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

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

Abstract

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.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Lycopersicon esculentum / enzymology
  • Lycopersicon esculentum / immunology
  • Lycopersicon esculentum / microbiology*
  • Peptide Hydrolases / genetics
  • Peptide Hydrolases / metabolism
  • Plant Diseases / immunology
  • Plant Diseases / microbiology*
  • Plant Leaves / enzymology
  • Plant Leaves / immunology
  • Plant Leaves / microbiology
  • Plant Proteins / genetics
  • Plant Proteins / metabolism
  • Protease Inhibitors
  • Pseudomonas syringae / genetics
  • Pseudomonas syringae / pathogenicity*
  • Pseudomonas syringae / physiology
  • Virulence
  • Virulence Factors / genetics
  • Virulence Factors / metabolism*

Substances

  • Bacterial Proteins
  • Plant Proteins
  • Protease Inhibitors
  • Virulence Factors
  • Peptide Hydrolases