Specificity of resistance to pea seed-borne mosaic potyvirus in transgenic peas expressing the viral replicase (Nlb) gene.

Transgenic pea lines carrying the replicase (NIb) gene of pea seed-borne mosaic potyvirus (PSbMV) were generated and used in experiments to determine the effectiveness of induced resistance upon heterologous isolates. Three pea lines showed inducible resistance in which an initial infection by the homologous isolate (PSbMV-DPD1) was followed by a highly resistant state. Resistance was observed in plants in either the homozygous or hemizygous condition and resulted in no overall yield loss despite the initial infection. Resistance was associated with a loss of both viral and transgene RNA, which is indicative of a mechanism based upon post-transcriptional gene silencing. There was no correlation between the steady-state levels of transgene RNA and ability of the plants to show resistance. To test the specificity of the resistance, plants were also inoculated with the most distantly related sequenced PSbMV isolate, NY. PSbMV-NY varied between experiments in its ability to induce resistance, suggesting that the sequence identity in the NIb gene is borderline for the specificity required for triggering gene silencing. Upon challenge inoculation of virus-free recovered leaves, the specificity of the induced resistance varied between the two isolates and indicated that the virus and transgene additively determined the resistant state. These results suggest that the sequence requirements for triggering gene silencing may differ from those involved in the degradation process.

A simple system for pea transformation.

The lateral cotyledonary meristems of germinatingPisum sativum cv. Puget seeds were used to develop a reproducibleAgrobacterium tumefaciens-mediated transformation system. This procedure exhibits distinct advantages over those previously reported, in that it uses dry seed as starting material, and the highly regenerable cotyledonary meristems rapidly produce transgenic shoots without an intermediate callus phase. This transformation regime facilitates the rapid generation of phenotypically normal, self-fertile plants containing functional transgenes inherited in a Mendelian fashion.