The effects of spacer sequences on silencing efficiency of plant RNAi vectors

RNA interference (RNAi) has been used to suppress gene expression in various eukaryotic organisms. In plants, RNAi can be induced by introduction of an RNAi vector that transcribes a self-complementary hairpin RNA. Most basic RNAi constructs have an inverted repeat interrupted with a spacer sequence. To test silencing capability of RNAi constructs, we developed an in vivo assay that is based on the RNAi-mediated changes of the α-linolenic acid content in hairy roots. A tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) is the main enzyme for production of α-linolenic acid of root membrane lipids. Tobacco hairy roots transformed with the RNAi vectors against the NtFAD3 gene showed a decrease in α-linolenic acid content. The frequency of RNA silencing was more affected by spacer sequence than by spacer length, at least between 100 and 1800 bp. Since significant amounts of hairpin RNA against the NtFAD3 gene remained in the transgenic plants displaying a weak silencing phenotype, low degree of silencing was attributed to low efficiency of hairpin RNA processing mediated by Dicer-like proteins. Our results show the possibility of producing a broad range of the RNAi-induced silencing phenotypes by replacing the spacer sequence of RNAi construct.     

Post-transcriptional Gene Silencing of GBSSI in Potato: Effects of Size and Sequence of the Inverted Repeats

In the past, silencing of granule-bound starch synthase (GBSSI) in potato was achieved by antisense technology, where it was observed that inclusion of the 3' end of the GBSSI coding region increased silencing efficiency. Since higher silencing efficiencies were desired, GBSSI inverted repeat constructs were designed and tested in potato. First, large inverted repeats comprising the 5' and the 3' half of the GBSSI cDNA were tested. The 5' IR construct gave a significantly higher silencing efficiency than the 3' IR construct. Since it was not known whether the observed difference was due to the sequence or the orientation of the inverted repeat, the GBSSI cDNA was divided into three regions, after which each region was tested in small inverted repeats in two orientations. To this end large numbers of independent transformants were produced for each construct. The results suggested that there was no effect of inverted repeat orientation on silencing efficiency. The percentage of transformants showing strong inhibition varied from 48% for a 3'-derived construct to 87% for a 5' as well as a middle region-derived construct. Similar to the large inverted repeats, the 3' sequences induced the least efficient silencing implying that the observed differences in silencing efficiency are caused by sequence differences. The small inverted repeat constructs with a repeat size of 500-600 bp and a spacer of about 150 bp were more efficient silencing inducers than the large inverted repeat constructs where the size of the repeat was 1.1 or 1.3 kb whilst the size of spacer was 1.3 or 1.1 kb. The results presented here show that size and sequence of the inverted repeat influenced silencing efficiency.     

Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules

We investigated the efficacy of self-complementary hairpin RNA (hpRNA) expression to induce RNA silencing in the roots and nodules of model legume Lotus japonicus, using hairy root transformation mediated by Agrobacterium rhizogenes. Transgenic lines that express beta-glucuronidase (GUS) by constitutive or nodule-specific promoters were supertransformed by infection of A. rhizogenes harboring constructs for the expression of hpRNAs with sequences complementary to the GUS coding region. GUS activity in more than 60% of the hairy roots was decreased or silenced almost completely. Silencing of the GUS gene was also observed in symbiotic nodules formed on hairy roots in both early and late stages of nodule organogenesis. These results indicate that transient RNA silencing by hairy root transformation provides a powerful tool for loss-of-function analyses of genes that function in roots and root nodules.     

Hairpin RNA-mediated strategies for silencing of tomato leaf curl virus AC1 and AC4 genes for effective resistance in plants

RNA interference (RNAi) using short interfering RNAs (siRNAs) has been widely explored for the suppression of intracellular viral target mRNAs. On the basis of our previous work with stable silencing of Tomato leaf curl virus, in vivo by the antisense replicase gene (AC1) of the virus and characterizing AC4, as a small RNA regulator, besides its role in pathogenicity, we used four different plasmid vector-based siRNA generation strategies to silence viral genes (AC1 and AC4) of tomato leaf curl viruses. The RNAi target sequence were chosen from DNA A of the Tomato leaf curl virus (ToLCV) on the basis of conserved regions in AC1 with an overlapping sequences of the AC4 gene. Different hairpin RNA-mediated strategies like antisense, self-complementary inverted repeats, intron-spliced hairpin RNAs, and small hairpin RNAs were deployed for efficient and predictable resistance to the viruses. Here we present that appropriately designed siRNAs not only prevents RNAi suppression but also help in developing trait-stable transgenics. These strategies imply that ToLCV rep-driven RNAi, targeting AC4 and conserved viral sequences, provides a promising approach to suppress a wide spectrum ToLCV infection in the tomato.     

RNA silencing of single and multiple members in a gene family of rice

RNA silencing with inverted repeat (IR) constructs has been used to suppress gene expression in various organisms. However, the transitive RNA-silencing effect described in plants may preclude the use of RNA silencing for a gene family. Here, we show that, in rice (Oryza sativa), transitive RNA silencing (spreading of double-stranded RNA along the target mRNA) occurred with the green fluorescent protein transgene but not with the endogenous phytoene desaturase gene. We fused IR copies of unique 3' untranslated regions derived from the rice OsRac gene family to a strong promoter and stably introduced them into rice. Each of the seven members of the OsRac gene family was specifically suppressed by its respective IR construct. We also examined IR constructs in which multiple 3' untranslated regions were fused and showed that three members of the OsRac gene family were effectively suppressed by a single construct. Using highly conserved regions of the two members of the OsRac gene family, we also suppressed the expression of all members of the gene family with variable efficiencies. These results suggest that RNA silencing is a useful method for the functional analysis of gene families in rice and other plants.