Comparison of pathogen-induced expression and efficacy of two amphibian antimicrobial peptides, MsrA2 and temporin A, for engineering wide-spectrum disease resistance in tobacco

The rapid accumulation of defensive transgene products in plants only on pathogen invasion has clear advantages over their constitutive synthesis. In this study, two antimicrobial peptides from the skin secretions of frogs, MsrA2 (N-methionine-dermaseptin B1) and temporin A, were evaluated for engineering pathogen-induced disease resistance in plants. Both peptides inhibited plant-specific pathogens in vitro at micromolar concentrations that were not toxic to plant protoplasts. The plant-optimized nucleotide sequences encoding MsrA2 and temporin A were transcriptionally fused to the inducible win3.12T poplar promoter, which had strong systemic activity in response to fungal infection, and introduced into tobacco (Nicotiana tabacum L. cv. Xanthi). Transgene expression was very low in the leaves of unstressed plants; however, it was strongly increased after pathogen challenge or wounding. The pathogen responsiveness of the win3.12T promoter was found to be universal rather than species specific, with high activity in response to all pathogens tested. On induction, the amount of MsrA2 was up to 6-7 mu g per gram of fresh leaf tissue. Most importantly, the induced accumulation of MsrA2 and temporin A in transgenic tobacco was sufficient to confer resistance to a variety of phytopathogenic fungi, such as Fusarium solani, F. oxysporum, Alternaria alternata, Botrytis cinerea, Sclerotinia sclerotiorum, the oomycete Pythium aphanidermatum and the bacterium Pectobacterium carotovorum. The accumulation of these peptides in transgenic plants did not alter the normal phenotype of tobacco. Thus, the expression of MsrA2 and temporin A in a pathogen-inducible manner enables the development of tobacco, and possibly other plant species, with wide-spectrum disease resistance, which can reduce the use of pesticides and the associated environmental risks.