The influence of small changes in transgene transcription on homology-dependent virus resistance and gene silencing

A tissue culture/regeneration scheme was employed to introduce epigenetic variation into tobacco lines that either do (T4) or do not (T19) exhibit post-transcriptional silencing of beta-glucuronidase (GUS) transgenes. Homology-dependent resistance to a potato virus X derivative carrying GUS (PVX-GUS), an indicator of post-transcriptional gene silencing, was used as a screen for epigenetic changes. Regenerant families derived from the high-expressing line T19 and nontransformed tobacco remained completely susceptible to PVX-GUS, like the progenitors. Twenty out of twenty T4 regenerant families were also susceptible to PVX-GUS and were therefore very distinct from the low-expressing, PVX-GUS-resistant T4 progenitor line. This change to PVX-GUS susceptibility was not associated with DNA structural changes, but was associated with slight increases in GUS mRNA accumulation, increased 35S promoter methylation and correspondingly decreased transcription of GUS, These results suggest that homology-dependent virus resistance and gene silencing are very sensitive to small changes in transgene transcription.