Applications of RNAi for Crop Improvement and Metabolic Engineering

In addition to crop protection, RNAi technology has been used successfully to modify agronomically relevant traits such as, e.g. nutritional or pharmaceutical value and crop toxicity (reviewed by Tang et al. 2007; Hebert et al. 2008). Many reports on the successful application of RNAi for crop improvement take advantage of the potential of RNAi to down-regulate multiple targets. For example, a nutritionally valuable high-lysine maize variant was produced by down-regulating the entire 22-kDa a-zein gene family (with the seven active members sharing >90% nucleotide sequence identity in the coding sequence; Segal et al. 2003). Similarly, a constitutively expressed RNAi effector transgene led to "decaffeination" of coffee plants by down-regulating multiple members of a caffeine biosynthesis gene family (Ogita et al. 2003, 2004). In another study, Allen et al. (2004) were able to generate opium poppy plants with high levels of the pharmaceutically valuable non-narcotic alkaloid reticuline by silencing the codeinone reductase multi-gene family that includes the key enzymes of morphine biosynthesis. Silencing of all gene family members was possible by a combination of targeting a highly conserved region among gene family members and incorporating a second gene fragment specific for a distinctive member into the same inverted repeat.

Some RNAi-based crop improvement strategies have taken advantage of the possibility to spatially restrict the induction of RNAi, e.g. by use of tissue-specific promoters. Examples include an alternative approach to engineering high-lysine maize by Houmard et al. (2007), who used endosperm-specific silencing of a lysine metabolism gene to confine increased lysine accumulation to the kernels, and the production of high-carotenoid and high-flavonoid tomato by RNAi of the photo-morphogenesis regulatory gene DET1 with fruit-specific promoters (Davuluri et al. 2005). In contrast to the spatial confinement of these compounds, overproduction of lysine also in other tissues (Houmard et al. 2007) and constitutive silencing of DET1 (Davuluri et al. 2004) caused severe developmental defects. In an exciting report on genetic engineering of cotton that opens a path for the use of this crop species both for fiber and for food production, Sunilkumar et al. (2006) disrupted the biosynthesis of the toxic terpenoid gossypol specifically in seeds. The observed significant reduction of gossypol in cottonseed greatly improves the value of cottonseed for human consumption, without abolishing the toxin's function in plant defense in other (non-edible) plant organs where the production of gossypol was not impaired (reviewed by Townsend and Llewellyn 2007). These reports clearly demonstrate the benefits of spatially restricted silencing. Importantly, although RNA silencing had been reported to be capable of spreading systemically, silencing targeted to either fruits or seeds did not spread to any significant extent beyond the targeted tissues to other parts of the plant. Finally, tissue-specific RNA silencing was also employed in recent "intragenic" approaches that only use sequence elements that are native to the target species for plant transformation (Rommens 2007). In one report, Rommens et al. (2008) constructed an inverted repeat driven by tuber-specific promoters to silence two asparagine synthesis genes in potato. The resulting transformants produced low-asparagine potatoes which after heat-processing accumulated as little as 5% of the suspected carcinogen acrylamide present in wild-type controls.

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