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First published online April 25, 2008; 10.1104/pp.108.118307 Plant Physiology 147:543-551 (2008) © 2008 American Society of Plant Biologists
Artificial trans-Acting siRNAs Confer Consistent and Effective Gene SilencingDuPont Crop Genetics Research, Experimental Station, Wilmington, Delaware 19880–0353
Manipulating gene expression is critical to exploring gene function and a useful tool for altering commercial traits. Techniques such as hairpin-based RNA interference, virus-induced gene silencing, and artificial microRNAs take advantage of endogenous posttranscriptional gene silencing pathways to block translation of designated transcripts. Here we present a novel gene silencing method utilizing artificial trans-acting small interfering RNAs in Arabidopsis (Arabidopsis thaliana). Replacing the endogenous small interfering RNAs encoded in the TAS1c gene with sequences from the FAD2 gene silenced FAD2 activity to levels comparable to the fad2-1 null allele in nearly all transgenic events. Interestingly, exchanging the endogenous miR173 target sequence in TAS1c with an miR167 target sequence led to variable, inefficient silencing of FAD2, suggesting a specific requirement for the miR173 trigger for production of small interfering RNAs from the TAS1c locus.
Loss-of-function mutations are the most basic tool of genetic analysis: The biological activity of a gene is inferred from the mutant phenotype. In traditional genetic analysis, a plant with a phenotype of interest is identified from a mutagenized population, and the gene responsible for the phenotype is identified by genetic mapping or by means of a T-DNA or transposon tag (Lukowitz et al., 2000  ). A complementary approach to investigating gene function, often called functional genomics, is to identify genes of interest based on their sequence and/or expression pattern, and then to use a transgenic approach to inactivate the gene to obtain a mutant phenotype. One strategy for functional genomics is to produce massive sequenced-indexed libraries of plants with genes randomly inactivated by T-DNA insertion (Sessions et al., 2002; Alonso et al., 2003). Although this approach has been of great value to the scientific community, T-DNA insertion sites are biased toward 5' and 3' untranslated region (UTR) sequences, and T-DNA insertions in a large proportion of genes, especially small genes, have never been recovered (Sessions et al., 2002; Alonso et al., 2003). In addition, indexed T-DNA insertion libraries are not available for many plant species used for basic research or for most agronomically important crop species. Thus, strategies to efficiently silence specific genes are essential.
Many different strategies have been used for gene silencing (for review, see Ossowski et al., 2008
All of these strategies rely on the genetic machinery of an endogenous phenomenon called posttranscriptional gene silencing (PTGS). PTGS was first recognized as a plant immune response to viral infection (Hamilton and Baulcombe, 1999
Generation of a third class of PTGS-associated small RNAs, called trans-acting siRNAs (ta-siRNAs), involves both siRNAs and miRNAs. ta-siRNAs differ from conventional siRNAs in that they target genes in trans (Peragine et al., 2004
In this study, we describe our efforts to extend previous silencing strategies by engineering the TAS1c (trans-acting siRNA1c) locus to silence the FAD2 gene in Arabidopsis (Arabidopsis thaliana). Processing of the TAS1c RNA is initiated by miR173-mediated cleavage, revealing six phased siRNAs downstream of the miR173 target site (Allen et al., 2005
Silencing with Artificial trans-Acting siRNAs
To determine if ta-siRNA loci could be engineered to silence genes of interest, we modified the TAS1c sequence to produce siRNAs targeting the FAD2 gene. FAD2 encodes an endoplasmic reticulum-localized We engineered a truncated TAS1c cDNA to silence FAD2 by replacing the sequence encoding the five native TAS1c siRNAs with a sequence encoding five identical siRNAs targeting base pairs 98 to 118 of the FAD2 coding sequence (CDS; 5XsiFAD2; Fig. 1A ). As a control, the miR173 binding site of 5XsiFAD2 was mutated to disrupt base pairing at base pairs 4, 10, and 11 of miR173, to produce mut-5XsiFAD2. Loss of the functional miR173 binding site in mut-5XsiFAD2 should prevent production of phased FAD2 siRNAs.
FAD2 expression is directly proportional to the amount of oleic to linoleic desaturase activity in a tissue (Okuley et al., 1994 ). Accordingly, silencing was quantified by expressing the degree of unsaturation of 18:1 to 18:2 and 18:3 fatty acids as oleic desaturation proportion (ODP; see "Materials and Methods"). Wild-type plants had an ODP of approximately 0.7 in seed, meaning that 70% of 18:1 fatty acids in seed are converted to 18:2 and 18:3 fatty acids (Fig. 1B). All 12 transgenic events we obtained for the 5XsiFAD2 construct showed strong silencing of the FAD2 gene. Ten of the 12 families had average ODP values between 0.1 and 0.2, while the remaining two families had ODP values of approximately 0.25 and 0.3 (Fig. 1B). T2 seed from each family showed the same degree of silencing whether they were homozygous or hemizygous for the 5XsiFAD2 transgene. The fad2-1 null allele had an ODP value of about 0.1 (Okuley et al., 1994; Stoutjesdijk et al., 2002), and thus the majority of our transgenic lines showed complete or almost complete silencing of the FAD2 gene, with the remaining two lines showing strong silencing. Plants harboring the mut-5XsiFAD2 construct had ODP values indistinguishable from the wild type (Fig. 1B), indicating that FAD2 silencing initiated by 5XsiFAD2 occurs through an miR173-triggered mechanism. We propagated three lines for several generations more and found that silencing was maintained in the T5 generation (Fig. 1B). To directly assay FAD2 gene expression in these lines, we determined the levels of FAD2 mRNA and siFAD2 by northern analysis of RNA from leaf tissue (Fig. 1D), and determined leaf fatty acid levels in the plants that we used for northern analysis (Fig. 1C). Using the same transgenic lines subjected to fatty acid analysis, we detected markedly decreased FAD2 mRNA levels in 5XsiFAD2 lines compared to wild-type plants. This decrease is consistent with the large decrease in ODP seen in these lines. Transgenic lines harboring the mut-5XsiFAD2 transgene showed no decrease in FAD2 mRNA levels. In small RNA northerns, siFAD2 siRNAs were observed in 5XsiFAD2 plants, yet they were absent from wild-type and mut-5XsiFAD2 plants (Fig. 1D). In addition, 5' RACE revealed cleavage at the miR173 site of 5XsiFAD2 (data not shown).
Next we sought to determine if a single copy of siFAD2 could effectively silence FAD2. We replaced the sequence encoding the native siRNA at the 3'D2+ position within the full-length TAS1c cDNA with a sequence encoding siFAD2, producing the construct 1XsiFAD2 (Fig. 2A ). As a control we engineered mut-1XsiFAD2, identical to 1XsiFAD2 except that the miR173 binding site was mutated to prevent miR173-induced cleavage.
When transformed into plants, 1XsiFAD2 decreased fatty acid ODP from about 0.75 in wild-type seed to an average of 0.18 in 10 independent T1 lines (Fig. 2B). All 10 independent transgenic lines showed approximately the same degree of FAD2 sense silencing: The SD in ODP score was only about 0.03. The silencing was heritable, as the majority of the T2 families examined showed ODP scores similar to those of T1 lines. Thus, engineering TAS1c with a single siFAD2 decreased ODP nearly as much as what was achieved with five copies of siFAD2. This level of silencing persisted in almost all lines in the T2 generation (Fig. 2B).
The experiments described above demonstrated that the TAS1c locus can be successfully modified to silence the FAD2 gene by substituting DNA encoding a deliberately designed FAD2 siRNA for a native TAS1c siRNA. However, it may not always be straightforward to design siRNAs targeting a gene of interest, as the characteristics of successful siRNAs are not completely known. For this reason, we determined if inserting a larger fragment of the FAD2 gene into TAS1c would effectively produce siRNAs. A 210-bp sense fragment of the FAD2 gene was inserted into the full-length TAS1c cDNA in place of the segment encoding the phased siRNAs 3'D2+ to 3'D6+ to produce the fragFAD2 construct (Fig. 3A ). As a control for miR173-dependent production of phased siRNAs, the miR173 target site was mutated to produce construct mut-fragFAD2.
As shown in Figure 3B, T1 transgenic plants containing the fragFAD2 construct had an average ODP of less than 0.1, similar to levels seen in the 5XsiFAD2 transgenic lines and in the fad2-1 mutant (Miquel and Browse, 1992 ). This level of silencing was maintained in six out of seven T2 transgenic lines. We checked ODP levels for three lines in the T3 generation and found that silencing was maintained. Transgenic lines containing the mut-fragFAD2 construct had ODP levels similar to the wild type, indicating that miR173-induced cleavage of fragFAD2 was required for siRNA production. To verify gene silencing, levels of FAD2 mRNA and FAD2 siRNAs were determined in fragFAD2 and mut-fragFAD2 transgenic plants (Fig. 3C). As expected, FAD2 mRNA levels were markedly decreased in fragFAD2 transgenic plants and were similar to the wild type in mut-fragFAD2 transgenic plants. In addition, FAD2 siRNAs were detected in fragFAD2 plants, but not in wild-type or mut-fragFAD2 transgenic plants. Thus, the substitution of native TAS1c siRNAs with a large fragment of the FAD2 gene leads to efficient and stable silencing of this target gene.
Whether each TAS locus requires its native miRNA trigger for proper function remains an open question. To address this, we substituted the endogenous miR173 target site in the 5XsiFAD2 construct with the target site for miR167, a highly expressed miRNA in leaves (data not shown) to produce miR167-5XsiFAD2 (Fig. 4A ). As a control to show that any siRNAs produced from this construct required miR167 for cleavage, we also made mut-miR167-5XsiFAD2, a construct with a mutated miR167 binding site.
When transformed into plants, miR167-5XsiFAD2 T1 seed had approximately half the ODP of the wild type, although there was considerable variation in the degree of silencing among T1 individuals, as shown by the large SD for the average ODP value of T1 seed (Fig. 4B). ODP levels climbed to approximately 75% of wild type in most T2 families. No difference was observed in ODP levels between mut-miR167-5XsiFAD2 control plants and the wild type. Such moderate and variable silencing was also noted in leaf tissue (Fig. 4C).
Modified 5' RACE showed a reduced frequency of cleavage at the miR167 site of miR167-5XsiFAD2 mRNA in leaf tissue. Out of 30 sequenced cleavage products, only two were cleaved at the miR167 target site (Fig. 4A). The remaining 28 5' RACE products showed cleavage throughout the miR167-5XsiFAD2 mRNA. For comparison, we performed 5' RACE on an endogenous target of miR167, ARF8. We detected robust miR167 cleavage events in ARF8 (data not shown), results similar to those published previously (Yang et al., 2006
Here we have described a novel method of PTGS utilizing the TAS1c locus in Arabidopsis. Engineering TAS1c to produce single or multiple copies of an artificial trans-acting siRNA (ata-siRNA) targeting the FAD2 gene, or with a 210-bp fragment of the FAD2 gene, resulted in consistent and very efficient silencing of FAD2. Notably, we also showed that miR167 was not an effective trigger for silencing in a TAS1c context, suggesting that miR173 may be specifically required to cleave TAS1c and/or may have a more sophisticated role in initiating phased siRNA from the TAS1c transcript.
TAS1c-mediated silencing consistently reaches complete phenotypic penetration. Nearly all T1 seed for constructs encoding single or multiple ata-siRNAs targeting FAD2 showed FAD2 silencing comparable to that of the fad2-1 null allele. In hairpinRNA-mediated silencing, a continuous spectrum of silencing was obtained in T1 plants with ODP scores ranging from similar to the fad2-1 mutant to as high as 50% of the wild type (Stoutjesdijk et al., 2002 In one important aspect, ata-siRNAs are similar to amiRNAs: Both methods produce 21-bp single-stranded RNAs that target a specific sequence. These 21-bp species can be used to target one gene among a cluster of tandemly repeated genes, a specific allele, or a splice variant. This is a distinct advantage over intron-spliced hairpin constructs where much longer sequences are used, and thus more off-target silencing is possible.
It has been suggested that it might be possible to design polycistronic amiRNAs with multiple stem-loops that can encode distinct amiRNAs, in order to target multiple genes from the same construct (Ossowski et al., 2008 Substituting miR167 for miR173 in TAS1c greatly reduces the ability of the 5XsiFAD2 construct to silence FAD2 and suggests that an intriguing new layer of ta-siRNA biogenesis remains to be explored. Even in leaves, where miR167 expression is high, the miR167-5XsiFAD2 construct showed inefficient FAD2 repression, precluding the possibility that weak silencing resulted from low miR167 expression. Instead, such poor silencing could indicate that TAS1c transcripts are not accessible to all miRNA. It is also possible that miR173 may have a specific role in promoting ta-siRNA biogenesis by recruiting an RNA-dependent RNA polymerase. In any case, once the hurdles to using miRNAs other than miR173 are overcome, one attractive possibility for silencing genes in a tissue-specific or temporally-specific manner would be to engineer TAS constructs triggered by miRNAs that are expressed in specific tissues or at specific times in development. The expression patterns of trigger miRNAs could be used to confer spatial or temporal specificity on TAS-induced silencing. In addition to providing an excellent tool for gene silencing, the consistency and efficacy of silencing the FAD2 gene with TAS1c could provide a valuable system for investigating the specificity of sequences and cofactors for the function of ta-siRNAs. We expect that refining the design of ata-siRNAs will go hand in hand with a deepening knowledge of the biology of ta-siRNAs.
Plant Material and Growth Conditions All experiments were performed on the Columbia (Col-0) ecotype of Arabidopsis (Arabidopsis thaliana) plants. Plants were grown in long-day conditions (16 h of light at 22°C, 8 h of dark at 20°C).
The artificial TAS1c constructs targeting the Arabidopsis FAD2 gene were made using a 21-bp or a 210-bp fragment of the 5' coding region of the gene. The 21-bp siFAD2 siRNA corresponds to base pairs 98 to 118 of the FAD2 CDS. These 21-bp sequences were selected by scanning the CDS for target regions that would be recognized by a ta-siRNA of sequence 5'-(T/A)NNNNNNNNNNNNNNNNN(C/G)NN-3' with GC content between 30% to 50%. Internal stability was analyzed by running the candidate 21mer through mFold (Zuker, 2003 5XsiFAD2 was made as follows: sense and antisense oligos for the 5' half and for the 3' half of the 484-nt piece were designed (FAD2TAS 5' sense, FAD2TAS 5' antisense, FAD2TAS 3' sense, FAD2TAS 3' antisense). The resulting DNA fragment contains the modified TAS1c fragment, flanked by attB sites and single-stranded overhangs (GATC and AATT). Oligos were annealed and ligated into pGEM7 (previously digested with BamHI and EcoRI). The 5XsiFAD2 in pGEM7 was then recombined with pBC vector, using Gateway reactions. This placed the 5XsiFAD2 fragment downstream of the 35S promoter and upstream of the phaseolin terminator. mut-5XsiFAD2 was made as above, except that oligos that contained mutations in the miR173 target site were used for the ligations (FAD2TASmut 3' sense, FAD2TASmut 3' antisense). To build the 1XsiFAD2 construct, we used primers TAS1c/FAD2-attB1 and TAS1c/FAD2-2R to generate the 5' fragment by PCR, and primers TAS1c/FAD2-3F and TAS1c/FAD2-attB-4 to generate the 3' fragment by PCR. The TAS1c/FAD2-2R and TAS1c/FAD2-3F fragments overlapped in the region of TAS1c that produces phased siRNAs, and modifications to the siRNAs were introduced using these long primers. We also modified 3'D5+ to target the unrelated AP1 gene. The 21-bp siAP1 sequence corresponds to base pairs 673 to 693 of the AP1 CDS. Silencing of AP1 was ineffective, likely due to the high intramolecular structure of the AP1 siRNA. The 5' and 3' fragments were gel purified and used together as a template to amplify the whole fragment by PCR with primers TAS1c/FAD2-attB1 and TAS1c/FAD2-attB-4. In a similar manner, construct miR167-5XsiFAD2 was generated using primers miR167/TAS1c/5XsiFAD2-attB1 and miR167/TAS1c/5XsiFAD2-2R to amplify the 5' fragment and miR167/TAS1c/5XsiFAD2-3F and miR167/TAS1c/5XsiFAD2-attB4 to amplify the 3' fragment. Both fragments were purified and used as a template to amplify the final product using primers miR167/TAS1c/5XsiFAD2-attB1 and miR167/TAS1c/5XsiFAD2-attB4. To build the fragFAD2 construct, we first amplified three separate fragments. Fragment 1 was amplified with primers TAS1c/FAD2-attB1 and TAS1c/FAD2-frag-2R; fragment 2 was amplified with primers TAS1c/FAD2-frag-3F and TAS1c/FAD2-frag-4R; and fragment 3 was amplified with primers TAS1c/FAD2-frag-5F and TAS1c/FAD2-attB-4. Then fragment 1 and fragment 2 were used as templates to amplify fragment 4 using primers TAS1c/FAD2-attB1 and TAS1c/FAD2-frag-4R. The complete construct was then amplified by combining fragment 4 and fragment 3 using primers TAS1c/FAD2-attB1 and TAS1c/FAD2-attB-4. The 210-bp FAD2 fragment corresponds to base pairs 1 to 210 of the FAD2 CDS.
To create the constructs with mutated miRNA binding sites, we used constructs 5XsiFAD2, 1XsiFAD, and fragFAD2 as templates for site-directed mutagenesis (Wang and Malcolm, 1999
The fragments of interest were amplified with attB-tagged primer pairs and cloned into the Gateway pDONR/Zeo (Invitrogen) vector to give rise to the entry clone. Sequences for all primers used in this study are listed in Table I. The pBC Yellow Gateway vector was used as a destination vector to generate the expression clone. pBC Yellow was generated from pBC (Aukerman and Sakai, 2003
Lipid compositional analyses were conducted on mature seeds or rosette leaves, collected from wild-type and transgenic plants. Lipids were extracted from single seeds by homogenization with a pestle in an Eppendorf tube in MeOH:CHCl3 (10 µL; 2:1, v/v). After a 30-min incubation, 50 µL of heptane was added. Samples were mixed thoroughly and spun at 10,000 rpm in a microfuge, and the organic layer was recovered and used for lipid compositional analyses as done by Damude et al. (2006)
Fatty acid profiles are expressed as ODP. Seed ODP is calculated as the percentage of 18:2 and 18:3 fatty acids out of the total amount of 18:1, 18:2, and 18:3 fatty acids, i.e. seed ODP = (%18:2 + %18:3)/(%18:1 + %18:2 + %18:3). Leaf ODP is calculated as the percentage of 18:2 fatty acids out of the total amount of 18:1 and 18:2 fatty acids, i.e. leaf ODP = (%18:2)/(%18:1 + %18:2). 18:3 fatty acids were not included in leaf ODP because the amount of 18:3 fatty acids in leaves is not dependent on FAD2 (Miquel and Browse, 1992
Total RNA was isolated from aerial tissues of adult wild-type and transgenic plants using TRIZOL reagent (Sigma). RNA was separated on agarose gels, blotted onto a Hybond N+ membrane (Amersham), and probed with 32P-labeled probes randomly primed with the RadPrime DNA labeling system (Invitrogen). Hybridization was carried out at 68°C using PerfectHyb Plus buffer (Sigma). Blots were washed once in 2x SSC and 0.1% SDS for 5 min at room temperature, twice in 0.5x SSC and 0.1% SDS for 20 min at 68°C, and once in 0.1x SSC, 0.1% SDS for 20 min at 68°C. The hybridization signal was detected with a Storm 860 (Molecular Dynamics).
Low-molecular-weight (LMW) RNA was purified from total RNA using the mirVANA miRNA isolation kit (Ambion). LMW RNA was separated on a 15% TBE-Urea Criterion gel (Bio-Rad) and transferred electrophoretically to Hybond N+ membrane (Amersham) using a TransBlot-SD apparatus (Bio-Rad). LMW blots were hybridized at 40°C using ULTRAhyb-oligo buffer (Ambion) with 32P-end-labeled oligonucleotide probes. Probes were labeled with [
Modified 5' RACE with the GeneRacer kit (Invitrogen) was adapted to validate the cleavage site determined by the miR167 target site. Primer TAS1c-2rev and nested primer FAD-TAS 1rev (miR167-5XsiFAD2) or ARF8 1Rev and ARF8 1Nested (ARF8) were used in PCRs, and the cleavage sites were revealed by sequence analyses of the PCR products.
Thanks to Leonard Farrell and Howard Damude for gas chromatography analysis of fatty acids, and to Manny Kiflemariam for growing plants for transformation. We greatly appreciate Enno Krebbers, Brian McGonigle, and Carl Falco for many helpful discussions. Additional thanks to Jeanne Wilson for critical comments on this manuscript, and to Mark Mucha for generating the pBC Yellow vector. Received February 23, 2008; accepted April 17, 2008; published April 25, 2008.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Robert W. Williams (robert-w2.williams{at}cgr.dupont.com). www.plantphysiol.org/cgi/doi/10.1104/pp.108.118307 * Corresponding author; e-mail robert-w2.williams{at}cgr.dupont.com.
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ABSTRACT
RESULTS
12 desaturase required for converting the monounsaturated oleic acid (18:1) to the polyunsaturated linoleic acid (18:2), itself the precursor to linoleic acid (18:3; Okuley et al., 1994
1) that had at least three unpaired nucleotides at the 5' and 3' ends. 
-32P]ATP using OptiKinase (USB). Blots were washed twice in 2x SSC and 0.5% SDS for 30 min at 38°C. 
