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.