**3. Methods of the induction of RNA silencing in soybean**

### **3.1. Transgene-induced RNA silencing**

Engineering novel traits through RNA silencing in soybean has been done using transgenes or virus vectors (Figure 1). RNA silencing in some transgenic soybean lines was induced by introducing a transgene that transcribes sense RNA homologous to a gene present in the plant genome, a phenomenon termed co-suppression [21]. This type of silencing was first discovered in transgenic petunia plants that had silencing of *CHS-A* for chalcone synthase [21, 22], in which mRNA transcribed from both *CHS-A* transgene and endogenous *CHS-A* gene was degraded. When sense transcripts from a transgene trigger RNA degradation, the pathway is also referred to as sense (S)-PTGS [19]. To obtain plants that have RNA silencing of a particular gene target, it is possible to generate co-suppressed plant lines as a byproduct of a transformation to overexpress the gene under the control of a strong promoter. Howev‐ er, a more promising method to induce RNA degradation is to transform plants with a con‐ struct comprising an inverted repeat (IR) sequence of the target gene, which forms dsRNA upon transcription (IR-PTGS) [39, 40]. This idea was based on the understanding of general mechanisms of RNA silencing in which dsRNA triggers the reaction of RNA degradation. The majority of transgene-induced RNA silencing in soybean have actually been done using such an IR construct. IR-PTGS can also be induced when multiple transgenes are integrated in the same site in the genome in an inverted orientation and fortuitous read-through tran‐ scription over the transgenes produces dsRNA.

An interesting finding reported in soybean is that RNA silencing is induced by a transgene that transcribes inverted repeats of a fatty acid desaturase *FAD2-1A* intron [41]. This result is contrary to the earlier belief that RNA silencing is a cytoplasmic event and intron does not trigger RNA degradation, which has been shown, for example, by using viral vector in plants [42] or by dsRNA injection to *C. elegans* cells [27], although irregular nuclear process‐ ing of primary transcripts associated with PTGS/RNAi has been reported previously [43]. The *FAD2-A1* intron-induced RNA silencing led to the understanding that RNA degrada‐ tion can take place in the nucleus [44]. Although whether RNA degradation in the nucleus is inducible for other genes or in other plants has not been known, this phenomenon is intrigu‐ ing because the involvement of nuclear events has been assumed for amplification of RNA silencing via transitivity [45] or intron-mediated suppression of RNA silencing [46].

Transcribing a transgene with a strong promoter tends to induce RNA silencing more fre‐ quently than that with a weak promoter [47]. For obtaining a higher level of transcription in soybean plants, the *Cauliflower mosaic virus* (CaMV) promoter has been used as in other plant species. Seed-specific promoters, such as those derived from the genes encoding subunits of β-conglycinin, glycinin, or Kunitz trypsin inhibitor, have also been used in soybean to in‐ duce seed-specific silencing, one feature that is exploited for metabolic engineering in soybean.

A gene construct that induces RNA silencing has been introduced to the soybean genome using either *Agrobacterium tumefaciens* infection or particle bombardment, which can pro‐ duce stable transgenic soybean lines that have altered traits. In addition, RNA silencing can be induced in soybean roots using *A. rhizogenes*-mediated transformation, which has been used for gene functional analysis. Methods for soybean transformation have been reviewed elsewhere [48].

### **3.2. Virus-induced gene silencing (VIGS)**

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‐

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

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

Engineering novel traits through RNA silencing in soybean has been done using transgenes or virus vectors (Figure 1). RNA silencing in some transgenic soybean lines was induced by introducing a transgene that transcribes sense RNA homologous to a gene present in the plant genome, a phenomenon termed co-suppression [21]. This type of silencing was first discovered in transgenic petunia plants that had silencing of *CHS-A* for chalcone synthase [21, 22], in which mRNA transcribed from both *CHS-A* transgene and endogenous *CHS-A* gene was degraded. When sense transcripts from a transgene trigger RNA degradation, the pathway is also referred to as sense (S)-PTGS [19]. To obtain plants that have RNA silencing of a particular gene target, it is possible to generate co-suppressed plant lines as a byproduct of a transformation to overexpress the gene under the control of a strong promoter. Howev‐ er, a more promising method to induce RNA degradation is to transform plants with a con‐ struct comprising an inverted repeat (IR) sequence of the target gene, which forms dsRNA upon transcription (IR-PTGS) [39, 40]. This idea was based on the understanding of general mechanisms of RNA silencing in which dsRNA triggers the reaction of RNA degradation. The majority of transgene-induced RNA silencing in soybean have actually been done using such an IR construct. IR-PTGS can also be induced when multiple transgenes are integrated in the same site in the genome in an inverted orientation and fortuitous read-through tran‐

tion are collectively called RNA silencing [34, 35].

Relationships

510

**3.1. Transgene-induced RNA silencing**

scription over the transgenes produces dsRNA.

producing new features in organisms including plants [36-38].

**3. Methods of the induction of RNA silencing in soybean**

RNA silencing has also been induced using a virus vector in soybean. Plants intrinsically have the ability to cope with viruses through the mechanisms of RNA silencing. When plants are infected with an RNA virus, dsRNA of the viral genome is degraded by the infect‐ ed plants [49, 50]. The dsRNA in the virus-infected cells is thought to be the replication in‐ termediate of the viral RNA [51] or a duplex structure formed within single-stranded viral RNA [52]. The viral genomic RNA can be processed into siRNAs, then targeted by the siR‐ NA/RNase complex. In this scenario, if a nonviral segment is inserted in the viral genome, siRNAs would also be produced from the segment. Therefore, if the insert corresponds to a sequence of the gene encoded in the host plant, infection by the virus results in the produc‐ tion of siRNAs corresponding to the plant gene and subsequently induces loss of function of the gene product (Figure 2). This fact led to the use of a virus vector as a source to induce silencing of a specific gene in the plant genome, which is referred to as virus-induced gene silencing (VIGS) [42, 53, 54]. So far, at least 11 RNA viruses and five DNA viruses were de‐ veloped as a plant virus vector for gene silencing, as listed previously [37]. Three vectors are now available in soybean: those based on *Bean pod mottle virus* (BPMV) [55], *Cucumber mosaic virus* (CMV) [56], and *Apple latent spherical virus* (ALSV) [57].

**Figure 2.** Virus-induced silencing of plant endogenous gene. When plants are infected with an RNA virus, dsRNA of the viral genome is degraded by the infected plants. The dsRNA in the virus-infected cells is thought to be the replica‐ tion intermediate or secondary-structured viral RNA. The viral genomic RNA can be processed into siRNAs. If a plant gene segment is inserted in the viral genome, siRNAs corresponding to the plant gene are produced and subsequently induce sequence-specific RNA degradation of the plant gene.
