Transcriptomic profiling of Burkholderia phymatum STM815, Cupriavidus taiwanensis LMG19424 and Rhizobium mesoamericanum STM3625 in response to Mimosa pudica root exudates illuminates the molecular basis of their nodulation competitiveness and symbiotic evolutionary history

Abstract

Background: Rhizobial symbionts belong to the classes Alphaproteobacteria and Betaproteobacteria (called "alpha" and "beta"-rhizobia). Most knowledge on the genetic basis of symbiosis is based on model strains belonging to alpha-rhizobia. Mimosa pudica is a legume that offers an excellent opportunity to study the adaptation toward symbiotic nitrogen fixation in beta-rhizobia compared to alpha-rhizobia. In a previous study (Melkonian et al., Environ Microbiol 16:2099-111, 2014) we described the symbiotic competitiveness of M. pudica symbionts belonging to Burkholderia, Cupriavidus and Rhizobium species.

Results: In this article we present a comparative analysis of the transcriptomes (by RNAseq) of B. phymatum STM815 (BP), C. taiwanensis LMG19424 (CT) and R. mesoamericanum STM3625 (RM) in conditions mimicking the early steps of symbiosis (i.e. perception of root exudates). BP exhibited the strongest transcriptome shift both quantitatively and qualitatively, which mirrors its high competitiveness in the early steps of symbiosis and its ancient evolutionary history as a symbiont, while CT had a minimal response which correlates with its status as a younger symbiont (probably via acquisition of symbiotic genes from a Burkholderia ancestor) and RM had a typical response of Alphaproteobacterial rhizospheric bacteria. Interestingly, the upregulation of nodulation genes was the only common response among the three strains; the exception was an up-regulated gene encoding a putative fatty acid hydroxylase, which appears to be a novel symbiotic gene specific to Mimosa symbionts.

Conclusion: The transcriptional response to root exudates was correlated to each strain nodulation competitiveness, with Burkholderia phymatum appearing as the best specialised symbiont of Mimosa pudica.

Keywords: Alpha-rhizobia; Beta-rhizobia; RNA-seq; Symbiosis; Transcriptome.

Fig. 1

a-b. Boxplots of Read counts number (Log10) per gene per condition for each bacterium (a), and of Log2 (Fold Change) of gene regulation per bacterium (b). BP, CT and RM: RNAseq data analysis from bacteria in broth culture sampled a 4.5, 4.5 and 6 h, respectively (corresponding to mid-exponential growth phase); BP-I, CT-I and RM-I are the same as previously mentioned, but mixed with Mimosa pudica root exudates

Fig. 2

% of DEG per genomes and replicons of bacteria induced by M. pudica root exudates. BP (B. phymatum STM815), CT (C. taiwanensis LMG19424) and RM (R. mesoamericanum STM3625). Replicons names according to Table 2

Fig. 3

a-b. Venn diagram of DEG orthologs among the 3 rhizobia (a) and their circular representation (b). On Venn diagram (a), numbers in black indicate the orthologs between bacterial genomes (at intersect) or their specific gene set. Numbers in colors indicate the number of up-regulated genes (red) or downregulated genes (green) among the orthologs at each intersection of the Venn diagram. Numbers in purple indicate gene orthologs regulated in opposite direction between strains. The circular representation (b) was made with RCircos and represent the bacterial replicons (outer circles), their up (red) and down (green) root exudate-regulated genes (log2(fold change) values), as well as links between co-regulated orthologs (green links, between all three rhizobia; blue lines between BP and CT (beta-rhizobia); red links between RM-BP; pink lines: RM-CT). Note the high number of blue lines depicting the high number of shared regulated genes between beta-rhizobial orthologs, while few were shared by all (green links) or between RM-CT and BP-RM (pink and red links)

Fig. 4

COG functional categories of differentially expressed genes after bacterial treatment by root exudates. Up-regulated COG categories are in red, downregulated in green

Fig. 5

Nodulation gene operons in BP, CT and RM, colored following their regulation level (fold change) in response to root exudates at 4.5, 4.5 and 6 h post induction, respectively. Black triangles indicate the presence of a conserved nodbox (AATNGATTGTTTNGATNNNNNNNAT). The regulation color scale is indicated on the right side of the figure

Fig. 6

Kinetics of nod (nodA, nodB and nodD) and fatty acid hydroxylase gene expression as measured by RT-qPCR. The uppS gene was used to normalise data, and the other reference (hisB) gene expression profile is shown

Fig. 7

Illustration of the main functions regulated in BP, CT and RM in the presence of root exudates from Mimosa pudica. Functions commonly up-regulated among the 3 rhizobia, or specific, are colored differently (red: in all 3 rhizobia, orange: BP specific, green: CT specific, blue: BP & CT, purple: RM specific, brown: BP&RM. The cross indicates down-regulated functions