Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence I
Small RNAs (sRNAs) are essential for normal plant development and range in size classes of 21-24 nucleotides.  The 22nt small interfering RNAs (siRNAs) and miRNAs are processed by Dicer-like 2 (DCL2) and DCL1 respectively and can initiate secondary siRNA production from the target transcript amplifying the silencing signal in plants.  22nt siRNAs are under-represented due to competition with DCL4, while only a small number of 22nt miRNAs exist due to the rare occurrence of an asymmetric bulge in the precursor miRNA stem. Here we report a strategy to produce abundant 22nt siRNAs and other desired siRNA size classes using long hairpin RNA (hpRNA) transgenes.  By introducing asymmetric bulges periodically into the antisense strand of hpRNA, we successfully shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs.  We showed that the asymmetric hpRNA constructs effectively silenced a ß-glucuronidase (GUS) reporter transgene and the endogenous ethylene insensitive-2 (EIN2) and chalcone synthase (CHS) genes. Furthermore, plants containing the asymmetric hpRNA transgenes targeting both GUS and EIN2 showed increased amount of 21nt siRNAs downstream of the hpRNA target site compared to plants with the traditional hpRNA transgenes.  This indicates that these asymmetric hpRNAs are more effective at inducing secondary siRNA production to amplify silencing signals. Consistent with the production of secondary siRNAs, the 22nt asymmetric hpRNA constructs increased gene silencing phenotypes and enhanced virus resistance in plants compared to the traditional hpRNA constructs. Overall design: To examine whether hpRNA transgenes containing asymmetric bulges could process different length siRNAs. Transgenic tobacco individuals containing a GUS transgene were transformed with different asymmetric bulge hpRNA transgenes (?22nt, ?23nt, ?24nt-1, ?24nt-2, ?24nt-3).  3 independent T0 individuals were sent for sRNA sequencing per construct