CRK12: A Key Player in Regulating the Phaseolus vulgaris-Rhizobium tropici Symbiotic Interaction.

Cysteine-rich receptor-like kinases (CRKs) are a type of receptor-like kinases (RLKs) that are important for pathogen resistance, extracellular reactive oxygen species (ROS) signaling, and programmed cell death in plants. In a previous study, we identified 46 CRK family members in the Phaseolus vulgaris genome and found that CRK12 was highly upregulated under root nodule symbiotic conditions. To better understand the role of CRK12 in the Phaseolus-Rhizobia symbiotic interaction, we functionally characterized this gene by overexpressing (CRK12-OE) and silencing (CRK12-RNAi) it in a P. vulgaris hairy root system. We found that the constitutive expression of CRK12 led to an increase in root hair length and the expression of root hair regulatory genes, while silencing the gene had the opposite effect. During symbiosis, CRK12-RNAi resulted in a significant reduction in nodule numbers, while CRK12-OE roots showed a dramatic increase in rhizobial infection threads and the number of nodules. Nodule cross sections revealed that silenced nodules had very few infected cells, while CRK12-OE nodules had enlarged infected cells, whose numbers had increased compared to controls. As expected, CRK12-RNAi negatively affected nitrogen fixation, while CRK12-OE nodules fixed 1.5 times more nitrogen than controls. Expression levels of genes involved in symbiosis and ROS signaling, as well as nitrogen export genes, supported the nodule phenotypes. Moreover, nodule senescence was prolonged in CRK12-overexpressing roots. Subcellular localization assays showed that the PvCRK12 protein localized to the plasma membrane, and the spatiotemporal expression patterns of the CRK12-promoter::GUS-GFP analysis revealed a symbiosis-specific expression of CRK12 during the early stages of rhizobial infection and in the development of nodules. Our findings suggest that CRK12, a membrane RLK, is a novel regulator of Phaseolus vulgaris-Rhizobium tropici symbiosis.

OnfD, an AraC-Type Transcriptional Regulator Encoded by Rhizobium tropici CIAT 899 and Involved in Nod Factor Synthesis and Symbiosis.

Rhizobium tropici CIAT 899 is a broad-host-range rhizobial strain that establishes symbiotic interactions with legumes and tolerates different environmental stresses such as heat, acidity, or salinity. This rhizobial strain produces a wide variety of symbiotically active nodulation factors (NF) induced not only by the presence of plant-released flavonoids but also under osmotic stress conditions through the LysR-type transcriptional regulators NodD1 (flavonoids) and NodD2 (osmotic stress). However, the activation of NodD2 under high-osmotic-stress conditions remains elusive. Here, we have studied the role of a new AraC-type regulator (named as OnfD) in the symbiotic interaction of R. tropici CIAT 899 with Phaseolus vulgaris and Lotus plants. We determined that OnfD is required under salt stress conditions for the transcriptional activation of the nodulation genes and therefore the synthesis and export of NF, which are required for a successful symbiosis with P. vulgaris Moreover, using bacterial two-hybrid analysis, we demonstrated that the OnfD and NodD2 proteins form homodimers and OnfD/NodD2 form heterodimers, which could be involved in the production of NF in the presence of osmotic stress conditions since both regulators are required for NF synthesis in the presence of salt. A structural model of OnfD is presented and discussed.IMPORTANCE The synthesis and export of rhizobial NF are mediated by a conserved group of LysR-type regulators, the NodD proteins. Here, we have demonstrated that a non-LysR-type regulator, an AraC-type protein, is required for the transcriptional activation of symbiotic genes and for the synthesis of symbiotically active NF under salt stress conditions.

Rhizobium tropici CIAT 899 copA gene plays a fundamental role in copper tolerance in both free life and symbiosis with Phaseolus vulgaris.

Rhizobium tropici CIAT 899 is a facultative symbiotic diazotroph able to deal with stressful concentrations of metals. Nevertheless the molecular mechanisms involved in metal tolerance have not been elucidated. Copper (Cu2+) is a metal component essential for the heme-copper respiratory oxidases and enzymes that catalyse redox reactions, however, it is highly toxic when intracellular trace concentrations are surpassed. In this study, we report that R. tropici CIAT 899 is more tolerant to Cu2+ than other Rhizobium and Sinorhizobium species. Through Tn5 random mutagenesis we identify a R. tropici mutant strain with a severe reduction in Cu2+ tolerance. The Tn5 insertion disrupted the gene RTCIAT899_CH17575, encoding a putative heavy metal efflux P1B-1-type ATPase designated as copA. Phaseolus vulgaris plants inoculated with the copA::Tn5 mutant in the presence of toxic Cu2+ concentrations showed a drastic reduction in plant and nodule dry weight, as well as nitrogenase activity. Nodules induced by the copA::Tn5 mutant present an increase in H2O2 concentration, lipoperoxidation and accumulate 40-fold more Cu2+ than nodules formed by the wild-type strain. The copA::Tn5 mutant complemented with the copA gene recovered the wild-type symbiotic phenotypes. Therefore, the copA gene is essential for R. tropici CIAT 899 to survive in copper-rich environments in both free life and symbiosis with P. vulgaris plants.

Genetic Interaction Studies Reveal Superior Performance of Rhizobium tropici CIAT899 on a Range of Diverse East African Common Bean (Phaseolus vulgaris L.) Genotypes.

We studied symbiotic performance of factorial combinations of diverse rhizobial genotypes (GR) and East African common bean varieties (GL) that comprise Andean and Mesoamerican genetic groups. An initial wide screening in modified Leonard jars (LJ) was followed by evaluation of a subset of strains and genotypes in pots (contained the same, sterile medium) in which fixed nitrogen was also quantified. An additive main effect and multiplicative interaction (AMMI) model was used to identify the contribution of individual strains and plant genotypes to the GL × GR interaction. Strong and highly significant GL × GR interaction was found in the LJ experiment but with little evidence of a relation to genetic background or growth habits. The interaction was much weaker in the pot experiment, with all bean genotypes and Rhizobium strains having relatively stable performance. We found that R. etli strain CFN42 and R. tropici strains CIAT899 and NAK91 were effective across bean genotypes but with the latter showing evidence of positive interaction with two specific bean genotypes. This suggests that selection of bean varieties based on their response to inoculation is possible. On the other hand, we show that symbiotic performance is not predicted by any a priori grouping, limiting the scope for more general recommendations. The fact that the strength and pattern of GL × GR depended on growing conditions provides an important cautionary message for future studies.IMPORTANCE The existence of genotype-by-strain (GL × GR) interaction has implications for the expected stability of performance of legume inoculants and could represent both challenges and opportunities for improvement of nitrogen fixation. We find that significant genotype-by-strain interaction exists in common bean (Phaseolus vulgaris L.) but that the strength and direction of this interaction depends on the growing environment used to evaluate biomass. Strong genotype and strain main effects, combined with a lack of predictable patterns in GL × GR, suggests that at best individual bean genotypes and strains can be selected for superior additive performance. The observation that the screening environment may affect experimental outcome of GL × GR means that identified patterns should be corroborated under more realistic conditions.

Revealing the roles of y4wF and tidC genes in Rhizobium tropici CIAT 899: biosynthesis of indolic compounds and impact on symbiotic properties.

Rhizobium tropici CIAT 899 is a strain known by its ability to nodulate a broad range of legume species, to synthesize a variety of Nod factors, its tolerance of abiotic stresses, and its high capacity to fix atmospheric N2, especially in symbiosis with common bean (Phaseolus vulgaris L.). Genes putatively related to the synthesis of indole acetic acid (IAA) have been found in the symbiotic plasmid of CIAT 899, in the vicinity of the regulatory nodulation gene nodD5, and, in this study, we obtained mutants for two of these genes, y4wF and tidC (R. tropiciindole-3-pyruvic acid decarboxylase), and investigated their expression in the absence and presence of tryptophan (TRP) and apigenin (API). In general, mutations of both genes increased exopolysaccharide (EPS) synthesis and did not affect swimming or surface motility; mutations also delayed nodule formation, but increased competitiveness. We found that the indole-3-acetamide (IAM) pathway was active in CIAT 899 and not affected by the mutations, and-noteworthy-that API was required to activate the tryptamine (TAM) and the indol-3-pyruvic acid (IPyA) pathways in all strains, particularly in the mutants. High up-regulation of y4wF and tidC genes was observed in both the wild-type and the mutant strains in the presence of API. The results obtained revealed an intriguing relationship between IAA metabolism and nod-gene-inducing activity in R. tropici CIAT 899. We discuss the IAA pathways, and, based on our results, we attribute functions to the y4wF and tidC genes of R. tropici.

The expression of an exogenous ACC deaminase by the endophyte Serratia grimesii BXF1 promotes the early nodulation and growth of common bean.

Ethylene acts as an inhibitor of the nodulation process of leguminous plants. However, some bacteria can decrease deleterious ethylene levels by the action of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase which degrades ACC, the ethylene precursor in all higher plants. Co-inoculation of rhizobia with endophytes enhances the rhizobial symbiotic efficiency with legumes, improving both nodulation and nitrogen fixation. However, not much is understood about the mechanisms employed by these endophytic bacteria. In this regard, the role of ACC deaminase from endophytic strains in assisting rhizobia in this process has yet to be confirmed. In this study, the role of ACC deaminase in an endophyte's ability to increase Rhizobium tropici nodulation of common bean was evaluated. To assess the effect of ACC deaminase in an endophyte's ability to promote rhizobial nodulation, the endophyte Serratia grimesii BXF1, which does not encode ACC deaminase, was transformed with an exogenous acdS gene. The results obtained indicate that the ACC deaminase-overexpressing transformant strain increased common bean growth, and enhanced the nodulation abilities of R. tropici CIAT899, in both cases compared to the wild-type non-transformed strain. Furthermore, plant inoculation with the ACC deaminase-overproducing strain led to an increased level of plant protection against a seed-borne pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: In this work, we studied the effect of ACC deaminase production by the bacterial endophyte Serratia grimesi BXF1, and its impact on the nodulation process of common bean. The results obtained indicate that ACC deaminase is an asset to the synergetic interaction between rhizobia and the endophyte, positively contributing to the overall legume-rhizobia symbiosis by regulating inhibitory ethylene levels that might otherwise inhibit nodulation and overall plant growth. The use of rhizobia together with an ACC deaminase-producing endophyte is, therefore, an important strategy for the development of new bacterial inoculants with increased performance.

Opening the "black box" of nodD3, nodD4 and nodD5 genes of Rhizobium tropici strain CIAT 899.

BACKGROUND: Transcription of nodulation genes in rhizobial species is orchestrated by the regulatory nodD gene. Rhizobium tropici strain CIAT 899 is an intriguing species in possessing features such as broad host range, high tolerance of abiotic stresses and, especially, by carrying the highest known number of nodD genes--five--and the greatest diversity of Nod factors (lipochitooligosaccharides, LCOs). Here we shed light on the roles of the multiple nodD genes of CIAT 899 by reporting, for the first time, results obtained with nodD3, nodD4 and nodD5 mutants. METHODS: The three nodD mutants were built by insertion of Ω interposon. Nod factors were purified and identified by LC-MS/MS analyses. In addition, nodD1 and nodC relative gene expressions were measured by quantitative RT-PCR in the wt and derivative mutant strains. Phenotypic traits such as exopolysaccharide (EPS), lipopolysaccharide (LPS), swimming and swarming motilities, biofilm formation and indole acetid acid (IAA) production were also perfomed. All these experiments were carried out in presence of both inducers of CIAT 899, apigenin and salt. Finally, nodulation assays were evaluated in up to six different legumes, including common bean (Phaseolus vulgaris L.). RESULTS: Phenotypic and symbiotic properties, Nod factors and gene expression of nodD3, nodD4 and nodD5 mutants were compared with those of the wild-type (WT) CIAT 899, both in the presence and in the absence of the nod-gene-inducing molecule apigenin and of saline stress. No differences between the mutants and the WT were observed in exopolysaccharide (EPS) and lipopolysaccharide (LPS) profiles, motility, indole acetic acid (IAA) synthesis or biofilm production, either in the presence, or in the absence of inducers. Nodulation studies demonstrated the most complex regulatory system described so far, requiring from one (Leucaena leucocephala, Lotus burtii) to four (Lotus japonicus) nodD genes. Up to 38 different structures of Nod factors were detected, being higher under salt stress, except for the nodD5 mutant; in addition, a high number of structures was synthesized by the nodD4 mutant in the absence of any inducer. Probable activator (nodD3 and nodD5) or repressor roles (nodD4), possibly via nodD1 and/or nodD2, were attributed to the three nodD genes. Expression of nodC, nodD1 and each nodD studied by RT-qPCR confirmed that nodD3 is an activator of nodD1, both in the presence of apigenin and salt stress. In contrast, nodD4 might be an inducer with apigenin and a repressor under saline stress, whereas nodD5 was an inducer under both conditions. CONCLUSIONS: We report for R. tropici CIAT 899 the most complex model of regulation of nodulation genes described so far. Five nodD genes performed different roles depending on the host plant and the inducing environment. Nodulation required from one to four nodD genes, depending on the host legume. nodD3 and nodD5 were identified as activators of the nodD1 gene, whereas, for the first time, it was shown that a regulatory nodD gene-nodD4-might act as repressor or inducer, depending on the inducing environment, giving support to the hypothesis that nodD roles go beyond nodulation, in terms of responses to abiotic stresses.

Isolation and the interaction between a mineral-weathering Rhizobium tropici Q34 and silicate minerals.

The purposes of this study were to isolate and evaluate the interaction between mineral-weathering bacteria and silicate minerals (feldspar and biotite). A mineral-weathering bacterium was isolated from weathered rocks and identified as Rhizobium tropici Q34 based on 16S rRNA gene sequence analysis. Si and K concentrations were increased by 1.3- to 4.0-fold and 1.1- to 1.7-fold in the live bacterium-inoculated cultures compared with the controls respectively. Significant increases in the productions of tartaric and succinic acids and extracellular polysaccharides by strain Q34 were observed in cultures with minerals. Furthermore, significantly more tartaric acid and polysaccharide productions by strain Q34 were obtained in the presence of feldspar, while better growth and more citric acid production of strain Q34 were observed in the presence of biotite. Mineral dissolution experiments showed that the organic acids and polysaccharides produced by strain Q34 were also capable of promoting the release of Si and K from the minerals. The results showed that the growth and metabolite production of strain Q34 were enhanced in the presence of the minerals and different mineral exerted distinct impacts on the growth and metabolite production. The bio-weathering process is probably a synergistic action of organic acids and extracellular polysaccharides produced by the bacterium.

High NaCl concentrations induce the nod genes of Rhizobium tropici CIAT899 in the absence of flavonoid inducers.

The nodulation (nod) genes of Rhizobium tropici CIAT899 can be induced by very low concentrations (micromolar to nanomolar range) of several flavonoid molecules secreted by the roots of leguminous plants under a number of different conditions. Some of these conditions have been investigated and appear to have a great influence on the concentration and the number of different Nod factors, which can induce root nodule primordia and pseudonodules in several leguminous plant roots. In one such condition, we added up to 300 mM NaCl to the induction medium of R. tropici CIAT899 containing the nod gene inducer apigenin. At the higher concentrations of NaCl, larger amounts and more different Nod factors were produced than in the absence of extra NaCl. To our surprise, under control conditions (300 mM NaCl without apigenin), some Nod-factor-like spots were also observed on the thin-layer plates used to detect incorporation of radiolabeled glucosamine into newly synthesized Nod factors. This phenomenon was further investigated with thin-layer plates, fusions of nod genes to the lacZ gene, high-performance liquid chromatography, mass spectrometry, and the formation of pseudonodules on bean roots. Here, we report that, in the absence of flavonoid inducers, high concentrations of NaCl induced nod genes and the production of Nod factors.

Transcript profiling of common bean nodules subjected to oxidative stress.

Several environmental stresses generate high amounts of reactive oxygen species (ROS) in plant cells, resulting in oxidative stress. Symbiotic nitrogen fixation (SNF) in the legume-rhizobia symbiosis is sensitive to damage from oxidative stress. Active nodules of the common bean (Phaseolus vulgaris) exposed to the herbicide paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride hydrate), which stimulates ROS accumulation, exhibited reduced nitrogenase activity and ureide content. We analyzed the global gene response of nodules subjected to oxidative stress using the Bean Custom Array 90K, which includes probes from 30,000 expressed sequence tags (ESTs). A total of 4280 ESTs were differentially expressed in stressed bean nodules; of these, 2218 were repressed. Based on Gene Ontology analysis, these genes were grouped into 42 different biological process categories. Analysis with the PathExpress bioinformatic tool, adapted for bean, identified five significantly repressed metabolic pathways related to carbon/nitrogen metabolism, which is crucial for nodule function. Quantitative reverse transcription (qRT)-PCR analysis of transcription factor (TF) gene expression showed that 67 TF genes were differentially expressed in nodules exposed to oxidative stress. Putative cis-elements recognized by highly responsive TF were detected in promoter regions of oxidative stress regulated genes. The expression of oxidative stress responsive genes and of genes important for SNF in bacteroids analyzed in stressed nodules revealed that these conditions elicited a transcriptional response.

Role of a LORELEI- like gene from Phaseolus vulgaris during a mutualistic interaction with Rhizobium tropici.

Reactive oxygen species (ROS), produced by NADPH oxidases known as RBOHs in plants, play a key role in plant development, biotic and abiotic stress responses, hormone signaling, and reproduction. Among the subfamily of receptor-like kinases referred to as CrRLK, there is FERONIA (FER), a regulator of RBOHs, and FER requires a GPI-modified membrane protein produced by LORELEI (LRE) or LORELEI-like proteins (LLG) to reach the plasma membrane and generate ROS. In Arabidopsis, AtLLG1 is involved in interactions with microbes as AtLLG1 interacts with the flagellin receptor (FLS2) to trigger the innate immune response, but the role of LLGs in mutualistic interactions has not been examined. In this study, two Phaseolus vulgaris LLG genes were identified, PvLLG2 that was expressed in floral tissue and PvLLG1 that was expressed in vegetative tissue. Transcripts of PvLLG1 increased during rhizobial nodule formation peaking during the early period of well-developed nodules. Also, P. vulgaris roots expressing pPvLLG1:GFP-GUS showed that this promoter was highly active during rhizobium infections, and very similar to the subcellular localization using a construct pLLG1::PvLLG1-Neon. Compared to control plants, PvLLG1 silenced plants had less superoxide (O2-) at the root tip and elongation zone, spotty hydrogen peroxide (H2O2) in the elongation root zone, and significantly reduced root hair length, nodule number and nitrogen fixation. Unlike control plants, PvLLG1 overexpressing plants showed superoxide beyond the nodule meristem, and significantly increased nodule number and nodule diameter. PvLLG1 appears to play a key role during this mutualistic interaction, possibly due to the regulation of the production and distribution of ROS in roots.

Genomic basis of broad host range and environmental adaptability of Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 which are used in inoculants for common bean (Phaseolus vulgaris L.).

BACKGROUND: Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 are α-Proteobacteria that establish nitrogen-fixing symbioses with a range of legume hosts. These strains are broadly used in commercial inoculants for application to common bean (Phaseolus vulgaris) in South America and Africa. Both strains display intrinsic resistance to several abiotic stressful conditions such as low soil pH and high temperatures, which are common in tropical environments, and to several antimicrobials, including pesticides. The genetic determinants of these interesting characteristics remain largely unknown. RESULTS: Genome sequencing revealed that CIAT 899 and PRF 81 share a highly-conserved symbiotic plasmid (pSym) that is present also in Rhizobium leucaenae CFN 299, a rhizobium displaying a similar host range. This pSym seems to have arisen by a co-integration event between two replicons. Remarkably, three distinct nodA genes were found in the pSym, a characteristic that may contribute to the broad host range of these rhizobia. Genes for biosynthesis and modulation of plant-hormone levels were also identified in the pSym. Analysis of genes involved in stress response showed that CIAT 899 and PRF 81 are well equipped to cope with low pH, high temperatures and also with oxidative and osmotic stresses. Interestingly, the genomes of CIAT 899 and PRF 81 had large numbers of genes encoding drug-efflux systems, which may explain their high resistance to antimicrobials. Genome analysis also revealed a wide array of traits that may allow these strains to be successful rhizosphere colonizers, including surface polysaccharides, uptake transporters and catabolic enzymes for nutrients, diverse iron-acquisition systems, cell wall-degrading enzymes, type I and IV pili, and novel T1SS and T5SS secreted adhesins. CONCLUSIONS: Availability of the complete genome sequences of CIAT 899 and PRF 81 may be exploited in further efforts to understand the interaction of tropical rhizobia with common bean and other legume hosts.

Variations in exopolysaccharide production by Rhizobium tropici.

Rhizobium tropici, a legume-symbiont soil bacterium, is known for its copious production of exopolysaccharide (EPS). Many aspects of this organism's growth and EPS production, however, remain uncharacterized, including the influence of environment and culturing conditions upon EPS. Here, we demonstrate that R. tropici EPS chemical composition and yield differ when grown with different substrates in a defined minimal medium in batch culture. Exopolysaccharide was quantified from R. tropici grown using arabinose, glucose, sucrose, mannitol, fructose, or glutamate as a sole carbon source. All tested substrates produced plenteous amounts of exopolysaccharide material. Variations in pH and carbon-to-nitrogen (C/N) ratio also resulted in assorted cell growth and exopolysaccharide production differences. We found that optimizing the C/N ratio has a greater impact upon R. tropici EPS production than upon R. tropici growth. A maximum EPS yield of 4.08 g/L was realized under optimized conditions, which is large even in comparison with other known rhizobia. We provide evidence that the chemical composition of R. tropici EPS can vary with changes to the growth environment. The composition of glucose-grown EPS contained rhamnose-linked residues that were not present in arabinose-grown EPS.

Production of extracellular biopolymers and identification of intracellular proteins and Rhizobium tropici.

The objective of this study was to identify species of rhizobia (from the IPA 403 and IPA 49 isolates), to assess the physico-chemical characteristics of the biopolymers produced by these rhizobia and to determine the soluble intracellular proteins that are present in these rhizobia. The polysaccharides containing acetyl and pyruvic acid groups that were produced by different strains that had been cultivated in yeast extract mannitol (YEM) medium for 132, 144, and 168 h were evaluated for yield, viscosity, and concentration. Based on the analysis of their partial 16S rDNA sequences, both isolates were identified as Rhizobium tropici. The polymers produced in liquid YEM medium were recovered, dried and weighed to determine culture yield. Soluble intracellular proteins were identified through the techniques of 2D-PAGE and mass spectrometry for cultures that were cultivated for 168 h. The largest biopolymer yield and the highest viscosity and concentration of acetyl and pyruvic acids were obtained from the IPA 403 isolate after 168 h of culture. The proteins that were identified for the CIAT 899 isolate included elongation factor TU, a chaperone; GroE/GroEs and a putative glycosyltransferase, all of which catalyze the production of polysaccharides. For the IPA 403 strain, dinitrogenase and nitrogenase iron proteins were found. In the IPA 49 strain, glyceraldehyde-3-phosphate dehydrogenase was found along with two other proteins, the beta subunit of an electron-transferring flavoprotein and a dehydrogenase.

Rhizobia exopolysaccharides: promising biopolymers for use in the formulation of plant inoculants.

Inoculants with beneficial microorganisms comprise both selected strains and carriers that ensure a favorable microenvironment for cell survival and stability. Formulations of inoculants using synthetic polymers as carriers are common. However, only a few studies are available in the literature regarding the formulation of inoculants using natural biomolecules as carriers. Exopolysaccharides (EPS) are biomolecules produced by a vast array of microbial species, including symbiotic nitrogen-fixing bacteria, commonly known as rhizobia. EPS perform several functions, such as the protection against the deleterious effects of diverse environmental soil stresses. Two Rhizobium tropici strains and one Paraburkholderia strain were selected after semiquantitative analysis by scanning electron microscopy (SEM) of their EPS production in liquid YMA medium. Their EPS were characterized through a series of analytical techniques, aiming at their use in the formulation of plant inoculants. In addition, the effect of the carbon source on EPS yield was evaluated. Multi-stage fragmentation analysis showed the presence of xylose, glucose, galactose, galacturonic acid, and glucuronic acid in EPS chemical composition, which was confirmed by FT-IR spectra and 13C NMR spectroscopy. Thermal stability (thermogravimetric) was close to 270 °C and viscosity ranged from 120 to 1053.3 mPa.s. Surface morphology (SEM) was rough and irregular, with a cross-linked spongy matrix, which, together with the hydrophilic functional groups, confers water holding capacity. The present study showed that the three EPS have potential as microorganism carriers for formulation of microbial inoculants to be applied in plants.

PvRACK1 loss-of-function impairs cell expansion and morphogenesis in Phaseolus vulgaris L. root nodules.

Receptor for activated C kinase (RACK1) is a highly conserved, eukaryotic protein of the WD-40 repeat family. Its peculiar ß-propeller structure allows its interaction with multiple proteins in various plant signal-transduction pathways, including those arising from hormone responses, development, and environmental stress. During Phaseolus vulgaris root development, RACK1 (PvRACK1) mRNA expression was induced by auxins, abscissic acid, cytokinin, and gibberellic acid. In addition, during P. vulgaris nodule development, PvRACK1 mRNA was highly accumulated at 12 to 15 days postinoculation, suggesting an important role after nodule meristem initiation and Rhizobium nodule infection. PvRACK1 transcript accumulation was downregulated by a specific RNA interference construct which was expressed in transgenic roots of composite plants of P. vulgaris inoculated with Rhizobium tropici. PvRACK1 downregulated transcript levels were monitored by quantitative reverse-transcription polymerase chain reaction analysis in individual transgenic roots and nodules. We observed a clear phenotype in PvRACK1-knockdown nodules, in which nodule number and nodule cell expansion were impaired, resulting in altered nodule size. Microscopic analysis indicated that, in PvRACK1-knockdown nodules, infected and uninfected cells were considerably smaller (80 and 60%, respectively) than in control nodules. In addition, noninfected cells and symbiosomes in silenced nodules showed significant defects in membrane structure under electron microscopy analysis. These findings indicate that PvRACK1 has a pivotal role in cell expansion and in symbiosome and bacteroid integrity during nodule development.

Phospholipid translocation captured in a bifunctional membrane protein MprF.

As a large family of membrane proteins crucial for bacterial physiology and virulence, the Multiple Peptide Resistance Factors (MprFs) utilize two separate domains to synthesize and translocate aminoacyl phospholipids to the outer leaflets of bacterial membranes. The function of MprFs enables Staphylococcus aureus and other pathogenic bacteria to acquire resistance to daptomycin and cationic antimicrobial peptides. Here we present cryo-electron microscopy structures of MprF homodimer from Rhizobium tropici (RtMprF) at two different states in complex with lysyl-phosphatidylglycerol (LysPG). RtMprF contains a membrane-embedded lipid-flippase domain with two deep cavities opening toward the inner and outer leaflets of the membrane respectively. Intriguingly, a hook-shaped LysPG molecule is trapped inside the inner cavity with its head group bent toward the outer cavity which hosts a second phospholipid-binding site. Moreover, RtMprF exhibits multiple conformational states with the synthase domain adopting distinct positions relative to the flippase domain. Our results provide a detailed framework for understanding the mechanisms of MprF-mediated modification and translocation of phospholipids.

Effect of plant age on endophytic bacterial diversity of balloon flower (Platycodon grandiflorum) root and their antimicrobial activities.

Balloon flower (Platycodon grandiflorum) is widely cultivated vegetable and used as a remedy for asthma in East Asia. Experiments were conducted to isolate endophytic bacteria from 1-, 3-, and 6-year-old balloon flower roots and to analyze the enzymatic, antifungal, and anti-human pathogenic activities of the potential endophytic biocontrol agents obtained. Total 120 bacterial colonies were isolated from the interior of all balloon flower roots samples. Phylogenetic analysis based on 16S rRNA gene sequences showed that the population of 'low G + C gram-positive bacteria' (LGCGPB) gradually increased 60.0-80.0% from 1 to 6 years balloon flower sample. On the other hand, maximum hydrolytic enzyme activity showing endophytic bacteria was under LGCGPB, among the bacterial strains, Bacillus sp. (BF1-1 and BF3-8), Bacillus sp. (BF1-2 and BF3-5), and Bacillus sp. (BF1-3, BF3-6, and BF6-4) showed maximum enzyme activities. Besides, Bacillus licheniformis (BF3-5 and BF6-6) and Bacillus pumilus (BF6-1) showed maximum antifungal activity against Phytophthora capsici, Fusarium oxysporum, Rhizoctonia solani, and Pythium ultimum. Moreover, Bacillus licheniformis was found in 3 and 6 years balloon flower roots, but Bacillus pumilus was found only in 6 years sample. It is presumed that older balloon flower plants invite more potential antifungal endophytes for there protection from plant diseases. In addition, Bacillus sp. (BF1-2 and BF3-5) showed maximum anti-human pathogenic activity. So, plant age is presumed to influence diversity of balloon flower endophytic bacteria.

An alternative succinate (2-oxoglutarate) transport system in Rhizobium tropici is induced in nodules of Phaseolus vulgaris.

The rhizobial DctA permease is essential for the development of effective nitrogen-fixing bacteroids, which was correlated with its requirement for growth on C(4)-dicarboxylates. A previously described dctA mutant of Rhizobium tropici CIAT899, strain GA1 (dctA), however, was unexpectedly still able to grow on succinate as a sole carbon source but less efficiently than CIAT899. Like other rhizobial dctA mutants, GA1 was unable to grow on fumarate or malate as a carbon source and induced the formation of ineffective nodules. We report an alternative succinate uptake system identified by Tn5 mutagenesis of strain GA1 that was required for the remaining ability to transport and utilize succinate. The alternative uptake system required a three-gene cluster that is highly characteristic of a dctABD locus. The predicted permease-encoding gene had high sequence similarity with open reading frames encoding putative 2-oxoglutarate permeases (KgtP) of Ralstonia solanacearum and Agrobacterium tumefaciens. This analysis was in agreement with the requirement for this gene for optimal growth on and induction by 2-oxoglutarate. The permease-encoding gene of the alternative system was also designated kgtP in R. tropici. The dctBD-like genes in this cluster were found to be required for kgtP expression and were designated kgtSR. Analysis of a kgtP::lacZ transcriptional fusion indicated that a kgtSR-dependent promoter of kgtP was specifically induced by 2-oxoglutarate. The expression of kgtPp was found in bacteroids of nodules formed with either CIAT899 or GA1 on roots of Phaseolus vulgaris. Results suggested that 2-oxoglutarate might be transported or conceivably exported in nodules induced by R. tropici on roots of P. vulgaris.

Osmotic stress activates nif and fix genes and induces the Rhizobium tropici CIAT 899 Nod factor production via NodD2 by up-regulation of the nodA2 operon and the nodA3 gene.

The symbiosis between rhizobia and legumes is characterized by a complex molecular dialogue in which the bacterial NodD protein plays a major role due to its capacity to activate the expression of the nodulation genes in the presence of appropiate flavonoids. These genes are involved in the synthesis of molecules, the nodulation factors (NF), responsible for launching the nodulation process. Rhizobium tropici CIAT 899, a rhizobial strain that nodulates Phaseolus vulgaris, is characterized by its tolerance to multiple environmental stresses such as high temperatures, acidity or elevated osmolarity. This strain produces nodulation factors under saline stress and the same set of CIAT 899 nodulation genes activated by inducing flavonoids are also up-regulated in a process controlled by the NodD2 protein. In this paper, we have studied the effect of osmotic stress (high mannitol concentrations) on the R. tropici CIAT 899 transcriptomic response. In the same manner as with saline stress, the osmotic stress mediated NF production and export was controlled directly by NodD2. In contrast to previous reports, the nodA2FE operon and the nodA3 and nodD1 genes were up-regulated with mannitol, which correlated with an increase in the production of biologically active NF. Interestingly, in these conditions, this regulatory protein controlled not only the expression of nodulation genes but also the expression of other genes involved in protein folding and synthesis, motility, synthesis of polysaccharides and, surprinsingly, nitrogen fixation. Moreover, the non-metabolizable sugar dulcitol was also able to induce the NF production and the activation of nod genes in CIAT 899.