Commentary Plant gene silencing regularized

Two surprising and potentially useful phenomena of transgenic plants originated from two distinct lines of research but may converge in underlying mechanism or mechanisms (1–3). Plant transformation technology, the staple of the crop biotechnology industry, overcomes the species barrier and allows introduction into plants of genes from other plant species, genes from species of other kingdoms, and even sequences derived from the DNA synthesizer. Subsequently, the introduced sequences often, but not always, are inherited as simple Mendelian characters and exhibit an expected phenotype. It is one group of annoying exceptions to expected phenotype and inheritance that constitutes the first surprising phenomenon. Investigators, seeking to increase the expression, and thereby enhance the phenotype, of an endogenous gene, introduced additional copies of the endogenous gene as a transgene. Usually the constructions use promoters more active than the promoter of the endogenous gene. In some instances, the result was not the overexpression of the gene, as expected, but a drastically reduced or undetected expression of both the endogenous and the introduced sequences (4, 5). Much of the early research used a chalcone synthase gene, the silencing of which sometimes resulted in flowers with spectacular patterns of nonpigmented and pigmented areas. The reduced expression phenomenon is termed gene silencing. Gene silencing results in no expression or very low expression of a gene or RNA sequence that formerly was expressed, or likely would be expressed, absent the gene-silencing phenomenon. Paradoxically, plants with multiple copies of the transgene andyor high levels of transgene transcription are more likely than plants with a single copy and low-level transcription to exhibit gene silencing (6). However, the correlation has not been consistent for some systems (7, 8). Gene silencing can be induced not only by expression of a transgene but also by transient expression from DNA geminivirus vectors (9) and RNA virus vectors (10, 11). Similarly, Agrobacterium tumefaciens-based transient expression systems can generate RNA targets for gene silencing (12, 13). Transient expression allows rapid testing of various sequences without the time-consuming process of plant transformation and regeneration. For transgenic plants, the phenotype of a silenced transgene usually is maintained through vegetative propagation or organ regeneration and can be transmitted through a graft (14, 15). However, transmission of gene silencing to progeny through meiosis, though often complicated by the effects of multiple inserts, is unpredictable, with silencing appearing among the progeny of a silenced plant with frequencies of 2–100%. Similarly, some progeny of a nonsilenced transgenic plant may be silenced (15–18). That is, gene silencing behaves as an epigenetic trait. The second phenomenon appeared in the course of experiments intended to exploit pathogen-derived resistance (19, 20). As pathogen-derived resistance is applied against plant viruses, a virus genome-derived sequence is incorporated into the plant transgene. For some gene constructions, researchers noticed an unexpected inverse relationship between the degree of protection shown by a particular transformed plant and the level of expression of the introduced, virus genome-derived sequence (3, 21). Resistance exhibited by plants with constructions designed to be translation competent, and by plants with constructions designed to generate RNA that is not translatable, generated similar resistance. This finding suggests that resistance did not require the synthesis of any virus-derived protein or protein fragment (22–24). Thus the term ‘‘RNAmediated resistance’’ applies. The resistance conferred by the expressed RNA usually is very robust, not being overcome by virus inoculum concentrations that are orders of magnitude greater than the concentrations that routinely infect wild-type plants. The resistance is maintained in protoplasts isolated from resistant plants (7, 22). The conferred resistance proved to be highly virus specific, failing even against viruses closely related to the virus that was the source of the transgene sequence. RNA-mediated resistance, like transgene-induced gene silencing, usually proved to be unpredictable for appearance after passing through meiosis (25). In this issue of the Proceedings, Waterhouse et al. (26) report a modified approach to gene silencing and RNA-mediated protection that generated transgenic plants and their progeny with predictable phenotypes. What are other similarities of RNA-mediated resistance and gene silencing (27)—of highly virus-resistant plants and, for example, plants with flowers showing unusual pigmentation? Two sorts of gene silencing have been documented for plants. In transcriptional silencing, messenger RNA synthesis is greatly reduced or absent (28). In posttranscriptional gene silencing (PTGS), messenger RNA, or messenger RNA precursors, are synthesized but apparently are degraded rapidly or improperly processed or both (7, 18, 25, 29, 30). PTGS was demonstrated for chalcone synthase by analyses of RNA synthesized by isolated nuclei, i.e., in ‘‘run-on’’ experiments (6, 8). The accumulation of transcripts in the nucleus but not in the cytoplasm (31) also suggests that transcription is not altered significantly and that PTGS may occur in the cytoplasm. Plant RNA viruses, which have been the usual targets of RNA-mediated resistance, replicate in the cytoplasm. The elegant experiments of English et al. (32) directly connected RNA-mediated resistance and PTGS. The RNA genome of potato virus X (PVX) is tolerant of the insertion of an additional gene, although the burden of the inserted nucleotide sequences slows the replication process (33). PVX with inserted Escherichia coli b-glucuronidase gene was infectious to wild-type tobacco plants but not to plants from transgenic tobacco lines that are posttranscriptionally silenced for b-glucuronidase. Results from experiments with PVX inserts corresponding to various fragments of the b-glucuronidaseencoding sequences allowed the target of the silencing to be mapped to the 39 region of b-glucuronidase ORF. PTGS, unlike transcriptional gene silencing, is correlated with RNAmediated resistance against RNA plant viruses (7). In both phenomena, although the targets are different, silencing affects both the target and the expressed sequence that triggered the silencing, justifying the term ‘‘cosuppression’’ (7, 34).