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House flies (Musca domestica) are widespread, synanthropic filth flies commonly found on decaying matter, garbage, and feces as well as human food. They have been shown to vector microbes, including clinically relevant pathogens. Previous studies have demonstrated that house flies carry a complex and variable prokaryotic microbiota, but the main drivers underlying this variability and the influence of habitat on the microbiota remain understudied. Moreover, the differences between the external and internal microbiota and the eukaryotic components have not been examined. To obtain a comprehensive view of the fly microbiota and its environmental drivers, we sampled over 400 flies from two geographically distinct countries (Belgium and Rwanda) and three different environments—farms, homes, and hospitals. Both the internal as well as external microbiota of the house flies were studied, using amplicon sequencing targeting both bacteria and fungi. Results show that the house fly’s internal bacterial community is very diverse yet relatively consistent across geographic location and habitat, dominated by genera Staphylococcus and Weissella. The external bacterial community, however, varies with geographic location and habitat. The fly fungal microbiota carries a distinct signature correlating with the country of sampling, with order Capnodiales and genus Wallemia dominating Belgian flies and genus Cladosporium dominating Rwandan fly samples. Together, our results reveal an intricate country-specific pattern for fungal communities, a relatively stable internal bacterial microbiota and a variable external bacterial microbiota that depends on geographical location and habitat. These findings suggest that vectoring of a wide spectrum of environmental microbes occurs principally through the external fly body surface, while the internal microbiome is likely more limited by fly physiology.

The Casparian strip (CS) constitutes a physical diffusion barrier to water and nutrients in plant roots, which is formed by the polar deposition of lignin polymer in the endodermis tissue. The precise pattern of lignin deposition is determined by the scaffolding activity of membrane-bound Casparian Strip domain proteins (CASPs), but little is known of the mechanism(s) directing this process. Here, we demonstrate that Endodermis-specific Receptor-like Kinase 1 (ERK1) and, to a lesser extent, ROP Binding Kinase1 (RBK1) are also involved in regulating CS formation, with the former playing an essential role in lignin deposition as well as in the localization of CASP1. We show that ERK1 is localized to the cytoplasm and nucleus of the endodermis and that together with the circadian clock regulator, Time for Coffee (TIC), forms part of a novel signaling pathway necessary for correct CS organization and suberization of the endodermis, with their single or combined loss of function resulting in altered root microbiome composition. In addition, we found that other mutants displaying defects in suberin deposition at the CS also display altered root exudates and microbiome composition. Thus, our work reveals a complex network of signaling factors operating within the root endodermis that establish both the CS diffusion barrier and influence the microbial composition of the rhizosphere. ispartof: PLANT AND CELL PHYSIOLOGY vol:62 issue:2 pages:248-261 ispartof: location:Japan status: published

BackgroundMineral P fertilisers must be used wisely in order to preserve rock phosphate, a limited and non-renewable resource. The use of bio-inoculants to improve soil nutrient availability and trigger an efficient plant response to nutrient deficiency is one potential strategy in the attempt to decrease P inputs in agriculture.MethodA gnotobiotic co-cultivation system was used to study the response ofBrachypodium distachyonto contrasted P supplies (soluble and poorly soluble forms of P) and inoculation with P solubilizing bacteria.Brachypodium’s responses to P conditions and inoculation with bacteria were studied in terms of developmental plasticity and P use efficiency.ResultsBrachypodiumshowed plasticity in its biomass allocation pattern in response to variable P conditions, specifically by prioritizing root development over shoot productivity under poorly soluble P conditions. Despite the ability of the bacteria to solubilize P, shoot productivity was depressed in plants inoculated with bacteria, although the root system development was maintained. The negative impact of bacteria on biomass production inBrachypodiummight be attributed to inadequate C supply to bacteria, an increased competition for P between both organisms under P-limiting conditions, or an accumulation of toxic bacterial metabolites in our cultivation system. Both P and inoculation treatments impacted root system morphology. The modulation ofBrachypodium’s developmental response to P supplies by P solubilizing bacteria did not lead to improved P use efficiency.ConclusionOur results support the hypothesis that plastic responses ofBrachypodiumcultivated under P-limited conditions are modulated by P solubilizing bacteria. The considered experimental context impacts plant–bacteria interactions. Choosing experimental conditions as close as possible to real ones is important in the selection of P solubilizing bacteria. Both persistent homology and allometric analyses proved to be useful tools that should be considered when studying the impact of bio-inoculants on plant development in response to varying nutritional context.

Bradyrhizobium japonicum E109 is a bacterium widely used for inoculants production in Argentina. It is known for its ability to produce several phytohormones and degrade indole-3-acetic acid (IAA). The genome sequence of B. japonicum E109 was recently analyzed and it showed the presence of genes related to the synthesis of IAA by indole-3-acetonitrile, indole-3-acetamide and tryptamine pathways. Nevertheless, B. japonicum E109 is not able to produce IAA and instead has the ability to degrade this hormone under saprophytic culture conditions. This work aimed to study the molecular and physiological features of IAA degradation and identify the genes responsible of this activity. In B. japonicum E109 we identified two sequences coding for a putative 3-phenylpropionate dioxygenase (subunits α and β) responsible for the IAA degradation that were homologous to the canonical cluster of iac C and iacD of Pseudomonas putida 1290. These genes form a separate cluster together with three additional genes with unknown functions. The degradation activity was found to be constitutively expressed in B. japonicum E109. As products of IAA degradation, we identified two compounds, 3-indoleacetic acid 2,3-oxide and 2-(2-hydroperoxy-3-hydroxyindolin-3-yl) acetic acid. Our report proposes, for the first time, a model for IAA degradation in Bradyrhizobium.

A phylogenomic analysis based on 107 single-copy core genes revealed that three strains from sugar-rich environments, i.e. LMG 1728T, LMG 1731 and LMG 22058, represented a single, novel Gluconacetobacter lineage with Gluconacetobacter liquefaciens as nearest validly named neighbour. OrthoANIu and digital DNA–DNA hybridization analyses among these strains and Gluconacetobacter type strains confirmed that the three strains represented a novel Gluconacetobacter species. Biochemical characteristics and MALDI-TOF mass spectra allowed differentiation of this novel species from the type strains of G. liquefaciens and other closely related Gluconacetobacter species. We therefore propose to classify strains LMG 1728T, LMG 1731 and LMG 22058 in the novel species Gluconacetobacter dulcium sp. nov., with LMG 1728T (=CECT 30142T) as the type strain.

Significance Plants are exposed to conflicting stresses simultaneously in nature. As stress responses are costly, plants likely coordinate these responses to minimize fitness costs. The nature and extent to which plants employ inducible mechanisms to cope with combined physical and biological stresses remains unknown. We identify a genetic mechanism by which leaves of distinct ages differentially control stress-response cross-talk. At the organism level, this mechanism balances stress-response trade-offs to maintain plant growth and reproduction during combined stresses. We also show that this leaf age-dependent stress-response prioritization influences the establishment of plant-associated leaf bacterial communities. This study illustrates the importance of active balancing of stress-response trade-offs for plant fitness maintenance and for interaction with the plant microbiota., In nature, plants must respond to multiple stresses simultaneously, which likely demands cross-talk between stress-response pathways to minimize fitness costs. Here we provide genetic evidence that biotic and abiotic stress responses are differentially prioritized in Arabidopsis thaliana leaves of different ages to maintain growth and reproduction under combined biotic and abiotic stresses. Abiotic stresses, such as high salinity and drought, blunted immune responses in older rosette leaves through the phytohormone abscisic acid signaling, whereas this antagonistic effect was blocked in younger rosette leaves by PBS3, a signaling component of the defense phytohormone salicylic acid. Plants lacking PBS3 exhibited enhanced abiotic stress tolerance at the cost of decreased fitness under combined biotic and abiotic stresses. Together with this role, PBS3 is also indispensable for the establishment of salt stress- and leaf age-dependent phyllosphere bacterial communities. Collectively, our work reveals a mechanism that balances trade-offs upon conflicting stresses at the organism level and identifies a genetic intersection among plant immunity, leaf microbiota, and abiotic stress tolerance.

The capacity of plant growth-promoting rhizobacteria (PGPR) – Bacillus amyloliquefaciens GB03 (BamGB03), B. megaterium SNji (BmeSNji), and Azospirillum brasilense 65B (Abr65B) – to enhance growth and nutrient uptake in wheat was evaluated under different mineral N fertilizer rates, in sterile and non-sterile soils, and at different developmental stages. In gnotobiotic conditions, the three strains significantly increased plant biomass irrespective of the N rates. Under greenhouse conditions using non-sterile soil, growth promotion was generally highest at a moderate N rate, followed by a full N dose, while no significant effect was observed for the inoculants in the absence of N fertilizer. At 50N, plant biomass was most significantly increased in roots (up to +45% with Abr65B) at stem-elongation stage and in the ears (+19–23% according to the strains) at flowering stages. For some nutrients (N, P, Mn, and Cu), the biomass increases in roots and ears were paralleled with reduced nutrient concentrations in the same organs. Nevertheless, growth stimulation resulted in a higher total nutrient uptake and higher nutrient uptake efficiency. Furthermore, Abr65B and BmeSNji counteracted the reduction of root development caused by a high N supply. Therefore, combining PGPR with a proper cultivated system, N rate, and plant stage could enhance their biostimulant effects.

Plant survival depends on the ability of roots to sense and acquire nutrients in soils, which harbor a rich diversity of microbes. A subset of this microcosm interacts with plant roots and collectively forms root-associated microbial communities, termed the root microbiota. Under phosphorus-limiting conditions, some plants can engage in mutualistic interactions, for example with arbuscular mycorrhizal fungi. Here, we describe how Arabidopsis thaliana, which lacks the genetic capacity for establishing the aforementioned symbiosis, interacts with soil-resident bacteria and fungi in soil from a long-term phosphorus fertilization trial. Long-term, contrasting fertilization regimes resulted in an ∼6-fold and ∼2.4-fold disparity in bioavailable and total phosphorous, respectively, which may explain differences in biomass of A. thaliana plants. Sequencing of marker genes enabled us to characterize bacterial and fungal communities present in the bulk soil, rhizosphere, and root compartments. Phosphorus had little effect on alpha- or beta-diversity indices, but more strongly influences bacterial and fungal community shifts in plant-associated compartments compared with bulk soil. The significant impact of soil P abundance could only be resolved at operational taxonomic unit level, and these subtle differences are more pronounced in the root compartment. We conclude that despite decades of different fertilization, both bacterial and fungal soil communities remained unexpectedly stable in soils tested, suggesting that the soil biota is resilient over time to nutrient supplementation. Conversely, low-abundance, root-associated microbes, which collectively represent 2 to 3% of the relative abundance of bacteria and fungi in the roots, exhibited a subtle, yet significant shift between the two soils. [Formula: see text] Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

The innate immune system of plants recognizes microbial pathogens and terminates their growth. However, recent findings suggest that at least one layer of this system is also engaged in cooperative plant-microbe interactions and influences host colonization by beneficial microbial communities. This immune layer involves sensing of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) that initiate quantitative immune responses to control host-microbial load, whereas diversification of MAMPs and PRRs emerges as a mechanism that locally sculpts microbial assemblages in plant populations. This suggests a more complex microbial management role of the innate immune system for controlled accommodation of beneficial microbes and in pathogen elimination. The finding that similar molecular strategies are deployed by symbionts and pathogens to dampen immune responses is consistent with this hypothesis but implies different selective pressures on the immune system due to contrasting outcomes on plant fitness. The reciprocal interplay between microbiota and the immune system likely plays a critical role in shaping beneficial plant-microbiota combinations and maintaining microbial homeostasis.

The Casparian Strip (CS) constitutes a physical diffusion barrier to water and nutrients in plant roots, and is formed by the polar deposition of lignin polymer in the endodermis. This precise pattern of lignin deposition is thought to be mediated by the scaffolding activity of membrane-bound Casparian Strip domain proteins (CASPs). However, we show that endodermis-specific receptor-like kinase 1 (ERK1) and ROP Binding Kinase1 (RBK1) are also involved in this intricate process, with the former playing an essential role both in the localization of CASP1 and in lignin deposition. We further characterised ERK1 and determined its subcellular localisation in the cytoplasm and nucleus of the endodermis, as well as provide evidence for its involvement in a signalling pathway together with the circadian clock regulator, Time for Coffee (TIC). We also show that disruption to CS organisation and increased suberisation in the endodermis due to loss of function of eitherERK1orTICcollectively leads to an altered root microbiome composition. Thus, our work reveals additional players in the complex cascade of signalling events operating in the root endodermis to establish both the CS diffusion barrier and the microbial composition of the rhizosphere.

Phosphate starvation response (PSR) in non-mycorrhizal plants comprises transcriptional reprogramming resulting in severe physiological changes to the roots and shoots and repression of plant immunity. Thus, plant-colonizing microorganisms – the plant microbiota – are exposed to direct influence by the soil’s phosphorous (P) content itself, as well as to the indirect effects of soil P on the microbial niches shaped by the plant. The individual contribution of these factors to plant microbiota assembly remains unknown. To disentangle these direct and indirect effects, we planted PSR-deficient Arabidopsis mutants in a long-term managed soil P gradient, and compared the composition of their shoot and root microbiota to wild type plants across different P concentrations. PSR-deficiency had a larger effect on the composition of both bacterial and fungal plant-associated microbiota composition than P concentrations in both roots and shoots. The fungal microbiota was more sensitive to P concentrationsper sethan bacteria, and less depended on the soil community composition.Using a 185-member bacterial synthetic community (SynCom) across a wide P concentration gradient in an agar matrix, we demonstrated a shift in the effect of bacteria on the plant from a neutral or positive interaction to a negative one, as measured by rosette size. This phenotypic shift is accompanied by changes in microbiota composition: the genusBurkholderiais specifically enriched in plant tissue under P starvation. Through a community drop-out experiment, we demonstrate that in the absence ofBurkholderiafrom the SynCom, plant shoots accumulate higher phosphate levels than shoots colonized with the full SynCom, only under P starvation, but not under P-replete conditions. Therefore, P-stressed plants allow colonization by latent opportunistic competitors found within their microbiome, thus exacerbating the plant’s P starvation.

The use of plant growth-promoting rhizobacteria as a sustainable alternative for chemical nitrogen fertilizers has been explored for many economically important crops. For one such strain isolated from rice rhizosphere and endosphere, nitrogen-fixing Pseudomonas stutzeri A15, unequivocal evidence of the plant growth-promoting effect and the potential contribution of biological nitrogen fixation (BNF) is still lacking. In this study, we investigated the effect of P. stutzeri A15 inoculation on the growth of rice seedlings in greenhouse conditions. P. stutzeri A15 induced significant growth promotion compared to uninoculated rice seedlings. Furthermore, inoculation with strain A15 performed significantly better than chemical nitrogen fertilization, clearly pointing to the potential of this bacterium as biofertilizer. To assess the contribution of BNF to the plant growth-promoting effect, rice seedlings were also inoculated with a nitrogen fixation-deficient mutant. Our results suggest that BNF (at best) only partially contributes to the stimulation of plant growth.

Phosphate starvation response (PSR) in nonmycorrhizal plants comprises transcriptional reprogramming resulting in severe physiological changes to the roots and shoots and repression of plant immunity. Thus, plant-colonizing microorganisms—the plant microbiota—are exposed to direct influence by the soil’s phosphorus (P) content itself as well as to the indirect effects of soil P on the microbial niches shaped by the plant. The individual contribution of these factors to plant microbiota assembly remains unknown. To disentangle these direct and indirect effects, we planted PSR-deficient Arabidopsis mutants in a long-term managed soil P gradient and compared the composition of their shoot and root microbiota to wild-type plants across different P concentrations. PSR-deficiency had a larger effect on the composition of both bacterial and fungal plant-associated microbiota than soil P concentrations in both roots and shoots. To dissect plant–microbe interactions under variable P conditions, we conducted a microbiota reconstitution experiment. Using a 185-member bacterial synthetic community (SynCom) across a wide P concentration gradient in an agar matrix, we demonstrated a shift in the effect of bacteria on the plant from a neutral or positive interaction to a negative one, as measured by rosette size. This phenotypic shift was accompanied by changes in microbiota composition: the genus Burkholderia was specifically enriched in plant tissue under P starvation. Through a community drop-out experiment, we demonstrated that in the absence of Burkholderia from the SynCom, plant shoots accumulated higher ortophosphate (Pi) levels than shoots colonized with the full SynCom but only under Pi starvation conditions. Therefore, Pi-stressed plants are susceptible to colonization by latent opportunistic competitors found within their microbiome, thus exacerbating the plant’s Pi starvation., This study shows that plants' response to phosphate starvation has more influence on plant microbiota than does the phosphate concentration itself; these changes are not necessarily beneficial for the plant, and can even exacerbate the phosphate starvation.

The aim of this research was to analyse the global indole-3-acetic acid (IAA) metabolism in three commercially used strains of Azospirillum brasilense. Methods and Results: Azospirillum brasilense Sp245, Az39 and Cd, containing a plasmid with the ipdC-gusA fusion (pFAJ64), were cultured in minimal medium MMAB with or without 10 mg l−1 of l-trp till exponential or stationary growth phase. The cultures were then split into 10 ml tubes and individually treated with 10 mg ml−1 IAA, IBA or NAA (auxin catabolism and homeostasis); IAPhe, IALeu, IAA-ala, IAA-glucose (IAA conjugate hydrolysis); or l-lys, l-leu, l-ileu, l-phe, l-ala, l-val, l-arg, l-glu, l-his, l-met, l-asp, l-cys, l-ser, l-pro, l-thr and l-trp (regulation of IAA biosynthesis and IAA conjugation). Bacterial growth, IAA production and ipdC expression were evaluated. None of the A. brasilense strains were able to hydrolyse IAA conjugates, catabolize auxins, or conjugate IAA with amino acids or glucose. l-amino acids l-met, l-val, l-cys and l-ser inhibited bacterial growth and decreased IAA biosynthesis. The expression of ipdC and IAA biosynthesis but not bacterial growth was affected by l-leu, l-phe, l-ala, l-ile, l-pro. l-arg, l-glu, l-his, l-lys, l-asp and l-thr did not affect any of the measured parameters. Conclusions: In this paper, we confirmed that A. brasilense produces IAA only in presence of l-trp is not able to degrade auxins, conjugate IAA with sugars and/or l-amino acids, or hydrolyse such conjugates to release free IAA. Finally, we found that bacterial growth and/or IAA biosynthesis were inhibited by the presence of several l-amino acids probably by diversion of the cellular metabolism. Significance and Impact of the Study: We propose a renewed model to explain IAA metabolism in A. brasilense, one of the most studied phytostimulatory bacteria. Fil: Rivera, Diego. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Ciencias Naturales; Argentina Fil: Mora, Maria Veronica. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; Argentina Fil: Lopez, Gaston Alberto. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; Argentina Fil: Rosas, Susana Beatriz. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Spaepen, S.. Katholikie Universiteit Leuven; Bélgica Fil: Vanderleyden, J.. Katholikie Universiteit Leuven; Bélgica Fil: Cassan, Fabricio Dario. Universidad Nacional de Río Cuarto; Argentina. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; Argentina

Plants and animals each have evolved specialized organs dedicated to nutrient acquisition, and these harbor specific bacterial communities that extend the host’s metabolic repertoire. Similar forces driving microbial community establishment in the gut and plant roots include diet/soil-type, host genotype, and immune system as well as microbe-microbe interactions. Here we show that there is no overlap of abundant bacterial taxa between the microbiotas of the mammalian gut and plant roots, whereas taxa overlap does exist between fish gut and plant root communities. A comparison of root and gut microbiota composition in multiple host species belonging to the same evolutionary lineage reveals host phylogenetic signals in both eukaryotic kingdoms. The reasons underlying striking differences in microbiota composition in independently evolved, yet functionally related, organs in plants and animals remain unclear but might include differences in start inoculum and niche-specific factors such as oxygen levels, temperature, pH, and organic carbon availability.

Plants differ from animals in their capability to easily regenerate fertile adult individuals from terminally differentiated cells [1]. This unique developmental plasticity is commonly observed in nature where many species can reproduce asexually through the ectopic initiation of organogenic or embryogenic developmental programs [2, 3]. However, it is not currently known if this developmental reprogramming is coupled to a global epigenomic resetting, or what impact it has on the phenotype of the clonal progeny. Here we show that plants asexually propagated via induction of a zygotic developmental program do not fully reset cell-specific epigenetic imprints. These imprints are instead inherited even over multiple rounds of sexual reproduction, becoming fixed in hybrids and resulting in heritable molecular and physiological phenotypes that depend on the founder cell used. Our results demonstrate how novel phenotypic variation in plants can be unlocked through the incomplete reprogramming of cell-specific epigenetic marks during asexual propagation.

Bacterial metabolism of phytohormones includes several processes such as biosynthesis, catabolism, conjugation, hydrolysis and homeostatic regulation. However, only biosynthesis and occasionally catabolism are studied in depth in microorganisms. In this work, we evaluated and reconsidered IAA metabolism in Bradyrhizobium japonicum E109, one of the most widely used strains for soybean inoculation around the world. The genomic analysis of the strain showed the presence of several genes responsible for IAA biosynthesis, mainly via indole-3-acetonitrile (IAN), indole-3-acetamide (IAM) and tryptamine (TAM) pathways. However; in vitro experiments showed that IAA is not accumulated in the culture medium in significant amounts. On the contrary, a strong degradation activity was observed after exogenous addition of 0.1 mM of IAA, IBA or NAA to the medium. B. japonicum E109 was not able to grow in culture medium containing IAA as a sole carbon source. In YEM medium, the bacteria degraded IAA and hydrolyzed amino acid auxin conjugates with alanine (IAAla), phenylalanine (IAPhe), and leucine (IAPhe), releasing IAA which was quickly degraded. Finally, the presence of exogenous IAA induced physiological changes in the bacteria such as increased biomass and exopolysaccharide production, as well as infection effectiveness and symbiotic behavior in soybean plants. Fil: Torres, Daniela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción Planta-microorganismo; Argentina Fil: Benavidez, Iliana. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción Planta-microorganismo; Argentina Fil: Donadío, Evelyn Florencia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción Planta-microorganismo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mongiardini, Elias Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina Fil: Rosas, Susana Beatriz. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción Planta-microorganismo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Spaepen, Stijn. Max Planck Institute for Plant Breeding Research; Alemania. Katholikie Universiteit Leuven; Bélgica Fil: Vanderleyden, Jozef. Katholikie Universiteit Leuven; Bélgica Fil: Pencík, Ales. Palacký University; República Checa Fil: Novák, Ondrej. Palacký University; República Checa Fil: Strnad, Miroslav. Palacký University; República Checa Fil: Frébortová, Jitka. Palacký University; República Checa Fil: Cassan, Fabricio Dario. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción Planta-microorganismo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The auxin-producing bacterium Azospirillum brasilense Sp245 can promote the growth of several plant species. The model plant Arabidopsis thaliana was chosen as host plant to gain an insight into the molecular mechanisms that govern this interaction. The determination of differential gene expression in Arabidopsis roots after inoculation with either A. brasilense wild-type or an auxin biosynthesis mutant was achieved by microarray analysis. Arabidopsis thaliana inoculation with A. brasilense wild-type increases the number of lateral roots and root hairs, and elevates the internal auxin concentration in the plant. The A. thaliana root transcriptome undergoes extensive changes on A. brasilense inoculation, and the effects are more pronounced at later time points. The wild-type bacterial strain induces changes in hormone- and defense-related genes, as well as in plant cell wall-related genes. The A. brasilense mutant, however, does not elicit these transcriptional changes to the same extent. There are qualitative and quantitative differences between A. thaliana responses to the wild-type A. brasilense strain and the auxin biosynthesis mutant strain, based on both phenotypic and transcriptomic data. This illustrates the major role played by auxin in the Azospirillum-Arabidopsis interaction, and possibly also in other bacterium-plant interactions.

Azospirillum influences growth and development of several crops by producing phytohormones such as auxins which have a major impact on root development. An improved root system leads to better water and nutrient uptake that in turn may influence yield positively. In this study, two agronomically contrasting sugarcane cvs R 570 and M 1176/77 adapted to different agroclimatic zones were inoculated with Azospirillum sp., with and without drought stress, to gauge how far they could benefit from this bacterial association. As early as 103 days after planting, cv M 1176/77 responded positively to inoculation with 15% improved growth (shoot height) and 75% more root dry mass when subjected to drought stress, whereas cv R 570 responded negatively particularly in the absence of drought stress. The significant interaction of cultivar x water regime x Azospirillum inoculation suggests a complex interplay of these factors, possibly involving the indigenous plant auxin pool. Therefore, plant genotype needs to be taken into account when recommending bacterial inoculation for direct plant growth promotion. Furthermore, enhanced growth under sub-optimal water conditions shows clearly the benefits that could be obtained in semi-arid conditions where water deficits frequently occur.

The second-order nonlinear optical properties of two monomeric red fluorescent proteins, mStrawberry and mCherry, have been experimentally determined by frequency-resolved femtosecond hyper-Rayleigh scattering. These proteins were found to exhibit a stronger nonlinear response than the previously described eGFP, eYFP and DsRed,1 confirming the trend that fluorophores with a more extended conjugated system, or a lower-energy band gap between ground and excited state, exhibit a larger static hyperpolarizability (β0). Furthermore, these experimental data were complemented with quantum chemical calculations. A discrepancy was observed between experimental and theoretical results, but this could be explained by the chromophore model extracted from the available X-ray diffraction data. While eGFP showed a larger dynamic experimental response (βHRS) due to the highest resonance enhancement, we measured an even higher signal for mCherry. Furthermore, mCherry also shows a better separation of the second harmonic signal and two-photon excited fluorescent signal, making this the preferred fluorescent protein for second harmonic imaging at 800 nm so far.

Aims: To assess the applicability of the 16S–23S rDNA internal spacer regions (ISR) as targets for PCR detection of Azospirillum ssp. and the phytostimulatory plant growth-promoting rhizobacteria seed inoculant Azospirillum lipoferum CRT1 in soil. Methods and Results: Primer sets were designed after sequence analysis of the ISR of A. lipoferum CRT1 and Azospirillum brasilense Sp245. The primers fAZO/rAZO targeting the Azospirillum genus successfully yielded PCR amplicons (400–550 bp) from Azospirillum strains but also from certain non-Azospirillum strains in vitro, therefore they were not appropriate to monitor indigenous Azospirillum soil populations. The primers fCRT1/rCRT1 targeting A. lipoferum CRT1 generated a single 249-bp PCR product but could also amplify other strains from the same species. However, with DNA extracts from the rhizosphere of field-grown maize, both fAZO/rAZO and fCRT1/rCRT1 primer sets could be used to evidence strain CRT1 in inoculated plants by nested PCR, after a first ISR amplification with universal ribosomal primers. In soil, a 7-log dynamic range of detection (102–108 CFU g−1 soil) was obtained. Conclusions: The PCR primers targeting 16S–23S rDNA ISR sequences enabled detection of the inoculant A. lipoferum CRT1 in field soil. Significance and Impact of the Study: Convenient methods to monitor Azospirillum phytostimulators in the soil are lacking. The PCR protocols designed based on ISR sequences will be useful for detection of the crop inoculant A. lipoferum CRT1 under field conditions.

The second-order nonlinear optical properties of three fluorescent proteins (FPs) (green, EGFP; yellow, EYFP; and red, DsRed) have been experimentally determined by frequency-resolved femtosecond hyper-Rayleigh scattering. As expected, DsRed, with its lower-energy bandgap between ground and excited state, exhibits the largest intrinsic hyperpolarizability. The anomalously low first hyperpolarizability for the yellow variant has been rationalized in terms of the centrosymmetrical arrangement between the phenolic Tyr203 (Tyr = tyrosine) residue and the chromophoric Tyr66 moiety, leaving the small imidazolinone moiety as the only effective non-centrosymmetric chromophore for second-order nonlinear effects. The experimental findings are corroborated by high-level computational results and suggest molecular engineering strategies to produce a full rainbow of FPs with enhanced nonlinear optical properties.

Azospirillum brasilense is a diazotrophic bacterium and one of the best studied plant-growth-promoting bacterium living in close association with several agronomically important crops. The production of plant-growth-regulating substances is a main mechanism of plant growth stimulation, although other mechanisms have also been proposed. Nitrogen transfer from the bacterium to the plant is one among the other possible mechanisms of plant growth stimulation. In this study, we investigated, by means of a greenhouse trial with winter wheat inoculation, the effect of a point mutation in the ammonium binding site of the A. brasilense glutamine synthetase. The glutamine synthetase is one of the main ammonium-assimilating enzymes and mutations in this enzyme generally result in the release of ammonium from the bacterium to its environment. The ammonium-excreting mutant used in this study was shown to perform better than the wild-type A. brasilense strain with respect to wheat growth parameters and yield. In the greenhouse conditions used, this effect was independent of the way fertilizer was applied.

In the rhizosphere and their interaction with plants rhizobia encounter many different plant compounds, including phytohormones like auxins. Moreover, some rhizobial strains are capable of producing the auxin, indole-3-acetic acid (IAA). However, the role of IAA for the bacterial partner in the legume– Rhizobium symbiosis is not known. To identify the effect of IAA on rhizobial gene expression, a transposon (mTn 5gusA - oriV ) mutant library of Rhizobium etli , enriched for mutants that show differential gene expression under microaerobiosis and/or addition of nodule extracts as compared with control conditions, was screened for altered gene expression upon IAA addition. Four genes were found to be regulated by IAA. These genes appear to be involved in plant signal processing, motility or attachment to plant roots, clearly demonstrating a distinct role for IAA in legume– Rhizobium interactions.

Strains Sp7 and Cd of Azospirillum brasilense, a plant growth-promoting rhizobacterium, differ in synthesis of carotenoids. While colonies of strain Sp7 have a white-cream colour on plates, colonies of strain Cd are orange-pink coloured because of the synthesis of carotenoids. Screening of a mini-Tn5 mutant library of A. brasilense Sp7 revealed two orange-pink-coloured mutants that produced carotenoids. Cloning and sequencing of the Tn5 flanking region in both the carotenoid-producing mutants of Sp7 revealed insertion of Tn5 in an ORF encoding anti-sigma factor, a ChrR-like protein. The upstream region of the Tn5-mutated ORF contained another ORF that encoded an extra-cytoplasmic function (ECF)-class sigma factor (sigma(E), RpoE). When the nucleotide sequences of the corresponding ORFs from the carotenoid-producing strain Cd were analysed, the sequence of the Cd sigma(E) was identical to that of the carotenoid non-producing strain Sp7, but the Cd anti-sigma(E) ORF had a deletion that caused frame shifting and creation of a stop codon. This resulted in the premature termination of the protein, which was about 7 kDa smaller than the Sp7 anti-sigma(E). Cloning of Sp7 anti-sigma(E) in a broad-host-range expression vector and expression in A. brasilense Cd and in the anti-sigma(E) knockout mutant of A. brasilense Sp7 resulted in the inhibition of carotenoid synthesis. Similarly, cloning and overexpression of A. brasilense Sp7 sigma(E) in A. brasilense Sp7 resulted in the production of carotenoids. These observations clearly indicate that carotenoid synthesis in A. brasilense is controlled by sigma(E) with its cognate anti-sigma(E).

The production of phytohormones by plant-growth promoting rhizobacteria is considered to be an important mechanism by which these bacteria promote plant growth. In this study the importance of indole-3-acetic acid (IAA) produced by Azospirillum brasilense Sp245 in the observed plant growth stimulation was investigated by using Sp245 strains genetically modified in IAA production. Firstly wild-type A. brasilense Sp245 and an ipdC knock-out mutant which produces only 10% of wild-type IAA levels (Vande Broek et al., J Bacteriol 181:1338–1342, 1999) were compared in a greenhouse inoculation experiment for a number of plant parameters, thereby clearly demonstrating the IAA effect in plant growth promotion. Secondly, the question was addressed whether altering expression of the ipdC gene, encoding the key enzyme for IAA biosynthesis in A. brasilense, could also contribute to plant growth promotion. For that purpose, the endogenous promoter of the ipdC gene was replaced by either a constitutive or a plant-inducible promoter and both constructs were introduced into the wild-type strain. Based on a greenhouse inoculation experiment it was found that the introduction of these recombinant ipdC constructs could further improve the plant-growth promoting effect of A. brasilense. These data support the possibility of constructing Azospirillum strains with better performance in plant growth promotion.

Phenylpyruvate decarboxylase (PPDC) of Azospirillum brasilense, involved in the biosynthesis of the plant hormone indole-3-acetic acid and the anti- microbial compound phenylacetic acid, is a thiamine diphosphate-depend- ent enzyme that catalyses the nonoxidative decarboxylation of indole- and phenylpyruvate. Analogous to yeast pyruvate decarboxylases, PPDC is sub- ject to allosteric substrate activation, showing sigmoidal v versus (S) plots. The present paper reports the crystal structure of this enzyme determined at 1.5 Aresolution. The subunit architecture of PPDC is characteristic for other members of the pyruvate oxidase family, with each subunit consisting of three domains with an open a ⁄b topology. An active site loop, bearing the catalytic residues His112 and His113, could not be modelled due to flexibility. The biological tetramer is best described as an asymmetric dimer of dimers. A cysteine residue that has been suggested as the site for regula- tory substrate binding in yeast pyruvate decarboxylase is not conserved, requiring a different mechanism for allosteric substrate activation in PPDC. Only minor changes occur in the interactions with the cofactors, thiamine diphosphate and Mg 2+ , compared to pyruvate decarboxylase. A greater diversity is observed in the substrate binding pocket accounting for the dif- ference in substrate specificity. Moreover, a catalytically important glutam- ate residue conserved in nearly all decarboxylases is replaced by a leucine in PPDC. The consequences of these differences in terms of the catalytic and regulatory mechanism of PPDC are discussed.

Background Plant growth-promoting rhizobacteria are increasingly being seen as a way of complementing conventional inputs in agricultural systems. The effects on their host plants are diverse and include volatile-mediated growth enhancement. This study sought to assess the effects of bacterial volatiles on the biomass production and root system architecture of the model grass Brachypodium distachyon (L.) Beauv. Results An in vitro experiment allowing plant-bacteria interaction throughout the gaseous phase without any physical contact was used to screen 19 bacterial strains for their growth-promotion ability over a 10-day co-cultivation period. Five groups of bacteria were defined and characterised based on their combined influence on biomass production and root system architecture. The observed effects ranged from unchanged to greatly increased biomass production coupled with increased root length and branching. Primary root length was increased only by the volatile compounds emitted by Enterobacter cloacae JM22 and Bacillus pumilus T4. Overall, the most significant results were obtained with Bacillus subtilis GB03, which induced an 81 % increase in total biomass, as well as enhancing total root length, total secondary root length and total adventitious root length by 88.5, 201.5 and 474.5 %, respectively. Conclusions This study is the first report on bacterial volatile-mediated growth promotion of a grass plant. Contrasting modulations of biomass production coupled with changes in root system architecture were observed. Most of the strains that increased total plant biomass also modulated adventitious root growth. Under our screening conditions, total biomass production was strongly correlated with the length and branching of the root system components, except for primary root length. An analysis of the emission kinetics of the bacterial volatile compounds is being undertaken and should lead to the identification of the compounds responsible for the observed growth-promotion effects. Within the context of the inherent characteristics of our in vitro system, this paper identifies the next critical experimental steps and discusses them from both a fundamental and an applied perspective. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0585-3) contains supplementary material, which is available to authorized users.

Roots and leaves of healthy plants host taxonomically structured bacterial assemblies, and members of these communities contribute to plant growth and health. We established Arabidopsis leaf- and root-derived microbiota culture collections representing the majority of bacterial species that are reproducibly detectable by culture-independent community sequencing. We found an extensive taxonomic overlap between the leaf and root microbiota. Genome drafts of 400 isolates revealed a large overlap of genome-encoded functional capabilities between leaf- and root-derived bacteria with few significant differences at the level of individual functional categories. Using defined bacterial communities and a gnotobiotic Arabidopsis plant system we show that the isolates form assemblies resembling natural microbiota on their cognate host organs, but are also capable of ectopic leaf or root colonization. While this raises the possibility of reciprocal relocation between root and leaf microbiota members, genome information and recolonization experiments also provide evidence for microbiota specialization to their respective niche.

Plant hormones or phytohormones are historically classified into five major classes: auxins, cytokinins, gibberellins, abscisic acid and ethylene. Nowadays, many other phytohormones have been identified. Diverse microbial species possess the ability to produce phytohormones, with most data accumulated for the production and role of auxin. In this chapter the microbial biosynthesis, its regulation and the role of the different phytohormones in the interaction with the plant are discussed. Microbial phytohormonal production is a potent mechanism to alter plant physiology, leading to diverse outcomes from pathogenesis to promotion of plant growth. However, genetic evidence for the role of many phytohormones in microbe-plant interactions is still lacking, thus questioning the importance of the microbial production. Targeted approaches focusing on genetic evidence for the role of phytohormones together with plant experiments in an agronomic setting will allow unraveling the importance and potential of this fascinating microbial trait.

We present the complete genome sequence of Azospirillum brasilense Az39, isolated from wheat roots in the central region of Argentina and used as inoculant in extensive and intensive agriculture during the last four decades. The genome consists of 7.39 Mb, distributed in six replicons: one chromosome, three chromids, and two plasmids.

The functional analysis of phytohormone production, interaction, and regulation in higher plants has re-emerged in the past 10 years due to spectacular advances in integrative study models. However, plants are not axenic in natural conditions and are usually colonized or influenced directly by different microorganisms such as rhizobacteria of which many have the ability to produce phytohormones. This review summarizes information related to the biosynthesis, metabolism, regulation, physiological role, and agronomical impact of phytohormones produced by the model plant-growth-promoting rhizobacteria (PGPR) belonging to the genus Azospirillum, considered to be one of the most representative PGPR. We include exhaustive information about the phytohormones auxins, gibberellins, cytokinins, ethylene, and abscisic acid, as well as the plant growth regulators polyamines and nitric oxide. We deal with their metabolism by Azospirillum sp. in chemically defined medium, in plant–microbe interactions, or in the context of the agronomical use of Azospirillum sp. Fil: Cassan, Fabricio Dario. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquímicas y Naturales. Departamento de Cs.naturales. Laboratorio de Fisiología Vegetal y de la Interacción Planta-microorganismo; Argentina Fil: Vanderleyden, Jos. Centre of Microbial and Plant Genetics. Heverlee; Bélgica Fil: Spaepen, Stijn. Centre of Microbial and Plant Genetics. Heverlee; Bélgica

Cell surface display of proteins can be used for several biotechnological applications such as the screening of protein libraries, whole cell biocatalysis and live vaccine development. Amongst all secretion systems and surface appendages of Gram-negative bacteria, the autotransporter secretion pathway holds great potential for surface display because of its modular structure and apparent simplicity. Autotransporters are polypeptides made up of an N-terminal signal peptide, a secreted or surface-displayed passenger domain and a membrane-anchored C-terminal translocation unit. Genetic replacement of the passenger domain allows for the surface display of heterologous passengers. An autotransporter-based surface expression module essentially consists of an application-dependent promoter system, a signal peptide, a passenger domain of interest and the autotransporter translocation unit. The passenger domain needs to be compatible with surface translocation although till now no general rules have been determined to test this compatibility. The autotransporter technology for surface display of heterologous passenger domains is critically discussed for various applications.

The auxin-producing bacterium Azospirillum brasilense Sp245 can promote the growth of several plant species. The model plant Arabidopsis thaliana was chosen as host plant to gain an insight into the molecular mechanisms that govern this interaction. The determination of differential gene expression in Arabidopsis roots after inoculation with either A. brasilense wild-type or an auxin biosynthesis mutant was achieved by microarray analysis. Arabidopsis thaliana inoculation with A. brasilense wild-type increases the number of lateral roots and root hairs, and elevates the internal auxin concentration in the plant. The A. thaliana root transcriptome undergoes extensive changes on A. brasilense inoculation, and the effects are more pronounced at later time points. The wild-type bacterial strain induces changes in hormone- and defense-related genes, as well as in plant cell wall-related genes. The A. brasilense mutant, however, does not elicit these transcriptional changes to the same extent. There are qualitative and quantitative differences between A. thaliana responses to the wild-type A. brasilense strain and the auxin biosynthesis mutant strain, based on both phenotypic and transcriptomic data. This illustrates the major role played by auxin in the Azospirillum-Arabidopsis interaction, and possibly also in other bacterium-plant interactions.

Lectin-like bacteriotoxic proteins, identified in several plant-associated bacteria, are able to selectively kill closely related species, including several phytopathogens, such as Pseudomonas syringae and Xanthomonas species, but so far their mode of action remains unrevealed. The crystal structure of LlpABW, the prototype lectin-like bacteriocin from Pseudomonas putida, reveals an architecture of two monocot mannose-binding lectin (MMBL) domains and a C-terminal β-hairpin extension. The C-terminal MMBL domain (C-domain) adopts a fold very similar to MMBL domains from plant lectins and contains a binding site for mannose and oligomannosides. Mutational analysis indicates that an intact sugar-binding pocket in this domain is crucial for bactericidal activity. The N-terminal MMBL domain (N-domain) adopts the same fold but is structurally more divergent and lacks a functional mannose-binding site. Differential activity of engineered N/C-domain chimers derived from two LlpA homologues with different killing spectra, disclosed that the N-domain determines target specificity. Apparently this bacteriocin is assembled from two structurally similar domains that evolved separately towards dedicated functions in target recognition and bacteriotoxicity., Author Summary In their natural environments, microorganisms compete for space and nutrients, and a major strategy to assist in niche colonization is the deployment of antagonistic compounds directed at competitors, such as secondary metabolites (antibiotics) and antibacterial peptides or proteins (bacteriocins). The latter selectively kill closely related bacteria, which is also the case for members of the LlpA family. Here, we investigate the structure-function relationship for the prototype LlpABW from a saprophytic plant-associated Pseudomonas whose genus-specific target spectrum includes several phytopathogenic pseudomonads. By determining the 3D structure of this protein, we could assign LlpA to the so-called monocot mannose-binding lectin (MMBL) family, representing its first prokaryotic member, and also add a new type of protective function, as the eukaryotic MMBL members have been linked with antiviral, antifungal, nematicidal or insecticidal activities. For the protein containing two similarly folded domains, we constructed site-specific mutants affected in carbohydrate binding and domain chimers from LlpA homologues to show that mannose-specific sugar binding mediated by one domain is required for activity and that the other domain determines target strain specificity. The strategy that evolved for these bacteriocins is reminiscent of the one used by mammalian bactericidal proteins of the RegIII family that recruited a C-type lectin fold to kill bacteria.

Autotransporters are a widespread family of proteins, generally known as virulence factors produced by Gram-negative bacteria. In this study, the esterase A (EstA) autotransporter of the rice root-colonizing beneficial bacterium Pseudomonas stutzeri A15 was characterized. A multiple sequence alignment identified EstA as belonging to clade II of the GDSL esterase family. Autologous overexpression allowed the investigation of several features of both autotransporter proteins and GDSL esterases. First, the correctly folded autotransporter was shown to be present in the membrane fraction. Unexpectedly, after separation of the membrane fraction, EstA was detected in the N -laurylsarcosine soluble fraction. However, evidence is presented for the surface exposure of EstA based on fluorescent labeling with EstA specific antibodies. Another remarkable feature is the occurrence of a C-terminal leucine residue instead of the canonical phenylalanine or tryptophan residue. Replacement of this residue with a phenylalanine residue reduced the stability of the β-barrel. Regarding the esterase passenger domain, we show the importance of the catalytic triad residues, with the serine and histidine residues being more critical than the aspartate residue. Furthermore, the growth of an estA -negative mutant was not impaired and cell mobility was not disabled compared to the wild type. No specific phenotype was detected for an estA -negative mutant. Overall, P. stutzeri A15 EstA is a new candidate for the surface display of proteins in environmentally relevant biotechnological applications.

The rhizosphere bacterium Azospirillum brasilense produces the auxin indole-3-acetic acid (IAA) through the indole-3-pyruvate pathway. As we previously demonstrated that transcription of the indole-3-pyruvate decarboxylase (ipdC) gene is positively regulated by IAA, produced by A. brasilense itself or added exogenously, we performed a microarray analysis to study the overall effects of IAA on the transcriptome of A. brasilense. The transcriptomes of A. brasilense wild-type and the ipdC knockout mutant, both cultured in the absence and presence of exogenously added IAA, were compared. Interfering with the IAA biosynthesis/homeostasis in A. brasilense through inactivation of the ipdC gene or IAA addition results in much broader transcriptional changes than anticipated. Based on the multitude of changes observed by comparing the different transcriptomes, we can conclude that IAA is a signaling molecule in A. brasilense. It appears that the bacterium, when exposed to IAA, adapts itself to the plant rhizosphere, by changing its arsenal of transport proteins and cell surface proteins. A striking example of adaptation to IAA exposure, as happens in the rhizosphere, is the upregulation of a type VI secretion system (T6SS) in the presence of IAA. The T6SS is described as specifically involved in bacterium–eukaryotic host interactions. Additionally, many transcription factors show an altered regulation as well, indicating that the regulatory machinery of the bacterium is changing.

International audience; Abstract The phytostimulatory properties of Azospirillum inoculants, which entail production of the phytohormone indole-3-acetic acid (IAA), can be enhanced by genetic means. However, it is not known whether this could affect their interactions with indigenous soil microbes. Here, wheat seeds were inoculated with the wild-type strain Azospirillum brasilense Sp245 or one of three genetically modified (GM) derivatives and grown for one month. The GM derivatives contained a plasmid vector harboring the indole-3-pyruvate/phenylpyruvate decarboxylase gene ipdC (IAA production) controlled either by the constitutive promoter PnptII or the root exudate-responsive promoter PsbpA, or by an empty vector (GM control). All inoculants displayed equal rhizosphere population densities. Only inoculation with either ipdC construct increased shoot biomass compared with the non-inoculated control. At one month after inoculation, automated ribosomal intergenic spacer analysis (ARISA) revealed that the effect of the PsbpA construct on bacterial community structure differed from that of the GM control, which was confirmed by 16S rDNA-based denaturing gradient gel electrophoresis (DGGE). The fungal community was sensitive to inoculation with the PsbpA construct and especially the GM control, based on ARISA data. Overall, fungal and bacterial communities displayed distinct responses to inoculation of GM A. brasilense phytostimulators, whose effects could differ from those of the wild-type.

The rhizosphere as compared to bulk soil is rich in nutrients because of root exudation and deposits. As a consequence, the number of bacteria surrounding plant roots is 10–100 times higher than in bulk soil. These rhizobacteria, based on their effects on plants, can be largely divided into beneficial, deleterious, or neutral bacteria. The beneficial bacteria, also called plant growth-promoting rhizobacteria (PGPR), exert their beneficial effect through either direct or indirect mechanisms or both. In this chapter, the different mechanisms of plant-growth promotion and their impact are discussed. The mechanisms comprise the production of plant growth-promoting substances, nitrogen transformations, increasing bioavailability of phosphate and micronutrients, and biological control, as the best documented cases. In addition, the bacterial production of some molecules with recently described plant growth-promoting effects is discussed. To indicate the impact of PGPR, the applications and relevance of these bacteria in agricultural practices are highlighted. The importance of the plant genotype, inoculum density and technology, and co‐inoculation practices, in terms of plant responsiveness are discussed. It is concluded that basic research should remain a priority in order to be able to develop performing and reliable bacterial inocula as a means to support sustainable agriculture.

2,6-Dibromophenol (DBP) was reported as an auxin-like molecule using molecular quantum similarity measures. in this study, the auxin activity of this molecule and its chlorinated homologue is further determined using a bacterial biosensor; the auxin-inducible ipdC promoter of Azospirillum brasilense. We were able to demonstrate that DBP can induce gene expression, but to a lesser extent than the auxin indole-3-acetic acid (IAA) and that DBP is not an antagonist for the IAA signalling pathway. To investigate the role of the bromine groups, the molecule 2,6-dichlorophenol (DCP) was also tested for gene expression induction. However, no induction could be observed. In a second part, DBP and other molecules were modelled in the auxin-binding pocket of the plant auxin receptor TIR1 to evaluate theoretical binding energies. Both DBP and its chlorinated homologue DCP are not strong ligands compared to other known auxins such as IAA. The importance of a carboxylated side chain for optimal binding (and probably auxin activity) was demonstrated. (C) 2008 Elsevier Masson SAS. All rights reserved.

Glutamine synthetase, a key enzyme in nitrogen metabolism of both prokaryotes and eukaryotes, is strictly regulated. One means of regulation is the modulation of activity through adenylylation catalyzed by adenylyltransferases. Using PCR primers based on conserved sequences in glutamine synthetase adenylyltransferases, we amplified part of the glnE gene of Azospirillum brasilense Sp7. The complete glnE sequence of A. brasilense Sp245 was retrieved from the draft genome sequence of this organism (http://genomics.ornl.gov/research/azo/). Adenylyltransferase is a bifunctional enzyme consisting of an N-terminal domain responsible for deadenylylation activity and a C-terminal domain responsible for adenylylation activity. Both domains are partially homologous to each other. Residues important for catalytic activity were present in the deduced amino acid sequence of the A. brasilense Sp245 glnE sequence. A glnE mutant was constructed in A. brasilense Sp7 by inserting a kanamycin resistance cassette between the two active domains of the enzyme. The resulting mutant was unable to adenylylate the glutamine synthetase enzyme and was impaired in growth when shifted from nitrogen-poor to nitrogen-rich medium.

Azospirillum brasilense belongs to the plant growth-promoting rhizobacteria with direct growth promotion through the production of the phytohormone indole-3-acetic acid (IAA). A key gene in the production of IAA, annotated as indole-3-pyruvate decarboxylase ( ipdC ), has been isolated from A. brasilense , and its regulation was reported previously (A. Vande Broek, P. Gysegom, O. Ona, N. Hendrickx, E. Prinsen, J. Van Impe, and J. Vanderleyden, Mol. Plant-Microbe Interact. 18:311-323, 2005). An ipdC -knockout mutant was found to produce only 10% (wt/vol) of the wild-type IAA production level. In this study, the encoded enzyme is characterized via a biochemical and phylogenetic analysis. Therefore, the recombinant enzyme was expressed and purified via heterologous overexpression in Escherichia coli and subsequent affinity chromatography. The molecular mass of the holoenzyme was determined by size-exclusion chromatography, suggesting a tetrameric structure, which is typical for 2-keto acid decarboxylases. The enzyme shows the highest k cat value for phenylpyruvate. Comparing values for the specificity constant k cat / K m , indole-3-pyruvate is converted 10-fold less efficiently, while no activity could be detected with benzoylformate. The enzyme shows pronounced substrate activation with indole-3-pyruvate and some other aromatic substrates, while for phenylpyruvate it appears to obey classical Michaelis-Menten kinetics. Based on these data, we propose a reclassification of the ipdC gene product of A. brasilense as a phenylpyruvate decarboxylase (EC 4.1.1.43).

Diverse bacterial species possess the ability to produce the auxin phytohormone indole-3-acetic acid (IAA). Different biosynthesis pathways have been identified and redundancy for IAA biosynthesis is widespread among plant-associated bacteria. Interactions between IAA-producing bacteria and plants lead to diverse outcomes on the plant side, varying from pathogenesis to phyto-stimulation. Reviewing the role of bacterial IAA in different microorganism-plant interactions highlights the fact that bacteria use this phytohormone to interact with plants as part of their colonization strategy, including phyto-stimulation and circumvention of basal plant defense mechanisms. Moreover, several recent reports indicate that IAA can also be a signaling molecule in bacteria and therefore can have a direct effect on bacterial physiology. This review discusses past and recent data, and emerging views on IAA, a well-known phytohormone, as a microbial metabolic and signaling molecule.

Background Solanum lycopersicum or tomato is extensively studied with respect to the ethylene metabolism during climacteric ripening, focusing almost exclusively on fruit pericarp. In this work the ethylene biosynthesis pathway was examined in all major tomato fruit tissues: pericarp, septa, columella, placenta, locular gel and seeds. The tissue specific ethylene production rate was measured throughout fruit development, climacteric ripening and postharvest storage. All ethylene intermediate metabolites (1-aminocyclopropane-1-carboxylic acid (ACC), malonyl-ACC (MACC) and S-adenosyl-L-methionine (SAM)) and enzyme activities (ACC-oxidase (ACO) and ACC-synthase (ACS)) were assessed. Results All tissues showed a similar climacteric pattern in ethylene productions, but with a different amplitude. Profound differences were found between tissue types at the metabolic and enzymatic level. The pericarp tissue produced the highest amount of ethylene, but showed only a low ACC content and limited ACS activity, while the locular gel accumulated a lot of ACC, MACC and SAM and showed only limited ACO and ACS activity. Central tissues (septa, columella and placenta) showed a strong accumulation of ACC and MACC. These differences indicate that the ethylene biosynthesis pathway is organized and regulated in a tissue specific way. The possible role of inter- and intra-tissue transport is discussed to explain these discrepancies. Furthermore, the antagonistic relation between ACO and E8, an ethylene biosynthesis inhibiting protein, was shown to be tissue specific and developmentally regulated. In addition, ethylene inhibition by E8 is not achieved by a direct interaction between ACO and E8, as previously suggested in literature. Conclusions The Ethylene biosynthesis pathway and E8 show a tissue specific and developmental differentiation throughout tomato fruit development and ripening.

Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.

Microbial synthesis of the phytohormone auxin has been known for a long time. This property is best documented for bacteria that interact with plants because bacterial auxin can cause interference with the many plant developmental processes regulated by auxin. Auxin biosynthesis in bacteria can occur via multiple pathways as has been observed in plants. There is also increasing evidence that indole-3-acetic acid (IAA), the major naturally occurring auxin, is a signaling molecule in microorganisms because IAA affects gene expression in some microorganisms. Therefore, IAA can act as a reciprocal signaling molecule in microbe-plant interactions. Interest in microbial synthesis of auxin is also increasing in yet another recently discovered property of auxin in Arabidopsis. Down-regulation of auxin signaling is part of the plant defense system against phytopathogenic bacteria. Exogenous application of auxin, e.g., produced by the pathogen, enhances susceptibility to the bacterial pathogen.