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

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.

An effector from the Huanglongbing-associated pathogen targets citrus proteases

“…tomato strain DC3000 ( Pto DC3000) was previously reported to produce a Sec-secreted protein called Cip1, which can inhibit the protease activity of tomato C14, a member of the RD21a subfamily of PLCPs 32 . A cip1 knockout mutant of Pto DC3000 exhibited reduced virulence, indicating that Cip1 contributes to bacterial infection, likely through its inhibitory effect on PLCP activities 32 . We examined whether SDE1 could complement the Cip1 virulence activity that was lost in the knockout mutant of Pto DC3000.…”

Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation

“…Also unexpected was the large repertoire of SSPs predicted in mycorrhizal fungi [ 72 , 73 ] . SSPs were also recently reported in bacteria such as the plant pathogen Pseudomonas syringae [ 74 ], but up to now no SSPs were described in oomycete genomes. Comparative fungal genomics studies showed evidences of rapid evolution of SSPs in related pathogens with different host ranges [ 70 , 75 ].…”

Transcriptional profiling of threePseudomonas syringaepv.actinidiaebiovars reveals different responses to apoplast-like conditions related to strain virulence

“…They subvert host cellular processes to support bacterial proliferation, thus promoting disease. TTSS components are encoded by hypersensitive reaction and pathogenicity (hrp) and hrp conserved (hrc) genes (8)(9)(10), which are present in almost all phytopathogenic bacteria including Psa (11,12). The expression of hrp/hrc genes is one of the most important events during infection, and is induced following contact with plant tissues or in minimal hrp-inducing medium (HIM) (13)(14)(15)(16).…”