Department of Plant Pathology, Cornell University, NYSAES, Geneva, NY 14456, USA

Background and objectives
Previously, we reported that a non-target DNA sequence, green fluorescent protein (GFP) gene, reduces the nucleocapsid (N) transgene length necessary for RNA-mediated tospovirus resistance [1]. Transgenic plants expressing large fragments (387-453 bp) displayed resistance through post-transcriptional gene silencing (PTGS). N transgene fragments smaller than 235 bp were ineffective when expressed alone, but conferred resistance through the PTGS mechanism when fused to the non-target GFP gene [1]. This study was initiated to determine if other DNA sequences (besides GFP) could be used to link the N gene of tomato spotted wilt tospovirus (TSWV) and still induce gene silencing. Additionally, if the silencing DNA is of viral origin, it could also serve as a viral transgene for imparting multiple resistance.

Materials and methods
Transgenic Nicotiana benthamiana plants with various N gene fragments, 2/2N (453 bp), 3/4N (218 bp), and 5/8N (118 bp) of TSWV fused to GFP conferred tospovirus resistance in both R0 and R1 progenies [1]. To determine whether these transgenic plants with GFP/N fusions can confer multiple resistance to TSWV as well as to a chimaeric virus with GFP sequence (TMV-GFP), R2 transgenic plants with 2/2N, 3/4N or 5/8N linked to GFP were inoculated with TSWV-BL or with infectious transcripts of TMV-GFP [2] or TMV-GFP-NP. Transgenic N. benthamiana plants containing 3/4N or 5/8N gene of TSWV linked to the coat protein (CP) gene of turnip mosaic potyvirus (TuMV) were generated. R1 progenies of transgenic plants with CP/N fusion were inoculated with TSWV-BL and TuMV-Esc8. The combination of nuclear run-off experiments and steady RNA analysis were utilized for assessing whether PTGS had occurred [1].

Results and conclusions
R2 transgenic plants with 2/2N, 3/4N, and 5/8N gene fragments of TSWV fused to GFP DNA displayed resistance to both TSWV and TMV-GFP. The R1 transgenic plants with 3/4N and 5/8N of TSWV linked to TuMV CP were resistant to TuMV and TSWV; four of 18 tested lines with CP-3/4N and one of 14 tested lines with CP-5/8N were resistant to TuMV as well as TSWV. These results demonstrate that (i) small fragment of viral genome sequence linked with a silencing DNA (GFP or TuMV CP) can trigger PTGS and confer multiple virus resistance; and (ii) the DNA that triggers silencing of the linked viral gene fragments can itself be a viral gene, as we have shown with TuMV. Therefore, our data serve as the basis to develop a simple and effective new strategy for engineering multiple virus resistance by linking several small viral DNA fragments with a silencer DNA (GFP, viral sequences or other genes from plants, animals or other organisms). These data also suggest the possibility of developing a simple and effective new strategy for controlling and regulating gene expression, enzyme or protein production and other phenotypes in transgenic plants.

1. Pang S-Z, Jan F-J, Gonsalves D, 1997. Proceedings of the National Academy of Sciences, USA 94, 8261-8266.
2. Casper SJ, Holt CA, 1996. Gene 173, 69-73.