Isolation and Characterization of Plant Metabolite Signals that Induce Type III Secretion by the Plant Pathogen Pseudomonas syringae

Abstract

Pseudomonas syringae is a bacterium that can cause disease on a wide range of plant species including important agricultural crops. A primary virulence mechanism used by P. syringae to infect host plants is the type III secretion system (T3SS), a syringe-like structure that delivers defense-suppressing proteins directly into plant cells. Genes encoding the T3SS are not transcribed in P. syringae prior to contact with a potential host plant and must be expressed during initial stages of infection. Specific organic and amino acids exuded by plants were recently identified as signals that can induce expression of T3SS-associated genes. Here we describe a technique to produce exudates from intact Arabidopsis seedlings and evaluate the exudates for the presence of these bioactive metabolites. We provide procedures for exudate production as well as downstream assays to assess T3SS gene expression using a GFP transcriptional reporter. We also describe methods for preparing high-quality protein and RNA from exudate-treated bacteria to directly assess changes in mRNA and protein abundance. These methods could be used to investigate mechanisms regulating P. syringae perception of plant metabolites as well as the release of these substances by the plant, and more generally to investigate host signals perceived by other phytopathogens.