Generation of siRNAs by T-DNA sequences does not require active transcription of homology to sequences in the plant

-Delivery into plants of T-DNAs containing promoter, terminator, or coding sequences generated small interfering RNAs (siRNAs) specific to each type of sequence. When both promoter and transcribed sequences were simultaneously present in the T-DNA, accumulation of siRNAs to transcribed sequences was favored over accumulation of siRNAs to the nontranscribed upstream promoter sequences. The generation of specific siRNA sequences occurred even in the absence of T-DNA homology to sequences in the plant. Delivery of T-DNA, with homology to the transgene limited to the nontranscribed cauliflower mosaic virus 35S promoter (35SP) and the transcribed nopaline synthase transcription termination (NosT)signal sequences, into transgenic plants expressing the green fluorescent protein (GFP), generated siRNAs in infiltrated tissues to both 35SP (35SsiRNAs) and NosT (NosTsiRNAs), but not to the GFP sequence (GFPsiRNAs). In infiltrated tissues, the 35SsiRNAs failed to trigger the transcriptional silencing of the transgene, accumulation of 35SsiRNAs could be prevented by the potyviral HC-Pro, and the NosTsiRNAs required an initial amplification to trigger efficient transgene silencing, which is mediated by transcripts from the exogenous T-DNA, and not from the transgene. In upper leaves, silencing correlated with the presence of GFPsiRNAs and the absence of 35SsiRNAs, confirming that its spread was posttranscriptionally mediated by the transgene mRNA.