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Tobacco plants over-expressing L-phenylalanine ammonia-lyase (PAL(+)) produce high levels of chlorogenic acid (CGA) and exhibit markedly reduced susceptibility to infection with the fungal pathogen Cercospora nicotianae, although their resistance to tobacco mosaic virus (TMV) is unchanged. Levels of the signal molecule salicylic acid (SA) were similar in uninfected PAL(+) and control plants and also following TMV infection. In crosses of PAL(+) tobacco with tobacco harboring the bacterial NahG salicylate hydroxylase gene, progeny harboring both transgenes lost resistance to TMV, indicating that SA is critical for resistance to TMV and that increased production of phenylpropanoid compounds such as CGA cannot substitute for the reduction in SA levels. In contrast, PAL(+)/NahG plants showed strongly reduced susceptibility to Cercospora nicotianae compared to the NahG parent line. These results are consistent with a recent report questioning the role of PAL in SA biosynthesis in Arabidopsis, and highlight the importance of phenylpropanoid compounds such as CGA in plant disease resistance.

Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.

Double-stranded RNA when introduced into cells results in severe reduction of the target mRNA. This phenomenon is known as posttranscriptional gene silencing in plants and RNA interference in animals. Hairpin RNA-mediated gene silencing exploits this cellular mechanism. A convenient way of generating hairpin constructs is to use generic vectors such as pHANNIBAL and pHELLSGATE, vectors based on the Gateway technology. These vectors are suitable for high-throughput gene silencing, and the silencing effect is stably inherited over many generations.

Hairpin (hpRNA)-mediated gene silencing exploits a cellular mechanism that recognizes double-stranded RNA (dsRNA) and subjects it and its corresponding mRNA to a sequence-specific degradation. This phenomenon is known as posttranscriptional gene silencing (PTGS) in plants and RNA interference (RNAi) in animals. dsRNA, when introduced into plant cells through hpRNA constructs, results in severe reduction of the target mRNA-the silencing effect being stably inherited over many generations. While hpRNA constructs can be made using conventional plasmids, use of generic vectors such as pHANNIBAL makes it more convenient to silence a number of genes simultaneously. Vectors, such as pHELLSGATE, that are based on the Gateway(R) technology are suitable for high-throughput gene silencing. The specificity of dsRNA silencing, it's ability to simultaneously silence multiple genes combined with the availability of high-throughput silencing vectors enables the researcher to generate custom knockout plants.

Over the last decade, both antisense gene constructs and sense co-suppression gene constructs have been invaluable in the molecular study of biological processes. In plant species ranging from Arabidopsis to rice, these types of constructs have been used to silence genes ranging from transcription factors to metabolic enzymes (1). However, it seems that they are about to be superseded by constructs that produce double-strand (ds) RNA. This method of silencing genes was only recognized in 1998 (2, 3, 4, 5), but it is fast becoming a standard investigative method in organisms ranging from mammals to fungi (6, 7, 8, 9, 10, 11). The way in which dsRNA induces gene silencing,bydirected RNA degradation, and the natural role of dsRNA in plants have been extensively reviewed (e.g., 12, 13, 14, 15) and are not the focus of this review, although the basic mechanism is described in Figure 1. Instead, we examine the more applied aspects of dsRNA-mediated post-transcriptional gene silencing (PTGS) in plants, including a number of examples of its use, and attempt to provide some design rules for the application of this technology.

A major challenge in the post-genome era of plant biology is to determine the functions of all genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study but is limited by gene redundancy, lethal knockouts, non-tagged mutants, and the inability to target the inserted element to a specific gene. The efficacy of gene silencing in plants using inverted-repeat transgene constructs that encode a hairpin RNA (hpRNA) has been demonstrated by a number of groups, and has several advantages over insertional mutagenesis. In this paper we describe two improved pHellsgate vectors that facilitate rapid generation of hpRNA-encoding constructs. pHellsgate 4 allows the production of an hpRNA construct in a single step from a single polymerase chain reaction product, while pHellsgate 8 requires a two-step process via an intermediate vector. We show that these vectors are effective at silencing three endogenous genes in Arabidopsis, FLOWERING LOCUS C, PHYTOENE DESATURASE and ETHYLENE INSENSITIVE 2. We also show that a construct of sequences from two genes silences both genes.