between the microalga Chlorella vulgaris with its natural associative bacterium Phyllobacterium myrsinacearum and with the plant growth-promoting bacterium Azospirillum brasilense[END_REF][START_REF] De-Bashan | Cell-cell interaction in the eukaryote-prokaryote model using the microalgae Chlorella vulgaris and the bacterium Azospirillum brasilense immobilized in polymer beads[END_REF], similar to what was observed during the attachment of Azospirillum spp. to cells of higher plants [START_REF] Bashan | Evidence for a weak active external adsorption of Azospirillum brasilense Cd to wheat roots[END_REF][START_REF] Bashan | Root surface colonization of non-cereal crop plants by pleomorphic Azospirillum brasilense Cd[END_REF]. However, we currently lack molecular details on how Azospirillum spp. efficiently colonizes microalgae and plants.

In this study, we show that the A. brasilense T6SS1 has a positive effect on growth of the microalgae Chlorella. We further demonstrate that a A. brasilense strain defective for T6SS1 presents a reduced enhancement of the production of microalgal metabolites such as lipids, carbohydrates, and photosynthetic pigments compared to wild-type A. brasilense. Finally, we demonstrate that the Azospirillum T6SS provides anti-bacterial activity against E. coli and several plant pathogens, including Agrobacterium tumefaciens, Pectobacterium carotovorum and Dickeya dadantii. These data support the idea that the T6SS is required for the establishment of an efficient synthetic mutualism between Azospirillum and Chlorella and confers protection of the microalgae -and potentially of plants -against pathogens.

Results

Construction of the A. brasilense Az39∆hcp-E mutant

The Hcp protein is an essential component of the T6SS and its encoding gene is present as a single copy in the T6SS1 gene cluster of A. brasilense Az39. It is located upstream of the tssE gene, which encodes an important component of the T6SS assembly baseplate [START_REF] Brunet | The Type VI secretion TssEFGK-VgrG phage-like baseplate is recruited to the TssJLM membrane complex via multiple contacts and serves as assembly platform for tail tube/sheath polymerization[END_REF] (Fig. 1). Both genes are in tandem in chromid 1 in A. brasilense Az39 (GenBank, CP007794.1, AbAz39_p1, WP_051658325.1) and share high identity to those encoded within the genomes of targeted mutagenesis to construct an isogenic mutant of Az39. For this, a genomic fragment overlapping with the hcp and tssE genes was replaced with a gentamycin-resistant cassette by homologous recombination (Fig. S1). The genomic structure of the wild-type strain and its isogenic ∆hcp-tssE (∆hcp-E) mutant was confirmed by PCR using several combinations of primers ( Fig. S1).

Phenotypic characterization of the A. brasilense Az39∆hcp-E strain for cell growth, IAA production, motility, cell aggregation and biofilm formation

To characterize the Az39∆hcp-E mutant strain, we measured its growth rate in rich and minimum media, its production of indole-3 acetic acid, and its capacity to swim, swarm and to form biofilm compared to the wild-type strain.

Growth rate -The growth rate and the number of colony-forming units (CFU) of the A. brasilense wild-type strain and its hcp-tssE mutant were compared in two different media. In both rich (LB) and minimal (MMAB) media, the two strains exhibited similar growth patterns for 80 hours (Fig. S2). Wild-type A. brasilense Az39 had the highest number of living cells (4.63×10 9 CFU×mL -1 in LB medium, and 1.85×10 9 CFU×mL -1 in MMAB) during the exponential growth phase (24 h after inoculation) and fewer cells (0.75×10 9 CFU×mL -1 in LB; 0.75×10 9 CFU×mL -1 in MMAB) during the stationary growth phase (>36 h after inoculation) (Fig S2). The A. brasilense Az39∆hcp-E mutant strain exhibited a similar pattern, with a higher number of cells during the exponential growth phase (2×10 9 CFU×mL -1 in LB; 1.6×10 9 CFU×mL -1 in MMAB) and fewer living cells during the stationary growth phase (1.5×10 9 CFU×mL -1 in LB; 1.06×10 9 CFU×mL -1 ) (Fig. S2).

Indole-3-acetic acid (IAA) production -In minimum medium, the mutant strain produced significantly less IAA during exponential growth compared to wild-type Az39, with a maximum difference at 24 h of culture (Az39∆hcp-E: 56.77 ± 2.59 µg•mL -1 ; Az39: 79.98 ± 2.14 µg•mL -1 ), but the accumulation of the phytohormone in the culture medium was restored to wild-type levels during the stationary growth phase (Az39∆hcp-E: 79.82 ± 1.60 µg•mL -1 , Az39: 79.45 ± 4.94 µg•mL -1 ), showing a saturation kinetic (Fig. 2).

Swimming, swarming, aggregation and biofilm formation -Previous reports have shown that mutation within T6SS gene clusters confer defects in swimming, swarming or biofilm formation [START_REF] Aschtgen | SciN is an outer membrane lipoprotein required for type VI secretion in enteroaggregative Escherichia coli[END_REF][START_REF] De Pace | The type VI secretion system plays a role in type 1 fimbria expression and pathogenesis of an avian pathogenic Escherichia coli strain[END_REF][START_REF] Decoin | A type VI secretion system is involved in Pseudomonas fluorescens bacterial competition[END_REF][START_REF] Bouteiller | Crosstalk between the type VI secretion system and the expression of class IV flagellar genes in the Pseudomonas fluorescens MFE01 strain[END_REF]. The Az39∆hcp-E mutant strain exhibited better swimming capacity compared to its parental strain in swim and MMAB culture media (Fig. 3a). By contrast, both strains swarmed at similar rates (Fig. 3b). Cell aggregation was reduced for the Az39∆hcp-E mutant in MMAB and LB culture media with an average reduction of 30% in comparison to the wild-type strain (Table 1). Finally, we observed that the Az39∆hcp-E mutant strain produced ~50 % less biofilm after 48 and 72 h of incubation, indicating a reduction in attachment capacity between cells. After 96 h of incubation, the mutant showed a reduction of 23.15 % of biofilm production compared to the wild-type strain, but this difference was not significant (Fig. 3c). Altogether, these data showed that the A. brasilense T6SS1 decreases the ability to swim and confers an increased ability to adhere to abiotic surfaces, at least in the early stages of the attachment phase.

The A. brasilense T6SS provides anti-bacterial activity against competitors, including phytopathogens.

A number of reports have demonstrated that the T6SS is involved in regulating bacterial communities by targeting effector toxins into competing bacteria sharing the same ecological niche [START_REF] Hood | A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria[END_REF][START_REF] Wexler | Human symbionts inject and neutralize antibacterial toxins to persist in the gut[END_REF][START_REF] Sana | Salmonella Typhimurium utilizes a T6SSmediated antibacterial weapon to establish in the host gut[END_REF][START_REF] Anderson | Shigella sonnei encodes a functional T6SS used for interbacterial competition and niche occupancy[END_REF][START_REF] Chassaing | Antibacterial weapons: targeted destruction in the microbiota[END_REF][START_REF] Allsopp | Causalities of war: The connection between type VI secretion system and microbiota[END_REF][START_REF] Wood | From welfare to warfare: the arbitration of hostmicrobiota interplay by the type VI secretion system[END_REF]Duran et al., 2021). We therefore asked whether the A. brasilense T6SS might confer protection to the microalgae or plants by delivering anti-bacterial toxins into phytopathogens. We performed in vitro competition experiments in minimal medium against Escherichia coli K-12, the soil bacterium Ralstonia eutropha and selected plant pathogens such as Agrobacterium tumefaciens, Pectobacterium carotovorum, Dickeya dadantii and Ralstonia solanacearum. After 8 h of co-incubation with wild-type A.

brasilense Az39 and its isogenic ∆hcp-E mutant, surviving prey cells were counted on selective medium. Fig. 4 shows that the wild-type A. brasilense strain has T6SS-dependent anti-bacterial activity against all the strains tested as only 7-15% of cells survive against the wild-type A.

brasilense compared to the ∆hcp-E mutant. The T6SS activity was significantly increased in presence of indole-3-acetic acid, previously shown to induce the T6SS gene cluster [START_REF] Van Puyvelde | Transcriptome analysis of the rhizosphere bacterium Azospirillum brasilense reveals an extensive auxin response[END_REF]. In presence of IAA, only 0.1-1% of the prey cells survived co-incubation with wildtype A. brasilense cells whereas their recovery was not affected when co-incubated with ∆hcp-E mutant cells (Fig. 4). We conclude that the A. brasilense T6SS has anti-bacterial activity against several strains and therefore might confer protection to plant and microalgae against bacterial pathogens.

Contribution of the T6SS for C. sorokiniana/A. brasilense association

Microalgae growth -To test the role of the A. brasilense T6SS for mutualistic growth of Chlorella, we followed the growth of the microalgae alone or in presence of the A. brasilense Az39 wild-type strain or its isogenic ∆hcp-E mutant (Fig. 5a). When cultured alone, the C. sorokiniana population increased for the first two days and then slowly decreased. After six days, the population reached its lowest numbers (Fig. 5a, open circles). In the presence of the A. brasilense wild-type or mutant strains, the Chlorella population increased for 4 days and then declined (Fig. 5a, closed squares and triangles, respectively). The growth rate of the microalgae after 4 days with either A. brasilense strain was ~3.5 higher than microalgae cultured alone (Fig. 5a). For 6 days, the interaction with both types of bacteria supported larger populations of microalgae, compared to the microalgae that were cultured alone.

Cell aggregation -Chlorella-Azospirillum association and aggregation were probed by fluorescence in situ hybridization (FISH) (Fig. 5b). Aggregates of the two bacterial strains grown with C. sorokiniana showed different dynamics throughout the experiment. The initial distribution of cells represents the outset of the microorganism's interaction (Fig. 5b panels a,e,i,m,q). In all cases, the aggregates are increasing in size from day 1 (Fig. 5b panels a-t; Fig. 5c). The difference in aggregates depending on the bacterial strain is evident at day 4. When C. sorokiniana was cocultured with A. brasilense Az39 large aggregates composed of bacteria and microalgae were found (Fig. 5b panel d; Fig. 5c). Co-cultures of C. sorokiniana with A. brasilense Az39∆hcp-E showed aggregates mainly composed of microalgae (Fig. 5b panel h) and significantly smaller than the aggregates formed with the wild-type Az39 strain (Fig. 5c). C. sorokiniana alone showed smaller aggregates (5b panel t; Fig. 5c). A. brasilense Az39 growing alone displayed aggregates increasing in size (Fig. 5b panels i-l; Fig. 5c); however, A. brasilense Az39∆hcp-E showed less aggregation than wild-type, maintaining a similar size of aggregates at days 2 and 4 (Fig. 5b panels m-p; Fig. 5c), in agreement with the defect in cell aggregation observed previously (Table 1).

Accumulation of carbohydrates, lipids and photosynthetic pigments -

To better understand the effect of the A. brasilense T6SS during mutualistic growth with C. sorokiniana, we measured the accumulation of carbohydrates, lipids and pigments in the microalgae alone or in presence of the A. brasilense Az39 wild-type strain or its isogenic ∆hcp-E mutant (Fig. 6 and7). Fig. 6a shows that co-culture of Chlorella with the wild-type A. brasilense Az39 strain significantly increased the accumulation of carbohydrates in the microalgae-bacteria association only at days 1 and 4 of co-culturing, while co-culture with A. brasilense Az39∆hcp-E cells had no effect on carbohydrates accumulation (Fig. 6a). Regarding lipids production, co-culture of C. sorokiniana with either A. brasilense strain significantly improved the production of lipids in the microalgae, although the wild-type induced higher production of lipids at days 1, 2, and 6, while it was higher with the mutant at day 4 (Fig. 6b). The effect of the A. brasilense T6SS on microalgae photosynthesis was indirectly measured by quantifying major and auxiliary photosynthetic pigments: chlorophyll a and b, violaxanthin, lutein and b-carotene (Fig. 7c-f). The accumulation of these photosynthetic pigments followed a similar pattern. After 1 day of co-culture, the presence of A. brasilense strains did not improve the synthesis of pigments. However, the presence of the Az39 wild-type strain increased the production of pigments by Chlorella on days 2, 4 and 6. By contrast, co-culture of Chlorella with the A. brasilense ∆hcp-E strain did not improve pigment production during the four first days.

Discussion

In this study, we provide the first characterization of a Type VI secretion system from the plant-growth promoting bacterium Azospirillum brasilense (Fig. 7). We show that the A. brasilense T6SS1 is involved in attachment to the microalgae Chlorella sorokiniana, participates to the formation of an efficient algae/bacterium consortium, and in the production of the IAA phytohormone. Through its role on attachment and IAA levels, which exert major changes on growth and the metabolism of the microalgae the T6SS indirectly allows increased lipid metabolism in the microalgae. We also reveal that the A. brasilense T6SS confers protection against bacterial phytopathogens. This study therefore demonstrates that the T6SS is an important beneficial determinant of Azospirillum/Chlorella interaction by mediating attachment, improvement of the metabolic performances and bio-control (Fig. 7).

We have shown that the T6SS participates to biofilm formation on abiotic surfaces and helps attachment of Azospirillum to Chlorella cells and formation of Chlorella clusters. In addition, we observed a role of the T6SS in IAA production. Attachment and IAA production are key in Azospirillum-plant cell interactions [START_REF] Bashan | How the plant growth-promoting bacterium Azospirillum promotes plant growth -a critical assessment[END_REF]. Attachment of Azospirillum to cells is a major, active and complex phenomenon [START_REF] Bashan | Evidence for a weak active external adsorption of Azospirillum brasilense Cd to wheat roots[END_REF][START_REF] Gafni | Adsorption of Azospirillum brasilense to corn roots[END_REF]). The initial interaction of Azospirillum spp. with plant cells it mainly mediated by attachment of the bacterium, which employs a variety of piliated appendages at the cell surface to promote stable interactions between the eukaryotic and prokaryotic cells [START_REF] Pereg | Assessment of affinity and specificity of Azospirillum for plants[END_REF]; Attachment between bacteria and microalgae was previously observed (de- [START_REF] Bashan | Development of two culture media for mass cultivation of Azospirillum spp. and for production of inoculants to enhance plant growth[END_REF][START_REF] Bashan | Increase in auxiliary photoprotective photosynthetic pigments in wheat seedlings induced by Azospirillum brasilense[END_REF][START_REF] Bashan | Chlorella sorokiniana (formerly C. vulgaris) UTEX 2714, a non-thermotolerant microalga useful for biotechnological applications and as a reference strain[END_REF], but the question remains on whether this attachment is significant for the development of this association by creating additional massive microalgae-bacteria aggregates.

Active attachment to plants and subsequent colonization were observed with the roots of cereals, tomato, pepper, cotton and soybean [START_REF] Levanony | Ultrastructural localization and identification of Azospirillum brasilense Cd on and within wheat root by immuno-gold labeling[END_REF]Bashan, Singh and Levanony 1989;[START_REF] Bashan | Root surface colonization of non-cereal crop plants by pleomorphic Azospirillum brasilense Cd[END_REF][START_REF] Levanony | Ultrastructural localization and identification of Azospirillum brasilense Cd on and within wheat root by immuno-gold labeling[END_REF][START_REF] Assmus | Situ localization of Azospirillum brasilense in the rhizosphere of wheat with fluorescently labeled, rRNA-targeted oligonucleotide probes and scanning confocal laser microscopy[END_REF]Pereg Gerk, Gilchrist and Kennedy 2000). The initial step of plant root colonization by Azospirillum spp.

necessitates flagella and exopolysaccharides to create very stable interaction between the eukaryotic and prokaryotic cells [START_REF] Pereg | Assessment of affinity and specificity of Azospirillum for plants[END_REF]. These structures are also needed for adhesion to non-living substrates [START_REF] Bashan | Active attachment of Azospirillum brasilense Cd to quartz sand and to a light-textured soil by protein bridging[END_REF]Bashan and Holguin 1993). The polar flagellum of A. brasilense, which is primarily used for swimming, is involved in the initial attachment process of the bacteria to wheat roots [START_REF] Croes | Identification and mapping of loci involved in motility, adsorption to wheat roots, colony morphology, and growth in minimal medium on the Azospirillum brasilense Sp7 90-MDa plasmid[END_REF]. Then the attachment process involves two distinct steps. The first stage is fast and corresponds to a weak adhesion and is mediated by host cell surface hydrophobicity, charges and lectins (Castellanos, Ascencio andBashan 1998, 2000). The second stage takes longer time but the attachment is irreversible. This stage is mediated by extracellular surface polysaccharides and mucigel-like substances [START_REF] Gafni | Adsorption of Azospirillum brasilense to corn roots[END_REF][START_REF] Bashan | Active attachment of Azospirillum brasilense Cd to quartz sand and to a light-textured soil by protein bridging[END_REF][START_REF] Bashan | Root surface colonization of non-cereal crop plants by pleomorphic Azospirillum brasilense Cd[END_REF][START_REF] Bashan | Root surface colonization of non-cereal crop plants by pleomorphic Azospirillum brasilense Cd[END_REF][START_REF] Michiels | Plasmid localization and mapping of two Azospirillum brasilense loci that affect exopolysaccharide synthesis[END_REF][START_REF] Puente | Root surface colonization of black mangrove seedlings by Azospirillum halofraeference and Azospirillum brasilense in seawater[END_REF]Pereg Gerk, Gilchrist and Kennedy 2000;[START_REF] Lerner | The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis[END_REF]. Other determinants of Azospirillum attachment are the flcA regulator [START_REF] Pereg Gerk | A transcriptional regulator of the LuxR-UhpA family, FlcA, controls flocculation and wheat root surface colonisation by Azospirillum brasilense Sp7[END_REF]) that is also involved in stress response and carbohydrate and nitrogen metabolism [START_REF] Hou | Cellular responses during morphological transformation in Azospirillum brasilense and its flcA knockout mutant[END_REF], chemotaxis pathways [START_REF] Bible | The Azospirillum brasilense Che1 chemotaxis pathway controls swimming velocity, which affects transient cell-to-cell clumping[END_REF][START_REF] Pereg | Assessment of affinity and specificity of Azospirillum for plants[END_REF], Cpa pili (Wisniewski-Dye et al. 2011) as well as genes related to the surface properties of Azospirillum, such as noeJ (mannose-6-phosphate isomerase) and noeL (GDPmannose 4,6-dehydratase) and pathways involved in lipopolysaccharide core processing (dTDPrhamnose biosynthesis pathway; [START_REF] Jofre | Disruption of dTDP-rhamnose biosynthesis modifies lipopolysaccharide core, exopolysaccharide production, and root colonization in Azospirillum brasilense[END_REF] or energy taxis (Greer-Phillips, Stephens and Alexandre 2004). Although the T6SS might be a new molecular determinant of the adhesion of Azospirillum to microalgae cells, it remains to define whether the effect of the T6SS mutation on adhesion is due to defect in flagellar synthesis or expression of attachment genes. While cross-talk between the T6SS and flagellar genes have been demonstrated in the P. fluorescens PGPB [START_REF] Bouteiller | Crosstalk between the type VI secretion system and the expression of class IV flagellar genes in the Pseudomonas fluorescens MFE01 strain[END_REF], the first hypothesis is unlikely as we have shown here that deletion of the hcp-tssE genes does not affect swarming rates and increases swimming rates. The second hypothesis merits investigation, as previous studies have shown that the inactivation of the T6SS may affect expression of fimbrial genes in pathogenic E. coli strains (de [START_REF] De Pace | The type VI secretion system plays a role in type 1 fimbria expression and pathogenesis of an avian pathogenic Escherichia coli strain[END_REF].

Previous studies have shown that A. brasilense enhance the growth and induce an increased accumulation of carbohydrates, starch (Choix et al., 2012a, b), fatty acids, total lipids [START_REF] Leyva | Accumulation of fatty acids in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoA carboxylase, temperature, and co-immobilization with Azospirillum brasilense[END_REF][START_REF] Peng | Indole-3-acetic acid from Azospirillum brasilense promotes growth in green algae at the expense of energy storage products[END_REF] and pigments (de- [START_REF] De-Bashan | Increased pigment and lipid content, lipid variety, and cell and population size of the microalgae Chlorella spp. when co-immobilized in alginate beads with the microalgae-growthpromoting bacterium Azospirillum brasilenses[END_REF][START_REF] Peng | Indole-3-acetic acid from Azospirillum brasilense promotes growth in green algae at the expense of energy storage products[END_REF] in Chlorella spp. The effect has been mostly attributed to the IAA production by the bacteria (de- Bashan and Bashan, 2008;[START_REF] De-Bashan | Cell-cell interaction in the eukaryote-prokaryote model using the microalgae Chlorella vulgaris and the bacterium Azospirillum brasilense immobilized in polymer beads[END_REF][START_REF] De-Bashan | Interaction of Azospirillum spp. with microalgae; a basic eukaryotic-prokaryotic model and its biotechnological applications[END_REF][START_REF] Peng | Indole-3-acetic acid from Azospirillum brasilense promotes growth in green algae at the expense of energy storage products[END_REF]. Since the concentration of IAA produced by the wild-type and T6SS A. brasilense strains is similar at the times of the experiments with the microalgae (after 30 hours), the reduction of lipids and photosynthetic pigments induced by the T6SS mutant can be attributed to its lower attachment capacity. It was previously shown that attachment is not essential for C and N compounds transfer between individual cells of C. sorokiniana and A. brasilense (de- [START_REF] Bashan | Chlorella sorokiniana (formerly C. vulgaris) UTEX 2714, a non-thermotolerant microalga useful for biotechnological applications and as a reference strain[END_REF]; however, the metabolites produced by Azospirillum, might be transient and probably a close cell proximity is required to achieve a more effective mass transfer [START_REF] Peng | Indole-3-acetic acid from Azospirillum brasilense promotes growth in green algae at the expense of energy storage products[END_REF].

We have also shown that mutation in the A. brasilense T6SS affects IAA production. IAA, as a phytohormone, has no important role in bacterial metabolism [START_REF] Spaepen | Auxin and plant-microbe interactions[END_REF], but is widespread among PGPB [START_REF] Duca | Indole-3-acetic acid in plantmicrobe interactions[END_REF] and increases plant growth. The production of IAA during the interaction between Azospirillum spp. and Chlorella spp. and its effect on growth was reported several times (de- Bashan et al., 2008;Palacios et al., 2016a, b, Peng et al., 2020).

The role of the T6SS in Azospirillum/plant interactions suggests that the expression of this cluster and/or the activity of the machine is responsive to environmental or plant cues. It would be therefore interesting to determine what are the environmental signals in addition to IAA [START_REF] Van Puyvelde | Transcriptome analysis of the rhizosphere bacterium Azospirillum brasilense reveals an extensive auxin response[END_REF], and to identify the regulators that control the expression of this T6SS gene cluster.

The role of the A. brasilense T6SS in plant/bacterium interactions is supported by the observation that T6SS genes are up-regulated in presence of the IAA phytohormone [START_REF] Van Puyvelde | Transcriptome analysis of the rhizosphere bacterium Azospirillum brasilense reveals an extensive auxin response[END_REF]. However, contrarily to T2SS or T3SS present in plant-associated bacteria, the A. brasilense T6SS is not deployed for pathogenic purposes. Interestingly, T6SS gene clusters are conserved in proteobacterial and bacteroidetes genomes, including non-pathogenic bacteria such as gut symbiots [START_REF] Wexler | Human symbionts inject and neutralize antibacterial toxins to persist in the gut[END_REF], soil and marine bacteria. The presence of a T6SS in these strains suggests that the T6SS could be used for promoting commensal or mutualistic relationships between bacteria and eukaryotes [START_REF] Jani | Type VI secretion: not just for pathogenesis anymore[END_REF][START_REF] Konovalova | Extracellular biology of Myxococcus xanthus[END_REF][START_REF] Jiménez-Guerrero | Bacterial protein secretion systems. Implications in beneficial associations with plants[END_REF]. Indeed, the Rhizobium leguminosarum bv. trifolii T6SS has a role in the nodulation process by blocking the colonization/infection process of nonhost peas [START_REF] Roest | A Rhizobium leguminosarum biovar trifolii locus not localized on the sym plasmid hinders effective nodulation on plants of the pea cross-inoculation group[END_REF][START_REF] Bladergroen | Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion[END_REF]. Whereas it has been proposed that RbsB is a T6SS effector delivered by R. leguminosarum into pea cells [START_REF] Bladergroen | Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion[END_REF], the effector(s) delivered by the A. brasilense T6SS into plant/algae cells remain to be identified. Finally, our data show that the T6SS provides antagonistic activities to A. brasilense against a number of bacterial strains including phytopathogens such as Agrobacterium, Pectobacterium, Dickeya and Ralstonia in vitro. This demonstrates that, in addition to promoting growth, A. brasilense likely confers protection against harmful pathogens in a T6SS-dependent manner.

Effectors with DNase, phospholipase and peptidoglycan hydrolase activities have been shown to be delivered into bacterial cells (Russel et al., 2014;Jurenas and Journet, 2021). The A. brasilense T6SS effector repertoire needs to be determined. It has been shown that the effector genes are usually located within the T6SS gene cluster or within hcp/vgrG islands scattered in the genome [START_REF] Durand | Tle, and beyond: the versatile arsenal of Type VI secretion effectors[END_REF]. Indeed, the vgrG gene encoded within the cluster is fused to an additional domain resembling Bacillus subtilis YwqJ, a potent toxin with putative deaminase activity, and is followed by a gene resembling its cognate YwqK antitoxin [START_REF] Brantl | Toxin-Antitoxin Systems in Bacillus subtilis[END_REF][START_REF] Kobayashi | Diverse LXG toxin and antitoxin systems specifically mediate intraspecies competition in Bacillus subtilis biofilms[END_REF] (Fig. 1).

In summary, the Chlorella spp.-Azospirillum spp. association has been used as a model system to study aggregation, metabolic, and molecular effect occurring during plant-bacteria interaction (de- Bashan and Bashan, 2008). The present work extends the potential to study a molecular mechanism occurring between a bacterium and a microalga, specifically by showing that a T6SS mutant is also deficient in attachment capabilities and produces less microalgal aggregation. This T6SS mutant, while promoting the growth of microalgae similar to its parental wild-type A. brasilense strain also impacts lipid, and pigment metabolism. This study has thus far broader impacts than observing interactions between microalgae and bacteria in vitro, by demonstrating that a widespread secretion system, known almost exclusively from studies of pathogenesis and antibacterial competition, is participating in processes that are beneficial to the plant/microalga cells.

Experimental procedures

Microorganisms and growth conditions

The unicellular microalga Chlorella sorokiniana Shih. et Krauss (UTEX 2714, University of Texas, Austin, TX; formerly C. vulgaris UTEX 2714, [START_REF] Bashan | Chlorella sorokiniana (formerly C. vulgaris) UTEX 2714, a non-thermotolerant microalga useful for biotechnological applications and as a reference strain[END_REF]) was used in this study.

C. sorokiniana was axenically grown at 27 ± 2 °C, with stirring at 120 rpm under a light intensity amplified by PCR from genomic DNA using primer pairs Az39-5′HcpFW/Az39-B-5′HcpRV and Az39-B-3′HcpFW/Az39-3′HcpRV, respectively (Table S2) cloned into the pGemT-Easy vector (Promega, Madison, WI) and sequenced. The upstream fragment was inserted into the downstream fragment-containing plasmid, using BamHI, and PstI restriction enzymes to yield the pGemT-Hcp-UD vector. Then, a gentamicin-resistance cassette (Gm R ), excised from pME3280a [START_REF] Zuber | GacS sensor domains pertinent to the regulation of exoproduct formation and to the biocontrol potential of Pseudomonas fluorescens CHA0[END_REF] with XhoI, was inserted in reverse orientation into the BamHI site of pGemT-Hcp-UD after blunting ends with T4 DNA polymerase (New England Biolabs, Ipswich, MA). The resulting upstream-Gm R -downstream construction was finally sub-cloned into pK18mobSacB [START_REF] Schäfer | Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum[END_REF], using PstI and SphI to obtain pK18-Hcp-UGD. This suicide plasmid, carrying the mutagenesis cassette, was mobilized into A. brasilense Az39 by biparental mating with Escherichia coli S17-1 as the donor [START_REF] Simon | A broad host range mobilization system for in vivo genetic-engineering -transposon mutagenesis in gram-negative bacteria[END_REF]. Double recombinant clones, resistant to gentamicin and sensitive to kanamycin, were recovered without the need for sacB counter selection. To confirm the correct replacement of the targeted genome region, PCR was used with several primer combinations, and the amplified fragments were visualized by TAE-agarose electrophoresis. The phenotypes of the mutant were analyzed in three independent clones (C1, C2, and C3) to exclude the presence of additional mutations (Fig. S1). Fig. S1 describes the genomic arrangement of the wild-type, the two possible simple recombinants (R1 or R2), and the double recombinant (R1+R2) of A. brasilense Az39. The site of annealing and the product length of the different diagnostic primers (P1, P2, P3, P4, P5, P6, GmR, and GmF) on the four possible arrangements are indicated. The inserted table indicates the PCR products expected for each strain, and the agarose gel shows the actual results of the PCR. A PCR was performed using P3 and P6 primers that uniquely anneal on the genomic region that flanks the modified region.

Bacterial Growth -Growth curves of A. brasilense Az39 and mutant strain Az39Dhcp-E were determined by measuring the turbidity of cell suspensions (OD595) and cell number (CFU.mL -1 ) over time. Sample of 0.5 mL of an overnight culture (OD595 = 1.0, ~10 9 CFU×mL -1 ) were inoculated to 200-mL Erlenmeyer flasks containing 50 mL of Luria-Bertani medium (LB) or MMAB minimal medium [START_REF] Vanstockem | Transposon mutagenesis of Azospirillum brasilense and Azospirillum lipoferum: physiological analysis of Tn5 and Tn5-mob insertional mutants[END_REF] supplemented with 100 µg×mL -1 of Ltryptophan. Cultures were incubated at 37 °C and constant stirring at 180 rpm for 80 h. Turbidity of cell suspensions was spectrophotometrically measured at 595 nm over time (ZelTec ZL5000P, Zel Technologies, Hampton, VA) at 5 h intervals. At the same time, cell counts were performed on LB agar plates supplemented with Congo red (1.5%, v:v), after incubation for 72 h at 37 °C.

Indole-3-acetic acid production -Pure cultures of A. brasilense Az39 and its ∆hcp-E derivative were cultivated in LB and MMAB media, supplemented with L-tryptophan, at 37 °C, under constant stirring at 180 rpm for 48 h corresponding to stationary stage of growth of this species.

Cultures were centrifuged at 15,650 × g for 10 min and cells were discarded. Quantification of IAA was performed by spectrophotometry [START_REF] Glickmann | A Critical Examination of the specificity of the Salkowski Reagent for indolic compounds produced by phytopathogenic bacteria[END_REF] and confirmed by HPLC [START_REF] Rivera | New insights into indole-3-acetic acid metabolism in Azospirillum brasilense[END_REF]. Briefly, aliquots of 1000 µL of bacterial culture were centrifuged at 11,300 × g for 10 min. Subsequently, samples were filtered (0.2 µm), and 500 µL of supernatant were mixed with 500 µL of Salkowski´s reagent (7.9 M H2SO4 and 12.5 g•L -1 FeCl3) and gently shaken in inverted position at least 10 times. Samples were incubated in the dark for 30 min and the absorbance at 530 nm was measured. An aliquot of filtered supernatants was injected with a final volume of 20 µL in an HPLC Waters 600-MS device (Waters Inc., USA) equipped with an U6K injector and C18 reverse phase column (Purospher STAR RP C-18 3 mm, Lichrocart 55-4) heated at 30°C, coupled to a system with UV-VIS Waters 486 detector (Waters Inc., USA) set at 265 nm. The elution was performed with a mixture of ethanol: acetic acid: water (Et-OH/H-Ac/H20) (12: 1: 87) as mobile phase at a flow rate of 1 mL•min-1 at 30°C. The retention time for IAA was 10.1-10.3 minutes and the quantification was performed by integration of the peak area corresponding to the retention time (RT) using an integration software (Waters Inc. USA). The IAA concentration was expressed in μg•mL -1 Motility -Motility was assayed under different concentrations of agar-agar: 1 µL of bacterial culture in late exponential phase was placed in the center of Petri dishes containing minimal swim motility medium [START_REF] Atkinson | Quorum sensing in Yersinia enterocolitica controls swimming and swarming motility[END_REF] and MMAB medium as previously described [START_REF] Vanstockem | Transposon mutagenesis of Azospirillum brasilense and Azospirillum lipoferum: physiological analysis of Tn5 and Tn5-mob insertional mutants[END_REF]. Minimal swim motility agar plates contained (g×L -1 ) tryptone (10.0) and NaCl (5.0). Both media were prepared with 0.3% (w/v) agar to observe swimming and 0.7% (w/v) agar for determining swarming. The plates were inverted and incubated at 28 ± 2 °C for 72 h to avoid drying the agar at higher temperatures. The diameter of the displacement halo was then measured [START_REF] Atkinson | A hierarchical quorum sensing system in Yersinia pseudotuberculosis is involved in the regulation of motility and clumping[END_REF][START_REF] Hall | Swarming of Azospirillum brasilense on solid media[END_REF].

Cell aggregation and biofilm production -Cell aggregation was measured according to [START_REF] Madi | Aggregation in Azospirillum brasilense Cd: conditions and factors involved in cell-to-cell adhesion[END_REF] with modifications [START_REF] Burdman | Aggregation in Azospirillum brasilense: effects of chemical and physical factors and involvement of extracellular components[END_REF]. Briefly, 5 mL of stationary cultures of both strains obtained in MMAB and LB culture medium at 37°C and constant stirring at 180 rpm for 48 hours were transferred to 10 mL capacity conical tubes and allowed to stand for 20 min.

Then, turbidity was measured at 540 nm using a Zeltec ZL5000P spectrophotometer (OD1). The culture was homogenized for 1 min and the turbidity was measured again (OD2). The aggregation percentage was calculated according to the following equation %AP (OD2 -OD1) × 100/OD2. Biofilm formation was measured according to O'Toole & Kolter (1998). Briefly, 13 µL of late exponential cultures were inoculated in hemolysis tubes containing 1,300 µL of sterile LB medium and incubated at 37 ± 1°C for 48, 72, and 96 h, without stirring. At each sampling time, the culture medium was carefully removed from the tube by micropipetting, leaving only the biofilm. The biofilm was rinsed three times with 1,300 µL of sterile 0.85 % saline solution. Once the culture medium was removed, the biofilm was stained with 0.1% crystal violet (C3886, Sigma-Aldrich) solution (1,300 µL for 15 min at room temperature, 24 ± 2 °C). The stained biofilm was rinsed three times with sterile distilled water. The biofilm was re-suspended in 1,300 µL of 96% ethanol containing three glass beads (3 mm diam) in each tube and stirred vigorously by a vortex for ~1 min. Finally, absorbance was measured at 560 nm by spectrophotometry (ZelTec ZL5000P, Zel Technologies).

Anti-bacterial competition assay.

Spontaneous Agrobacterium tumefaciens, Pectobacterium carotovorum, Dickeya dadantii, Ralstonia eutropha and Ralstonia solanacearum strains resistant to nalidixic acid were obtained after 5 consecutive liquid growth cultures in presence of increasing concentrations of nalidixic acid (2, 5, 10, 20 and 30 µM) and plating on agar plates supplemented with 40 µM nalidixic acid.

Azospirillum brasilense and nalidixic acid-resistant recipient cells were grown in MMAB supplemented -or not -with 400 µM indole-3-acetic acid (IAA) to an OD595 ~1 and concentrated in MMAB to a final OD595 of 10. A. brasilense attacker cells were mixed with nalidixic resistant recipient cells at a 4:1 ratio and 15-µl drops of the mixture were spotted on dried minimum MMAB or MMAB/IAA agar plates. After incubation for 8 h at 28°C, the spots were scratched off, cells were re-suspended in LB to an OD595 of 0.5, and the surviving recipient cells were counted after platting serial dilutions on LB agar plates supplemented with 40 µM nalidixic acid.

Immobilization of microorganisms in alginate beads

Before immobilization in alginate beads, 10 mL of axenic microalgae culture was inoculated into 90 mL sterile C30 medium and incubated at 27 ± 2 °C, with stirring at 120 rpm under 60 µmol photons×m -2 ×s -1 light intensity for six days. The two strains of A. brasilense were grown in BTB culture medium containing gluconic acid as a carbon source [START_REF] Bashan | Development of two culture media for mass cultivation of Azospirillum spp. and for production of inoculants to enhance plant growth[END_REF] for 18 h, at 35 ± 2 °C and stirred at 120 rpm. Immobilization procedure was done as described by [START_REF] De-Bashan | Interaction of Azospirillum spp. with microalgae; a basic eukaryotic-prokaryotic model and its biotechnological applications[END_REF]. Briefly, after growth period, each culture was harvested by centrifugation (Hemle Z 200A, Wehingen, Germany) at 2,000 × g, washed twice, resuspended in sterile saline solution (0.85% NaCl) and cell density was adjusted to 5×10 6 cells•mL -1 for microalgae and ~10 9 CFU× mL -1 (OD540 nm = 1) for bacteria. Then, 40 mL of axenic cultures of C. sorokiniana or A. brasilense Az39 or A. brasilense Az39∆hcp-E were mixed with 160 mL of sterile 1.5% alginate solution (~1200 cP, #05218295, MP Biomedicals, Santa Ana, CA) and stirred for 15 min until the alginate slurry was completely dissolved. Beads (highly homogenous; 3 mm diam) were produced in 2% CaCl2, using automatic bead-forming equipment under constant pressure to ensure uniformity of each bead (de- [START_REF] Bashan | How the plant growth-promoting bacterium Azospirillum promotes plant growth -a critical assessment[END_REF]. The beads were cured for 30 min in the same 2% CaCl2 solution to improve hardening. They were washed in sterile 0.85% saline solution. This procedure routinely produces ~1×10 6 cells×bead -1 for each organism with low variability (de- [START_REF] De-Bashan | Microalgae growthpromoting bacteria as "helpers" for microalgae: a novel approach for removing ammonium and phosphorus from municipal wastewater[END_REF]. For microalgae and bacteria co-immobilized in the same bead, 20 mL of each culture was mixed in 160 mL alginate to form the beads, as previously described. Because immobilization usually reduces the number of A. brasilense in beads, the beads were incubated again for 24 h in 10% (v/v) diluted nutrient broth (N7519, Sigma-Aldrich) to reach an initial population of 1×10 6 cells×bead -1 .

Experimental conditions to assess the performance of microalgae.

After the second incubation, beads were washed twice with sterile saline solution. In preparation of each experiment, 15 g of beads from each treatment (microorganisms immobilized alone or coimmobilized) were added to 500 mL Erlenmeyer flasks containing 200 mL of synthetic growth medium (SGM) with the following ingredients (in mg•L -1 ): NaCl (7), CaCl2 (4), MgSO4•7H2O

(2), K2HPO4 (217), KH2PO4 (8.5), Na2HPO4 (33.4),NH4Cl (191) and incubated under 90 µmol photon•m -2 s -1 light intensity and stirred at 122 rpm at 29 ± 2 °C for 6 d.

Quantifying microalgae cells

In each experiment for each sampling time, and each experimental replicate, three beads per flask were solubilized in 1 mL of 4% NaHCO3 solution for 20 min at ambient temperature (25 ± 4°C).

C. sorokiniana cells were counted under a light microscope, using a Neubauer hemocytometer (Bright line counting chamber, Hausser Scientific, Horsham, PA) connected to an image analyser (Image ProPlus 6.3, Media Cybernetics, Silver Spring, MD). Growth rate of C. sorokiniana (µ) was defined as: µ = (ln Nt1 -ln Nt0) / (t1 -t0), where Nt1 is the number of cells at sampling time, and Nt0 is the number of cells at the beginning of the experiment, t1 is sampling time and t0 the beginning of the experiment [START_REF] Oh-Hama | Chlorella. In Micro-algae Biotechnology[END_REF].

Analytical methods

Total carbohydrates

For each replicate of each treatment (n = 15), carbohydrates extraction and analysis were done following Choix et al. (2012a). Briefly, 1 g of alginate beads were sampled after 1, 2, 4, and 6 d, washed in distilled water, dried at 80 •C for 12 h, and ground with a mortar and pestle, which yielded 10 mg samples. These 10 mg ground samples were re-suspended in 5 mL of H2SO4 1 M and sonicated for 4 min at 22.5 kHz with an ultrasonic cell disruptor (Misonix, Farmingdale, NY).

Carbohydrates were extracted by acid hydrolysis of the sample at 100ºC for 60 min. The quantification was done by the phenol-sulfuric method adapted to microplate [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF][START_REF] Masuko | Carbohydrate analysis by a phenol-sulfuric acid method in microplate format[END_REF].

Total lipid analysis

Lipid extraction followed the method of [START_REF] Leyva | Accumulation of fatty acids in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoA carboxylase, temperature, and co-immobilization with Azospirillum brasilense[END_REF], which was developed for microalgae and based on [START_REF] Bligh | A rapid method for total lipid extraction and purification[END_REF]. Quantification of total lipids was done as previously described [START_REF] Pande | Microdetermination of lipids and serum total fatty acids[END_REF].

Pigment analysis

Extraction of pigments (chlorophyll a and b, violaxanthin, and lutein) was performed with HPLC grade methanol, after dissolving the beads. Identification of pigments used the HPLC method [START_REF] Vidussi | Determination of chlorophylls and carotenoids of marine phytoplankton: separation of chlorophyll a from divinyl-chlorophyll a and zeaxanthin from lutein[END_REF]. The HPLC system (Agilent 1100; Agilent Technologies, Santa Clara, CA) was equipped with a reverse phase column (Hypersil BDS C8, 5 µm particle size, 100×45 mm; Thermo Scientific, Waltham, MA) and was run isocratically. The mobile phase included two solutions. Solution A was a mixture of 70:30 methanol:1N ammonium acetate. Solution B was 100% HPLC grade methanol. Injection volume was 20 µL; flow rate was 0.5 mL•min -1 . The detector was a diode array with a wavelength of 190-900 nm, with the capacity to detect at five fixed wavelengths. Identification criteria for pigments were retention time of the standard pigments (International Agency for 14 C determinations, Denmark, DHI-Denmark, PPS-CHLA) and absorbance at 350-750 nm from the diode detector. Results are expressed as µg×g -1 .

Fluorescent in situ hybridization (FISH)

The FISH procedure described by [START_REF] Palacios | Early changes in nutritional conditions affect formation of synthetic mutualism between Chlorella sorokiniana and the bacterium Azospirillum brasilense[END_REF], was used to analyze the patterns of cell aggregation during the interaction of C. sorokiniana and A. brasilense strains. Approximately 100 images were acquired with a digital camera (Evolution FV Cooled Color; Media Cybernetics).

Five random images per treatment were used for quantification and measurement of clusters with an Image ProPlus 4.5 software (Media Cybernetics).

Experimental design and statistical analyses

  Two types of experiments were conducted. (a) In vitro characterization of the two bacteria, strains were performed in batch culture in four replicates. Each experiment was independently repeated three times. (b) The setup of the experiments of microalgae-bacteria was by batch culturing in Erlenmeyer flasks in five replicates, where one flask served as a replicate. Each of these experiments was independently repeated two times. Each experimental setup contained the following treatments in beads containing: (1) A. brasilense Az39, (2) A. brasilense Az39∆hcp-E, (3) C. sorokiniana, (4) co-immobilization of C. sorokiniana with A. brasilense Az39, (5) coimmobilization of C. sorokiniana with the mutant strain Az39∆hcp-E (n = 20). The results from each treatment from two repetitions were combined. For characterization of the mutant strain, each pair of values at every sampling time were analyzed by Student's t-test. For analysis of the effect of A. brasilense strains, the data were first analyzed by one-way ANOVA and then by HSD

Fig. 1 .

 1 Fig. 1. Schematic representation of the Azospirillum brasilense Az39 T6SS1 gene cluster.

Fig. 3 .

 3 Fig. 3. Swimming and swarming motility, biofilm formation and cell aggregation. Swimming

Table 1 . Aggregation percentage of A. brasilense Az39 wild-type and its isogenic Az39Δhcp-E mutant.

 1 Cell aggregation was measured after growth for 48 h in LB or MMAB culture medium. The reduction of the aggregation percentage in the Az39Δhcp-E relative to its parental Az39 strain is indicated below. Different letters differ significantly at P<0.05 (Tukey's post-hoc test).

	Strain

*

Acknowledgments

Funding

This study was supported by the CONICET (PPI-112-201001-00454-KA1) and FONCyT (PICT-2012-1051) of Argentina; by Consejo Nacional de Ciencia y Tecnología of Mexico (CONACYT-Basic Science-2015, contract 251102 and CONACYT Basic Science-2017, contract 284562), and by the Bashan Institute of Science of the USA. Work in EC laboratory is supported by the CNRS, the Aix-Marseille University, and the Fondation Bettencourt-Schueller.

CA), Prism 5.0 (GraphPad Software, La Jolla, CA), and Sigmaplot 13.0 (Systat Software, Chicago, IL).

Disclosure Statement

No potential conflict of interest was reported by the authors. Treatments analyzed at each hour denoted by different lowercase letter differ significantly at P < 0.05 in one-way analysis of variance, according to Tukey's post hoc analysis. Different capital letter at each day of incubation differs significantly at P < 0.05 in Tukey's post hoc analysis. Azospirillum/Azospirillum interaction: the T6SS is required for biofilm formation and indole-3acetic acid (IAA, purple triangles) production. IAA positively regulates the expression of the A. brasilense T6SS1 gene cluster. Azospirillum/bacteria interactions: A. brasilense uses its T6SS to eliminate bacteria sharing the same niche (blue cell). By targeting plant pathogens, it confers protection to plants and microalgae.

Author's contribution