The RPG gene of Medicago truncatula controls Rhizobium-directed polar growth during infection

Jean-François Arrighi, Olivier Godfroy, Françoise de Billy, Olivier Saurat, Alain Jauneau, and Clare Gough
PNAS July 15, 2008 105 (28) 9817-9822; https://doi.org/10.1073/pnas.0710273105

  1. Edited by Rafael Palacios, National University of Mexico, Cuernavaca, Morelos, Mexico, and approved April 14, 2008 (received for review October 30, 2007)

Article Figures & SI

Figures

  • Fig. 1.
    Fig. 1.

    ITs, nodules, and RHC in response to S. meliloti expressing lacZ, 5 (A–D and F), 21 (E, G, and H), and 3 (I–L) dpi. (A and B) RH infection in the rpg mutant (A) and in WT (B). (C and D) Cortical infection in the rpg mutant, showing an enlarged sac-like structure (arrow) (C) and in WT (D). (E) Nodule-like structure on the rpg mutant showing infection (blue coloration) limited to the epidermis (arrowhead). (F) An infected nodule primordium in WT. (G and H) Nodule sections in the rpg mutant (G) and WT (H). (I–L) RHC [loose and incomplete with RH outgrowth (arrow)] in the rpg mutant (I–K) and (tight) in WT (L). [Scale bars: 10 μm (A–D and I–L) and 100 μm (E–H).]

  • Fig. 2.
    Fig. 2.

    RH infection and cortical cell responses. (A–F) Confocal images of S. meliloti expressing GFP. ITs in the rpg mutant (A–C) and WT (D–F) are shown in 3D representations (B and E), and confocal sections (C and F) show bacterial chambers (arrowheads) and probable restricted entry sites (arrows). (G–K) Root sections. (G–I) Cytoplasmic bridge (arrow in G) in an outer cortical cell overlying cell divisions in the rpg mutant, 4 dpi. (H) Immunolocalization of α-tubulin showing parallel microtubules (arrow). (I) Nucleus (arrow) visualized by using DAPI. (J and K) Infected cortical RHs 7 dpi, with toluidine blue coloration, in rpg inoculated with S. meliloti WT (J) and in WT inoculated with S. meliloti exoA (K). (Scale bars: 10 μm.)

  • Fig. 3.
    Fig. 3.

    Subcellular localization of RPG in Nicotiana benthamiana leaf epidermal cells. (A and B) The p35S::GFP control showing nuclear and cytoplasmic GFP. (C and D) p35S::GFP::RPG showing nuclear localization of GFP. Stars indicate nuclei. (Scale bars: 20 μm.)

  • Fig. 4.
    Fig. 4.

    Spatiotemporal analysis of RPG gene expression by using an RPG promoter-GUS fusion. Shown is GUS coloration of whole roots (A, C, D, K, and L) or sections (B, E–J, and M), after inoculation with S. meliloti lacZ (A–J) or after NF/control treatment (K–M). GUS coloration is magenta (A–I) or blue (J–M), and bacteria are colored blue (E–I) or magenta (J). (A and B) RH cells at a lateral root apex, 2 dpi. (C–F) Deformed and infected (arrows) RHs, 3 dpi. Sectioning shows GUS expression limited to the infected cell (E and F). (G) A developing nodule primoridium, 5 dpi. (H and I) Young nodule section 8 dpi (I is a zoom of H). (J) Section of a mature nodule 18 dpi, showing GUS coloration in the infection zone. (K–M) Without (K) or with (L and M) 10−8 M NF treatment. Sectioning shows that NF induction is localized in RH cells and the central vascular system (M). [Scale bars: 300 μm (A, C, and J–M), 100 μm (B, H, and I), 10 μm (D–G).]

  • Fig. 5.
    Fig. 5.

    Predicted secondary structure of RPG homologs. Proteins are drawn to scale and aa lengths are given. Sequence conservation is given as percentage identity values. RRP, RRP domains (gray); D, disordered regions (striped); α-helix, coiled-coil regions (black). Because of low sequence conservation, At1g22060 and Os10g21940 (a) and RRP1 (b) were excluded from the analysis of sequence conservation. 1NLS sequence at the start of RPG. 2Signal peptide and transmembrane domain at the start of Os10g21940. 3NLS sequence within the RRP domain of Os10g36060.

  • Fig. 6.
    Fig. 6.

    Unrooted phylogenetic tree of RPG homologs based on the RRP domain. One thousand bootstrap replicates were performed and percentage bootstrap supports are given. Family clades are circled.

Tables

  • Table 1.

    Expression analysis of RPG and M. truncatula RRP genes by quantitative RT-PCR

    GeneExpression levels
    RootNoduleLeafStemFlower
    RPG0.02 ± 0.001 ± 0.35*0.05 ± 0.000.04 ± 0.010.01 ± 0.00
    RRP11*0.06 ± 0.010.84 ± 0.03*0.70 ± 0.29*0.06 ± 0.01
    RRP20.61 ± 0.13*0.04 ± 0.011 ± 0.32*0.53 ± 0.04*0.13 ± 0.01
    RRP30.36 ± 0.00*0.40 ± 0.01*1 ± 0.15*0.55 ± 0.08*0.40 ± 0.02*
    RRP41 ± 0.12*0.51 ± 0.20*0.88 ± 0.23*0.87 ± 0.28*0.82 ± 0.06*
    RRP50.68 ± 0.19*0.80 ± 0.05*0.62 ± 0.03*0.81 ± 0.25*1 ± 0.17*

    • Values are ratios relative to the sample having the highest expression level, ± SEMs.

    • ↵*Preferential expression pattern.

  • Table 2.

    Expression analysis of RPG by quantitative RT-PCR at early time points after rhizobial inoculation of wild-type (WT) and M. truncatula hcl and lin mutants

    PlantExpression levels
    0 dpi1 dpi3 dpi
    WT0.03 ± 0.000.34 ± 0.141 ± 0.18
    hcl0.03 ± 0.01ND0.14 ± 0.01
    lin0.01 ± 0.00ND0.13 ± 0.01

    • Values are ratios relative to the sample having the highest expression level, ±SEMs.

Data supplements

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The RPG gene of Medicago truncatula controls Rhizobium-directed polar growth during infection
Jean-François Arrighi, Olivier Godfroy, Françoise de Billy, Olivier Saurat, Alain Jauneau, Clare Gough
Proceedings of the National Academy of Sciences Jul 2008, 105 (28) 9817-9822; DOI: 10.1073/pnas.0710273105
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