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Title: Antimicrobial Activity of snakin-1 and defensin as a fusion protein in vitro

Author
item KOVALSKAYA, NATALIA - MOSCOW RUSSIA
item Zhao, Yan
item Hammond, Rosemarie

Submitted to: BARC Poster Day
Publication Type: Abstract Only
Publication Acceptance Date: 3/26/2007
Publication Date: 3/26/2007
Citation: Kovalskaya, N.Y., Zhao, Y., Hammond, R. 2007. Antimicrobial Activity of snakin-1 and defensin as a fusion protein in vitro. BARC Poster Day. Proceedings. pag. 27.

Interpretive Summary:

Technical Abstract: Phytopathogenic bacteria and fungi cause significant losses in important agricultural crop yields. This problem can be overcome by genetic engineering of plants with abroad range of disease resistance. It is known that some microorganisms, plants and animals can produce a large variety of small proteins (<100 amino acids) and peptides with antimicrobial activity. Utilization of such peptides for expression in transgenic plants is a promising new approach for plant protection. However, it is important to test the selected protein(s) on a broad range of phytopathogens prior to performing the plant transformation experiments. In the present study, we tested two naturally occurring peptides possessing antimicrobial (antibacterial and antifungal) activity, snakin-1 (SN1) and defensin (PTH1), produced in potato tubers. It has been shown that the spectrum of the SN1 activity is distinct from that of the PTH1 and simultaneous expression of both peptides in plants greatly expands the range of antimicrobial resistance. To test this assumption we constructed an expression cassette in a pET-based vector for simultaneous production of the two peptides as a single fusion protein capable of undergoing a self-cleavage by means of an embedded 2A protease of the foot-and-mouth disease virus (FMDV). However, no expected self-cleavage of the fusion expressed in the host E.coli strain BL21 (DE3) was seen. Moreover, we found that when the SN1-2A-PTH1 (SAP) fusion protein was expressed in prokaryotic cells, it was localized in inclusion bodies. The SAP fusion protein solubilized from the inclusion bodies was tested for its antimicrobial activity in vitro against phytopathogenic bacteria Pseudomonas syringae pv. syringae and Pseudomonas syringae pv. tabaci as well as fungi Botrytis cinerea, and Colletotrichum sp. by the methods of plating of bacteria on solid medium, and fungal spore germination inhibition assays, respectively. The fungus Colletotrichum sp. has been found the most sensitive to the SAP protein with IC50 about 40µg/ml. The inhibition of spore germination of this fungus reached almost 100% at the protein concentration of 100µg/ml, in contrast to the fungus Botrytis cinerea, that was not sensitive to SAP at any concentration tested. Both bacterial cultures showed low susceptibility to the SAP protein, with the maximum inhibitory effect of 10-13% at the highest concentration. To determine the reason for low SAP activity observed in our experiments, we have recently constructed expression cassettes for individual production of each of the two peptides (SN1 and PTH1) in a pET-based vector. This approach will allow us to test the antimicrobial activity of the each individual protein as well as their combination on phytopathogenic bacteria and fungi and compare it to that of the fusion protein. This research was supported by USDA ARS CRIS Project No. 1275-22000-206-00D.