1.12.20
INVESTIGATIONS INTO THE USE AND MECHANISMS OF GENE SILENCING

A HAMILTON and D BAULCOMBE

Sainsbury Laboratory, John Innes Centre, Norwich, UK

Background and objectives
Post-transcriptional gene silencing (PTGS) is characterized by the severe reduction of steady-state levels of specific RNAs without a perceptible effect on transcription after the introduction of homologous sequences. As well as efficiently suppressing the expression of nuclear genes, these silencing transgenes confer extreme resistance to RNA viruses if they share sequence homology. Probably this is the mechanism behind most examples of pathogen-derived resistance in transgenic plants [1]. Our first objective is to discover how this sequence-specific signal is generated by activators of PTGS, the nature of this signal, and how it exerts its effect(s) upon targets of PTGS.

Recombinant potato virus X (PVX) can act not only as a target of PTGS, but also an activator of silencing (on Nicotiana benthamiana) if it contains sequences homologous with an endogenous gene. Our second objective described here is to determine the range of genes which are susceptible to virus-induced gene silencing in order to assess its potential as a new method for quickly and simultaneously cloning and assigning function to uncharacterized plant genes.

Results and conclusions
We have been attempting to detect unusual RNA species homologous to genes undergoing PTGS. Several PTGS systems are being analysed: (i) silencing of endogenous nuclear genes by transgenes with and without internal repeats (ACC-oxidase); (ii) silencing of transgenes by other transgenes or transgene self-silencing (35S-GUS; 35S-GFP); (iii) silencing of viruses by transgenes (PVX, ACC-oxidase, GUS, GFP); (iv) silencing induced by bombardment (GFP); (v) silencing transmitted by grafting (GFP); (vi) silencing of plants genes by recombinant RNA viruses (RUBISCO, PVX). The data presented will concern possible silencing-specific RNAs and their potential role in gene silencing will be discussed.

In pursuit of our second objective, the following genes have been tested for their susceptibility to virus-induced gene silencing: GFP, phytoene desaturase, glutamate semialdehyde amino transferase, RUBISCO small subunit, cyclin A, 26S ribosomal RNA, Nfl (homologue of Floricaula), auxin-binding protein, zeaxanthin epoxidase, acetyl CoA carboxylase, and glucan synthase. Where gene silencing was observed the phenotypes obtained developed progressively from about 2 weeks after inoculation and culminated in severe suppression of that gene.

References
1. Baulcombe DCB, 1996. Plant Molecular Biology 32, 79-88.