Role of RNAi in crop improvement
Authors: Amandeep Kaur and Pradeep Sharma ( corresponding author: Email-
Crop Improvement Division, ICAR-Directorate of Wheat Research, Karnal-132001, India

RNA interference (RNAi) has emerged as one of the reverse genetics tool in the regulation of gene expression by different types of RNA in plants. RNA is an intermediate product from the process of decoding the information on a DNA to coding it on a protein. But this is not always the case, as some RNA, known as non-coding RNAs (ncRNAs), does not code for any peptide or protein and have good regulatory roles in cellular processes. These non-coding RNAs range from very well-known transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) to recently discovered regulatory RNAs, such as small interfering RNAs (siRNAs), microRNAs (miRNAs), trans-acting siRNAs (ta-siRNAs).

RNAi is a phenomenon which evolved naturally in plants in defence of viral infections. Now, it is well known for their many cellular functions in plants as well as in animals and fungus. It is also known by other names, co-suppression, post-transcriptional gene silencing (PTGS) and quelling. It involves two types of single-stranded non-coding small RNA molecules, siRNA and miRNA, that regulate gene expression by cleaving targeted mRNA molecules or by interfering with translation.

The siRNAs are rarely conserved, ~21nts long, non-coding, single stranded, small RNAs that are derived from exogenous double stranded RNAs (dsRNAs) such as viruses, transposons and transgenes by the Dicer-like enzyme. On the other hand, miRNAs are mostly conserved, ~21nts long, non-coding, single stranded, small RNAs and endogenously derived from a primary-miRNA (pri-miRNA) having a stem-loop structure. Dicer processed pri-miRNA into precursor-miRNA (pre-miRNA) and miRNA::miRNA duplex. These short RNA duplexes (sense and antisense strands) are unwound to produce a single guide strand. The siRNA or miRNA guide strand interacts with the RNA-induced silencing complex (RISC) in the cytoplasm. RISC contains a subunit Argonaute (Ago) that help to recognise single stranded RNA and has an endonuclease domain that cleaves target RNA having complementary sequences to small RNAs. Therefore, it leads to a sequence-specific transcriptional or translational inhibition of the genes and can be used to engineer crops to improve quality and quantity.

The process of RNAi was first observed by Andrew Fire and Craig C. Mello in 1998 in a nematode, Caenorhabditis elegans, and later they awarded Nobel Prize 2006 in Physiology or Medicine. After that RNAi technology had come into existence to produce transgenic crops with improved nutritional and physiological characters. The most common method of gene silencing is based on the introduction of dsRNAs homologous to target gene that will produce siRNAs to silence the gene. Its excellent product was Flavr-savr tomato in which a polygalacturonase gene that codes for the enzyme that degrade pectin present in the cell wall and lead to early ripening of tomatoes, had been silenced by expressing an antisense transcript of this gene. The endogenous sense transcript of the gene and exogenous expressed antisense transcript form a double stranded secondary structure that ultimately degraded by the RNA interference machinery. RNA-mediated gene silencing was successfully performed to silence two fatty acid desaturase genes, ghSAD-1-encoding stearoyl-acyl-carrier protein 9-desaturase and ghFAD2-1-encoding oleoyl-phosphatidylcholine 6-desaturase, to increase the stearic acid and oleic acid content in cotton seeds (Liu et al., 2002). Similarly, the nutritional contents of carotenoid and flavonoid had been significantly increased by suppressing an endogenous photomorphogenesis regulatory gene, DET1 by insertion of a DET1-derived inverted-repeat construct in tomato (Davuluri et al., 2005). The high amylose cereals have potential health benefits for humans over certain chronic diseases such as cancer and diabetes. Hence, two different isoforms of starch-branching enzyme II (SBEIIa and SBEIIb) in wheat (Regina et al., 2006) and a SBEIIb gene in rice (Butardo et al., 2011) had been silenced by hairpin RNA (hp-RNA) to increase relative content of amylose as compare to amylopectin. A multidrug resistance-associated protein ATP-binding cassette transporter had been silenced in an embryo-specific manner to produce transgenic maize and soybean seeds to reduce phytic acid (Shi et al., 2007). The ability of gene silencing to produce virus resistant plants was successfully exploited in sugarcane by transferring a hairpin interference sequence against the coat protein gene of sorghum mosaic virus. Consequently, it is a good technology to produce transgenic crops with improved qualities. However, it has some drawbacks as well as it could be act on off-targets to silence multiple genes. Locally induced post-transcriptional gene silencing in plants could spread in the whole plant and do not provide a tissue specific gene silencing. Moreover, now a day, artificial miRNAs has been used to overcome these problems.

Artificial miRNA (amiRNA) is natural endogenous miRNA gene that is genetically engineered to produce a transgene in which the endogenous miRNA in the precursor is replaced with one that is complementary to the targeted messenger RNA. Thus, they have highly specific to endogenous miRNA as well as target tissue and do not show off-target effects. Qu et al. (2007) used successfully used an amiRNA to inhibit the expression of 2b of Cucumber mosaic virus to make virus resistant transgenic tobacco plants. Recently, an another class of long non-coding RNAs known as miRNA decoys has been identified that carry a short stretch of sequence having homology to miRNA binding sites in endogenous targets and are able to sequester and inactivate miRNA function (Bank et al., 2012). A miRNA decoy can degrade more than one miRNA in a sequence-dependent manner and can be potentially very useful tool for crop improvement.

The potential of RNAi has been utilized in various plants till date and it can be exploited in future for many purposes like higher yield of cereals and grains, alteration in plant architecture, abiotic stress tolerance, biotic stress resistance, nutritional improvement, deletion of allergens and toxic substances, prolongation of shelf-life, modulation of flower colours and scents, enhancement of secondary metabolites, seedless fruit and male sterile lines development (Jagtap et al., 2011). Therefore, RNAi has a great potential for crop improvement.


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