Epimutant Induction as a New Plant Breeding Technology

A small interfering RNA (siRNA) can be custom-produced in plant cells when a transgene comprising an inverted-repeat sequence is used to form the corresponding double-stranded (ds)RNA. This siRNA induces methylation of the homologous DNA through the RNA-directed DNA methylation (RdDM) pathway. As methylation of the promoter region causes transcriptional gene silencing (TGS), dsRNA for the promoter sequence of a gene can induce TGS of the gene, accompanied by modification of chromatin. Such epigenetic variation provides a novel technique to induce silencing of a target gene. Furthermore, once epigenetic variation has occurred, the siRNA need not be present to maintain the stable repression of transcription even in the subsequent generation. Thus, the induction of epigenetic changes is a potentially new plant-breeding technology to improve crops. Discipline: Plant breeding Additional key words: DNA methylation, RNA silencing, transcriptional gene silencing, siRNA longdistance transport *Corresponding author: e-mail tharada@cc.hirosakiu. ac.jp Received 10 March 2014; accepted 9 January 2015. Introduction Napoli et al. (1990) and van der Krol et al. (1990) reported an RNA interference (RNAi) phenomenon for the first time. They overexpressed a transgene of chalcone synthase (CHS), a key enzyme in flavonoid biosynthesis and the rate-limiting enzyme in anthocyanin biosynthesis, to generate a darker violet petunia flower, but unexpectedly obtained a white flower. Because the CHS transcription level was 50-fold lower than that in the wild-type flower, they concluded that as well as the transgene, the endogenous CHS gene had also been silenced. A similar phenomenon in a fungus (Neurospora crassa), termed “quelling”, was reported by Romano & Macino (1992). Furthermore, in an animal species (Caenorhabditis elegans), it was reported that introducing sense or antisense RNA of a gene to cells resulted in the mRNA degrading (Guo et al. 1995). Fire et al. (1998), Nobel Prize winner in 2006, clarified the RNAi mechanism by which dsRNA could cause endogenous mRNA to degrade. A number of subsequent studies revealed that dsRNA initiates gene silencing through inhibition at the post-transcriptional level (post-transcriptional gene silencing; PTGS) or the transcriptional level (transcriptional gene silencing; TGS). In the latter case, a small interfering RNA (siRNA) 24 nt long induces methylation of the fifth carbon cytosine residue within the region of sequence identity between the triggering RNA and the homologous DNA (Matzke et al. 2004). Although cytosine methylation is mostly limited to CG dinucleotide sequence contexts in animal genomes, in plants the methylation occurs not only in the symmetrical CG context but also in the CHG and CHH sequence contexts (where H=A, C or T) throughout their genomes. The cytosine methylation of each context is performed by respective methylation enzymes. The process of de novo DNA methylation is triggered by 24 nt siRNAs produced by RdDM. Furthermore, two plant-specific RNA polymerases, PolIV and PolV and the RDR2, DCL3 and AGO4 proteins operate in this pathway (Matzke et al. 2009, Haag & Pikaad 2011). Chemical modifications of DNA influence chromatin structure through histone methylation, while the methylation of histone H3 lysine (H3K9) plays an important role in establishing and maintaining DNA methylation (Grewal & Jia 2007). Epigenetic changes play pivotal roles in the developmental responses of plants. Recent studies have suggested