Post-transcriptional silencing of a neomycin phosphotransferase II transgene correlates with the accumulation of unproductive RNAs and with increased cytosine methylation of 3' flanking regions

Abstract

The transgenic tobacco plant GVCHS(320)-1 harbours several T-DNAs with the neomycin phosphotransferase II (nptII)-encoding chimeric gene under control of the cauliflower mosaic virus 35S promoter (CaMV 35S). These TDNAs are distributed over two loci, named A and B. The primary transformant GVCHS(320)-1 had substantially reduced steady-state nptII transcript levels due to the activity of a post-transcriptional silencing mechanism. Silencing of the nptII-encoding transgenes in the progeny of GVCHS(320)-1 requires the presence of the A locus that consists of two physically separated T-DNAs. Although the B locus contains three T-DNAs in the same orientation, it gives rise to high steady-state nptII mRNA levels and NPTII protein levels even in homozygous condition. The B locus only becomes silenced in trans in the presence of a silencing locus. The data suggest that silencing of the nptII transgenes is reinforced by ageing. It is also suggested that silencing is correlated with a reduced NPTII protein/nptII mRNA ratio, which may be interpreted as the accumulation of unproductive nptII RNA molecules in silenced plants. The data further demonstrate that the silenced state is correlated with extensive C-methylation of diagnostic sites in the 3' three-quarters of the coding sequence and in the 3' region up to 1400 bp downstream of the polyadenylation signal.