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.

Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris

“…Enhanced resistance to pathogen infections has also been reported in plants that express snakin genes [28,29]. Snakin peptides are thus considered important candidates for producing biotic stress tolerance in crops.…”

“…Both cause rapid aggregation of pathogens, although this response is not directly related with their antimicrobial activity [11,26]. Transgenic plants over-expressing the SN-1 and SN-2 genes have been shown to exhibit increased resistance to a wide range of invading phytopathogens [28,29].…”

One new kind of phytohormonal signaling integrator: Up-and-coming GASA family genes

“…Antibacterial experiments and in vivo experiments showed that potato Snakin-1 and Snakin-2 proteins have the anti-bacterial and -fungi capacity. 16,17,48,49,50 The CaSn protein of the pepper has the ability to anti-root-knot nematode (Meloidogyne spp.). 30 French bean Snakin-2-like protein may be formed an approximately 42 kDa protein complex (FBCBP) with the proline-rich protein PRP to participate in the plant defense process.…”

Tomato Fruit and Seed Colonization by Clavibacter michiganensis subsp. michiganensis through External and Internal Routes

“…A 1-cm stem section above the inoculation site was aseptically removed from the harvested plants and homogenized in 400 l of sterile 10 mM MgCl 2 using a Retsch MM400 Tissuelyser (Newton, PA). After complete homogenization, 600 l of sterile 10 mM MgCl 2 was added for a final volume of 1 ml (25). The solution was spun down at 400 ϫ g for 3 min (16).…”