Virus Resistance in Transgenic Plants That Express a Functional Potyvirus P1 Proteinase Gene
Indu B. Maiti, Carol Von Lanken, Sitakanta Pattanaik and Arthur G. Hunt
The first 390 amino acids encoded by the potyvirus tobacco vein mottling virus (TVMV), which encompass the entire P1 polypeptide and about 1/3 of the helper component gene (HC*) were fused with either the TVMV coat protein genes or the E. coli glutamine binding protein gene (glnH) and expressed in transgenic tobacco (Nicotiana tabacum cv burley 21). In both constructions, the cleavage site between the P1 and HC polypeptides was retained so that authentic P1 and HC-CP or HC-glutamine binding protein (GBP) fusion proteins were generated. Several independent transgenic lines were developed carrying either P1-HC*-CP or P1-HC*-glnH constructs. RT-PCR analysis of total RNA isolated from transgenic seedlings (R1 and R2 progeny) showed expected size of transcripts. Western blot analysis showed the expression of P1 protein of appropriate size; expression levels were about 0.12% of total soluble leaf protein. Two polypeptides of about 41 kD and 24 kD, immunoreactive to GBP antisea, were observed in plants carrying the P1-HC*-glnH construction; these correspond to the HC*-GBP fusion protein and to GBP whose bacterial signal peptide (a part of the glnH gene) had apparently been recognized by the protein localization apparatus in the transgenic tobacco plants. In P1-HC*-CP lines, a polypeptide of approximately 44kD immunoreactive to TVMV-CP antiseara was detected and this corresponds to HC*-CP fusion protein. When challenged with TVMV, plants that carried the P1-HC*-glnH construction displayed a significant delay in the development of disease symptoms. Plants that expressed the P1-HC*-CP gene displayed a transient infection after inoculation with TVMV. This latter group of plants were protected against the potyvirus tobacco etch virus as well. We conclude from these studies that the TVMV P1 gene, or its product, can delay disease development after inoculation with TVMV, and that a modified CP with a substantial N-terminal extension can confer upon tobacco protection characteristics similar or identical to those seen in TVMV CP-containing plants.