Organization, not duplication, triggers silencing in a complex transgene locus in rice

Despite the presence in nature of many functional gene families that contain several to many highly similar sequences, the presence of identical DNA sequence repeats is widely thought to predispose transgene inserts to homology dependent gene silencing (HDGS). The induction of transcriptional gene silencing (TGS) by RNAs homologous to promoter sequences has been reported recently in Arabidopsis and humans. However, mechanisms for TGS have not been studied in detail for rice, the most widely cultivated crop plant. Taking advantage of a well-characterized homozygous silenced transgenic rice line (siJKA), supertransformation was performed with a binary vector bearing mUbi1 and 35S promoter sequences identical to those in the resident transgenes. Analysis of the incoming and resident transgenes in the supertransformants revealed that the incoming mUbi1 transgene promoter was not silenced whereas the incoming 35S transgene promoter was silenced. That the resident silenced mUbi1-bar was not reactivated in these experiments as a result of passage through tissue culture and regeneration was established by the finding that regenerants from siJKA immature embryos were all silenced for mUbi1-bar. In a parallel experiment, when wild type rice calli were transformed with the same binary vector, neither of the incoming transgene promoters was silenced. Following 5-azacytidine (5-azaC) treatment of siJKA, aberrant RNA species corresponding to the 35S promoter, but not to the mUbi1 promoter, were detected. Nevertheless, no 21-25 nt RNAs corresponding to the 35S promoter sequence were detected. These results, together with detailed analyses of the progenies from the primary transformants and supertransformants, revealed that HDGS of the resident silenced locus was caused not by simple transgene duplication, but by aberrant transcripts derived from rearranged promoters present in siJKA. Practical consequences of this study include a justification for the use of multiple copies of a given promoter for transformation without inducing silencing, provided that their genomic integration does not result in aberrant transcription of the promoters.