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

Background and objectives
Post-transcriptional gene silencing (PTGS) has recently been found to be the cause of RNA-mediated virus resistance in transgenic plants. This type of resistance is also referred to as homology-dependent resistance. This study was initiated to investigate the effect of transgene length and non-target DNA sequence on RNA-mediated virus resistance. Experiments were also designed to determine the minimum DNA length that triggers effective PTGS and to determine the minimum length of the nucleocapsid (N) gene of tomato spotted wilt tospovirus (TSWV) that is required for trans-inactivation of the incoming tospovirus genome.

Materials and methods
Transgenic Nicotiana benthamiana plants containing gene constructs ranging from about 1/2N (400 bp) to 1/32N (28 bp) of TSWV alone or linked to the jellyfish green fluorescent protein (GFP) gene were generated using Agrobacterium-mediated transformation. The combination of nuclear run-off experiments and steady RNA analysis were utilized to assess whether PTGS had occurred [1]. TSWV-BL [1] and infectious transcripts of TMV-GFP [2] or TMV-GFP-NP were used as inoculum.

Results and conclusions
We have generated a number of plant lines that contain gene constructs containing about 1/2 to 1/32 of the N gene of TSWV alone or fused with a non-viral sequence, GFP. Transgenic plants expressing large N transgene fragments (387-453 bp) displayed resistance through PTGS. Small N transgene fragments (235-110 bp) were ineffective when expressed alone, but conferred resistance through PTGS mechanism when fused to the non-target GFP DNA. These results showed that the inability of the small N transgenes alone to induce homology-dependent virus resistance was not due to insufficient lengths of homology to the silenced transgene, but because they are incapable of inducing gene silencing. Thus this study differentiates the ability to induce transgene silencing from the ability to provide homology-dependent trans-inactivation. In addition, the fusions of N gene segments larger than 110 bp with GFP resulted in plants with both genes silenced, and those were resistant to TSWV and a chimaeric virus, TMV-GFP. However, transgenic plants with smaller N gene segments (56 or 28 bp) linked with GFP conferred resistance only to TMV-GFP or TMV-GFP-NP but not to TSWV. These results demonstrate that (i) a critical length (236-387 bp) rather than the specific N gene sequences is required for transgene silencing and virus resistance; (ii) the non-target DNA sequence, GFP, reduced the transgene length necessary for RNA-mediated tospovirus resistance; (iii) TMV-GFP infectious transcripts can be a useful tool to identify the PTGS state in the transgenic plants expressing GFP or GFP-N fusions; (iv) a critical length of the homologous transgene (56-110 bp) in transgenic plants with PTGS state is required to target the incoming virus and virus resistance; and (v) small fragments of viral gene linked to GFP can confer multiple resistance.

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.