Abstract
Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker of tomato. Currently, no Solanum lycopersicum resistant varieties are commercially available, but some degree of Cmm resistance has been identified in Solanum peruvianum. Previous research showed up-regulation of a SUMO E2 conjugating enzyme (SCEI) transcript in S. peruvianum compared to S. lycopersicum following infection with Cmm. In order to test the role of SCEI in resistance to Cmm, a fragment of SCEI from S. peruvianum was cloned into a novel virus-induced gene-silencing (VIGS) vector based on the geminivirus, Tomato Mottle Virus (ToMoV). Using biolistic inoculation, the ToMoV-based VIGS vector was shown to be effective in S. peruvianum by silencing the magnesium chelatase gene, resulting in leaf bleaching. VIGS with the ToMoV_SCEI construct resulted in ~61% silencing of SCEI in leaves of S. peruvianum as determined by quantitative RT-PCR. The SCEI-silenced plants showed unilateral wilting (15 dpi) and subsequent death (20 dpi) of the entire plant after Cmm inoculation, whereas the empty vector-treated plants only showed wilting in the Cmm-inoculated leaf. The SCEI-silenced plants showed higher Cmm colonization and an average of 4.5 times more damaged tissue compared to the empty vector control plants. SCEI appears to play an important role in the innate immunity of S. peruvianum against Cmm, perhaps through the regulation of transcription factors, leading to expression of proteins involved in salicylic acid-dependent defense responses.
Highlights
Clavibacter michiganensis subsp. michiganensis (Cmm) is a Gram-positive plant bacterial pathogen belonging to the order Actinomycetales in family Microbacteraceae (Gartemann et al, 2008)
In order to develop a virus-induced gene-silencing (VIGS) vector based on Tomato Mottle Virus (ToMoV), the coat protein contained in component A of ToMoV was removed, and a cloning site was added at the same location (Figure 1)
For S. lycopersicum, there have been several viruses used for VIGS, such as Potato Virus X (Giliberto et al, 2005) and DNA1 component of tobacco curly shoot virus (TbCSV) (Huang et al, 2009), but the most commonly used VIGS vector has been based on Tobacco Rattle Virus (TRV) (Liu et al, 2002)
Summary
Clavibacter michiganensis subsp. michiganensis (Cmm) is a Gram-positive plant bacterial pathogen belonging to the order Actinomycetales in family Microbacteraceae (Gartemann et al, 2008). Michiganensis (Cmm) is a Gram-positive plant bacterial pathogen belonging to the order Actinomycetales in family Microbacteraceae (Gartemann et al, 2008). It is the causal agent of bacterial wilt and canker of tomato (Solanum lycopersicum), which occurs worldwide (Eichenlaub and Gartemann, 2011). Bacterial wilt and canker has been reported to cause losses of tomato production as high as 84% in the U.S.A. and Canada (Strider, 1969; Gleason et al, 1993). Cankers on young plants are damaging as they can result in plant death. Cmm infects fruit, which results in necrotic spots called bird’s eyes (Gartemann et al, 2003)
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