The Pseudomonas syringae HopPtoV protein is secreted in culture and translocated into plant cells via the type III protein secretion system in a manner dependent on the ShcV type III chaperone

Abstract

The bacterial plant pathogen Pseudomonas syringae depends on a type III protein secretion system and the effector proteins that it translocates into plant cells to cause disease and to elicit the defense-associated hypersensitive response on resistant plants. The availability of the P. syringae pv. tomato DC3000 genome sequence has resulted in the identification of many novel effectors. We identified the hopPtoV effector gene on the basis of its location next to a candidate type III chaperone (TTC) gene, shcV, and within a pathogenicity island in the DC3000 chromosome. A DC3000 mutant lacking ShcV was unable to secrete detectable amounts of HopPtoV into culture supernatants or translocate HopPtoV into plant cells, based on an assay that tested whether HopPtoV-AvrRpt2 fusions were delivered into plant cells. Coimmunoprecipitation and Saccharomyces cerevisiae two-hybrid experiments showed that ShcV and HopPtoV interact directly with each other. The ShcV binding site was delimited to an N-terminal region of HopPtoV between amino acids 76 and 125 of the 391-residue full-length protein. Our results demonstrate that ShcV is a TTC for the HopPtoV effector. DC3000 overexpressing ShcV and HopPtoV and DC3000 mutants lacking either HopPtoV or both ShcV and HopPtoV were not significantly impaired in disease symptoms or bacterial multiplication in planta, suggesting that HopPtoV plays a subtle role in pathogenesis or that other effectors effectively mask the contribution of HopPtoV in plant pathogenesis.

FIG. 1.

Organization of ORF6 (shcV) and ORF17 (hopPtoV) within a pathogenicity island in the chromosome of P. syringae pv. tomato DC3000. ORF6 (shcV), a candidate chaperone gene, and ORF17 (hopPtoV), its cognate effector gene, are predicted to be part of the same transcriptional unit and are within a pathogenicity island containing other type III-related genes. ORF6 and ORF17 are depicted as white boxes, and the confirmed effector genes hopPtoA2 (7), hopPtoD2 (27), and hopPtoG (61) are represented as hatched boxes. An insertion sequence-interrupted avrPphD gene is depicted as horizontally striped boxes, and the apparent borders of this type III-related region are delimited by cmaTU and a DNA helicase gene, both represented as grey boxes. Other ORFs in the region are represented as stippled boxes. With the exception of ORF6 and ORF17, the numbers represent the ORF numbers given when the genome was annotated and should be preceded by the prefix PSPTO. Insertion sequences are depicted as diagonally striped boxes. The arrow indicates the predicted direction of ORF6 and ORF17 transcription, and the black box represent a putative HrpL-dependent (type III) promoter.

FIG. 2.

HopPtoV is secreted in culture via the DC3000 TTSS in an ShcV-dependent manner. (A) Cultures of wild-type (WT) P. syringae pv. tomato DC3000 and a DC3000 TTSS-defective mutant (hrcC) were grown in hrp-inducing medium and separated into cell-bound (C) and supernatant (S) fractions. The cell-bound and supernatant fractions were concentrated 13.3-fold and 133-fold, respectively, relative to the initial culture volumes. The samples were separated by SDS-PAGE and immunoblotted. HopPtoV and β-lactamase, which was used as a lysis control, were detected with anti-HA and anti-β-lactamase antibodies, respectively. Wild-type DC3000 strains containing plasmid pLN127 (phopPtoV-ha) or pLN517 (pshcV/hopPtoV-ha) secreted HopPtoV-HA into the supernatant fraction. However, much more HopPtoV-HA was found in the supernatant fractions from DC3000(pLN517), which overexpressed ShcV. (B) Cultures of DC3000 shcV (UNL120) and hopPtoV (UNL125) insertion mutants containing plasmid pLN127 (phopPtoV-ha) or pLN517 (pshcV/hopPtoV-ha) were grown in hrp-inducing medium, separated into cell-bound (C) and supernatant (S) fractions, and separated by SDS-PAGE. Immunoblotted proteins were detected with anti-HA or anti-NPTII antibodies. Plasmid-encoded NPTII should remain cell bound and was used as a lysis control. The shcV mutant UNL120 was unable to secrete HopPtoV-HA unless shcV was also provided in trans.

FIG. 3.

AvrRpt2 translocation assays indicate that HopPtoV is translocated into plant cells in an ShcV-dependent manner. P. syringae pv. phaseolicola NPS3121 carrying plasmids that encoded either full-length AvrRpt2, N-terminally truncated AvrRpt2 (′AvrRpt2), or a HopPtoV-truncated AvrRpt2 fusion (HopPtoV-′AvrRpt2) with and without ShcV were infiltrated into A. thaliana Col-0 (RPS2) plants at an OD₆₀₀ of 0.4. Plants were scored for production of a hypersensitive response (HR) after 48 h, and representative leaves were photographed. Ten leaves were infiltrated for each bacterial strain, and the number of times that the pictured result was observed over the total number of samples is indicated as a fraction under each picture.

FIG. 4.

HopPtoV interacts with ShcV in coimmunoprecipitation experiments. Soluble protein samples from sonicated E. coli DH5α(pLN127, pLN688), which expressed HopPtoV-HA and His-ShcV, were mixed with anti-HA affinity matrix as described in Materials and Methods. Aliquots from the soluble total protein (lysate), the protein sample post-anti-HA matrix treatment (unbound), the final wash, and the proteins bound to the matrix (matrix) were separated by SDS-PAGE and immunoblotted. HopPtoV-HA and His-ShcV were detected with anti-HA and anti-His antibodies, respectively. Also included were samples that contained constructs that expressed either ShcV-HA or His-ShcV and a vector control (either pQE30 and pML123), demonstrating that both proteins needed to be present to detect His-ShcV in the matrix sample.

FIG. 5.

ShcV interacts with the N-terminal third of HopPtoV in LexA yeast two-hybrid assays, with the strongest binding occurring between amino acids 76 and 125 of HopPtoV. (A) Schematic representation of the HopPtoV fragments that were fused to the DNA-binding domain (DBD) and used in the LexA yeast two-hybrid interaction assay. The top white box represents the full-length HopPtoV protein (391 amino acids). The lower black bars represent a series of HopPtoV fragments that were fused to the DBD and tested in the yeast two-hybrid assay to determine if they interacted with ShcV fused to the transcriptional activation domain (AD). A representation of the results is shown: +, strong interaction; −, no detectable interaction; +/−, weak interaction. (B) Yeast strains carrying pJG4-5::shcV (producing AD-ShcV) and pEG202 with different hopPtoV fragments (producing DBD fusions) were grown at 30°C for 2 days on the appropriate selective medium containing X-Gal and either galactose (Gal) or glucose (Glu). Results were scored based on the amount of blue pigmentation produced by the colonies: dark blue, strong interaction; pale blue, weak interaction; and white, no detectable interaction. Analogous experiments measuring the rescue of leucine auxotrophy, the other reporter of this yeast two-hybrid system, showed similar results (data not shown).

FIG. 6.

ShcV and HopPtoV do not contribute measurably to the ability of DC3000 to grow in Arabidopsis leaf tissue. The following strains were dip inoculated into A. thaliana Col-0 as described in Materials and Methods: wild-type DC3000 (WT); wild-type DC3000 with the empty vector pML123; DC3000(pLN517) expressing shcV and hopPtoV; the DC3000 shcV mutant UNL120; and the DC3000 hopPtoV mutant UNL125. Leaf tissue was harvested at days 0, 2, and 4 and enumerated by plating dilutions on KB plates with the appropriate antibiotics. Each assay was done at least three times, and error bars indicate standard deviations. These results suggest that ShcV and HopPtoV do not contribute significantly to growth in planta. The disease symptoms produced by these strains were similar (data not shown).