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. 2016 Sep 7;12(9):e1005874.
doi: 10.1371/journal.ppat.1005874. eCollection 2016 Sep.

Screen of Non-annotated Small Secreted Proteins of Pseudomonas Syringae Reveals a Virulence Factor That Inhibits Tomato Immune Proteases

Free PMC article

Screen of Non-annotated Small Secreted Proteins of Pseudomonas Syringae Reveals a Virulence Factor That Inhibits Tomato Immune Proteases

Takayuki Shindo et al. PLoS Pathog. .
Free PMC article


Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is an extracellular model plant pathogen, yet its potential to produce secreted effectors that manipulate the apoplast has been under investigated. Here we identified 131 candidate small, secreted, non-annotated proteins from the PtoDC3000 genome, most of which are common to Pseudomonas species and potentially expressed during apoplastic colonization. We produced 43 of these proteins through a custom-made gateway-compatible expression system for extracellular bacterial proteins, and screened them for their ability to inhibit the secreted immune protease C14 of tomato using competitive activity-based protein profiling. This screen revealed C14-inhibiting protein-1 (Cip1), which contains motifs of the chagasin-like protease inhibitors. Cip1 mutants are less virulent on tomato, demonstrating the importance of this effector in apoplastic immunity. Cip1 also inhibits immune protease Pip1, which is known to suppress PtoDC3000 infection, but has a lower affinity for its close homolog Rcr3, explaining why this protein is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of P. syringae, indicating that also P. syringae secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes.

Conflict of interest statement

The authors have declared that no competing interests exist.


Fig 1
Fig 1. In silico selection of small, secreted, non-annotated proteins of PtoDC3000.
(A) Selection of small proteins. 5616 proteins encoded by the PtoDC3000 genome were ranked based on amino acid length. 2420 proteins with 50–260 amino acid length were selected. (B) Selection of secreted proteins. 2420 small proteins were plotted with their SignalP scores for both Hidden Markov (HM) and Neural Network (NN). 243 proteins with HM+NN>1.1 were selected. (C) Selection of significant signal peptide (SP) prediction scores. For each of the 243 proteins, significance scores for both HM and NN algorithms were added up and 200 proteins with scores of 5 or higher were selected. (D) Selection of non-annotated proteins. Annotations in the genome database were used to select 131 non-annotated proteins. (E) Number of Cys residues in the selected 131 mature proteins. The predicted signal peptide was removed from the sequence and the number of Cys residues in the remaining protein was counted and plotted in a frequency histogram.
Fig 2
Fig 2. Occurence of small, secreted non-annotated proteins of PtoDC3000 in other Pseudomonas species.
BLAST scores were generated for each of the 131 small secreted, non-annotated proteins against each of the protein databases of 24 sequenced Pseudomonas species. BLAST scores were presented in shades of red, and black boxes represent no significant BLAST score. Blast scores are clustered over both the species and proteins. Conservation of the proteins occurs in five groups (1–5). Representative putative proteins having the highest SP scores were picked from each of these classes and produced and purified (black boxes on the bottom). See S1 Fig for more details.
Fig 3
Fig 3. Expression and presence of putative effectors in the apoplast.
The closest PsyB728a-homologs of the 43 small secreted, nonannotated proteins of PtoDC3000 proteins (bottom), were identified in the PsyB728a genome. The respective expression levels of these genes during colonization of PsyB728a in the apoplast were extracted from the microarray database. For comparison, also the expression levels of 25 type-III (T3) effectors of PsyB728a are shown on the right. Error bars indicate standard error of n = 4 biological replicates. PtoDC3000 proteins that have been detected in the apoplast by mass spectrometry are marked with an asterisk (dark grey bars).
Fig 4
Fig 4. Heterologous production of secreted proteins using a Gateway-modified vector.
(A) Gateway compatible vector pTSGATE1 for the expression of secreted FLAG/His-tagged proteins in Escherichia coli. The vector carries a Gateway cloning cassette with the ccdB suicide gene and the attR1 and attR2 recombination sites, preceded by a sequence encoding a secretion peptide (SP), a FLAG epitope tag for detection, and a Histidine-tag for purification. (B) Examples of secreted proteins purified from the medium of E.coli cells carrying pTSGATE1 expressing genes of interest. E. coli cultures were induced with IPTG, cleared by centrifugation and His-tagged proteins were purified from the medium by Nickel affinity chromatography.
Fig 5
Fig 5. Competitive ABPP screen reveals inhibitor of immune protease C14.
Protein extracts of leaves transiently overexpressing the C14 immune protease were pre-incubated with or without 50 μM E-64 or with 1 μg purified Avr2, EpiC1 or Epic2B (A) or 1 μg purified putative effector protein (B) or for 30 minutes and then incubated for one hour with 1 μM MV201 to label the non-inhibited proteases. Proteins were separated on a 12% protein gel and scanned for fluorescently labeled proteins. The signals were quantified and plotted in a bar graph. The region corresponding to 0.5 to 1.5 times the no-inhibitor-control is shaded grey. This screen was repeated twice with a similar outcome.
Fig 6
Fig 6. Cip1 is an extracellular virulence factor that is expressed during Infection.
(A) The Δcip1 mutant (UNL231) shows reduced bacterial growth in susceptible tomato plants when compared to wild type and cip1-complemented strains. Error bars represent standard errors of n = 4 biological replicates. A repetition experiment had a similar outcome. (B) The Δcip1a mutant (UNL231) has reduced bacterial speck symptoms when compared to wild type and complemented strains. Pictures were taken three days after bacterial infiltration with 105 CFU/ml. (C) Cip1 is expressed when bacteria are grown in minimal medium and in planta. RNA was isolated from PtoDC3000 and a Δcip1 mutant (UNL232) grown in minimal medium (MM) containing mannitol-glutamate or isolated from apoplastic fluids (AF) at two days after infection, and used as template for PCR with and without reverse transcriptase (RT) pretreatment. Primer pairs were used to amplify Cip1, the two flanking genes and RecA as a control. (D) Cip1 protein is detected I the supernatant of cultures of bacteria grown in minimal medium. Wild-type, Δcip1 (UNL231(a) and UNL232(b)) and ΔhopQ1-1 mutant bacteria were grown in minimal medium (MM), centrifuged and the supernatant was used for western blot analysis using a specific antibody raised against the Cip1 protein produced in E. coli. *, background signal showing equal loading.
Fig 7
Fig 7. Cip1 is a Chagasin-like protease inhibitor.
(A) Alignment of chagasins showing conserved chagasin motifs NPTTG, GxGG and RPW (red). Amino acid identity with Cip1 is shown next to each of the protein sequences. (B) Cip1 is a competitive inhibitor of papain. Papain was incubated without or with 7.46 or 3.98 nM Cip1 and assayed at increasing substrate concentrations. Fluorescence values were plotted against the inverse velocity (V) and inverse substrate concentration [S] in a Lineweaver- Burk plot. Ki = 3.98 nM. (C) Cip1 mutant proteins are soluble and stable. The NPTTG motif was mutated by removing the two threonines (ΔT) or replacing them by alanines (AA). Mutant and wild-type (WT) Cip1 proteins were expressed in E.coli as His-FLAG-tagged protein, purified, separated on SDS-PAGE and stained by coomassie. (D) Mutagenesis of the NPTTG motif affects inhibition of papain. 4.1 μM papain was incubated with 0.56 μM (mutant) Cip1 inhibitor or chicken cystatin and 0.2 mM BAPNA. Substrate conversion was monitored by increased fluorescence at 410 nm over time. (E) Protease activity profiling of papain (left) and C14 (right) in the presence of (mutant) Cip1 using pure papain or apoplastic fluids from leaves overexpressing C14 were preincubated with 100 μM E-64 or (mutant) Cip1 for 30 minutes, and then labeled with 1 μM MV201 for 1 hour.
Fig 8
Fig 8. Cip1 blocks all three immune proteases of tomato with different affinities and escapes recognition by Rcr3/Cf-2.
(A) Cip1 suppresses active site labeling of C14, Pip1 and Rcr3. Extracts containing the proteases were pre-incubated with 7.8 μM Cip1 and then labeled with MV201, a fluorescent probe for PLCPs. *, endogenously biotinylated protein. Abbreviations: i, iC14; m, mC14; p, Pip1; r, Rcr3. (B) Cip1 has highest affinity for Pip1 and C14. Extracts containing the proteases were pre-incubated with various concentration of Cip1 and then labeled with MV201. Labeled proteins were quantified from gel and plotted against the Cip1 concentration. (A-B) C14, Pip1 and Rcr3 were transiently overexpressed by agroinfiltration and proteomes were isolated and pre-incubated with and without various concentrations of Cip1 at pH 6.2 for 30 minutes and then labeled with 1 μM MV201 for one hour. Labeled proteins were detected by in-gel fluorescence scanning. (C) Cip1 does not trigger hypersensitive cell death in Cf2/Rcr3 tomato plants. 100 nM Avr2 or 1 μM Cip1 were injected into leaflets of MM-Cf2 and MM-Cf0 tomato plants and photographs were taken five days after the injection. (D) The presence of Rcr3 does not affect bacterial growth of PtoDC3000. Cf2/Rcr3 and Cf2/rcr3-3 tomato plants were spray-inoculated with PtoDC3000 and the bacterial populations were determined at 0, 2 and 4 days-post-inoculation. Error pars represent SEM for n = 4 different leaf samples taken during the same assay. This experiment was repeated four times with similar results.

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