Publishable summary- Investigating molecular determinants involved in the interaction between Clavibactermichiganensissubsp. 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 proteinaceousCmm 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, using global proteomics techniques, 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-typeCmm382 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 intracellularCmm 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 VATR2and 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.
In summary, we showed that the plant mounts a basal defense response during Cmm infection, which implicates PAMP perception. We have characterized this defense response and identified components of it that are likely to play a role in the interaction with the bacterium. These proteins are candidates for further follow up work, as mentioned in the 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.
The Marie Curie grant has allowed me to carry out an extensive research, using the most advanced technologies available (e.g. global proteomics and RNA-Seq experiments), and helped in establishing my scientific career in Israel. At the end of the research period I was hired by the Weizmann Institute of Science in Rehovot, Israel, as a proteomics specialist in the Nancy & Stephen Grand Israel National Center for Personalized Medicine, much thanks to the research I have done under the Marie Curie grant.