1The Sainsbury Laboratory, Norwich Research Park, Colney Lane, Norwich NR7 4UH, UK

Background and objectives
Studying gene function in plants by conventional approaches utilizing insertional mutagenesis has significant disadvantages. The specific genes of interest cannot be targeted and therefore the number of transformants required for efficient mutant screens may be difficult to obtain in recalcitrant species. These obstacles can potentially be overcome in a range of plant species by employing amplicons, defined as transcribed and replicating virus-based transgenes. Using a potato virus X (PVX) amplicon carrying a GUS reporter gene insert, the consistent post-transcriptional gene silencing of transient GUS expression in Nicotiana tabacum cv. Petite Havana has been reported earlier [1]. Further findings suggest that PVX amplicons can also efficiently target homologous sequences in the host genome for amplicon-mediated co-suppression [unpublished data]. Cytoplasmic viral replication is considered the crucial determinant for consistent gene silencing conferred by the PVX amplicon. Neither the PVX coat protein (CP) nor the triple gene block (TGB) proteins are directly involved in replication and predominantly serve systemic and cell-to-cell movement of the virus in the host plant.

Our aim was to minimize the PVX amplicon to facilitate its future use as a research tool. We therefore focused on the removal of the PVX CP and its TGB region to establish whether these modifications maintain viral replication and thus consistent gene silencing.

Results and conclusions
N. benthamiana leaves were agro-infiltrated with a 35S-driven PVX construct carrying the jellyfish green fluorescent protein (GFP) as a replacement for the coat protein gene (PVX/TGB/GFP). Viral replication and production of subgenomic RNAs were detected by bright green fluorescence on infiltrated leaves and confirmed by Northern analysis. For modified constructs lacking the 5' end of GFP and all or a major part of the TGB (PVX/FP), Northern analysis also indicated replication and subgenomic RNA production. Analogous constructs expressing a beta-glucuronidase (GUS) reporter gene (PVX/GUS) supported this finding with spots of GUS activity in infiltrated leaves.

On transformation of transgenic 35S-GFP Arabidopsis thaliana plants with PVX/TGB/GFP, the primary transformants displayed a silenced phenotype for GFP. This is in line with observations in crosses of A. thaliana plants homozygous for a 35SGFP transgene with amplicon plants expressing a wild-type PVX transgene with a GFP insert (PVX/TGB/GFP/CP). The F1 progeny of these crosses were uniformly silenced for GFP. PVX/FP constructs and replication-deficient controls for PVX/TGB/GFP and PVX/FP are currently being transformed into 35S-GFP A. thaliana plants and the progeny of the PVX/TGB/GFP transformants awaited for further analyses.

1. Angell SM, Baulcombe DC, 1997. EMBO Journal 16, 3675-3684.