title: Over-expression of snakin-2 and extensin-like protein genes restricts pathogen invasiveness and enhances tolerance to Clavibacter michiganensis subsp. michiganensis in transgenic tomato (Solanum lycopersicum).
abstract: Two tomato proteins were evaluated by over-expression in transgenic tomato for their ability to confer resistance to Clavibacter michiganensis subsp. michiganensis (Cmm). Snakin-2 (SN2) is a cysteine-rich peptide with broad-spectrum antimicrobial activity in vitro while extensin-like protein (ELP) is a major cell-wall hydroxyproline-rich glycoprotein linked with plant response to pathogen attack and wounding. Tomato plants, cultivar Mountain Fresh, were transformed via Agrobacterium tumefaciens harboring a binary vector for expression of the full-length SN2 gene or ELP cDNA under the regulation of the CaMV 35S promoter. Molecular characterization of PCR-positive putative T(0) transgenic plants by Northern analysis revealed constitutive over-expression of SN2 and ELP mRNA. Junction fragment analysis by Southern blot showed that three of the four SN2 over-expressing T(0) lines had single copies of complete T-DNAs while the other line had two complete T-DNA copies. All four ELP over-expressing T(0) lines had a single copy T-DNA insertion. Semi-quantitative RT-PCR analysis of T(1) plants revealed constitutive over-expression of SN2 and ELP. Transgenic lines that accumulated high levels of SN2 or ELP mRNA showed enhanced tolerance to Cmm resulting in a significant delay in the development of wilt symptoms and a reduction in the size of canker lesions compared to non-transformed control plants. Furthermore, in transgenic lines over-expressing SN2 or ELP bacterial populations were significantly lower (100-10,000-fold) than in non-transformed control plants. These results demonstrate that SN2 and ELP over-expression limits Cmm invasiveness suggesting potential in vivo antibacterial activity and possible biotechnological application for these two defense proteins.
authors.affiliation: Department of Plant Pathology and Plant-Microbe Biology, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456, USA.
authors.forename: Vasudevan
authors.initials: V
authors.lastname: Balaji
authors.forename: Christine D
authors.initials: CD
authors.lastname: Smart
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D004269:DNA, Bacterial; D060467:Disease Resistance / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D018551:Solanum lycopersicum / Q000235:genetics* / Q000382:microbiology; D008835:Micrococcaceae / Q000472:pathogenicity*; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000235:genetics* / Q000382:microbiology*; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000382:microbiology; D011198:Solanum tuberosum / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D010940:Plant Proteins; C036483:T-DNA