HOMOLOGY-DEPENDENT GENE SILENCING IN TRANSGENIC PLANTS - EPISTATIC SILENCING LOCI CONTAIN MULTIPLE COPIES OF METHYLATED TRANSGENES

Previous work has shown that two homologous, unlinked transgene loci c an interact in plant nuclei, leading to non-reciprocal trans-inactivat ion and methylation of genes at one locus. Here, we report the structu re and methylation of different transgene loci that contain the same c onstruct but are variably able to inactivate and methylate a partially homologous, unlinked target locus. Silencing loci comprised multiple, methylated copies of the transgene construct, whereas a non-silencing locus contained a single, unmethylated copy. The correspondence betwe en strength of silencing activity and copy number/degree of methylatio n was further demonstrated by producing novel alleles of a strong sile ncing locus: reducing the transgene copy number and methylation within this silencing locus decreased its ability to inactivate the target l ocus. The strong silencing locus, which was located close to a telomer e, trans-inactivated various structural variants of the original targe t construct, regardless of their location in the genome. This suggests that the silencing locus can scan the entire genome for homologous re gions, a process possibly aided by its telomeric location. Our data su pport the idea that epistatic trans-inactivation of unlinked, homologo us transgenes in plants results from a pre-existing epigenetic differe nce between transgene loci, which is subsequently equalized by ''epige ne conversion'' involving DNA-DNA pairing.