Genetically modified plants and the 35S promoter: assessing the risks and enhancing the debate

The 35S promoter, derived from the common plant virus, cauliflower mosaic virus (CaMV), is a component of transgenic constructs in more than 80% of genetically modified (GM) plants. Alarming reports have suggested that the 35S promoter might cause accidental activation of plant genes or endogenous viruses, promote horizontal gene transfer, or might even recombine with mammalian viruses such as HIV, with unexpected consequences. In this article, we discuss the properties of CaMV and the 35S promoter and the potential risks associated with the use of the promoter in GM plants, concluding that any risks are no greater than those encountered in conventional plant breeding. Introduction In a recent article, Ho et al. (1999) suggested that the widespread use of the 35S promoter of cauliflower mosaic virus (CaMV) in transgenic plants is “a recipe for disaster”. Ho et al. (1999) base their arguments on three considerations, a) that the CaMV 35S promoter has a hotspot for recombination (Kohli et al., 1999); b) that the 35S promoter has several domains with different tissue specificities and c) that the 35S promoter is very efficient and can function in a wide range of organisms, not only plants but also bacteria and animals. From this they deduce that the 35S promoter could recombine to activate dormant viruses, create new viruses and “cause cancer by the overexpression of normal genes”. As scientists who have worked on CaMV for up to 25 years and have contributed much to the understanding of its molecular biology we wish to put these scenarios in the correct context. The first plant promoter Over 15 years ago, CaMV was one of several plant genetic systems being studied for its potential use in plant transformation (Hull, 1983; 1984; 1985). As part of these studies, much basic research went on into understanding the genetic organisation of CaMV and the means by which its genes are expressed and regulated. The CaMV genome was the first significant piece of plant DNA to be completely sequenced (Frank et al., 1980) and the two CaMV promoters, the 35S and 19S promoters, were the first plant promoters identified (Covey et al., 1981; Hull and Covey, 1983c; Odell et al., 1995). Because of the latter discovery, and the finding that the 35S promoter was active in directing heterologous expression of plant genes in a variety of plants, its use in the development of GM plants for research and agronomic applications became widespread. The virus CaMV can infect a wide range of crucifers (see Schoelz and Bourque, 1999) and is commonly found in cabbages, cauliflowers, oilseed rape, mustard and other brassicas in temperate countries (Tomlinson, 1987). A survey of a local market, as part of a risk assessment exercise for the Ministry of Agriculture, Fisheries and Food (the UK regulatory authority for biosafety of genetic manipulation of plant pests) in the late 1980s, showed that about 10% of the cauliflowers and cabbages were infected with CaMV. The virus is transmitted in nature by aphids (see Schoelz and Bourque, 1999) and it might be expected that organically grown crucifers, on which the aphids have not been controlled by insecticides, would have higher rates of infection. Infection early in the plant growth might affect the quality, especially of cauliflowers, but later infections show leaf symptoms but little other overt effects. The virus infects most cells of the plant and produces about 10 particles per cell. Each particle contains one molecule of the viral genome, an 8 kbp circular double-stranded DNA with one copy each of the two promoters. The replication cycle of the virus has two phases (see Hohn, 1999), the first in the nucleus where the viral genome is uncoated, forms a minichromosome and is transcribed to give two RNA species, the 35S RNA (using the 35S promoter) and the 19S RNA (using another promoter). These RNAs pass to the cytoplasm where the 35S RNA acts as a template for reverse transcription as well as a template for translation of some gene products; the 19S RNA is the template for the translation of just one gene product. Various unencapsidated replication intermediates are found in infected cells (Hull and Covey, 1983a) which are estimated to give a further 10 CaMV molecules per cell. CaMV was the first plant virus shown to involve reverse transcription in its replication (Hull and Covey, 1983b; Pfeiffer and Hohn, 1983) but has now been joined by more than a thirty other plant viruses which have the same replication mechanism. Reverse transcribing elements