Identification of protein interaction partners of two-component system CvsSR in Pseudomonas syringae pathovar tomato DC3000.

Abstract

Pseudomonas syringae pathovar tomato (Pto) is a hemibiotrophic plant pathogen that proliferates aggressively in the tomato plant’s apoplast to cause disease. The Pto two-component system (TCS) CvsSR is essential for virulence in the apoplast, regulating numerous virulence genes. Its transmembrane histidine kinase CvsS senses environmental signals, and then activates its cognate cytoplasmic response regulator CvsR by phosphorylation. The activated CvsR subsequently acts as a transcription factor, affecting gene expression. In addition to the histidine kinase and response regulator interaction, some TCSs interact with other signaling systems, which increases the extent of their regulation. Because CvsSR affects the expression of genes belonging to regulatory pathways of other signaling systems, we hypothesized that it may be regulating these systems directly. To determine if CvsSR engages in regulatory interactions with other proteins, I performed a series of experiments to develop a functional oimmunoprecipitation protocol. First, I tested and confirmed the expression of plasmids transformed into Pto that contained the genes encoding CvsS or CvsR modified to include a targetable FLAG sequence. I then attempted various growth and immunoprecipitation conditions and identified the ones that produced protein interaction partners. The interaction partners of CvsS or CvsR were separated by SDS-PAGE, which revealed a single ~17 kDa band in both that was enriched for relative to the control. The protein band was identified by liquid chromatography coupled with dual mass spectrometry as 30S Ribosomal Subunit S5. The result suggests the discovery of a new type of interaction between a TCS and the ribosome.