RNAi for Crop Improvement
Authors: Deepak Vishwanath Pawar1 Rakesh Kumar Prajapat1, and Ashish Marathe2
1ICAR-NRC on Plant Biotechnology, ICAR-IARI, New Delhi-1110012
2Division of Biochemistry, IARI, New Delhi- 110012.

RNA interference (RNAi) is a method of blocking gene function by inserting short sequences of ribonucleic acid (RNA) that match part of the target gene’s sequence and thus producing no proteins. RNAi has emerged as the method for researchers studying the structure and function of genes. RNAi has a huge potential as a powerful approach in targeted and personalized medicine as well as in agriculture. RNAi has provided a way to control pests and diseases, introduce traits and increase crop yield. Scientists have developed novel crops such as nicotine-free tobacco, and nutrient fortified maize through RNAi.

Discovery of RNAi

Scientists believed they could produce any gene product simply by introducing foreign genes in plants (1). In a previous study, biologists introduced multiple copies of the gene for purple petunia flowers to create deep purple flowers. This instead resulted in plants with white or variegated flowers. The transgenes were silenced as well as the plant’s ‘purple-flower’ gene (2,3). The mechanism that caused this effect was discovered by Andrew Fire and Craig Mello when their injection of double stranded ribonucleic acids (dsRNA) into the worm Caenorhabditis elegans triggered silencing of genes with sequences identical to that of the dsRNA (4). They called the phenomenon RNA interference. Fire and Mello were awarded the 2006 Nobel prize for Physiology or Medicine for their discovery.

In addition to its roles in regulating gene expression, RNAi is used as an immune response to infection5 and as a natural defense mechanism against molecular parasites such as jumping genes and viral genetic elements that affect genome stability (6). Specific types of bacteria have also been shown to trigger the RNAi pathway in plants.

How RNAi works

1. The entry of any long double stranded RNA triggers the RNAi pathway of cells. This results in the recruitment of the enzyme Dicer (Figure 1).

2. Dicer cleaves the dsRNA into short, 20-25 base pair-long fragments, called small interfering RNA (siRNA).

3. An RNA-induced silencing complex (RISC) then separates the siRNA strands into two: sense or antisense strand. The sense strands, or those with exactly the same sequence as the target gene, are degraded.

4. The antisense strands are incorporated to the RISC and are used as guide to target messenger RNAs (mRNA).

5. mRNA, which codes for amino acids, are cleaved by RISC. The activated RISC can repeatedly participate in mRNA degradation, inhibiting protein synthesis.

Disease and pathogen resistance

Gene silencing was first used to develop virus-resistant varieties. This was first demonstrated in Potato Virus Y- resistant plants with RNA transcripts of a viral proteinase gene (7,8). Plants can also be modified to produce dsRNAs that silence genes in crop pests. This was used to develop resistance to root-knot nematode (9), corn rootworm (10) and cotton bollworm (11).

Male sterility

RNAi has also been used to develop male sterility, which is vital in the hybrid seed industry. Genes expressed solely in tissues involved in pollen production are targeted through RNAi. Scientists developed male sterile tobacco by inhibiting the expression of TA29, a gene necessary for pollen development (12).

Plant functional genomics

RNAi offers specificity and efficacy in silencing members of a gene or multiple gene family to characterize a gene function. The expression of dsRNAs with inducible promoters can also control the extent and timing of gene silencing, resulting in silenced genes at a certain growth stage or plant organ (13,14). There are several ways of activating the RNAi pathway in plants such as the use of hairpin RNA-expressing vectors, particle bombardment, Agrobacterium mediated transformation and virus-induced gene silencing (VIGS) (15).

Engineering plant metabolic pathways

RNAi has been used to modify plant metabolic pathways to enhance nutrient content and reduced toxin production (Table 1). The technique uses heritable and stable RNAi phenotypes in plants.

Prospects for RNAi

RNAi presents the possibility of targeting multiple genes for silencing using a thoroughly-designed single transformation construct. It can also provide resistance against a wide range of pathogens.9 Studies have also used it in plant stress adaptation.

Table 1. Examples of novel plant traits engineered through RNAi

Trait Target Gene Host Application
Enhanced nutrient content Lyc Tomato Increased concentration of lycopene (carotenoid antioxidant)
DET1 Tomato Higher flavonoid and b-carotene contents
SBEII Wheat, Sweet potato, Maize Increased levels of amylose for glycemic management and digestive health
FAD2 Canola, Peanut, Cotton Increased oleic acid content
SAD1 Cotton Increased stearic acid content
ZLKR/SDH Maize Lysine-fortified maize
Reduced alkaloid production CaMXMT1 Coffee Decaffeinated coffee
COR Opium poppy Production of non-narcotic alkaloid, instead of morphine
CYP82E4 Tobacco Reduced levels of the carcinogen nornicotine in cured leaves
Heavy metal accumulation ACR2 Arabidopsis Arsenic hyperaccumulation for phytoremediation
Reduced polyphenol production s-cadinene synthase gene Cotton Lower gossypol levels in cottonseeds, for safe consumption
LeETR4 Tomato Early ripening tomatoes
Ethylene Sensitivity ACC oxidase gene Tomato Longer shelf life because of slow ripening
Reduced Allergenicity Arah2 Peanut Allergen-free peanuts
Lolp1, Lolp2 Ryegrass Hypo-allergenic ryegrass
Reduced production of lachrymatory factor synthase lachrymatory factor synthase gene Onion "Tearless" onion

1. Tang, G., G. Galili, and X. Zhuang. 2007. RNAi and MicroRNA: Breakthrough Technologies for the Improvement of Plant Nutritional Value and Metabolic Engineering. Metabolomics 3(3): 357-369.

2. Novina, C.D. and P.A. Sharp. 2004. The RNAi Revolution. Nature 430 (6996): 161-164.

3. Napoli, C., C. Lemieux, and R. Jorgensen. 1990. Introduction of a Chimeric Chalcone Synthase Genes into Petunia Results in Reversible Co-Suppression of Homologous Genes in Trans. The Plant Cell 2(4): 279-289.

4. Fire, A., S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, and C.C. Mello. 1998. Potent and Specific Genetic Interference by Double-Stranded RNA in Caenorhabditis elegans. Nature 391(6669): 806-811.

5. Fritz, J.S., S. Girardin, and D. Philpott. 2006. Innate Immune Defense through RNA Interference. Science’s STKE 339: pe27.

6. Gupta, P.K. 2006. RNA Interference - Gene Silencing by Double Stranded RNA: The 2006 Nobel Prize for Physiology or Medicine. Current Science 91(11): 1443-1446.

7. Mansoor, S., I. Amin, M. Hussain, Y. Zafar, and R.W. Briddon. 2006. Engineering Novel Traits in Plants through RNA Interference. Trends in Plant Science 11(11): 559-565.

8. Qu, J., J. Ye, and R. Fang. 2007. Artificial MicroRNA-mediated Virus Resistance in Plants. Journal of Virology 81(12): 6690-6699.

9. Huang, G., R. Allen, E.L. Davis, T.J. Baum, and R.S. Hussey. 2006. Engineering Broad Root-Knot Resistance in Transgenic Plants by RNAi Silencing of a Conserved and Essential Root-Knot Nematode Parasitism Gene. PNAS 103(39): 14302-1406.

10. Mao, Y.B., W.J. Cai, J.W. Wang, G.J. Hong, X.Y. Tao, L.J. Wang, Y.P. Huang, and X.Y. Chen. 2007. Silencing a Cotton Bollworm P450 Monooxygenase Gene by Plant- Mediated RNAi Impairs Larval Tolerance of Gossypol. Science 25(11): 1307-1313.

11. Baum, J.A., T. Bogaert, W. Clinton, G.R. Heck, P. Feldman, O. Ilagan, S. Johnson, G. Plaetinck, T. Munyikwa, M. Pleau, T. Vaughn, and J. Roberts. 2007. Control of Coleopteran Insect Pests through RNA Interference. Nature Biotechnology 25(11):1322-1326.

12. Nawaz-Ul-Rehman, M.S., S. Mansoor, A.A. Khan, Y. Zafar, and R.W. Briddon. 2007. RNAi-Mediated Male Sterility of Tobacco by Silencing TA29. Molecular Biotechnology 36: 159-165.

13. Matthew, L. 2004. RNAi for Plant Functional Genomics. Comparative and Functional Genomics 5(3): 240-244.

14. Sato, F. 2005. RNAi and Functional Genomics. Plant Biotechnology 22(5): 431-442.

15. Burch-Smith T.M., J.C. Anderson, G.B. Martin, and S.P. Dinesh-Kumar. 2004. Applications and Advantages of Virus-Induced Gene Silencing for Gene Function Studies in Plants. The Plant Journal 39(5): 734-746.

About Author / Additional Info:
I am PhD research scholar, pursuing PhD at IARI, New Delhi in the discipline of Molecular Biology and Biotechnology. I am working on blast disease resistance in O. sativa