A branched pathway for transgene-induced RNA silencing in plants

In plants, RNA silencing can be induced by highly transcribed sense transgenes (S-PTGS) [1, 2] or by transgene loci producing double-stranded RNA (dsRNA) due to the presence of inverted repeats (IR-PTGS) [3, 4, 5, 6]. Both phenomena correlate with accumulation of 21-25 nt sense and anti-sense RNA homologous to the silent gene (7] and with methylation of the coding sequence [3, 4, 6, 8]. We have challenged IR-PTGS with four viruses known to inhibit S-PTGS: CMV, TuMV, TVCV, and TCV ([9, 10, 11] this work) and in sgs2, sgs3, and ago1 mutants impaired in S-PTGS [8, 11, 12, 13, 14]. Surprisingly, whereas the four viruses inhibit IR-PTGS, IR-PTGS and methylation of a GUS trangene and IR-PTGS of three endogeneous genes occur in the sgs2, sgs3, and ago1 mutations. Based on these results, we propose a branched pathway for RNA silencing in plants. RNA silencing would occur via the action of dsRNA produced either via the action of SGS2 (also known as SDE1), SGS3, and AGO1 on the S-PTGS branch or by transgenes arranged as inverted repeats on the IR-PTGS branch. Moreover, transgene methylation would result from production or action of dsRNA, since it does not require SGS2/SDE1, SGS3, and AGO1.