Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence.

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. 22nt siRNAs are under-represented due to competition between DCL2 and DCL4, while only a small number of 22nt miRNAs exist. Here we produce abundant 22nt siRNAs and other siRNA size classes using long hairpin RNA (hpRNA) transgenes. By introducing asymmetric bulges into the antisense strand of hpRNA, we shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs. The asymmetric hpRNAs 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 showed increased amounts 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. The 22nt asymmetric hpRNA constructs enhanced virus resistance in plants compared to the traditional hpRNA constructs.

Nucleotide mismatches prevent intrinsic self-silencing of hpRNA transgenes to enhance RNAi stability in plants.

Hairpin RNA (hpRNA) transgenes are the most successful RNA interference (RNAi) method in plants. Here, we show that hpRNA transgenes are invariably methylated in the inverted-repeat (IR) DNA and the adjacent promoter, causing transcriptional self-silencing. Nucleotide substitutions in the sense sequence, disrupting the IR structure, prevent the intrinsic DNA methylation resulting in more uniform and persistent RNAi. Substituting all cytosine with thymine nucleotides, in a G:U hpRNA design, prevents self-silencing but still allows for the formation of hpRNA due to G:U wobble base-pairing. The G:U design induces effective RNAi in 90-96% of transgenic lines, compared to 57-65% for the traditional hpRNA design. While a traditional hpRNA transgene shows increasing self-silencing from cotyledons to true leaves, its G:U counterpart avoids this and induce RNAi throughout plant growth. Furthermore, siRNAs from G:U and traditional hpRNA show different characteristics and appear to function via different pathways to induce target DNA methylation.

Development of a Brassica napus (Canola) Crop Containing Fish Oil-Like Levels of DHA in the Seed Oil.

Plant seeds have long been promoted as a production platform for novel fatty acids such as the ω3 long-chain (≥ C20) polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) commonly found in fish oil. In this article we describe the creation of a canola (Brassica napus) variety producing fish oil-like levels of DHA in the seed. This was achieved by the introduction of a microalgal/yeast transgenic pathway of seven consecutive enzymatic steps which converted the native substrate oleic acid to α-linolenic acid and, subsequently, to EPA, docosapentaenoic acid (DPA) and DHA. This paper describes construct design and evaluation, plant transformation, event selection, field testing in a wide range of environments, and oil profile stability of the transgenic seed. The stable, high-performing event NS-B50027-4 produced fish oil-like levels of DHA (9-11%) in open field trials of T3 to T7 generation plants in several locations in Australia and Canada. This study also describes the highest seed DHA levels reported thus far and is one of the first examples of a deregulated genetically modified crop with clear health benefits to the consumer.

Comparison of the Substrate Preferences of ω3 Fatty Acid Desaturases for Long Chain Polyunsaturated Fatty Acids.

Omega-3 long chain polyunsaturated fatty acids (ω3 LC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5ω3) and docosahexaenoic acid (DHA; 22:6ω3) are important fatty acids for human health. These ω3 LC-PUFAs are produced from their ω3 precursors by a set of desaturases and elongases involved in the biosynthesis pathway and are also converted from ω6 LC-PUFA by omega-3 desaturases (ω3Ds). Here, we have investigated eight ω3-desaturases obtained from a cyanobacterium, plants, fungi and a lower animal species for their activities and compared their specificities for various C18, C20 and C22 ω6 PUFA substrates by transiently expressing them in Nicotiana benthamiana leaves. Our results showed hitherto unreported activity of many of the ω3Ds on ω6 LC-PUFA substrates leading to their conversion to ω3 LC-PUFAs. This discovery could be important in the engineering of EPA and DHA in heterologous hosts.

Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol yields from plant leaves.

High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co-expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild-type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil-processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications.