Crop protection and productivity with less investment has a great impact on world economy. Several biotechnological approaches already have been implemented for the crop improvement programme. Among several biotechnological techniques, The RNAi mechanism has a great potential to produce disease resistant and different abiotic stress tolerant plants. RNAi mediated genetic engineering also helps to enhance nutrient content as well as helps to reduce toxin production in plants. Generally RNAi or RNA interference involves small RNA mediating gene silencing without affecting the other genes.
This review focuses the biogenesis, mechanism of action and the application of RNAi mediating silencing phenomena in different crop varieties for improvement of crop quality in agricultural field.
RNAi, also known as RNA interference, is commonly applied in the field of plant biotechnology to reduce the expression of harmful or undesirable gene and to characterize the function of a novel gene. This advance technology can be used in all eukaryotes, from yeast to mammals. This phenomenon was first established by Fire and Mello in 1998.
They introduced a long double stranded RNA (dsRNA) in Caenorhabditis elegans (Fire et al., 1998) and led to degradation of homologous host mRNA by ubiquitination (Lee et al., 1993). This dsRNA was found to be more useful for gene silencing mechanism than sense or antisense single stranded RNA. In plants, co-suppression and virus induced gene silencing are also elucidated by the principles of RNA interference.
In plants, the gene silencing was first reported on Petunia hybrida L. (Van der krol et al., 1990) when chalcone synthase gene (chsA) was introduced to enhance anthocyanin pigment (Napoli et al.
, 1990). Interestingly, in transgenic Petunia, the variegated flower color was observed because of the cosupression of the other endogenous loci which encodes crucial enzymes of anthocyanin biosynthesis pathway. RNAi is also referred as post transcriptional gene silencing (PTGS) (Bernstein et al., 2001; Jakowitch et al., 1999), transgene silencing or quelling (Klahre et al., 2002). Generally, it plays many important roles in post-transcriptional gene regulation, transposon regulation and plant-virus interaction. Specifically, RNAi negatively regulates the parasitic genetic elements in some eukaryotes thus helps the host to defend against RNA viruses and transgenes in plants. In addition, RNAi also regulates DNA methylation in plants.
In plants, RNAi also has a major role in crop improvement (Jagtab et al., 2011) by down regulating the expression of undesirable gene without affecting the expression of other genes. Gene modification by RNAi is one of the ways through which multiple characters can be modulated including increased resistance against different environmental stresses, improvement of nutritional quality of crops, suppression of toxic or allergen production, regulation of secondary metabolite production, alterations of male sterility or morphological features of plants (Saurabh et al. 2014).
Generally two main types of small RNAs are categorizeded as RNAi system (Bosher et al., 2000). They are small interfering RNA (siRNA) and microRNA (miRNA).The main and basic fundamental principle of RNAi is the degradation of mRNA upon binding with homologous dsRNA (18-24 nucleotide long) and repression of translation process by imperfect binding of miRNAs (20-25 nucleotide long) with coding sequence or 3′ untranslated region of target mRNA. In principle, miRNAs are generally endogenously synthesized in plants while siRNAs are externally designed and introduced in plants to modulate a specific gene expression. Generally the mode of function of RNAi is divided into two processes.
In this pathway, the dsRNAs are processed by the help of dicer enzyme into siRNA (in the duplex form) and is composed of two ~21 nucleotides strands with end overhangs (Bernstein et al., 2001). In plants, methylation stabilizes the overhang which is unlikely to animals. Dicer is an ATP-dependent ribonucleases, the member of RNAse III family (dsRNA specific) (Bernstein et al., 2001). During gene silencing mechanism, siRNA forms RISC loading complex by interaction with dsRNA binding protein (Agrawal et al., 2003). One of the strand of siRNA duplex act as guide strand binds with target gene for degradation (Kim et al., 2007) while other strand is degraded during RISC activation, and is known as passenger strand. The target mRNA strand which is nearly perfect complementary to bound siRNA, is cleaved by argonaute proteins, which itself acts an endonuclease (active component of RISC).
In addition, some other classes of small RNAs are also found in plants and known as repeat-associated small interfering RNAs (ra-siRNAs), heterochromatic small interfering RNAs (he-siRNAs), trans-acting small interfering RNAs (ta-siRNAs), secondary transitive siRNAs, primary siRNAs, and long small interfering RNAs (lsiRNAs). Among these, lsiRNAs is generated by DCL4 and AGO7 members of ARGONAUTE subfamily protein. This differs from the formation of 25 to 31 nucleotide long animals PIWI interacting RNAs. The biogenesis of PIWI RNAs are independent of Dicer and AGO subfamily protein.
microRNAs are small, highly conserved, 20-25 nucleotides long RNAs that regulate gene expression at post transcriptional level by binding to the UTR regions of target mRNAs. During mature miRNAs biogenesis, pri-miRNAs are first synthesized from MIR gene in the nucleus by RNA polymerase II (Jones- Rhoades et al., 2006). Then, an RNase III enzyme, named drosha cleaves pri- miRNA to form pre- miRNA. This pre- miRNA is exported from the nucleus to cytosol. In cytosol, dicer cuts pre- miRNA into miRNA, for incorporation into RISC.
Briefly, in plants, DCL1 catalyzes the formation of pre-miRNAs from pri-miRNAs by the help of two proteins like HYPONASTIC LEAVES1 (HYL1) and SERRATE (SE) proteins. The hairpin precursor like pre-miRNA is processed into 20 to 22-nt miRNA/miRNA* duplexes by the help of DCL1, HYL1 and SE proteins (Datta and Paul., 2015).
A wide range of techniques are available to introduce RNAi or activating RNAi pathway in plants. However, we will highlight the particle bombardment, Agrobacterium mediated transformation, virus induced gene silencing (VIGS) and hairpin RNA expression vector mediated transformation in this study.
Particle bombardment is a powerful tool for development of transgenic plants and the study of gene expression in transgenic plants. In this technology, siRNA or dsRNA constructs (DNAs) are coated with micro or nano particle (gold or tungsten) and are bombarded on explants to activate RNAi mediated silencing pathway. Synthetic siRNAs or dsRNAs have been delivered into the nucleus of plants by a low pressure helium pulse (gene gun or biolistic technology) (Kim et al., 2007). Generally silencing process starts after bombardment and continues up to next four to five days post bombardment. After two weeks, the silencing of the desired gene is validated by RNA blot hybridization.
According to Hilly and Liu, 2007, agro inoculation is a powerful mechanism by which Agrobacterium harbouring RNAi construct is injected into the intracellular spaces of leaves to generate RNAi mediated gene silencing process. In this Agro infiltration process first a gene of interest is cloned into a suitable vector and introduced into Agrobacterium strain. Then the bacterial solution is injected into the abaxial side of the leaf.
In this technique, a modified or recombinant virus which carries the target gene sequence is used to infect plants. There are so many different DNA and RNA viruses which have been modified to serve as VIGS or virus induced gene silencing, they are tobacco mosaic virus (TMV), tobacco rattle virus (TRV) and potato virus X (PVX). Among them all RNA virus mediated gene silencing vectors are not suitable because they contain anti silencing proteins such as tobacco etch virus (TEV) which has a tendency to interfere with host silencing machinery. On contrary, DNA viruses are rarely used as expression or silencing vectors due to their large size that hampers the movement. For silencing endogenous plant genes, the homologous gene fragments are required to be cloned and incorporated inside the virus. This process was first observed in RNA viruses (inserting sequences into the TMV). The two genes such as phytoene desaturase (PDS) and chalcone synthase (CHS) have been used to identify the effect of VIGS in plants. Generally PDS gene has a function on antenna complex in thylakoid membranes and protects chlorophyll from photo oxidation mediated degradation. By silencing of PDS gene, a drastic fall in leaf carotene content causes photo bleaching in plants while the over expression of CHS gene caused albino phenotype due to co-suppression of other genes of different loci.
This powerful RNAi technique is successfully applied in a wide variety of species for crop improvement programme (Zhang et al., 2015). It can increase nutritional value in edible seeds as well as reduce toxin production by modifying the metabolic pathway of plants. Many novel traits are also engineered in plants through RNAi. They are enlisted in this study in Table 1-3.
Table 1: RNAi in different trait modification
Food security is a major challenging issue in modern day agriculture. In the last decade, different biotechnological techniques have been implemented successfully in crop improvement programme. Among them, RNAi was also used widely to offer many advantages in crop production, nutrient enrichment and to enhance tolerance against environmental threats (Senthil and Mysore., 2010). Recently CRISPR (clustered regularly interspaced short palindromic repeat) has been proposed a silver bullet in agricultural field and CRISPR-Cas9 technology is used to modulate the gene expression including activation or down regulation (knock in or knock out) in plants. Several advancement of CRISPR-Cas mediated genome editing technology also will help to improve crop quality and production during any environmental stress in future.