**7. Methods to induce selective RNA silencing of duplicated genes**

When duplicated genes are subfunctionalized with only limited nucleotide changes and are expressed in overlapping tissues, specific silencing of each gene will be necessary for under‐ standing their function(s). Silencing a specific copy of duplicated genes can be achieved by targeting a gene portion whose nucleotide sequence is differentiated between the duplicated genes. A condition that allows this type of silencing involves a lack of silencing of the other copy of duplicated genes even when they have the same sequence in the other portions.

In plants, miRNAs or siRNAs promote production of secondary siRNAs from the 5′ up‐ stream region and/or the 3′ downstream region of the initially targeted region via produc‐ tion of dsRNA by RdRP. These secondary siRNAs can lead to silencing of a secondary target that is not directly targeted by the primary silencing trigger [124]. Studies so far have indi‐ cated that such a spread of RNA silencing, called transitive RNA silencing, does not occur with the majority of endogenous genes, although it can happen to a transgene [45; and refer‐ ences therein]. Assuming the lack of transitive RNA silencing, it is possible to induce silenc‐ ing of a specific copy of a duplicated gene. Targeting a region specific for each copy, e.g., the 3′ untanslated region (UTR), can induce silencing of the gene copy only, whereas targeting a region conserved in duplicated gene copies can induce silencing of the multiple gene copies simultaneously (Figure 3). Such selective RNA silencing was successful in a gene family of rice [125] and this strategy may work for analyzing functional diversification of duplicated genes in any plant species.

An alternative approach to suppress gene expression in plants is the use of artificial miR‐ NAs (Figure 4) (amiRNAs; also called synthetic miRNAs) [38, 126]. This approach involves modification of plant miRNA sequence to target specific transcripts, originally not under miRNA control, and downregulation of gene expression via specific cleavage of the target RNA. Melito *et al*. have used amiRNA to downregulate the leucine-rich repeat transmem‐ brane receptor-kinase gene in soybean [99]. miRNA has been extensively studied in soybean [127-130], information of which may be useful for designing amiRNAs. Because of its specif‐ icity, this method will be useful for silencing a limited copy of duplicated genes in soybean.

Induction of TGS by targeting dsRNA to a gene promoter can also be the method of choice. Gene silencing through transcriptional repression can be induced by dsRNA targeted to a gene promoter (Figure 4). However, until recently, no plant has been produced that harbors an endogenous gene that remains silenced in the absence of promoter-targeting dsRNA. We have reported for the first time that TGS can be induced by targeting dsRNA to the endoge‐ nous gene promoters in petunia and tomato plants, using a *Cucumber mosaic virus* (CMV) based vector and that the induced gene silencing is heritable. Efficient silencing depended on the function of the 2b protein encoded in the vector, which facilitates epigenetic modifi‐ cations through the transport of siRNA to the nucleus [131, 132]. The progeny plants do not have any transgene because the virus is eliminated during meiosis. Therefore, plants that are produced by this system have altered traits but do not carry a transgene, thus constituting a novel class of modified plants [131, 132]. We have also developed *in planta* assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcription‐ ally silenced by an epigenetic mechanism [133]. Using these systems, we found that genis‐ tein, a major isoflavonoid compound rich in soybean seeds, inhibits cytosine methylation and restores the transcription of epigenetically silenced genes [133]. Whether developing soybean seeds are resistant (or susceptible) to epigenetic modifications is an interesting issue in terms of both developmental control of gene expression and intentionally inducing TGS through epigenetic changes.

In plants, miRNAs or siRNAs promote production of secondary siRNAs from the 5′ up‐ stream region and/or the 3′ downstream region of the initially targeted region via produc‐ tion of dsRNA by RdRP. These secondary siRNAs can lead to silencing of a secondary target that is not directly targeted by the primary silencing trigger [124]. Studies so far have indi‐ cated that such a spread of RNA silencing, called transitive RNA silencing, does not occur with the majority of endogenous genes, although it can happen to a transgene [45; and refer‐ ences therein]. Assuming the lack of transitive RNA silencing, it is possible to induce silenc‐ ing of a specific copy of a duplicated gene. Targeting a region specific for each copy, e.g., the 3′ untanslated region (UTR), can induce silencing of the gene copy only, whereas targeting a region conserved in duplicated gene copies can induce silencing of the multiple gene copies simultaneously (Figure 3). Such selective RNA silencing was successful in a gene family of rice [125] and this strategy may work for analyzing functional diversification of duplicated

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

An alternative approach to suppress gene expression in plants is the use of artificial miR‐ NAs (Figure 4) (amiRNAs; also called synthetic miRNAs) [38, 126]. This approach involves modification of plant miRNA sequence to target specific transcripts, originally not under miRNA control, and downregulation of gene expression via specific cleavage of the target RNA. Melito *et al*. have used amiRNA to downregulate the leucine-rich repeat transmem‐ brane receptor-kinase gene in soybean [99]. miRNA has been extensively studied in soybean [127-130], information of which may be useful for designing amiRNAs. Because of its specif‐ icity, this method will be useful for silencing a limited copy of duplicated genes in soybean.

Induction of TGS by targeting dsRNA to a gene promoter can also be the method of choice. Gene silencing through transcriptional repression can be induced by dsRNA targeted to a gene promoter (Figure 4). However, until recently, no plant has been produced that harbors an endogenous gene that remains silenced in the absence of promoter-targeting dsRNA. We have reported for the first time that TGS can be induced by targeting dsRNA to the endoge‐ nous gene promoters in petunia and tomato plants, using a *Cucumber mosaic virus* (CMV) based vector and that the induced gene silencing is heritable. Efficient silencing depended on the function of the 2b protein encoded in the vector, which facilitates epigenetic modifi‐ cations through the transport of siRNA to the nucleus [131, 132]. The progeny plants do not have any transgene because the virus is eliminated during meiosis. Therefore, plants that are produced by this system have altered traits but do not carry a transgene, thus constituting a novel class of modified plants [131, 132]. We have also developed *in planta* assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcription‐ ally silenced by an epigenetic mechanism [133]. Using these systems, we found that genis‐ tein, a major isoflavonoid compound rich in soybean seeds, inhibits cytosine methylation and restores the transcription of epigenetically silenced genes [133]. Whether developing soybean seeds are resistant (or susceptible) to epigenetic modifications is an interesting issue in terms of both developmental control of gene expression and intentionally inducing TGS

genes in any plant species.

Relationships

516

through epigenetic changes.

**Figure 3.** Selective RNA silencing of duplicated genes. The gene 1 and gene 2 are produced as a consequence of gene duplication. They share a highly conserved nucleotide sequence in the 5′ region, while they have a different sequence in the 3′ region. When siRNAs corresponding to the conserved region are produced, they can induce RNA degradation of the transcripts from both genes (A). On the other hand, siRNAs corresponding to the 3′ region can induce gene 1 specific or gene 2-specific RNA degradation (B). A combination of these different approaches enables functional anal‐ ysis of duplicated genes.

**Figure 4.** Various pathways of RNA silencing that can be intentionally induced to suppress gene expression in plants. Transcripts from transgenes that have an IR sequence of a plant gene segment or viral genomic RNA that carries the segment can form dsRNA. These dsRNAs are subsequently processed into siRNAs in the cytoplasm. Similarly, amiRNA precursors transcribed from the amiRNA gene are processed into amiRNAs. These small RNAs can cause degradation of target gene transcripts, a process termed PTGS (A). When siRNAs corresponding to a gene promoter are produced, they can induce RdDM in the nucleus, thereby TGS of the target gene can be induced (B). Abbreviations: amiRNA, arti‐ ficial microRNA; PTGS, posttranscriptional gene silencing; RdDM, RNA-directed DNA methylation; TGS, transcriptional gene silencing.
