Financial support: This work was supported by Grant PUCV-DI 122-769- Chile.

Keywords: antibacterial peptide, disease resistance, in vivo expression, Nicotiana tabacum L., Pseudomonassyringae pv. syringae, transgenic tobacco plants.

Antibacterial peptides are small molecules which are part of the innate response and as such constitute the first line of defence against microbial infections. They have been reported at all levels, from mammals to insects, marine invertebrates and plants. At present, more that 500 different molecules have been reported of which defensins constitutes the most important peptide family characterized.

There have been a number of attempts to use both heterologous and even homologous peptides as tools to increase disease-resistance in transgenic plants. Unfortunately although most of them have been unsuccessful some of them have been inspiring in the search for new alternatives. Some of the reasons for the lack of success in the past have been estimated to be due to either a low level of expression of the transgene, a putative inactivation of the gene product by constitutive proteases in the new host environment, or a low half-life of the expressed peptide in the host plant.

Using a natural peptide from molluscs belonging to the defensin family class, in this report we wanted to contribute to evaluate the impact of family-related peptides in commercially relevant plant species which might be optimized though their enhanced disease-resistance potential. The rationale was the following: a) that the transgene product is a defensin-like molecule expressing in a system with has natural counterparts of antimicrobial peptides; b) assuming that endogenous proteases would not destroy it; c) that the transgene product could act synergically with endogenous AMPs, natural defensins and/ or alikes, which might explain the enhanced expected over protective effect over challenged plants.

We have obtained a clone which is a “defensin-like” antibacterial peptide from immune cells recovered from the Chilean mussel Mytilus edulis chilensis, through a standard procedure in which primers specially designed which allow to recover the nucleotide sequence (gene) codifying for the functional molecule. The gene was incorporated into a vector which allows it dissemination into different cellular systems. Since we wanted to express this new gene in plant tissues to provide protection to pathogenic bacteria, we inserted the gene into a binary new vector for expression in plants. The new clone was electroporated into A. tumefaciens to transform tobacco plants. To test that the new vector is indeed inside the plants cells, molecular techniques were used to detect its presence as well as to demonstrate it is being expressed. To determine if transformed tobacco plants were more resistant than not transformed plants, tobacco leaves were infiltrated with Pseudomonassyringae pv. syringae and visual lesions determined post-exposure up to seven days post-infection .Of seven plants exposed, four gave variable protection n while one of them appears to be fully protected. These results suggest that defensin-like antimicrobial peptides from molluscs are a good source to provide resistance of tobacco plants to Pseudomonasyringae pv. syringae.