A tobacco calmodulin-related protein suppresses sense transgene-induced RNA silencing but not inverted repeat-induced RNA silencing

A tobacco calmodulin-related protein, rgs-CaM, interacts with viral suppressors of RNA silencing and modulates host RNA silencing. Plants overexpressing the rgs-CaM gene were crossed with plants exhibiting sense transgene-induced RNA silencing (S-PTGS) or inverted repeat-induced RNA silencing (IR-PTGS). S44 plants harboring a sense transgene encoding a tobacco microsomal ω-3 fatty acide desaturase (NtFAD3) exhibited the S-PTGS phenotype. The frequency of the S-PTGS phenotype incidence was nearly 100 % in the hemizygous S44 plants, but was reduced to 30 % in crossbred plants with an rgs-CaM-overexpressing transgenic line. The remaining 70 % of crossbred plants successfully overexpressed the NtFAD3 transgene, and the amount of NtFAD3 small interfering RNAs (siRNAs) was largely decreased. In contrast, overexpression of rgs-CaM did not suppress siRNA production in the IR-PTGS that targeted the NtFAD3 gene. These results indicated that rgs-CaM suppresses RNA silencing at a step upstream of siRNA production and does not interfere with the later steps of RNA silencing, including siRNA-mediated RNA degradation.     

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.     

Preferential spreading of RNA silencing into the 3’ downstream region of the transgene in tobacco

The amplification mechanism of short interfering RNAs (siRNAs) along the transgene sequence exists in RNA interference (RNAi). The RNA-dependent RNA polymerase synthesizes complementary RNAs by using the transgene mRNA as a template, and the secondary siRNAs are generated from the outside of primary RNAi target. Four independent RNAi vectors which produced primary siRNAs against distinct regions of the tobacco endoplasmic reticulum ω-3 fatty acid desaturase gene (NtFAD3) were transiently expressed in leaves of theNtF4D3-overexpressed transgenic plants. Regardless of the RNAi vector used, the secondaryNtFAD3 siRNAs were generated preferentially from the 3’ downstream region of the transgene. Secondary siRNAs from the 5’ upstream region adjacent to the annealing site of primary siRNAs accumulated under the detection level. Our results suggest that different regulatory mechanisms are involved in the spreading of RNA silencing into 5’ upstream and 3’ downstream regions of the target sequence, respectively.     

Enzymatically Produced Pools of Canonical and Dicer-Substrate siRNA Molecules Display Comparable Gene Silencing and Antiviral Activities against Herpes Simplex Virus

RNA interference (RNAi)-based sequence-specific gene silencing is applied to identify gene function and also possesses great potential for inhibiting virus replication both in animals and plants. Small interfering RNA (siRNA) molecules are the inducers of gene silencing in the RNAi pathway but may also display immunostimulatory activities and promote apoptosis. Canonical siRNAs are 21 nucleotides (nt) in length and are loaded to the RNA Induced Silencing Complex when introduced into the cells, while longer siRNA molecules are first processed by endogenous Dicer and thus termed Dicer-substrate siRNA (DsiRNA). We have applied RNA polymerases from bacteriophages T7 and phi6 to make high-quality double-stranded RNA molecules that are specific for the UL29 gene of herpes simplex virus (HSV). The 653 nt long double-stranded RNA molecules were converted to siRNA and DsiRNA pools using Dicer enzymes originating from human or Giardia intestinalis, producing siRNAs of approximately 21 and 27 nt in length, respectively. Chemically synthesised 21 and 27 nt single-site siRNA targeting the UL29 were used as references. The impact of these siRNAs on cell viability, inflammatory responses, gene silencing, and anti-HSV activity were assayed in cells derived from human nervous system and skin. Both pools and the canonical single-site siRNAs displayed substantial antiviral activity resulting in four orders of magnitude reduction in virus titer. Notably, the pool of DsiRNAs caused lower immunostimulation than the pool of canonical siRNAs, whereas the immunostimulation effect was in relation to the length with the single-site siRNAs. Our results also propose differences in the processivity of the two Dicers.     

Silencing of Gm<Emphasis Type="Italic">FAD3</Emphasis> gene by siRNA leads to low α-linolenic acids (18:3) of <Emphasis Type="Italic">fad3</Emphasis>-mutant phenotype in soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (Merr.)]

RNA interference (RNAi) has been recently employed as an effective experimental tool for both basic and applied biological studies in various organisms including plants. RNAi deploys small RNAs, mainly small interfering RNAs (siRNAs), to mediate the degradation of mRNA for regulating gene expression in plants. Here we report an efficient siRNA-mediated gene silencing of the omega-3 fatty acid desaturase (FAD3) gene family in a complex genome, the soybean (Glycine max). The FAD3 enzyme is responsible for the synthesis of alpha-linolenic acids (18:3) in the polyunsaturated fatty acid pathway. It is this fatty acid that contributes mostly to the instability of soybean and other seed oils. Therefore, a significant reduction of this fatty acid will increase the stability of the seed oil, enhancing the seed agronomical value. A conserved nucleotide sequence, 318-nt in length, common to the three gene family members was used as an inverted repeat for RNA interference. The RNAi expression cassette was driven by a seed-specific promoter. We show that the transgene-produced siRNA caused silencing of FAD3 that was comparable to the fad3 mutant phenotype and, furthermore, that such a silencing is stably inherited in engineered soybean lines. Since the pool size of the alpha-linolenic acids is small relative to the other polyunsaturated fatty acids in soybean, the significant reduction of this fatty acid suggests a role and great potential for the siRNA strategy in silencing gene families in a complex genome.     

Host sunflower-induced silencing of parasitism-related genes confers resistance to invading Orobanche cumana

Orobanche cumana is a holoparasitic plant that attaches to host–plant roots and seriously reduces the yield of sunflower (Helianthus annuus L.). Effective control methods are lacking with only a few known sources of genetic resistance. In this study, a seed-soak agroinoculation (SSA) method was established, and recombinant tobacco rattle virus vectors were constructed to express RNA interference (RNAi) inducers to cause virus-induced gene silencing (VIGS) in sunflower. A host target gene HaTubulin was systemically silenced in both leaf and root tissues by the SSA–VIGS approach. Trans-species silencing of O. cumana genes were confirmed for 10 out of 11 target genes with silencing efficiency of 23.43%–92.67%. Knockdown of target OcQR1, OcCKX5, and OcWRI1 genes reduced the haustoria number, and silencing of OcEXPA6 caused further phenotypic abnormalities such as shorter tubercles and necrosis. Overexpression of OcEXPA6 caused retarded root growth in alfalfa (Medicago sativa). The results demonstrate that these genes play an important role in the processes of O. cumana parasitism. High-throughput small RNA (sRNA) sequencing and bioinformatics analyses unveiled the distinct features of target gene-derived siRNAs in O. cumana such as siRNA transitivity, strand polarity, hotspot region, and 21/22-nt siRNA predominance, the latter of which was confirmed by Northern blot experiments. The possible RNAi mechanism is also discussed by analyzing RNAi machinery genes in O. cumana. Taken together, we established an efficient host-induced gene silencing technology for both functional genetics studies and potential control of O. cumana. The ease and effectiveness of this strategy could potentially be useful for other species provided they are amenable to SSA.

Knockdown of several parasitism-related genes endows sunflower with resistance to invading broomrape.     

Dual-target gene silencing by using long,synthetic siRNA duplexes without triggering antiviral responses

Chemically synthesized small interfering RNAs (siRNAs) can specifically knock-down expression of target genes via RNA interference (RNAi) pathway. To date, the length of synthetic siRNA duplex has been strictly maintained less than 30 bp, because an early study suggested that double-stranded RNAs (dsRNAs) longer than 30 bp could not trigger specific gene silencing due to the induction of nonspecific antiviral interferon responses. Contrary to the current belief, here we show that synthetic dsRNA as long as 38 bp can result in specific target gene silencing without nonspecific antiviral responses. Using this longer duplex structure, we have generated dsRNAs, which can simultaneously knock-down expression of two target genes (termed as dual-target siRNAs or dsiRNAs). Our results thus demonstrate the structural flexibility of gene silencing siRNAs, and provide a starting point to construct multifunctional RNA structures. The dsiRNAs could be utilized to develop a novel therapeutic gene silencing strategy against diseases with multiple gene alternations such as viral infection and cancer.     

Simple gene silencing using the trans‐acting siRNA pathway

In plants, particular micro‐RNAs (miRNAs) induce the production of a class of small interfering RNAs (siRNA) called trans‐acting siRNA (ta‐siRNA) that lead to gene silencing. A single miRNA target is sufficient for the production of ta‐siRNAs, which target can be incorporated into a vector to induce the production of siRNAs, and ultimately gene silencing. The term miRNA‐induced gene silencing (MIGS) has been used to describe such vector systems in Arabidopsis. Several ta‐siRNA loci have been identified in soybean, but, prior to this work, few of the inducing miRNAs have been experimentally validated, much less used to silence genes. Nine ta‐siRNA loci and their respective miRNA targets were identified, and the abundance of the inducing miRNAs varies dramatically in different tissues. The miRNA targets were experimentally verified by silencing a transgenic GFP gene and two endogenous genes in hairy roots and transgenic plants. Small RNAs were produced in patterns consistent with the utilization of the ta‐siRNA pathway. A side‐by‐side experiment demonstrated that MIGS is as effective at inducing gene silencing as traditional hairpin vectors in soybean hairy roots. Soybean plants transformed with MIGS vectors produced siRNAs and silencing was observed in the T1 generation. These results complement previous reports in Arabidopsis by demonstrating that MIGS is an efficient way to produce siRNAs and induce gene silencing in other species, as shown with soybean. The miRNA targets identified here are simple to incorporate into silencing vectors and offer an effective and efficient alternative to other gene silencing strategies.