**4. Examples of RNA silencing reported in soybean**

### **4.1. Metabolic engineering by transgene-induced RNA silencing**

To the authors' knowledge, 28 scientific papers that describe metabolic engineering by transgene-induced RNA silencing in soybean have been published up to 2011 [58]. Because soybean seeds are valued economically for food and oil production, most modifications to transgenic soybean plants using RNA silencing are focused on seed components. Metabolic pathways in developing seeds have been targeted in terms of altering nutritional value for human or animals, e.g., changing seed storage protein composition [59, 60], reducing phytic acids [61, 62], saponin [63] or allergens [64], and increasing isoflavone [65]. Metabolic engi‐ neering has also targeted oil production [66-72]. These modifications were done by inhibit‐ ing a step in a metabolic pathway to decrease a product or by blocking a competing branch pathway to increase a product.

RNA silencing can be induced efficiently in soybean roots using *A. rhizogenes*-mediated root transformation. This method has been used for analyzing roles of gene products in nodule development and/or function, which occurs as a consequence of interaction between legume plants and the nitrogen-fixing symbiotic bacterium *Bradyrhizobium japonicum* [73-78]. The hairy root system was also used for analyzing roles of a MYB transcription factor in isoflavo‐ noid biosynthesis [79].

Transgene-induced RNA silencing has also been induced in leaf tissues for the β-glucuroni‐ dase gene [80] or the senescence-associated receptor-like kinase gene [81] and in calli for the amino aldehyde dehydrogenase gene to induce the biosynthesis of 2-acetyl-1-pyrroline [82].

### **4.2. Disease resistance acquired by transgene-induced RNA silencing**

Another focus of modifying soybean plants through RNA silencing is resistance against dis‐ eases, particularly to those caused by viruses. Resistance to viruses was achieved by trans‐ forming plants with genes or segments of genes derived from viruses and was referred to as pathogen-derived resistance [23, 24, 83, 84]. The resistance did not need protein translated from the transgene [85-87], which led to the understanding that RNA is the factor that con‐ ferred resistance to the plants and that the enhanced resistance is acquired via a mechanism analogous to that involved in co-suppression. Using this strategy, soybean plants resistant to *Soybean mosaic virus* (SMV) [88-90], or *Soybean dwarf virus* [91, 92] have been produced.

In addition to resistance against a virus, transgenic soybean plants resistant to cyst nema‐ tode (*Heterodera glycines*) have also been produced using RNA silencing [93], in which an in‐ verted repeat of the major sperm protein gene from cyst nematode was transcribed from the transgene. RNA silencing was elicited in cyst nematode after nematode ingestion of dsRNA molecules produced in the soybean plants; as a consequence, reproductive capabilities of the cyst nematode were suppressed. The effects of RNA silencing on controlling *H. glycines* [94] or root-knot nematode (*Meloidogyne incognita*) [95] infection have been assayed in soybean roots using *A. rhizogenes*-mediated transformation. On the other hand, this root transforma‐ tion method has also been used for analyzing a role of host genes in resistance against dis‐ eases caused by *Phytophthora sojae* [96, 97], *Fusarium solani* [98] or cyst nematode [99].

#### **4.3. Gene functional analysis by VIGS**

Viral RNA

Relationships

512

dsRNA

induce sequence-specific RNA degradation of the plant gene.

pathway to increase a product.

**4. Examples of RNA silencing reported in soybean**

**4.1. Metabolic engineering by transgene-induced RNA silencing**

siRNA CH3

CH3 CH3

Sequence-specific RNA degradation

**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

To the authors' knowledge, 28 scientific papers that describe metabolic engineering by transgene-induced RNA silencing in soybean have been published up to 2011 [58]. Because soybean seeds are valued economically for food and oil production, most modifications to transgenic soybean plants using RNA silencing are focused on seed components. Metabolic pathways in developing seeds have been targeted in terms of altering nutritional value for human or animals, e.g., changing seed storage protein composition [59, 60], reducing phytic acids [61, 62], saponin [63] or allergens [64], and increasing isoflavone [65]. Metabolic engi‐ neering has also targeted oil production [66-72]. These modifications were done by inhibit‐ ing a step in a metabolic pathway to decrease a product or by blocking a competing branch

RNA silencing can be induced efficiently in soybean roots using *A. rhizogenes*-mediated root transformation. This method has been used for analyzing roles of gene products in nodule development and/or function, which occurs as a consequence of interaction between legume plants and the nitrogen-fixing symbiotic bacterium *Bradyrhizobium japonicum* [73-78]. The

CH3

CH3 CH3

Replication

Secondary structure

Target gene segment

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

An advantage of VIGS is its ease for making a gene construct and introducing nucleic acids to cells. In addition, the effect of silencing can be monitored within a short time after inocu‐ lating plants with the virus. Because of these features, VIGS is suitable for gene function analysis [51, 100, 101] and has been used for gene identification via downregulating a candi‐ date gene(s) responsible for a specific phenomenon in soybean. VIGS was used to demon‐ strate that genes present in the genetically identified loci actually encode the genes responsible for the phenotype: VIGS of the putative *flavonoid 3′-hydroxylase* (*F3′H*) gene re‐ sulted in a decrease in the content of quercetin relative to kampferol, which indicated that the putative gene actually encodes the F3′H protein [56]; VIGS of the *GmTFL1b* gene, a soy‐ bean orthologue of *Arabidopsis TERMINAL FLOWER1* (*TFL1*) and a candidate gene for the genetically identified locus *Dt1*, induced an early transition from vegetative to reproductive phases, which indicated the identity between *Dt1* and *GmTFL1b* [102]. VIGS has also been used to identify genes involved in resistance of soybean plants against pathogens such as SMV, BPMV, *Pseudomonas syringae* or *Phakopsora pachyrhizi* [103-107].

### **4.4. Naturally occurring RNA silencing**

In addition to artificially induced RNA silencing, naturally occurring RNA silencing has also been known in soybean. Naturally occurring RNA silencing, involving mRNA degradation induced as a consequence of certain genetic changes, has been detected based on phenotypic changes. Most commercial varieties of soybean produce yellow seeds due to loss of pigmen‐ tation in seed coats, and this phenotype has been shown to be due to PTGS of the *CHS* genes [108, 109]. In cultivated soybean, there are varieties producing seeds with yellow seed coats and those producing seeds with brown or black seed coats in which anthocyanin and proan‐ thocyanidin accumulate. In contrast, wild soybeans (*Glycine soja*), an ancestor of the cultivat‐ ed soybean, have exclusively produced seeds with pigmented seed coats in thousands of accessions from natural populations in East Asia that we have screened (unpublished data). Thus, the nonpigmented seed coat phenotype was probably generated after domestication of soybean, and humans have maintained the plant lines that have *CHS* RNA silencing. The genetic change that induced *CHS* RNA silencing has been attributed to a structural change in the *CHS* gene cluster, which allows production of inverted repeat *CHS* RNA [110].

The occurrence of RNA silencing that leads to changes in pigmentation of plant tissues has also been reported for the *CHS* genes in maize [111] and petunia [112]. In petunia, a variety 'Red Star' produces flowers having a star-type red and white bicolor pattern, which resem‐ bles the flower-color patterns observed in transgenic petunias with co-suppression of the *CHS* genes [113], and in fact, the phenotype was demonstrated to be due to RNA silencing of the *CHS* genes in the white sectors [112]. Breeding of petunia was launched in the 1830s by crossing among wild species. The generation of the star-type petunia plants as a conse‐ quence of hybridizations between plant lines suggests that RNA silencing ability can be con‐ ferred via shuffling of genomes that are slightly different from each other. These phenomena also resemble the RNA silencing in a seed storage protein gene in rice, which is associated with a structural change in the gene region induced by mutagenesis [114], a case of RNA silencing in nontransgenic plants.
