**2. Mechanisms and diverse pathways of RNA silencing**

Gene silencing is one of the regulatory mechanisms of gene expression in eukaryotes, which refers to diverse RNA-guided sequence-specific inhibition of gene expression, either at the posttranscriptional or transcriptional level [19, 20]. Post-transcriptional gene silencing (PTGS) was first discovered in transgenic petunia plants whose flower color pattern was changed as a consequence of overexpression of a gene that encodes the key enzyme for an‐ thocyanin biosynthesis in 1990 [21, 22]. Similar phenomena have also been reported for plants transformed with various genes, which include virus resistance of plants that have gene or gene segments derived from the viral genome [23, 24]. Because of these findings, gene silencing is thought to have developed to defend against viruses. Several lines of re‐ search in plants indicated that double-stranded RNA (dsRNA) is crucial for RNA degrada‐ tion [25, 26]. The potency of dsRNA to induce gene silencing was demonstrated in *Caenorhabditis elegans* by injecting dsRNA into cells in 1998 [27], and the phenomenon was termed RNA interference (RNAi).

functions of the single gene progenitor, an evolutionary consequence for duplicated loci referred to as subfunctionalization [4, 11, 13]. In addition to this notion, models involving epigenetic silencing of duplicate genes [14] or purifying selection for gene balance [15, 16] have also been proposed. In soybean, differential patterns of expression have often been de‐ tected between homoeologous genes [17, 18], which indicates that subfunctionalization has

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

When the extent of subfunctionalization is limited, mutations in only one of multiple cog‐ nate gene copies do not often result in phenotypic changes. Therefore, methods that allow suppression of all copies of the duplicated gene are required for analyzing gene function or engineering novel traits. RNA silencing refers collectively to diverse RNA-mediated path‐ ways of nucleotide-sequence-specific inhibition of gene expression, either at the posttran‐ scriptional or transcriptional level, which provides a powerful tool to downregulate a gene or a gene family [19, 20]. Suppression of gene expression through RNA silencing is particu‐ larly useful for analyzing the function(s) of duplicated genes or engineering novel traits be‐ cause it allows silencing of multiple cognate genes having nucleotide sequence identity. In fact, to produce soybean lines that have a novel trait, researchers have frequently used RNA

In this review, we describe application of RNA silencing to understand the roles of genes or engineering novel traits in soybean. We describe methods to induce simultaneous silencing of duplicated genes and selective silencing of each copy of duplicated genes through RNA silencing. In addition to intentionally induced RNA silencing, we also refer to naturally oc‐ curring RNA silencing. Based on our knowledge of RNA silencing in soybean, we propose a hypothesis that plants may have used subfunctionalization of duplicated genes as a means to avoid the occurrence of simultaneous silencing of duplicated genes, which could be dele‐

Gene silencing is one of the regulatory mechanisms of gene expression in eukaryotes, which refers to diverse RNA-guided sequence-specific inhibition of gene expression, either at the posttranscriptional or transcriptional level [19, 20]. Post-transcriptional gene silencing (PTGS) was first discovered in transgenic petunia plants whose flower color pattern was changed as a consequence of overexpression of a gene that encodes the key enzyme for an‐ thocyanin biosynthesis in 1990 [21, 22]. Similar phenomena have also been reported for plants transformed with various genes, which include virus resistance of plants that have gene or gene segments derived from the viral genome [23, 24]. Because of these findings, gene silencing is thought to have developed to defend against viruses. Several lines of re‐ search in plants indicated that double-stranded RNA (dsRNA) is crucial for RNA degrada‐ tion [25, 26]. The potency of dsRNA to induce gene silencing was demonstrated in *Caenorhabditis elegans* by injecting dsRNA into cells in 1998 [27], and the phenomenon was

**2. Mechanisms and diverse pathways of RNA silencing**

occurred in these genes.

Relationships

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silencing induced by a transgene.

terious to the organism.

termed RNA interference (RNAi).

**Figure 1.** Pathways of RNA silencing used to downregulate a target gene through RNA degradation. Posttranscrip‐ tional gene silencing is triggered by dsRNA. Transcripts from transgenes that have an IR sequence can form dsRNA. Sense transcripts can produce dsRNA through the synthesis of complementary strand by RdRP. The replication inter‐ mediate or duplex structures formed within single-stranded RNA of the viral genome can also provide dsRNA. These dsRNAs are processed into siRNAs by the endonuclease Dicer. The siRNA is loaded into the RISC complex that contains AGO and guides the RISC complex to the mRNA by base-pairing. The RISC complex cuts the mRNA, which is subse‐ quently degraded. Abbreviations: IR, inverted repeat; RdRP, RNA-dependent RNA polymerase; dsRNA, double-strand‐ ed RNA; siRNA, short interfering RNA; RISC, RNA-induced silencing complex; AGO, Argonaute.

Subsequent genetic and biochemical analyses in several organisms revealed that PTGS and RNAi share the same pathway and consist of two main processes: (i) processing of dsRNA into 20–26-nt small RNA molecules (short interfering RNA; siRNA) by an enzyme called Dicer that has RNaseIII-like endonuclease activity; (ii) cleavage of RNA guided by siRNA at a complementary nucleotide sequence in the RNA-induced silencing complex (RISC) con‐ taining the Argonaute (AGO) protein (Figure 1) [28]. The formation of dsRNA from singlestranded sense RNA was explained by the synthesis of its complementary strand by RNAdependent RNA polymerase (RdRP). This process provides templates for Dicer cleavage that produces siRNAs and consequently allows amplification of silencing [29]. siRNA is re‐ sponsible for not only induction of sequence-specific RNA degradation but also epigenetic changes involving DNA methylation and histone modification in the nucleus, which leads to transcriptional gene silencing (TGS) [30]. It has become evident that siRNA plays a role in systemic silencing as a mobile signal [31, 32]. In addition to siRNA, small RNA molecules called micro RNAs (miRNAs) are also involved in negative regulation of gene expression [33]. These gene silencing phenomena that are induced by sequence-specific RNA interac‐ tion are collectively called RNA silencing [34, 35].

RNA silencing plays an important role in many biological processes including development, stability of the genome, and defense against invading nucleic acids such as transgenes and viruses [20, 29, 30]. It can also be used as a tool for analyzing specific gene functions and producing new features in organisms including plants [36-38].
