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
- Francisco Pérez-Montaño†1Email authorView ORCID ID profile,
- Pablo del Cerro†1,
- Irene Jiménez-Guerrero1,
- Francisco Javier López-Baena1,
- Maria Teresa Cubo1,
- Mariangela Hungria2,
- Manuel Megías1 and
- Francisco Javier Ollero1
© Pérez-Montaño et al. 2016
Received: 15 October 2015
Accepted: 25 February 2016
Published: 8 March 2016
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 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.
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.
Rhizobia comprise a group of α- and β-proteobacteria known for their ability to establish symbioses with several leguminous species. The rhizobium-legume interaction, characterized by an exchange of signal molecules from both partners, culminates in the formation of specific structures, called nodules, where biological nitrogen fixation takes place [1–5]. This molecular dialogue begins with the exudation of flavonoids by the host legume roots that are recognized by a LysR-type transcriptional regulator in the bacterium, the NodD protein, which triggers the expression of the so-called nodulation (nod) genes by binding to specific sequences, nod boxes (NB), located upstream of these genes. Their cognate enzymes are implied in the production of lipochitooligosaccharides, also known as Nod factors (NF), which in turns induce the formation of root nodule primordia and play an essential role in the infection process. A part from flavonoids, other rhizobial nod gene inducers have been identified, such as betaines , but they are required at higher concentrations .
Rhizobium tropici CIAT 899 (hereafter CIAT 899) is a broad host-range rhizobial strain isolated from tropical acid soils of South America that effectively nodulates several legumes, including Phaseolus vulgaris, Macroptilium atropurpureum, and Leucaena leucocephala [8, 9]. Main characteristics of this strain includes its high tolerance to several environmental stresses such as high temperature, acidity or salinity and its capacity to producing a large variety of NF in the presence of inducer flavonoids, such as apigenin [8, 10, 11]. It is remarkable that under acidity or salt stress conditions the synthesis of NF in CIAT 899 is also induced, resulting in increased diversity and concentration of these molecules in comparison to non-stressing conditions [12, 13]. Interestingly, Guasch-Vidal et al.  demonstrated that, even in the absence of flavonoids, CIAT 899 is able of synthesizing NF in the presence of high concentrations of salt, and the biological activity of these NF was confirmed. Moreover, the activation under salt stress is independent of NodD1 . It has been reported that initial steps of rhizobium-legume symbioses are very sensitive to salt stress. However, the ability to form root nodules on their host legume species under saline conditions has been described for many rhizobia . In general, rhizobial strains use distinct mechanisms for osmotic adaptation under salt stressing conditions during the free-lifestyle, such as the intracellular accumulation of osmolytes and specific ions, modification in cell surface polysaccharides or the synthesis of certain ABC membrane transporters [15, 16]. However, the synthesis of salt-induced NF has been only reported in CIAT 899.
Genome sequencing of CIAT 899 revealed five different nodD genes and three different nodA genes in the symbiotic plasmid . NodA catalyzes the transfer of the fatty acyl group from an acyl carrier protein to a terminal n-glucosamine residue previously deacetyled by NodB, on the chitin oligomer . The nodA1 gene is located adjacent to nodD1, whose encoded protein seems to be the major regulator of NF synthesis upon induction with flavonoid [19, 20] and together with nodBC compose an operon responsible for the synthesis of the NF core. The nodA2 gene is part of a gene cluster including hsnT and nodFE, implied in unsaturated fatty acid incorporation into NF molecules and is located close to the nodD2 gene. Curiously, a previous study  showed that, apparently, the activation of the expression of the nodC gene under salt stress is lower in a nodD2 mutant than in both a nodD1 mutant and the wild-type strain. Finally, nodA3 is located downstream the nodD3 gene but no other symbiotic-related genes have been identified in its vicinities .
The main objective of this study was to identify genes of CIAT 899 that are differentially expressed in the presence of the nod-gene inducer molecules apigenin and salt by RNA-seq analysis. In addition, the promoter regions of the symbiotic genes that were up-regulated in both conditions were studied to determine possible conserved promoter consensus motifs. Our results showed similar patterns of expression for the differentially expressed genes of the symbiotic plasmid replicon in the presence of both apigenin and salt, indicating that the NF synthesis was carried out following the same pathway, independently of the inducer molecule. The biological significance of the CIAT 899 transcriptomic response under salt condition was discussed.
Results and discussion
Identification of the differentially expressed genes
RNA-seq data validation using qRT-PCR. Fold-change values were calculated using the ∆∆Ct method and normalized to the reference gene RNA 16S for 20 differentially expressed genes. HP: gene that codes for a hypothetical protein
Functions associated to responses to nod gene-inducing molecules
Apigenin activates the transcription of nod gene operons and the synthesis of indole-3-acetic acid
Common responses: synthesis of nodulation molecules and identification of nod boxes
Fold-change expression values of the R. tropici CIAT 899 nod genes on the RNA-seq analysis. Fold-change values followed by an asterisk (*) are significantly over-expressed
Gene name/Locus tag
Up-regulation of the R. tropici CIAT 899 pRtCIAT899b genes located downstream nod boxes. Transcriptional activation (4-fold induction with respect to control cultures) of several nod box controlled operons was demonstrated by RNA-seq data in the presence of both inducer molecules. HP: gene that codes for a hypothetical protein
Cds number/Locus tag/Gene name
1 (270 pb)
cds216 to 227/RTCIAT899_ PB01300 to RTCIAT899_ PB01345/nodABCSUIJHPQ1Q2
8.73 to 1.28
13.66 to 2.54
2 (250 pb)
cds177 to 180/RTCIAT899_ PB01095 to RTCIAT899_ PB01110/nodA2hsnTnodFE
10.3 to 10.37
9.81 to 11.94
3 (180 pb)
cds450 to 449/RTCIAT899_ PB02710 to RTCIAT899_ PB02705/nodM
2.43 to 3.18
5.85 to 8.49
4 (680 pb)
cds95 to 93/RTCIAT899_PB00575 to RTCIAT899_PB00565/y4wEF
Synthesis of IAA
8.57 to 1.63
12.17 to 3.27
5 (38 pb)
cds263 to 262/RTCIAT899_PB01550 to RTCIAT899_PB01545/HP
6.75 to 4.26
28.65 to 14.37
6 (70 pb)
cds61 to 62/RTCIAT899_PB00370 to RTCIAT899_PB00375/HP
1.80 to 2.11
2.17 to 1.64
7 (191 pb)
8 (470 pb)
9 (979 pb)
10 (292 pb)
Altogether, these results suggest that when CIAT 899 was induced either by apigenin or salt, genes related to the synthesis of NF and IAA were activated through the recognition of specific nod boxes (Table 2). In S. fredii NGR234, nineteen nod boxes have been identified and eighteen of them were inducible with flavonoids via NodD1. In addition, S. fredii NGR234 carries two copies of the nodD gene and four of these nod boxes are controlled by NodD2 .
Salt stress response: a pathway towards symbiosis?
Chromosomal RNA-seq analysis showed, under salt stress, an up-regulation of genes whose products are implied in the formation of ribosomes (both 30 and 50S ribosomal proteins coded by the rpl and rps genes, respectively) and in the utilization of the disaccharide trehalose (thuAB genes), an osmotic stabilizer [27, 28]. Instead, down-regulation was detected in genes that encode proteins involved in chemotaxis (cheXYAWRBYD genes) , formation of Flp-type pili (type IVb protein secretion system; tadBCD and cpaABCDEF genes) , or ABC-type transport (several genes: e.g. phn genes). In addition, a set of genes located in pRtrCIAT899c and involved in the export of capsular polysaccharide across the inner membrane (genes of the family bexC/ctrB/kpsE) was also down-regulated (Fig. 4, Additional file 5) .
Our work shows that in the presence of the nodulation inducing molecules apigenin (3.7 μM) and salt (300 mM), R. tropici CIAT 899 increases the transcription of the same set of genes (slightly higher expression upon salt treatment), whose encoded proteins are involved in the synthesis of symbiotic molecules. Besides, the salt-dependent production of these symbiotic molecules could be a CIAT 899 strategy to ensure nodulation under salt stress.
Culture conditions and RNA extraction
R. tropici CIAT 899 was grown for 72 h at 28 °C on tryptone yeast (TY) medium , supplemented with apigenin 3.7 μM or NaCl 300 mM when necessary (induction of the synthesis of NF) [14, 20]. Total RNA was isolated using a High Pure RNA Isolation Kit (Roche), according to the manufacturer’s instructions. Verification of the amount and quality of total RNA samples was carried out using a Nanodrop 1000 spectrophotometer (Thermo Scientific) and a Qubit 2.0 Fluorometer (Invitrogen). Two independent total RNA extractions were obtained for each condition.
Quantitative reverse transcription PCR
Result obtained in the RNA-seq analysis were validated by quantitative reverse transcription PCR (qRT-PCR) of 20 selected genes, which represented differentially and non-differentially expressed genes in the presence of apigenin and salt. Total RNA was isolated using a High Pure RNA Isolation Kit (Roche) and RNAase Free DNA Set (Qiagen), according to the manufacturer’s instructions. This (DNA-free) RNA was reverse transcribed into cDNA using a QuantiTec Reverse Transcription Kit (Qiagen). Quantitative PCR was performed using a LightCycler 480 (Roche) with the following conditions: 95 °C, 10 min; 95 °C, 30 s; 50 °C, 30 s; 72 °C, 20 s; forty cycles, followed by the melting curve profile from 60 to 95 °C to verify the specificity of the reaction. The R. tropici CIAT 899 16S rRNA gene was used as an internal control to normalize gene expression. The fold-changes of two biological samples with three technical replicates of each condition were obtained using the ∆∆Ct method . Selected genes and primers are listed in Additional file 2.
Ribosomal RNA was depleted using a MICROB Express Bacterial mRNA Purification kit (Ambion), following the manufacturer’s protocol. Integrity and quality of the ribosomal depleted RNA was checked with Agilent Bioanalyzer 2100 (Agilent Technologies). RNA sequencing was carried out by Sistemas Genómicos (https://www.sistemasgenomicos.com/web_sg/) with the Next Generation Sequence (NGS) platform Illumina using the Illumina HiSeq 2000 sequencing instrument (Illumina). Ribosomal-depleted samples were used to generate whole transcriptome libraries following the manufacturer's recommendations for sequencing on this NGS platform. Amplified cDNA quality was analyzed by the Bioanalyzer 2100 DNA 1000 kit (Agilent Technologies) and quantified using the Qubit 2.0 Fluorometer (Invitrogen).
Mapping of the RNA-seq data
The initial whole transcriptome paired-end reads obtained from sequencing were mapped against the latest version of the R. tropici CIAT 899 genome (http://www.ncbi.nlm.nih.gov/genome/?term=Rhizobium_tropici_CIAT_899) using the the Life Technologies mapping algorithm version 1.3 (http://www.lifetechnologies.com/). Low-quality reads were eliminated using Picard Tools software version 1.83, remaining only high quality reads.
Assessment of differentially expressed genes
Gene prediction was estimated using the cufflinks method  and the expression levels were calculated using the htseq software, version 0.5.4p3 . This method eliminates multimapped reads, considering only unique reads for the gene expression estimation. The edge method version 3.2.4 was applied for differential expression analysis among conditions . This method uses a Poisson model to estimate the variance of the RNA-seq data for differential expressions, and relies on different normalized processes based on depth global samples, CG composition and length of genes. Differentially expressed genes were established in those genes with a fold-change lower or higher than−4 or 4, respectively, with a p value adjust to 0.7.
Functional categorization of genes
In order to assign the statistical over-represented functional categories in the presence of both nod gene-inducing molecules, an enrichment functional study was performed. Thus, genes were annotated using Uniprot databases and a hypergeometrical test using all genes as background and differential gene expression as interesting group of genes was applied . This statistical test calculates the statistical significance using p value , being in this case evaluated the significance of functional categories. Those functional categories (biological processes) with a p value inferior to 0.15 were considered over-represented.
The program fuzznuc of the EMBOSS package was used to identify nod box-like sequences. Hits were selected based on their conservation to known NB sequences [44, 45]. Thus, the search pattern used was at[ct][cg][ag]n(5)[tc][ga][ga]atn(7)at[ct]caaacaatc[ga]attttncn(2)at, allowing a maximum of 3 mismatches. Searching of these consensus sequences was carried out on the whole genome of CIAT 899. Alignment of the NB sequences was performed using ClustalW at EMBnet.
RNA-seq data accession number
The RNA-seq data discussed in this publication have been deposited in the Sequence Read Archive of NCBI under the accession number SRP067561.
We would like to thank the Ministerio de Economía y Competitividad of the Spanish government (project AGL2012-), the Junta de Andalucía (project P11-CVI-7050) and the University of Seville (VPPI) for funding this work. Mr. Del Cerro is recipient of an FPU fellowship of the Ministerio de Economía y Competitividad. Dr. Pérez-Montaño is recipient of a post-doctoral contract of the VPPI of the University of Seville. Dr. Hungria is a research fellow of CNPq-Brazil. We also would like to acknowledge Dr. Juan Carlos Treviño (Sistemas Genómicos S.L.) for his help during all the bioinformatic analysis and Dr. Jonathan Trow (Sequence Read Archive from NCBI) for his technical assistance during the RNA-seq data submission. We thank the Servicio General de Biología of the CITIUS from the University of Seville for allowing us to use their laboratory equipment.
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