**5. Diagnosis of an RNA silencing-induced phenotype using viral infection**

In the course of the analysis of *CHS* RNA silencing, the function of a virus-encoded protein called suppressor protein of RNA silencing was used to visually demonstrate the occurrence of RNA silencing [108, 111, 112, 115]. These suppressor proteins affect viral accumulation in plants. The ability of the suppressor protein to allow viral accumulation is due to its inhibi‐ tion of RNA silencing by preventing the incorporation of siRNAs into RISCs or by interfer‐ ing with RISCs [116]. Because of these features, RNA silencing can be suppressed in plants infected with a virus that carries the suppressor protein. When a soybean plant that has a yellow seed coat is infected with CMV, the seed coat restores pigmentation [108]. This phe‐ nomenon is due to the activity of gene silencing suppressor protein called 2b encoded by the CMV. This example typically indicates that, using the function of viral suppressor protein, we can "diagnose" whether an observed phenotypic change in a plant is caused by RNA si‐ lencing. A similar phenomenon has also been detected in maize [111] and petunia [112] lines, both of which have phenotypic changes through naturally occurring RNA silencing of an endogenous *CHS* gene, or a transgenic petunia line that has *CHS* co-suppression [115].

**4.4. Naturally occurring RNA silencing**

Relationships

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silencing in nontransgenic plants.

**infection**

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seeds with pigmented seed coats in thousands of accessions from natural populations in East Asia that we have screened (unpublished data).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

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

in the *CHS* gene cluster, which allows production of inverted repeat *CHS* RNA [110].

**5. Diagnosis of an RNA silencing-induced phenotype using viral**

In the course of the analysis of *CHS* RNA silencing, the function of a virus-encoded protein called suppressor protein of RNA silencing was used to visually demonstrate the occurrence of RNA silencing [108, 111, 112, 115]. These suppressor proteins affect viral accumulation in plants. The ability of the suppressor protein to allow viral accumulation is due to its inhibi‐ tion of RNA silencing by preventing the incorporation of siRNAs into RISCs or by interfer‐ ing with RISCs [116]. Because of these features, RNA silencing can be suppressed in plants infected with a virus that carries the suppressor protein. When a soybean plant that has a yellow seed coat is infected with CMV, the seed coat restores pigmentation [108]. This phe‐ nomenon is due to the activity of gene silencing suppressor protein called 2b encoded by the CMV. This example typically indicates that, using the function of viral suppressor protein,

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
