Screen of Non-annotated Small Secreted Proteins of \u3ci\u3ePseudomonas syringae\u3c/i\u3e Reveals a Virulence Factor That Inhibits Tomato Immune Proteases

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

Screen of Non-annotated Small Secreted Proteins of \u3ci\u3ePseudomonas syringae\u3c/i\u3e Reveals a Virulence Factor That Inhibits Tomato Immune Proteases

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

Screen of Non-annotated Small Secreted Proteins of \u3ci\u3ePseudomonas syringae\u3c/i\u3e Reveals a Virulence Factor That Inhibits Tomato Immune Proteases

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

Screen of Non-annotated Small Secreted Proteins of \u3ci\u3ePseudomonas syringae\u3c/i\u3e Reveals a Virulence Factor That Inhibits Tomato Immune Proteases

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

Where Brain, Body and World Collide

The production cross section of electrons from semileptonic decays of beauty hadrons was measured at mid-rapidity (|y| < 0.8) in the transverse momentum range 1 < pt < 8 Gev/c with the ALICE experiment at the CERN LHC in pp collisions at a center of mass energy sqrt{s} = 7 TeV using an integrated luminosity of 2.2 nb^{-1}. Electrons from beauty hadron decays were selected based on the displacement of the decay vertex from the collision vertex. A perturbative QCD calculation agrees with the measurement within uncertainties. The data were extrapolated to the full phase space to determine the total cross section for the production of beauty quark-antiquark pairs

Screen of Non-annotated Small Secreted Proteins of <i>Pseudomonas syringae</i> Reveals a Virulence Factor That Inhibits Tomato Immune Proteases

<div><p><i>Pseudomonas syringae</i> pv. <i>tomato</i> 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. <i>Cip1</i> 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 <i>Cf-2</i> resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of <i>P</i>. <i>syringae</i>, indicating that also <i>P</i>. <i>syringae</i> secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes.</p></div

Cip1 is a Chagasin-like protease inhibitor.

<p>(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. K_i = 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 <i>E</i>.<i>coli</i> 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.</p

Cip1 blocks all three immune proteases of tomato with different affinities and escapes recognition by Rcr3/Cf-2.

<p>(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.</p

Occurence of small, secreted non-annotated proteins of PtoDC3000 in other <i>Pseudomonas</i> species.

<p>BLAST scores were generated for each of the 131 small secreted, non-annotated proteins against each of the protein databases of 24 sequenced <i>Pseudomonas</i> 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 <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005874#ppat.1005874.s001" target="_blank">S1 Fig</a></b> for more details.</p