Hairpin transcription does not necessarily lead to efficient triggering of the RNAi pathway

Abstract

Previously, we had shown that stable expression of a hairpin RNA sharing homology with the coat protein (CP) of the Cucumber mosaic virus (CMV) (hpRNA(CMV)) produced CMV resistant Nicotiana tabacum plants. However, only 17% of the hpRNA(CMV)-expressing plants generated substantial amounts of siRNAs that mediated CMV resistance (siRNAs(CMV)). Here, we demonstrate that the transcription of a hpRNA(CMV) per se is not sufficient to trigger cytoplasmic and nuclear RNAi. A multiple-transgene copy line showed a strong resistance phenotype. Segregation of individual copies revealed that in one locus, the transgene-produced hpRNA(CMV) transcript was processed into 21-nt and 24-nt siRNAs(CMV) and lines containing this locus were resistant. At a second locus, where the transgene was shown to be transcribed, no siRNAs(CMV) were produced and lines harbouring only this locus were susceptible. In addition, the second locus failed to trigger de novo RNA-directed DNA methylation (RdDM) in cis, of its cognate sequence. However, after being induced in trans, methylation in the transcribed region of the transgene was maintained in both CG and CHG residues. Sequence-specific maintenance of methylation in transcribed regions, as well as diverse RNA degradation pathways in plants are discussed in view of our observations.

Resistance to multiple viruses in transgenic tobacco expressing fused, tandem repeat, virus-derived double-stranded RNAs

“…2b), consistent with previous studies [6,14,51,52]. Moreover, as described in [53], the use of constructs expressing hairpin RNAs is not sufficient to ensure the production of siRNAs. Thus, not all such lines will produce siRNAs and not all the lines producing siRNAs will show resistance to the corresponding target viruses.…”

Generation of Marker-free Transgenic Plants Concurrently Resistant to a DNA Geminivirus and a RNA Tospovirus

“…In plants, RNAs that are inducers of PTGS can also mediate sequence-specific DNA methylation274649. We also observed that the double stranded RNA from the transgene hpIGR designed for inducing PTGS also triggered RdDM and methylated transgene sequences.…”

“…CHH methylation is mostly conducted by DRM2 and requires frequent methylation signals provided by the de novo methylation pathway43. Thus, methylated CHH is an indication for the ongoing de novo RdDM444546. Our data showed that the AYVV-resistant lines has, but susceptible lines lack, this competent CHH methylation and de novo RdDM signal.…”

Transcriptional silencing of RNAi constructs against nematode genes in Arabidopsis

“…A similar study by Dalakouras et al (2011) showed that siRNAs derived from a hairpin transgene construct triggered translational inhibition of that construct in tobacco. Although we only studied and confirmed a negative correlation between promoter methylation and construct expression for one of the constructs, a high extent of TGS is probably the main reason for the largely variable levels of resistance with the hairpin constructs against the three, targeted, nematodes that were observed in the current study.…”