Virulence-Related Assays for Investigation of the Acidovorax citrulli-Cucurbitaceae Pathosystem.

Acidovorax citrulli is one of the most important pathogens of cucurbit crops, mainly melon and watermelon. Although A. citrulli is able to infect all aerial parts of the plant, fruits are highly sensitive to the bacterium. Therefore, the disease is known as bacterial fruit blotch (BFB). The unavailability of effective tools for managing BFB, including the lack of resistant varieties, exacerbates the threat this disease poses to the cucurbit industry. However, despite the economic importance of BFB, still little is known about basic aspects of A. citrulli-plant interactions. Here, we present diverse techniques that have recently been developed for investigation of basic aspects of BFB, including identification of virulence determinants of the pathogen.

Microscope Subcellular Localization of Plant-Interacting Bacterial Effectors in Animal Cell Cultures.

Eukaryote-interacting bacteria have developed along the evolution of an arsenal of tools to interact with potential hosts and to evade their defensive responses. Among these tools, the effector proteins are gaining a special importance due to the high diversity of molecular actions that they play in the host cell, with the final aim of taking the control over the cell. Bacteria inject these effectors into the cytosol of the host cells through distinct ways, as the type III secretion system. The study of the effectors' molecular roles inside the host cell is challenging, due in part to the lack of traceability of such proteins once they are delivered by the bacteria. Here, we describe in depth a methodology that combines the increase of the bacterial effector concentration by protein expression systems with the use of heterologous hosts to facilitate the visualization of the subcellular targeting of the effector inside the host cell by fluorescence microscopy.

Isolation, Quantification, and Visualization of Extracellular Membrane Vesicles in Rhizobia Under Free-Living Conditions.

Rhizobia are a group of soil proteobacteria that are able to establish a symbiotic interaction with legumes. These bacteria are capable to fix atmospheric nitrogen into ammonia within specific plant root organs called nodules. The rhizobia-legume interaction is established by a complex molecular dialogue that starts with flavonoids exudated by the plant roots. In response, signaling molecules known as Nod factors (NFs) are secreted by the bacteria. These factors are sensed by specific plant receptors that trigger a downstream signaling cascade leading to rhizobium-specific intracellular colonization of the root hair via the formation of infection threads and the eventual development of nodules on roots. In these organs, rhizobia can fix nitrogen from the atmosphere for the plant in exchange for photosynthates and the appropriate environment for nitrogen fixation. Recently, it has been demonstrated that extracellular membrane vesicles (EMVs) produced by some rhizobia carry NFs. EMVs are proteolipidic structures that are secreted to the milieu from the bacterial membranes and are involved in several important biological processes, including intercellular communication. Thus far, little is known about rhizobia vesicles, and further studies are needed to understand their functions, including their role as transporting vessels of signaling molecules during the process of symbiosis. Here, we present a detailed protocol to isolate high-purity EMVs from free-living cultured rhizobia, test their integrity, and quantify their abundance.

Isolation of Rhizobial Extracellular Membrane Vesicles from Bacteroids.

Extracellular-membrane vesicles (EMVs) are spherical buds of the extracellular membrane, commonly produced by Gram-negative bacteria, known to mediate intricate inter-kingdom communication. In this context, comprehensive research dissecting the role of EMVs in one of the most complex nature-occurring molecular dialogues, rhizobium-legume symbiosis, has been so far neglected. During the different stages of the symbiotic process, rhizobia and their host plants establish a very specific and controlled intercellular trafficking of signal molecules. Thus, as conveyors of a broad range of molecules into the target cell, EMVs are gaining weight in the field. Here, we describe a detailed protocol to isolate EMVs from bacteroids of legume nodules, opening a new door for discovering new authors of the symbiotic process.

Natural variation in a short region of the Acidovorax citrulli type III-secreted effector AopW1 is associated with differences in cytotoxicity and host adaptation.

Bacterial fruit blotch, caused by Acidovorax citrulli, is a serious disease of melon and watermelon. The strains of the pathogen belong to two major genetic groups: group I strains are strongly associated with melon, while group II strains are more aggressive on watermelon. A. citrulli secretes many protein effectors to the host cell via the type III secretion system. Here we characterized AopW1, an effector that shares similarity to the actin cytoskeleton-disrupting effector HopW1 of Pseudomonas syringae and with effectors from other plant-pathogenic bacterial species. AopW1 has a highly variable region (HVR) within amino acid positions 147 to 192, showing 14 amino acid differences between group I and II variants. We show that group I AopW1 is more toxic to yeast and Nicotiana benthamiana cells than group II AopW1, having stronger actin filament disruption activity, and increased ability to induce cell death and reduce callose deposition. We further demonstrated the importance of some amino acid positions within the HVR for AopW1 cytotoxicity. Cellular analyses revealed that AopW1 also localizes to the endoplasmic reticulum, chloroplasts, and plant endosomes. We also show that overexpression of the endosome-associated protein EHD1 attenuates AopW1-induced cell death and increases defense responses. Finally, we show that sequence variation in AopW1 plays a significant role in the adaptation of group I and II strains to their preferred hosts, melon and watermelon, respectively. This study provides new insights into the HopW1 family of bacterial effectors and provides first evidence on the involvement of EHD1 in response to biotic stress.

Membrane vesicle engineering with "à la carte" bacterial-immunogenic molecules for organism-free plant vaccination.

The United Nations heralds a world population exponential increase exceeding 9.7 billion by 2050. This poses the challenge of covering the nutritional needs of an overpopulated world by the hand of preserving the environment. Extensive agriculture practices harnessed the employment of fertilizers and pesticides to boost crop productivity and prevent economic and harvest yield losses attributed to plagues and diseases. Unfortunately, the concomitant hazardous effects stemmed from such agriculture techniques are cumbersome, that is, biodiversity loss, soils and waters contaminations, and human and animal poisoning. Hence, the so-called 'green agriculture' research revolves around designing novel biopesticides and plant growth-promoting bio-agents to the end of curbing the detrimental effects. In this field, microbe-plant interactions studies offer multiple possibilities for reshaping the plant holobiont physiology to its benefit. Along these lines, bacterial extracellular membrane vesicles emerge as an appealing molecular tool to capitalize on. These nanoparticles convey a manifold of molecules that mediate intricate bacteria-plant interactions including plant immunomodulation. Herein, we bring into the spotlight bacterial extracellular membrane vesicle engineering to encase immunomodulatory effectors into their cargo for their application as biocontrol agents. The overarching goal is achieving plant priming by deploying its innate immune responses thereby preventing upcoming infections.

Silencing of SbPPCK1-3 Negatively Affects Development, Stress Responses and Productivity in Sorghum.

Phosphoenolpyruvate carboxylase (PEPC) plays central roles in photosynthesis, respiration, amino acid synthesis, and seed development. PEPC is regulated by different post-translational modifications. Between them, the phosphorylation by PEPC-kinase (PEPCk) is widely documented. In this work, we simultaneously silenced the three sorghum genes encoding PEPCk (SbPPCK1-3) by RNAi interference, obtaining 12 independent transgenic lines (Ppck1-12 lines), showing different degrees of SbPPCK1-3 silencing. Among them, two T2 homozygous lines (Ppck-2 and Ppck-4) were selected for further evaluation. Expression of SbPPCK1 was reduced by 65% and 83% in Ppck-2 and Ppck-4 illuminated leaves, respectively. Expression of SbPPCK2 was higher in roots and decreased by 50% in Ppck-2 and Ppck-4 in this tissue. Expression of SbPPCK3 was low and highly variable. Despite the incomplete gene silencing, it decreased the degree of phosphorylation of PEPC in illuminated leaves, P-deficient plants, and NaCl-treated plants. Both leaves and seeds of Ppck lines had altered metabolic profiles and a general decrease in amino acid content. In addition, Ppck lines showed delayed flowering, and 20% of Ppck-4 plants did not produce flowers at all. The total amount of seeds was lowered by 50% and 36% in Ppck-2 and Ppck-4 lines, respectively. The quality of seeds was lower in Ppck lines: lower amino acid content, including Lys, and higher phytate content. These data confirm the relevance of the phosphorylation of PEPC in sorghum development, stress responses, yield, and quality of seeds.

Multitask Approach to Localize Rhizobial Type Three Secretion System Effector Proteins Inside Eukaryotic Cells.

Rhizobia can establish mutually beneficial interactions with legume plants by colonizing their roots to induce the formation of a specialized structure known as a nodule, inside of which the bacteria are able to fix atmospheric nitrogen. It is well established that the compatibility of such interactions is mainly determined by the bacterial recognition of flavonoids secreted by the plants, which in response to these flavonoids trigger the synthesis of the bacterial Nod factors that drive the nodulation process. Additionally, other bacterial signals are involved in the recognition and the efficiency of this interaction, such as extracellular polysaccharides or some secreted proteins. Some rhizobial strains inject proteins through the type III secretion system to the cytosol of legume root cells during the nodulation process. Such proteins, called type III-secreted effectors (T3E), exert their function in the host cell and are involved, among other tasks, in the attenuation of host defense responses to facilitate the infection, contributing to the specificity of the process. One of the main challenges of studying rhizobial T3E is the inherent difficulty in localizing them in vivo in the different subcellular compartments within their host cells, since in addition to their low concentration under physiological conditions, it is not always known when or where they are being produced and secreted. In this paper, we use a well-known rhizobial T3E, named NopL, to illustrate by a multitask approach where it localizes in heterologous hosts models, such as tobacco plant leaf cells, and also for the first time in transfected and/or Salmonella-infected animal cells. The consistency of our results serves as an example to study the location inside eukaryotic cells of effectors in distinct hosts with different handling techniques that can be used in almost every research laboratory.

A complex regulatory network governs the expression of symbiotic genes in Sinorhizobium fredii HH103.

Introduction: The establishment of the rhizobium-legume nitrogen-fixing symbiosis relies on the interchange of molecular signals between the two symbionts. We have previously studied by RNA-seq the effect of the symbiotic regulators NodD1, SyrM, and TtsI on the expression of the symbiotic genes (the nod regulon) of Sinorhizobium fredii HH103 upon treatment with the isoflavone genistein. In this work we have further investigated this regulatory network by incorporating new RNA-seq data of HH103 mutants in two other regulatory genes, nodD2 and nolR. Both genes code for global regulators with a predominant repressor effect on the nod regulon, although NodD2 acts as an activator of a small number of HH103 symbiotic genes. Methods: By combining RNA-seq data, qPCR experiments, and b-galactosidase assays of HH103 mutants harbouring a lacZ gene inserted into a regulatory gene, we have analysed the regulatory relations between the nodD1, nodD2, nolR, syrM, and ttsI genes, confirming previous data and discovering previously unknown relations. Results and discussion: Previously we showed that HH103 mutants in the nodD2, nolR, syrM, or ttsI genes gain effective nodulation with Lotus japonicus, a model legume, although with different symbiotic performances. Here we show that the combinations of mutations in these genes led, in most cases, to a decrease in symbiotic effectiveness, although all of them retained the ability to induce the formation of nitrogen-fixing nodules. In fact, the nodD2, nolR, and syrM single and double mutants share a set of Nod factors, either overproduced by them or not generated by the wild-type strain, that might be responsible for gaining effective nodulation with L. japonicus.

The Rhizobial Type 3 Secretion System: The Dr. Jekyll and Mr. Hyde in the Rhizobium-Legume Symbiosis.

Rhizobia are soil bacteria that can establish a symbiotic association with legumes. As a result, plant nodules are formed on the roots of the host plants where rhizobia differentiate to bacteroids capable of fixing atmospheric nitrogen into ammonia. This ammonia is transferred to the plant in exchange of a carbon source and an appropriate environment for bacterial survival. This process is subjected to a tight regulation with several checkpoints to allow the progression of the infection or its restriction. The type 3 secretion system (T3SS) is a secretory system that injects proteins, called effectors (T3E), directly into the cytoplasm of the host cell, altering host pathways or suppressing host defense responses. This secretion system is not present in all rhizobia but its role in symbiosis is crucial for some symbiotic associations, showing two possible faces as Dr. Jekyll and Mr. Hyde: it can be completely necessary for the formation of nodules, or it can block nodulation in different legume species/cultivars. In this review, we compile all the information currently available about the effects of different rhizobial effectors on plant symbiotic phenotypes. These phenotypes are diverse and highlight the importance of the T3SS in certain rhizobium-legume symbioses.

The Rhizobium tropici CIAT 899 NodD2 protein promotes symbiosis and extends rhizobial nodulation range by constitutive nodulation factor synthesis.

In the symbiotic associations between rhizobia and legumes, the NodD regulators orchestrate the transcription of the specific nodulation genes. This set of genes is involved in the synthesis of nodulation factors, which are responsible for initiating the nodulation process. Rhizobium tropici CIAT 899 is the most successful symbiont of Phaseolus vulgaris and can nodulate a variety of legumes. Among the five NodD regulators present in this rhizobium, only NodD1 and NodD2 seem to have a role in the symbiotic process. However, the individual role of each NodD in the absence of the other proteins has remained elusive. In this work, we show that the CIAT 899 NodD2 does not require activation by inducers to promote the synthesis of nodulation factors. A CIAT 899 strain overexpressing nodD2, but lacking all additional nodD genes, can nodulate three different legumes as efficiently as the wild type. Interestingly, CIAT 899 NodD2-mediated gain of nodulation can be extended to another rhizobial species, since its overproduction in Sinorhizobium fredii HH103 not only increases the number of nitrogen-fixing nodules in two host legumes but also results in nodule development in incompatible legumes. These findings potentially open exciting opportunities to develop rhizobial inoculants and increase legume crop production.

The GDSL-Lipolytic Enzyme Lip1 Is Required for Full Virulence of the Cucurbit Pathogenic Bacterium Acidovorax citrulli.

Bacterial fruit blotch caused by Acidovoraxcitrulli is a serious disease of cucurbit crops. Here we report characterization of a mutant strain of A. citrulli M6 defective in lip1, a gene encoding a lipolytic enzyme. The M6-lip1- mutant was detected in a mutant library screen aimed at identifying M6 mutants with altered levels of twitching motility. In this screen M6-lip1- was the only mutant that showed significantly larger twitching motility haloes around colonies than wild-type M6. Sequence analyses indicated that lip1 encodes a member of the GDSL family of secreted lipolytic enzymes. In line with this finding, lipolytic assays showed that the supernatants of M6-lip1- had lower lipolytic activity as compared with those of wild-type M6 and a lip1-complemented strain. The mutant was also affected in swimming motility and had compromised virulence on melon seedlings and on Nicotiana benthamiana leaves relative to wild-type and complemented strains. Lip1 contains a predicted N-terminal signal sequence for type II secretion. Evidence from our study confirms Lip1 is indeed secreted in a type II secretion-dependent manner, and this is required for full virulence of A. citrulli. To the best of our knowledge this is the first study reporting contribution of lipolytic activity to virulence of a plant-pathogenic Acidovorax species.

One door closes, another opens: when nodulation impairment with natural hosts extends rhizobial host-range.

The rhizobium-legume symbiosis is the best-understood plant-microbe association. The high degree of specificity observed in this relationship is supported by a complex exchange of signals between the two components of the symbiosis. Findings reported in last years indicate that multiple molecular mechanisms, such as the production of a particular set of nodulation factors at a very specific concentration or a suitable arsenal of effectors secreted through the type III secretion system, have been adjusted during evolution to ensure and optimize the recognition of specific rhizobial strains by its legume host. Qualitative or quantitative changes in the production of these symbiotic molecular determinants are detrimental for nodulation with its natural host but, in some cases, can also result beneficial for the rhizobium since it extends the nodulation host-range to other legumes. Potential repercussion of the extension in the nodulation host-range of rhizobia is discussed.

The Sinorhizobium fredii HH103 type III secretion system effector NopC blocks nodulation with Lotus japonicus Gifu.

The broad-host-range bacterium Sinorhizobium fredii HH103 cannot nodulate the model legume Lotus japonicus Gifu. This bacterium possesses a type III secretion system (T3SS), a specialized secretion apparatus used to deliver effector proteins (T3Es) into the host cell cytosol to alter host signaling and/or suppress host defence responses to promote infection. However, some of these T3Es are recognized by specific plant receptors and hence trigger a strong defence response to block infection. In rhizobia, T3Es are involved in nodulation efficiency and host-range determination, and in some cases directly activate host symbiosis signalling in a Nod factor-independent manner. In this work, we show that HH103 RifR T3SS mutants, unable to secrete T3Es, gain nodulation with L. japonicus Gifu through infection threads, suggesting that plant recognition of a T3E could block the infection process. To identify the T3E involved, we performed nodulation assays with a collection of mutants that affect secretion of each T3E identified in HH103 RifR so far. The nopC mutant could infect L. japonicus Gifu by infection thread invasion and switch the infection mechanism in Lotus burttii from intercellular infection to infection thread formation. Lotus japonicus gene expression analysis indicated that the infection-blocking event occurs at early stages of the symbiosis.

Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III-secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus.

The cucurbit pathogenic bacterium Acidovorax citrulli requires a functional type III secretion system (T3SS) for pathogenicity. In this bacterium, as with Xanthomonas and Ralstonia spp., an AraC-type transcriptional regulator, HrpX, regulates expression of genes encoding T3SS components and type III-secreted effectors (T3Es). The annotation of a sequenced A. citrulli strain revealed 11 T3E genes. Assuming that this could be an underestimation, we aimed to uncover the T3E arsenal of the A. citrulli model strain, M6. Thorough sequence analysis revealed 51 M6 genes whose products are similar to known T3Es. Furthermore, we combined machine learning and transcriptomics to identify novel T3Es. The machine-learning approach ranked all A. citrulli M6 genes according to their propensity to encode T3Es. RNA-Seq revealed differential gene expression between wild-type M6 and a mutant defective in HrpX: 159 and 28 genes showed significantly reduced and increased expression in the mutant relative to wild-type M6, respectively. Data combined from these approaches led to the identification of seven novel T3E candidates that were further validated using a T3SS-dependent translocation assay. These T3E genes encode hypothetical proteins that seem to be restricted to plant pathogenic Acidovorax species. Transient expression in Nicotiana benthamiana revealed that two of these T3Es localize to the cell nucleus and one interacts with the endoplasmic reticulum. This study places A. citrulli among the 'richest' bacterial pathogens in terms of T3E cargo. It also revealed novel T3Es that appear to be involved in the pathoadaptive evolution of plant pathogenic Acidovorax species.

Complete Assembly of the Genome of an Acidovorax citrulli Strain Reveals a Naturally Occurring Plasmid in This Species.

Acidovorax citrulli is the causal agent of bacterial fruit blotch (BFB), a serious threat to cucurbit crop production worldwide. Based on genetic and phenotypic properties, A. citrulli strains are divided into two major groups: group I strains have been generally isolated from melon and other non-watermelon cucurbits, while group II strains are closely associated with watermelon. In a previous study, we reported the genome of the group I model strain, M6. At that time, the M6 genome was sequenced by MiSeq Illumina technology, with reads assembled into 139 contigs. Here, we report the assembly of the M6 genome following sequencing with PacBio technology. This approach not only allowed full assembly of the M6 genome, but it also revealed the occurrence of a ∼53 kb plasmid. The M6 plasmid, named pACM6, was further confirmed by plasmid extraction, Southern-blot analysis of restricted fragments and obtention of M6-derivative cured strains. pACM6 occurs at low copy numbers (average of ∼4.1 ± 1.3 chromosome equivalents) in A. citrulli M6 and contains 63 open reading frames (ORFs), most of which (55.6%) encoding hypothetical proteins. The plasmid contains several genes encoding type IV secretion components, and typical plasmid-borne genes involved in plasmid maintenance, replication and transfer. The plasmid also carries an operon encoding homologs of a Fic-VbhA toxin-antitoxin (TA) module. Transcriptome data from A. citrulli M6 revealed that, under the tested conditions, the genes encoding the components of this TA system are among the highest expressed genes in pACM6. Whether this TA module plays a role in pACM6 maintenance is still to be determined. Leaf infiltration and seed transmission assays revealed that, under tested conditions, the loss of pACM6 did not affect the virulence of A. citrulli M6. We also show that pACM6 or similar plasmids are present in several group I strains, but absent in all tested group II strains of A. citrulli.

Sinorhizobium fredii HH103 nolR and nodD2 mutants gain capacity for infection thread invasion of Lotus japonicus Gifu and Lotus burttii.

Sinorhizobium fredii HH103 RifR , a broad-host-range rhizobial strain, forms ineffective nodules with Lotus japonicus but induces nitrogen-fixing nodules in Lotus burttii roots that are infected by intercellular entry. Here we show that HH103 RifR nolR or nodD2 mutants gain the ability to induce infection thread formation and to form nitrogen-fixing nodules in L. japonicus Gifu. Microscopy studies showed that the mode of infection of L. burttii roots by the nodD2 and nolR mutants switched from intercellular entry to infection threads (ITs). In the presence of the isoflavone genistein, both mutants overproduced Nod-factors. Transcriptomic analyses showed that, in the presence of Lotus japonicus Gifu root exudates, genes related to Nod factors production were overexpressed in both mutants in comparison to HH103 RifR . Complementation of the nodD2 and nolR mutants provoked a decrease in Nod-factor production, the incapacity to form nitrogen-fixing nodules with L. japonicus Gifu and restored the intercellular way of infection in L. burttii. Thus, the capacity of S. fredii HH103 RifR nodD2 and nolR mutants to infect L. burttii and L. japonicus Gifu by ITs and fix nitrogen L. japonicus Gifu might be correlated with Nod-factor overproduction, although other bacterial symbiotic signals could also be involved.

Transcriptomic Studies of the Effect of nod Gene-Inducing Molecules in Rhizobia: Different Weapons, One Purpose.

Simultaneous quantification of transcripts of the whole bacterial genome allows the analysis of the global transcriptional response under changing conditions. RNA-seq and microarrays are the most used techniques to measure these transcriptomic changes, and both complement each other in transcriptome profiling. In this review, we exhaustively compiled the symbiosis-related transcriptomic reports (microarrays and RNA sequencing) carried out hitherto in rhizobia. This review is specially focused on transcriptomic changes that takes place when five rhizobial species, Bradyrhizobium japonicum (=diazoefficiens) USDA 110, Rhizobium leguminosarum biovar viciae 3841, Rhizobium tropici CIAT 899, Sinorhizobium (=Ensifer) meliloti 1021 and S. fredii HH103, recognize inducing flavonoids, plant-exuded phenolic compounds that activate the biosynthesis and export of Nod factors (NF) in all analysed rhizobia. Interestingly, our global transcriptomic comparison also indicates that each rhizobial species possesses its own arsenal of molecular weapons accompanying the set of NF in order to establish a successful interaction with host legumes.

The Sinorhizobium (Ensifer) fredii HH103 Nodulation Outer Protein NopI Is a Determinant for Efficient Nodulation of Soybean and Cowpea Plants.

The type III secretion system (T3SS) is a specialized secretion apparatus that is commonly used by many plant and animal pathogenic bacteria to deliver proteins, termed effectors, to the interior of the host cells. These effectors suppress host defenses and interfere with signal transduction pathways to promote infection. Some rhizobial strains possess a functional T3SS, which is involved in the suppression of host defense responses, host range determination, and symbiotic efficiency. The analysis of the genome of the broad-host-range rhizobial strain Sinorhizobium fredii HH103 identified eight genes that code for putative T3SS effectors. Three of these effectors, NopL, NopP, and NopI, are Rhizobium specific. In this work, we demonstrate that NopI, whose amino acid sequence shows a certain similarity with NopP, is secreted through the S. fredii HH103 T3SS in response to flavonoids. We also determined that NopL can be considered an effector since it is directly secreted to the interior of the host cell as demonstrated by adenylate cyclase assays. Finally, the symbiotic phenotype of single, double, and triple nopI, nopL, and nopP mutants in soybean and cowpea was assayed, showing that NopI plays an important role in determining the number of nodules formed in both legumes and that the absence of both NopL and NopP is highly detrimental for symbiosis.IMPORTANCE The paper is focused on three Rhizobium-specific T3SS effectors of Sinorhizobium fredii HH103, NopL, NopP, and NopI. We demonstrate that S. fredii HH103 is able to secrete through the T3SS in response to flavonoids the nodulation outer protein NopI. Additionally, we determined that NopL can be considered an effector since it is secreted to the interior of the host cell as demonstrated by adenylate cyclase assays. Finally, nodulation assays of soybean and cowpea indicated that NopI is important for the determination of the number of nodules formed and that the absence of both NopL and NopP negatively affected nodulation.

RNA-seq analysis of the Rhizobium tropici CIAT 899 transcriptome shows similarities in the activation patterns of symbiotic genes in the presence of apigenin and salt.

BACKGROUND: Rhizobium tropici strain CIAT 899 establishes effective symbioses with several legume species, including Phaseolus vulgaris and Leucaena leucocephala. This bacterium synthesizes a large variety of nodulation factors in response to nod-gene inducing flavonoids and, surprisingly, also under salt stress conditions. The aim of this study was to identify differentially expressed genes in the presence of both inducer molecules, and analyze the promoter regions located upstream of these genes. RESULTS: Results obtained by RNA-seq analyses of CIAT 899 induced with apigenin, a nod gene-inducing flavonoid for this strain, or salt allowed the identification of 19 and 790 differentially expressed genes, respectively. Fifteen of these genes were up-regulated in both conditions and were involved in the synthesis of both Nod factors and indole-3-acetic acid. Transcription of these genes was presumably activated through binding of at least one of the five NodD proteins present in this strain to specific nod box promoter sequences when the bacterium was induced by both apigenin and salt. Finally, under saline conditions, many other transcriptional responses were detected, including an increase in the transcription of genes involved in trehalose catabolism, chemotaxis and protein secretion, as well as ribosomal genes, and a decrease in the transcription of genes involved in transmembrane transport. CONCLUSIONS: To our knowledge this is the first time that a transcriptomic study shows that salt stress induces the expression of nodulation genes in the absence of flavonoids. Thus, in the presence of both nodulation inducer molecules, apigenin and salt, R. tropici CIAT 899 up-regulated the same set of symbiotic genes. It could be possible that the increases in the transcription levels of several genes related to nodulation under saline conditions could represent a strategy to establish symbiosis under abiotic stressing conditions.