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Adaptation of Rhizobium leguminosarum to pea, alfalfa and sugar beet rhizospheres investigated by comparative transcriptomics

Vinoy K Ramachandran1, Alison K East2, Ramakrishnan Karunakaran2, J Allan Downie2 and Philip S Poole2*

Author Affiliations

1 School of Biological Sciences, University of Reading, Reading, RG6 6AJ, UK

2 Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK

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Genome Biology 2011, 12:R106  doi:10.1186/gb-2011-12-10-r106

Published: 21 October 2011

Additional files

Additional file 1:

Figure S2 - effect of length of incubation in the pea rhizosphere. (a, b) Venn diagrams of Rlv3841 genes up-regulated (a) and down-regulated (b) in the pea rhizosphere at 1, 3 and 7 dpi of 7-day-old plants. Total genes differentially regulated in each rhizosphere are shown in brackets. Venn diagrams were drawn in GeneSpring by selecting differentially regulated genes (by three-fold or more, filtered on confidence P ≤ 0.05) for each condition.

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Additional file 2:

Figure S3 - effect of plant age. (a, b) Venn diagrams of Rlv3841 genes up-regulated (a) and down-regulated (b) in the rhizosphere at 1 dpi of 7-, 14- and 21-day-old pea plants. Total genes differentially regulated in each rhizosphere are shown in brackets. Venn diagrams were drawn in GeneSpring following selection of differentially regulated genes (by three-fold or more, filtered on confidence P ≤ 0.05) for each condition.

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Additional file 3:

Figure S4 - effect of size of inoculation of the pea rhizosphere. (a, b) Venn diagrams of Rlv3841 genes up-regulated (a) and down-regulated (b) in the pea rhizosphere at 1 dpi of 7-day-old plants with 103 and 108 CFU. Total genes differentially regulated in each rhizosphere are shown in brackets. Venn diagrams were drawn in GeneSpring by selecting differentially regulated genes by three-fold or more, filtered on confidence P ≤ 0.05) for each condition.

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Additional file 4:

Figure S1 - Venn diagrams of differentially regulated Rlv3841 genes (by three-fold or more, P ≤ 0.05). (a) An indirect comparison of rhizospheres in which pea, alfalfa and sugar beet rhizospheres are investigated using comparison with glucose-grown free-living cells (i) up-regulated and (ii) down-regulated. (b) Up-regulated in direct comparison with (i) pea, alfalfa and sugar beet rhizospheres and (ii) legume and sugar beet rhizospheres. Total genes differentially regulated in each rhizosphere are shown in brackets and listed for (a) in Additional file 7. Of the 106 genes up-regulated by all three plant rhizospheres (a(i); Additional file 7), 66% are annotated as hypothetical and 15% are membrane proteins or concerned with cell surface. The 184 genes down-regulated in all rhizospheres (a(ii); Additional file 7) include those for proteins involved in general cellular functions of bacterial motility and chemotaxis (6%), tRNA and DNA synthesis, chromosome and plasmid replication, cell division, protein export by the Sec system, electron transport and formation of ATP. In addition, 12% are annotated as hypothetical, 13% are membrane proteins or concerned with cell surface and 16% are ribosomal proteins. Components of two ABC transport systems that import glucose (RL3624-5, RL4252) [8] are down-regulated in all rhizospheres in comparison with glucose-grown cells (Additional file 7). From direct comparison of the pea rhizosphere with those of alfalfa and sugar beet (b(i)) there are 30 pea rhizosphere-specific genes (listed in Additional file 7). There are 9 legume rhizosphere-specific genes up-regulated in both the pea and alfalfa rhizosphere compared to that of sugar beet (b(ii)). Abbreviations: SB, sugar beet.

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Additional file 5:

Figure S5 - experimental design for direct comparison of Rlv3841 grown in three different rhizospheres. (a) A single set for the direct comparison experiment; two biological replicates from each rhizosphere sample were extracted and amplified. From each amplified RNA sample, an equal amount (15 μg) of amplified RNA was taken and labeled with Cy3 and Cy5 separately. Equal amounts of labeled cDNAs were used for each microarray experiment. A second set was performed before analysis, yielding four biological replicates per rhizosphere. (b) Design for direct two-color experiments. (c) A table summarizing rhizosphere microarray experiments.

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Additional file 6:

Table S1 - genes whose expression was differentially regulated by three-fold or more compared to free-living Rlv3841 isolated from various conditions. The conditions from which Rlv3841 were isolated were: the presence of hesperetin or pea exudate; the rhizospheres of 7-, 14- and 21-day-old pea plants at 1 dpi; the rhizosphere of 7-day-old pea plants at 1 and 3 dpi; and the rhizosphere of 7-day-old pea plants at 7 dpi inoculated with 103 CFU. The table also lists genes whose expression was differentially regulated by three-fold or more in the rhizosphere at 7 dpi of 7-day-old plants of pea versus alfalfa, pea versus sugar beet and alfalfa versus sugar beet.

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Additional file 7:

Table S2 - genes whose expression was differentially regulated by three-fold or more in the rhizospheres of pea, alfalfa and sugar beet at 7 dpi of 7-day-old plants compared to free-living Rlv3841. In addition, genes specifically up-regulated in the pea rhizosphere are shown compared to free-living cells and those in the alfalfa rhizosphere and the sugar beet rhizosphere.

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Additional file 8:

Table S4 - data used to draw Figures 1, 2 and 3and results for competition in pea and alfalfa rhizospheres of mutants compared with Rlv3841.

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Additional file 9:

Table S3 - genes whose expression was differentially regulated by three-fold or more compared to free-living Rlv3841 when grown in the presence of formate, protocatechuate, 4-hydroxybenzoate, phenylalanine, proline, L-arabinose, galactose, arabinogalactan and under conditions of N-limitation.

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Additional file 10:

Table S5 - identification of putative substrates for ABC, TRAP and MFS transporters of R. leguminosarum 3841.

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Additional file 11:

Table S6 - strains, plasmids and primers.

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Additional file 12:

Table S7 - real time-quantitative reverse transcription PCR and log fold values calculated by the comparative CT method.

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