Genetic analysis of gene silencing in Arabidopsis thaliana

Gene silencing frequently occurs in transgenic plants, the mechanism of which underlines an epigenetic control of plant genome structure and expression, but is poorly understood. A system of gene silencing was established in Arabidopsis thaliana. Transgenic plants expressing a fusion protein between either beta-glucuronidase (GUS) or green fluorescent protein (GFP) and CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COPl) displayed three types of gene silencing phenotypes, type L (late onset), type E (early onset) and type C (complex), which could be distinguished based on the gene dosage dependence and developmental timing of silencing, post-transcriptional versus transcriptional control, and the extent of endogene cosuppression. Chromosomal flanking sequences of seventeen transgene loci were isolated and characterized. Type Land type E loci had one and two T-DNAs, respectively, and were silenced post-transcriptionally. Gene dosage alone was important and allelic interaction was not required for type L silencing. On the other hand, the early onset endogene silencing in type E lines was determined by both gene dosage and the arrangement of T-DNAs in the transgene loci. Type C loci contained two or more T-DNAs, and were transcriptionally silenced. Two type C loci were able to heritably suppress cosuppression in type L and type E loci, a phenomenon reminiscent of paramutation. The paramutated state of a type Land a type E locus was reversible, and was not correlated with DNA methylation on the transgene coding sequence. One paramutated type L locus acquired the ability to suppress silencing of a paramutable locus in trans and might have become paramutagenic. In summary, gene silencing phenotypes were strongly correlated with transgene locus structure, while the effect of genomic environment on gene silencing was not significant. The possibility that PTGS could mature into TGS during plant development or through generations was suggested by the epigenetic transition of type C lines and of transgenic lines harboring more than two transgene loci. Ecotype effects on PTGS in the system were discovered between Nossen and Enkheim ecotypes, and between Columbia and Landsberg ecotypes, which leads to a new way of identifying genes in charge of PTGS.