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We developed a novel, two-component transient gene silencing system in which the satellite tobacco mosaic virus (STMV) is used as vector for the delivery of inhibitory RNA into tobacco plants and the tobacco mosaic virus strain U2 (TMV-U2) is used as helper virus for supplying replication and movement proteins in trans. The main advantage of the system is that by uncoupling virus replication components from silencing induction components, the intensity of silencing becomes more pronounced. We call this system satellite virus-induced silencing system (SVISS) and will demonstrate here its robustness, speed and effectiveness. We were able to obtain pronounced and severe knockout phenotypes for a range of targeted endogenous genes belonging to various biochemical pathways and expressed in different plant tissues, such as genes involved in leaf and flower pigmentation, genes for cell wall synthesis in leaf, stem and root tissues or a ubiquitous RNA polymerase gene. By tandem insertion of more than one target gene sequence into the vector, we were able to induce simultaneous knockouts of an endogenous gene and a transgene. SVISS is the first transient gene silencing system for Nicotiana tabacum, which is a genetically well-characterized bridging species for the Solanaceae plant family.

Summary In the transgenic tobacco line T17, plants homozygous for the gn1 transgene display developmentally regulated post-transcriptional silencing of basic β-1,3-glucanase genes. Previously, it has been shown that silencing involves a markedly increased turnover of silencing-target glucanase mRNAs. Using a two-component viral reporter system facilitated a comparison, in a quantitat- ive manner, of the relative silencing efficiencies of various sequences derived from the gn1 transgene. The results show that target sites for the silencing mechanism are present throughout the coding region of the gn1 mRNA. Similar-sized coding region sequences along the entire gn1 mRNA display a similar susceptibility to the silencing mechanism. The susceptibility to silencing increases as the coding region elements increase in size. Relative to internal sequences, the 5′ and 3′ terminal regions of the gn1 mRNA are inefficient targets for the silencing machinery. Importantly, sequences of the gn1 transgene that are not part of the mature gn1 mRNA are not recognized by the silencing machinery when expressed in chimeric viral RNAs. These results show that the glucanase silencing mechanism in T17 plants is primarily directed against gn1 mRNA-internal sequences and that terminal sequences of the gn1 mRNA are relatively unaffected by the silencing mechanism.

Post-transcriptional gene silencing of beta-1,3 glucanase genes in the transgenic tobacco line T17 is characterised by an increased turnover and, as a consequence, reduced levels of gn1 transgene and endogenous beta-1,3 glucanase mRNAs, Here, additional gn1 RNAs, both larger and smaller than the full-length messenger, are shown to accumulate in silenced plants of the transgenic tobacco line T17, The longer-than-full-length gn1 RNAs are the result of cryptic processing of the gn1 messenger, The small gnl RNAs in silenced plants correspond to distal and proximal parts of the mature gn1 messenger. The proximal RNA products are intact at their 5' extremity, but terminate at different positions at the 3'-end. The distal RNA products contain a poly(A) tail and are truncated to various positions at the 5'-end, These observations indicate that degradation of the mature gnl transcript does not start at the 5'- or 3'-end, but rather are consistent with degradation of the gnl transcript starting with an endonucleolytic cleavage followed by internal exonuclease digestion. Importantly, the truncated products are more abundant in silenced plants than in expressing plants, This suggests, together with the previously reported silencing-related increased gnl mRNA turnover and the similar rates of gnl transcription in silenced and expressing T17 plants, that the predominant decay route for the gnl transcripts differs between silenced and expressing conditions.

Summary In monocots, transgene expression can be stimulated by over two magnitudes by including an intron in the 5′ untranslated region (UTR). The underlying mechanism is presently unknown. Inclusion of the salT intron into the 5′ UTR of cat and bar genes stimulated expression of the first gene only, indicating that intron-mediated enhancement of expression (IME) is gene-dependent. Stimulation was associated with increased cat RNA levels, which did not result from a reduced cytoplasmic turnover and were not associated with increased translation. This implies that IME acts in the nucleus. Importantly, the cytoplasmic accumulation of spliced cat transcripts, even with IME, is less than that encoded by the intronless bar gene. As the cat and bar genes were flanked by identical regulatory signals, and the transcripts had a similar cytoplasmic stability, it may mean that IME rescues rather than stimulates gene expression.

Chimaeric PCaMV35S cry genes direct in tobacco mesophyll protoplasts mRNA levels of less than one transcript per cell. We provide evidence that this low cytoplasmic cry IA(b) mRNA level is not due to a rapid turnover but rather results from a marginal import flow of cry messenger into the cytoplasm. Run-on assays indicate that the frequency of transcription initiation is not limiting. However, the cry precursor mRNA carries at least three regions that are recognized as introns. The absence of high cytoplasmic levels of spliced cry mRNAs suggests that these mRNAs are unstable and/or not efficiently made. Point mutations in the 5′ splice site of the most distal intron allows high accumulation levels of the full-length mRNA. This implies that the inefficient formation of full-size mRNA is a major cause of the low expression level of chimaeric cry IA(b) genes in tobacco.

6' gentamicin acetyltransferases detoxify aminoglycoside antibiotics containing a 6' amino group. We tested whether a 6' gentamicin acetyltransferase gene (6' gat) of Shigella sp. is suitable as selectable gene in plant transformation using kanamycin (Km) as a substrate. A comparative transformation experiment using Nicotiana tabacum SR1 protoplasts showed that 6' gat is as effective for selection of transformants as the commonly used neomycin phosphotransferase II (nptII). In stably transformed plants we detected moderate levels of the 6' gat mRNA. An enzymatic assay was developed with which the acetyltransferase activity of the protein is easily demonstrated.

The effect of anti-sense RNA on the expression of the bialaphos resistance (bar) gene which encodes phosphinothricin acetyl transferase (PAT), was analysed in tobacco. Transient expression studies revealed that an anti-bar RNA with sequence complementarity to the complete bar coding region, inhibits PAT synthesis. To quantify the phenomenon, SR1 tobacco cells were transformed twice to introduce first a hybrid bar gene with a reporter gene and in a second instance an anti-bar gene. A first cycle transformant and a double transformant derived herefrom in which PAT synthesis was reduced to only 8%, were studied in detail. The interference of the anti-sense gene with the expression of the bar gene is manifested at least two levels. First, the bar mRNA steady state level is significantly reduced relative to the parental whereas the transcript level of the reporter gene is unchanged. Comparison of bar mRNA levels in total and single stranded (ss) RNA preparations demonstrated that little if any stably base-pairing bar and anti-bar RNA accumulates. Secondly, a three fold reduction of PAT synthesis per bar mRNA is observed. This supposes that because of unstable interactions with the complementary anti-bar RNA either a substantial part of the bar mRNA detected does not enter the cytoplasm and/or that in the cytoplasm the bar mRNA is less efficiently translated. It is not clear if or how the reduced bar mRNA level is related to such unstable interactions.

The plastid psbA promoter of tobacco was used with the aim to construct plastid specific marker genes. Upon transfer to the tobacco nuclear genome the plastid promoter fragment appeared to specify a messenger RNA. Placed 5' to the bar or nptII coding sequences the level of expression is sufficient to obtain a selectable phenotype. The transcription start site in the nucleus is site specific and is located 4 nucleotides downstream relative to the start site used in plastids. Translational fusions of the psbA coding sequence with the nptII and bar coding sequences revealed that the psbA leader sequence and the psbA translation start codon, being the second ATG codon, are recognized by the plant cytoplasmic translation apparatus. A promoter cassette utilisable in both E. coli and tobacco, was obtained by placing the CaMV 35S enhancer 5' to the psbA promoter.

In transgenic tobacco, anti-sense control of the chimaeric bar (bialophos resistance) gene is manifested by a reduced bar transcript level and a reduced synthesis of bar translational product (PAT, phosphinothricin acetyl transferase) per transcript (1). This study shows that the anti-bar gene controls the transcript level and the translation efficiency of the bar mRNA through independent events and at different cellular sites. On the basis of these results a model for the mechanism of anti-sense control is proposed.