Authors: Gayacharan1*, Nupur Mondal2
1ICAR-National Bureau of Plant Genetic Resources, New Delhi-110012
2Shivaji College, University of Delhi, New Delhi-110027
What is RNAi?
RNA interference (RNAi) is a sequence specific RNA degradation process that is triggered by the formation of double stranded RNA that can be introduced by virus or transgenes. Double stranded 21- nucleotide (nt) RNAs with symmetric 2-nt 3’overhangs are introduced into the cell which mediates the degradation of targeted mRNA. Therefore, the corresponding protein is not synthesized which leads to development of altered phenotype. This cellular mechanism is also known by other names like co-suppression, post-transcriptional gene silencing (PTGS), and quelling.
The RNA interference (RNAi) technology recently has become popular tool among plant scientists to understand the functions of individual genes, and is being used in trait improvement or new trait development. For genetic improvement of crop plants, RNAi have advantages over antisense-mediated gene silencing and co-suppression, in terms of its efficiency and stability. The common goal of all RNAi technologies is to produce sufficient amounts of double-stranded RNA (dsRNA) within plants which has homology with target endogenous messenger RNAs, so it can trigger the gene silencing mechanism. The most efficient delivery methods for dsRNA in plants today are inoculation of plants with engineered plant viruses which produce in their life cycles dsRNA intermediates (Robertson, 2004) and transformation of plants with transgene constructs from which the RNA transcripts are folded into dsRNA structures (Waterhouse et al. 1998). In both approaches accumulating dsRNA activates the plant dsRNA-specific RNase “DICER” which cleaves the dsRNA into small RNA fragments with a size of ~21-24 nucleotides. These small RNAs which are called small interfering RNAs (siRNAs) are integrated into an enzyme complex called RISC (RNA-induced silencing complex) and guide the complex to all homologous plant RNAs for degradation (Siomi and Siomi, 2009). The degree of degradation of the targeted plant RNAs can vary from partial to complete degradation and depends on exogenous factors, e.g. temperature as well as endogenous factors, e.g. the physiological status of the plant. This enables studies of the effect of down-regulating the expression of a gene at various levels.
Molecular biologists had tried lot to knockout gene expression at the mRNA level. Several efforts were made on similar aspects like using antisense sequences, ribozymes and chimeric oligonucleotides. Antisense technologies are relatively straight forward techniques for understanding gene functions; however, these technologies are not efficient enough to manipulate or knockout gene function. Moreover, due to lack of desired specificity often a weak suppression of gene function is was obtained (Guru, 2000). The first attempts to induce knockdown gene expression in plants were based on observations made during 1980s i.e. inhibitory effect of long antisense RNAs on corresponding mRNAs in animal cells (Izant and Weintraub, 1984). Later experiments with transient or stable expressed antisense RNAs resulted in successful suppression of accumulation of the targeted mRNA, which lead to conclusion that dsRNA acts as a template for RNA degradation (Mol et al., 1988). It was also observed that strong overexpression of sense transgenes sometimes results in co-suppression of both the transgene and the homologous endogenous gene (Napoli et al., 1990). PTGS is the process of down-regulation of a gene expression at the post transcriptional level, by targeting specific RNAs for degradation. RNAi was first described in detail in worms ( Caenorhabditis elegans) in 1998 by Andrew Fire and Craig C. Mello for which they shared the 2006 Nobel Prize in Physiology and Medicine. RNAi operates in plants, fungi, flies and mammals. PTGS of genes takes place that share significant sequence homology with the targeted silenced genes. Long molecules of double stranded RNA (dsRNA) trigger the process. Naturally the dsRNA which comes from virus and transposon activity can trigger RNAi process. Artificially dsRNA can be injected in the cells in experimental processes (Elbashir et al., 2001). The strand of the dsRNA that is identical in sequence to a region in target mRNA molecule is called the sense strand, and the other strand which is complimentary is termed the antisense strand. An enzyme complex called DICER (in D. melanogaster), thought to be similar to RNase III recognizes dsRNA, and cuts it into approximately 22- nucleotide long fragments. These fragments termed siRNAs (small interfering RNAs) which remain in double stranded duplexes with very short 3’ overhangs then act as templates for the RNAi inducing silencing complex to destroy the homologous mRNAs, thus specifically suppressing particular gene(s) expression.
Salient features of RNAi
- Double stranded RNA rather than single-stranded antisense RNA is the interfering agent.
- The silencing mechanism is systemic.
- High degree of specific gene silencing is achieved with comparative less effort.
- It requires only a few dsRNA molecules per cell for effective RNAi.
- Gene silencing can be done at any developmental stage.
- It can avoids associated developmental abnormalities caused by a knocked out gene in early stages.
- This silencing effect inherits through generations.
The RNAi technologies have been applied in several important crop plant species which are amenable to transformation and regeneration. First time the technology was used Petunia hybrida L. plants to enhance anthocyanin pigment through introducing chalcone synthase gene ( chsA) (Napoli et al 1990). The gene was overexpressed with the objective to increase its color intensity, but new pattern of flower color was observed. RNAi-induced gene silencing is emerging as a very effective technology to engineer pathogen resistant plants. And generally, host gene silencing-hair pin RNAi (HGS-hpRNAi) is reported as more stable gene silencing method in plants. There are several examples of its utilization in Arabidopsis, rice, wheat tomato, barley, etc. to provide resistance against targeted pathogens (Younis et al. 2014). Similarly VIGs (virus induced gene silencing) is another method of RNAi used to protect crops from infection using RNA and DNA viruses. Transgenic RNA silencing based resistance to a commercially important disease caused by a RNA virus is exemplified by the control of Sharka or plum pox in transgenic Prunus species (Ravelonandro et al., 2000). PTGS based resistance to a DNA virus has been achieved in cassava (Chellappan et al. 2004). The AC1 coding sequence is a moderately conserved sequence among these Gemini viruses is used for this purpose. AC1 gene-based RNA silencing appears to be a promising strategy for developing durable and broad Gemini virus resistance. Tyagi et al., 2008 reported on the use of RNAi to confer resistance against RTBV in rice. Wang et al (2000) reported the transformation of barley plants with a construct that encodes an hpRNA containing the polymerase gene sequences of BYDV-PAV confers immunity to BYDV-PAV on the plants. Potentially the RNAi technologies can be used for insect resistance, abiotic stress resistance, improving nutritional traits, in removing food allergens, etc. (Younis et al. 2014) across any crop species which are known to possess endogenous functional RNAi mechanism. Recent advances in the RNAi technologies is making great impact on crop improvement programs. Perhaps the most important applications will be in altering crop pest interactions so that plants are protected from insects, nematodes or pathogens.
Limitations of RNAi
It can have off target activity due to partial homology between the siRNA and other complementary mRNA. siRNAs, like most RNA molecules, are readily degraded by RNases, which are ubiquitous both in the extracellular and the intracellular. RNAi may also activate the interferon response, "a nonspecific viral defense mechanism," and thus makes RNAi ineffective.
Prospects of utilizing RNAi techniques
Lathyrus sativus is a leguminous crop and contains a neurotoxin called β-oxalyl aminoalanine-L-alanine (BOAA). After long time consumption of this legume, people can get paralytic disease called, lathyrism. The RNAi technology can be successfully used to silence the gene(s) responsible for production of BOAA. If the trait is associated with other desired traits like drought tolerance in the crop, bringing down the levels of BOAA to a safe concentration can have good impact. The technology can be used to control Banana Bract Mosaic Virus (BBrMV), a most devastating disease of banana in Southeast Asia and India. A possible application of RNAi can be the down regulation of a key enzyme (CoA ligase) in the biosynthetic pathway of lignin in the two economically important Corchorus species, i.e. C. capsularis and C. olitorius.
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