Alternative silencing effects involve distinct types of non-spreading cytosine methylation at a three-gene, single-copy transgenic locus in rice

We investigated transgene silencing in a line of rice plants that carries a single-copy 6.6-kb transgenic locus comprising three heterologous transgenes: bar, hpt and gusA. We identified at least three distinct types of silencing effects associated with different methylation patterns. including a novel form of transcriptional silencing involving methylation of cytosine residues only at non-conventional acceptor sites in the coding region. Silencing arose de novo in individual R1, R2 and R3 plants despite the stability of the transgenic locus, although the basic structure of the locus, transgene dosage and position effects remained constant within the line. We found that different silencing effects could occur concurrently in adjacent heterologous transgenes in the same plant, with no evidence for spreading of silenced states or methylation patterns from one transgene to another.