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. 2014 Dec 23;5(6):e02065-14.
doi: 10.1128/mBio.02065-14.

A Ralstonia solanacearum type III effector directs the production of the plant signal metabolite trehalose-6-phosphate

M Poueymiro  1 , A C Cazalé, J M François, J L Parrou, N Peeters  2 , S Genin  2
Affiliations

A Ralstonia solanacearum type III effector directs the production of the plant signal metabolite trehalose-6-phosphate

M Poueymiro et al. mBio. .

Abstract

The plant pathogen Ralstonia solanacearum possesses two genes encoding a trehalose-6-phosphate synthase (TPS), an enzyme of the trehalose biosynthetic pathway. One of these genes, named ripTPS, was found to encode a protein with an additional N-terminal domain which directs its translocation into host plant cells through the type 3 secretion system. RipTPS is a conserved effector in the R. solanacearum species complex, and homologues were also detected in other bacterial plant pathogens. Functional analysis of RipTPS demonstrated that this type 3 effector synthesizes trehalose-6-phosphate and identified residues essential for this enzymatic activity. Although trehalose-6-phosphate is a key signal molecule in plants that regulates sugar status and carbon assimilation, the disruption of ripTPS did not alter the virulence of R. solanacearum on plants. However, heterologous expression assays showed that this effector specifically elicits a hypersensitive-like response on tobacco that is independent of its enzymatic activity and is triggered by the C-terminal half of the protein. Recognition of this effector by the plant immune system is suggestive of a role during the infectious process.

Importance: Ralstonia solanacearum, the causal agent of bacterial wilt disease, infects more than two hundred plant species, including economically important crops. The type III secretion system plays a major role in the pathogenicity of this bacterium, and approximately 70 effector proteins have been shown to be translocated into host plant cells. This study provides the first description of a type III effector endowed with a trehalose-6-phosphate synthase enzymatic activity and illustrates a new mechanism by which the bacteria may manipulate the plant metabolism upon infection. In recent years, trehalose-6-phosphate has emerged as an essential signal molecule in plants, connecting plant metabolism and development. The finding that a bacterial pathogen could induce the production of trehalose-6-phosphate in plant cells further highlights the importance of this metabolite in multiple aspects of the molecular physiology of plants.

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Figures

FIG 1
FIG 1
ripTPS encodes a T3E homologous to trehalose phosphate synthases. (A) Enzymatic reactions catalyzed by trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) in the trehalose biosynthetic pathway. (B) Schematic representation of RipTPS functional domains and some of the conserved residues predicted to be essential for activity. (C) The concentrations of cyclic AMP (cAMP) were measured in N. tabacum leaves after infiltration of R. solanacearum strains with or without a functional T3SS (GMI1000 and hrcV, respectively) expressing the RipTPS N-terminal domain (first 88 amino acids) fused to the calmodulin-dependent adenylate cyclase. Standard errors of the results of three independent experiments are shown in brackets.
FIG 2
FIG 2
Bacterial TPS phylogeny. The accession numbers of the proteins aligned in this phylogeny can be found in the legend to Fig. S1 in the supplemental material. The outgroup Arabidopsis thaliana TPS1 (accession number NP_177979) was added in this analysis. Sequences were aligned with MUSCLE and curated with GBlocks, and the phylogeny was reconstructed with PhyML. All analysis was run using the http://phylogeny.fr webserver (58). Bootstrap values are indicated in red. Scale is number of substitutions per site.
FIG 3
FIG 3
Complementation of the growth defect of the S. cerevisiae tps1 tps2 strain on fructose by R. solanacearum ripTPS. RipTPS with or without an HA tag was expressed in the tps1 tps2 yeast mutant, and the growth of the strain was compared to that of strains carrying an empty vector or expressing E. coli OtsA with an HA tag or one of the three catalytic mutants of RipTPS (Y154V, W163S, and D208G) with an HA tag. Amounts of 5 µl of liquid cultures were spotted as a series of 10-times dilutions on 2% galactose or 2% fructose SD-LWU plates. The control yeast strain (WT) was grown on plates with LWU. Pictures of a representative experiment at day 2 are shown.
FIG 4
FIG 4
Production of trehalose-6-phosphate in yeast tps1 tps2 mutant expressing R. solanacearum ripTPS or one of its catalytic mutants. (A) RipTPS with an HA tag and its catalytic mutants were expressed in the tps1 tps2 yeast, and the levels of Tre6P production were compared to the levels in strains carrying an empty vector or expressing E. coli OtsA with an HA tag or RipTPS without a tag. All samples were analyzed five times, generating biologically independent replicates. The P value groupings indicate which strains cannot be distinguished (at a P value of <5%) using a nonparametric Wilcoxon matched-pairs signed-rank test, i.e., group a: the results for the empty vector pVTU and the three catalytic mutants cannot be distinguished and are all significantly different from the results for the other strains. (B) Western blot analysis of the expression of the HA-tagged TPS variants in the yeast protein extracts. A corresponding Coomassie-stained gel was used as a loading control. The black arrowhead indicates RipTPS (67 kDa).
FIG 5
FIG 5
Agrobacterium-mediated transient expression of RipTPS or the C-terminal domain of RipTPS in N. tabacum leaves elicits an HR-like necrotic response. (A) Schematics of the structures of the RipTPS proteins expressed, including WT RipTPS (a) and the three catalytic mutants of RipTPS-HA (b, c, and d). Non-RipTPS proteins used in the experiments are not depicted; these were OtsA-HA (e), GUS-HA (negative control) (f), and AvrA (positive HR control) (g). (B and D) N. tabacum leaf pictures taken 3 days after infiltration of Agrobacterium strains, allowing the expression of the different recombinant proteins. (C and E) Western blotting with anti-HA antibody and Coomassie staining of the protein extracts from leaves infiltrated like those shown in panels B and D, respectively. The black arrowheads indicate RipTPS (67 kDa). The asterisks indicate protein that is visible but at low abundance.
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References

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