ABSTRACT
Small non-protein coding RNAs, generated as short double-stranded duplexes, consisting of “guide” and “passenger” strands play a pivotal role in regulating the expression of target genes. The guide strand is loaded onto a multi-protein complex called RNA-induced silencing complex (RISC) and RNA induced transcriptional silencing (RITS). RISC is a multi-protein complex consisting of an Argonaute protein with Piwi/Argonaute/Zwille (PAZ) domain, which binds to small RNA, and Piwi domain that is responsible for endonucleolytic activity causing the cleavage of small RNA-messenger RNA (mRNA) complex and mediate regulation of target sequences. RITS is RNAi effector complex that leads to the heterochromatin assembly by histone modification. RITS consists of the chromodomain protein, Chp1, and the other components including Ago1 and Tas3. Short interfering RNA (siRNA) loaded in this complex interacts with dg-dh repeats of centromere and cause methylation of lysine 9 of histone H3 via Clr4 methyltransferase. Several types of small RNAs like micro RNAs (miRNAs), siRNAs, tasi-RNAs, and Piwi-interacting RNAs (piRNAs), differ from each other in their biogenesis, mode of action and function. Among them miRNA and siRNA are important riboregulators and have been extensively studied in eukaryotes. miRNAs act post-transcriptionally in mRNA degradation and translational repression via partial or full complementary base pairing at spatial and temporal level. siRNAs can act in a similar manner as miRNAs, but in addition have a role in DNA methylation, histone modification and mRNA degradation via specific base pairing allowing no mismatches. The mechanism of action of siRNA and miRNA are termed as auto-silencing and hetero-silencing, respectively. Understanding of the biogenesis and mode of action including composition of RISC complex have led to the development of various gene silencing strategies such as virus induced gene silencing (VIGS), RNAi and artificial miRNAs with their inherent advantages and disadvantages. VIGS is a recent approach in which the function of a gene in question can be characterized exploiting RNA-mediated antiviral defense machinery in plants. This technique has been studied well in Arabidopsis thaliana, Nicotiana benthamiana, tomato, and barley. It has proved to be efficient in gaining knowledge about entire gene families. VIGS, as compared to other functional genomics approaches, is rapid, overcomes functional redundancy of genes, avoids plant transformation, is less expensive and can be used to test gene function in multiple genetic backgrounds. Apart from targeted silencing for genetic manipulation for crop improvement; gene silencing has also been applied to understand plant development and adaptation through generation of loss-of-function, knock-out/knock-down mutants. RNAi technology is being employed to manipulate important agronomic traits such as height (miR167 in tomato), root (miR171 in A. thaliana), leaf morphology (miR319, miR164), flower development (miR167 in tomato, miR159 in A. thaliana, etc.), seed productivity (OsmiR156), seed development (miR397 and miR328 in Rice), oil quality (miR156, miR167, and miR6029in Brassica napus), viral and fungal (miR156) tolerance, and many others such as fruit shelf life (miR156/miR157), metabolite content (miR393), and anthocyanin content (miR156)in major crop improvement programs. This chapter will summarize how endogenous/native and artificially synthesized small RNAs are being used to manipulate traits of agricultural significance.
