Active root-inhabiting microbes identified by rapid incorporation of plant-derived carbon into RNA

Abstract

Plant roots harbor a large diversity of microorganisms that have an essential role in ecosystem functioning. To better understand the level of intimacy of root-inhabiting microbes such as arbuscular mycorrhizal fungi and bacteria, we provided (13)CO(2) to plants at atmospheric concentration during a 5-h pulse. We expected microbes dependent on a carbon flux from their host plant to become rapidly labeled. We showed that a wide variety of microbes occurred in roots, mostly previously unknown. Strikingly, the greatest part of this unsuspected diversity corresponded to active primary consumers. We found 17 bacterial phylotypes co-occurring within roots of a single plant, including five potentially new phylotypes. Fourteen phylotypes were heavily labeled with the (13)C. Eight were phylogenetically close to Burkholderiales, which encompass known symbionts; the others were potentially new bacterial root symbionts. By analyzing unlabeled and (13)C-enriched RNAs, we demonstrated differential activity in C consumption among these root-inhabiting microbes. Arbuscular mycorrhizal fungal RNAs were heavily labeled, confirming the high carbon flux from the plant to the fungal compartment, but some of the fungi present appeared to be much more active than others. The results presented here reveal the possibility of uncharacterized root symbioses.

Fig. 1.

Isotopic signatures (δ ¹³C‰) of roots after ¹³CO₂ pulse labeling. Each dot represents a mean of four measures (i.e., not true replicates). Results of δ ¹³C‰ were obtained for T. repens (squares) and A. capillaris (circles) in experiment A and from A. stolonifera (diamonds) in experiment B. Fitted regression curves are shown. Natural values of δ ¹³C‰ (control before labeling) were −35 for T. repens roots, −28 for A. capillaris, and −30 for A. stolonifera.

Fig. 2.

RT-PCR products from each fraction collected from the CsTFA gradient. RNA extracts from T. repens were amplified by RT-PCR specific for AM fungi, using samples before (A0), immediately after (A1), and 5 h after (A2) the 5-h ¹³CO₂ labeling period.

Fig. 3.

Phylogenetic affinities of SSU rRNA representing Glomeromycota (AM fungi). Sequences amplified from T. repens, A. capillaris, and A. stolonifera roots are represented, along with representatives of the relevant known groups. The outgroup is a putative choanoflagellate Corallochytrium limacisporum (L42528). The tree was computed by NJ. (Scale bar: 0.1 substitutions per site.) The data were additionally analyzed by using MP and ML. Branches in bold were congruent among the three phylogenetic reconstructions (at least two bootstrap values of >70%). Bootstrap values of >50% are indicated at the nodes (NJ/MP/ML estimated from 1,000/500/100 iterations, respectively). Phylotypes are presented as colored triangles proportional to the number of clones found and their phylogenetic depth: black, DNA fraction; yellow, heavy RNA fraction; blue, light RNA fraction; green, sequences found in both RNA fractions. A, experiment A; B, experiment B.

Fig. 4.

Phylogenetic affinities of bacterial SSU rRNA sequences and statistical analyses of sampling effort. (A) Tree derived by ML analysis of all 151 bacterial sequences amplified from A. stolonifera roots, with representatives of major bacterial groups and the highest BLAST hit of each phylotype. The outgroup is an uncultured crenarchaeote (AJ347774). Branches in bold were congruent among MP, ML, and NJ phylogenetic reconstructions (at least two bootstrap values of >70%). Bootstrap values of >50% are indicated at the nodes (NJ/MP/ML estimated from 1,000/500/100 iterations, respectively). (Scale bar: 0.1 substitutions per site.) Phylotypes are presented as colored triangles proportional to the number of clones found and their phylogenetic depth: yellow, heavy RNA fraction; blue, light RNA fraction; green, sequences found in both RNA fractions. Asterisks indicate phylotypes in which all sequences have >97% identity. (B) Estimates of bacterial community diversity as a function of sampling effort for A. stolonifera root samples. Rarefaction curves are computed from 100 replicates of bootstrap (blue), and the expected richness function (red) is the number of phylotypes estimated (random sampling without replacement) from the sequences analyzed for heavy SSU RNA (i.e., ¹³C-labeled). (C) Estimates of bacterial community diversity for light SSU RNA.