Publishable summary- Investigating molecular determinants involved in the interaction between Clavibacter michiganensis subsp. michiganensis (Cmm) and tomato At the beginning of the project, 4 objectives were set for the research: 1) Characterization of basal defense responses induced by Cmm in tomato plants and suspension cells 2) Identification of tomato genes playing a role in activation of basal defense responses to Cmm pathogen associated molecular patterns (PAMPs) 3) Isolation and identification of proteinaceous Cmm PAMP(s) 4) Identification of Cmm secreted proteins involved in pathogenicity At the start of the project, we set up a system of tomato suspension cell cultures to be tested for basal defense responses triggered by Cmm. While this system proved to be overly sensitive, we were able to show using 3′3-diaminobenzidine (DAB) staining that reactive oxygen species (ROS) accumulation was significantly higher in Cmm-infected plants than in the control plants. As ROS production is a hallmark characteristic of the plant basal defense response, this finding reveals that the tomato plant senses the invading Cmm and mounts a basal defense response against it. Next, we carried out proteomics experiments at the Oak Ridge National Laboratory (ORNL) in Tennessee, U.S.A.. Here, by using multidimensional proteins identification technology (MudPIT) in conjunction with mass spectrometry, we were able to quantitatively analyze thousands of tomato and Cmm proteins. We showed that tomato plants sense the invading bacterium, transduce signal, and mount a basal defense response, although partial, as evident from induction of multiple defense-related tomato proteins. We showed that the virulent wild-type Cmm382 triggers ethylene biosynthesis in the plant by specifically inducing a single tomato enzyme in the ethylene biosynthesis pathway, ACC oxidase, which leads to disease symptom development. We identified several defense related tomato proteins, including 4 class III secreted plant peroxidases, that were unexpectedly down-regulated in Cmm infected plants, and may be targets of suppression by Cmm. On the bacterium side, we showed that during infection, Cmm senses the plant environment, transduces signal, and secretes multiple hydrolytic enzymes aimed at degrading plant molecules. We also identified 12 intracellular Cmm proteins involved in signal transduction that may play a role in pathogenicity. We selected a few of them for mutagenesis of their respective genes, and tested their effect on the ability of the bacterium to cause disease. Mutants in two of these genes, CMM_2645 and CMM_2969, which we subsequently named vatr1 and vatr2, respectively (for virulence associated transcriptional regulators), showed significant reduction in pathogenicity, as evident from the delayed or abolished wilting of infected plants (Figure 1). To further characterize VATR1 and VATR2 and reveal their involvement in pathogenicity, several physiological, biochemical, and molecular experiments were carried out, including a large scale RNA-Seq study. We showed that these putative transcriptional regulators affect expression of virulence factors during infection, along with other signal transducing proteins, membrane proteins and secreted proteins. The Δvatr1 and Δvatr1 mutants lead to reduced tomato ACC-oxidase activation and reduced ethylene production in infected plants compared to the wild-type bacterium, and consequently lead to reduced symptom development. Figure 1- Δvatr1 and Δvatr2 mutants cause In summary, we showed that the plant mounts reduced wilting in infected plants. A. a basal defense response during Cmm Twenty-four 5-week old tomato plants infection, which implicates PAMP perception. were inoculated with either WT Cmm382, Δvatr1, or Δvatr2, and were monitored for We have characterized this defense response wilting of the first leaf. B. An example of and identified components of it that are likely typical wilting of infected tomato plants 10 to play a role in the interaction with the days post-inoculation with Cmm382 or with bacterium. These proteins are candidates for the mutants. The red arrow in the vatr1 further follow up work, as mentioned in the panel points to a wilted leaf among others that had not wilted yet. original grant proposal. On the beacterium side, we identified both secreted and intracellular proteins that play a part in Cmm pathogenicity, and characterized components of this molecular virulence pathway. Taken together, the objectives mentioned above were accomplished in large part. The identification and characterization of the molecular components of the tomato-Cmm interaction, both of the bacterium and the plant, greatly improves our understanding of the tomato wilt and canker disease, and provides an important step towards devising future strategies of combating this important plant disease.