A bacterial sensor of plant cell contact controls the transcriptional induction of Ralstonia solanacearum pathogenicity genes

Abstract

The hrp genes of the plant pathogen Ralstonia solanacearum are key pathogenicity determinants; they encode a type III protein secretion machinery involved in the secretion of mediators of the bacterium-plant interaction. These hrp genes are under the genetic control of the hrpB regulatory gene, expression of which is induced when bacteria are co-cultivated with plant cell suspensions. In this study, we used hrp-gfp transcriptional fusions to demonstrate that the expression of the hrpB and type III secretion genes is specifically induced in response to the bacterium-plant cell contact. This contact-dependent induction of hrpB gene expression requires the outer membrane protein PrhA, but not a functional type III secretion apparatus. Genetic evidence indicates that PrhA constitutes the first example of a bacterial receptor for a non-diffusible signal present in the plant cell wall and which triggers the transcriptional activation of bacterial virulence genes.

Fig. 1. Model of regulation of the R.solanacearum Hrp type III secretion system in response to plant signal(s). Above, genetic organization of the hrp gene cluster, with grey arrows showing the orientation and length of the hrp transcriptional units. A new nomenclature has distinguished hrp and hrc (for hrp-conserved) genes, the latter are homologues of type III secretion genes of animal pathogens and are predicted to encode components of the type III secretion apparatus. Four proteins, the harpin PopA, PopB, PopC and HrpY have been described to be secreted by the Hrp pathway into the extracellular medium. HrpY is the structural component of the Hrp pilus, an essential extracellular structure involved in PopA secretion. The outer membrane protein PrhA controls the plant-responsive regulatory cascade composed of PrhJ, HrpG and HrpB, the final activator of hrp transcription units 1–4 and 7. Two additional regulatory components (dotted lines) have recently been identified (B.Brito and S.Genin, unpublished). The HrpG protein integrates another hrp-inducing signal pathway, which is dependent on the nutrient/metabolic status of the bacterium. This model is based on data compiled from Arlat et al. (1994), Marenda et al. (1998), Brito et al. (1999), Guéneron et al. (2000) and Van Gijsegem et al. (2000).

Fig. 2. Induction of hrp gene expression in response to bacterium–plant cell contact. Interaction of R.solanacearum and A.thaliana cells observed by epifluorescence microscopy (A–E and G–I) or by confocal microscopy (F) after 16 h of co-cultivation. The fluorescence and phase contrast images have been overlaid in (B) and (H) [(A) + (C) and (G) + (I), respectively] in order to co-localize induced bacteria and plant cell surfaces. Images were acquired with the same parameters to allow comparisons between the emitted fluorescence level in the different frames. The bar represents 20 µm, except in F (10 µm). (A–C) Co-cultivation of GMI1000/pSG261 with Arabidopsis cells. Strongly induced bacteria expressing the hrpB–gfp fusion are found in contact with plant cells; some uninduced bacteria present in the medium are indicated by arrowheads (B). The few strongly induced bacteria observed in the medium are assumed to have previously been in close association with plant cells. (D and E) Basal level of hrpB–gfp expression obtained when strain GMI100/pSG261 is grown in Arabidopsis-conditioned medium. (F) Detail of the interaction between strain GMI1000/pSG261 and Arabidopsis cell surface observed by confocal microscopy. The bacteria in contact with the plant cell surface exhibit a high level of GFP fluorescence (red colour); non-induced bacteria are visible at the upper right. (G–I) Co-cultivation of strain GMI1000/pSG282 (hrpY–gfp) with Arabidopsis cells, showing that the hrpY gene follows the same expression pattern as hrpB.

Fig. 3. Contact-dependent induction of hrpB gene expression requires components of the plant-responsive regulatory cascade. Epifluorescence microscopy study after 16 h of co-cultivation of R.solanacearum and Arabidopsis cells. The fluorescence and phase contrast images have been overlaid in (B), (D) and (F). The bar represents 20 µm. (A and B) Strain GMI1575/pSG261 carrying a prhA mutation. (C and D) Strain GMI1579/pSG261 carrying a prhJ mutation. (E and F) Strain GMI1425/pSG261 carrying an hrpG mutation.

Fig. 4. PrhA is not involved in the attachment of bacteria to plant cell surfaces. (A and B) Confocal analysis of the interaction between Arabidopsis cells and (A) strain GMI1600 carrying a constitutive gfp reporter fusion or (B) strain GMI1601, which carries, in addition, a prhA mutation. In both cases, adherence and polar attachment of the bacteria (in red) to the plant cell surface can be observed, as has been described for the wild-type strain GMI1000 (Van Gijsegem et al., 2000). The bar represents 15 µm. (C) Time course of attachment of ³²P-radiolabelled bacteria (wild-type strain GMI1000 and prhA mutant strain GMI1575) to Arabidopsis cells grown under continuous agitation.

Fig. 5. Transcriptional induction of hrpB gene expression in response to plant cell contact is not type III secretion dependent. Observations by epifluorescence microscopy after 16 h of co-cultivation of Arabidopsis cells with the R.solanacearum hrpY mutant strain GMI1410/pSG261 (A) and the hrcC mutant strain GMI1462/pSG261 (B), both carrying an hrpB–gfp reporter fusion. The same experimental conditions were used for strain GMI1410/pSG282, carrying an hrpY–gfp reporter fusion (C). The bar represents 20 µm.

Fig. 6. Different plant cell species induce hrpB gene expression in a PrhA-dependent manner. Illustrations are an overlay of the fluorescence and visible images. The bar represents 20 µm. (A and D) Co-cultivation of tobacco cells with R.solanacearum strain GMI1000/pSG261 (A) or the prhA mutant strain GMI1575/pSG261 (D). (B and E) Co-cultivation of M.truncatula cells with GMI1000/pSG261 (B) or GMI1575/pSG261 (E). (C and F) Co-cultivation of tomato cells with GMI1000/pSG261 (C) or GMI1575/pSG261 (F).

Fig. 7. The plant signal recognized by PrhA that induces hrpB gene expression is present in the Arabidopsis cell wall. Epifluorescence microscopy observations of R.solanacearum strains grown in the presence of Arabidopsis cell wall material. Illustrations (C–F) are overlays of the fluorescence and visible light microscopy images. The bar represents 20 µm. (A) Strain GMI1000/pSG261 co-cultivated for 4 h with cell wall material. (B–D) Strain GMI1000/pSG261 (B and C) and the prhA mutant strain GMI1575/pSG261 (D) co-cultivated for 16 h with cell wall material. (E) Strain GMI1000/pSG261 with heat-treated cell wall material (15 min, 100°C). (F) Strain GMI1000/pSG261 with proteinase K-treated cell wall material.