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In plants, small-interfering RNAs (siRNAs) serve as key regulators of gene expression. Although 24-nucleotide siRNAs are produced by DCL3 and mediate transcriptional silencing of transposons and pericentromeric chromatin through RNA-directed DNA methylation (Borges and Martienssen, 2015), 22-nucleotide siRNAs are processed by DCL2 and participate in transgene silencing and viral defense (Parent et al., 2015; Wang et al., 2018). However, because 22-nucleotide siRNAs are relatively rare in wild-type Arabidopsis (Arabidopsis thaliana; Henderson et al., 2006), and the loss of DCL2 doesn't produce obvious development defects (Henderson et al., 2006; Wang et al., 2018), their role is still largely unknown. Crops such as soybean (Glycine max) exhibit abundant 22-nucleotide siRNAs and are ideal systems to study their biogenesis, targeting, and functions in genome activity.
In a new study, Jinbu Jia and colleagues (Jia et al., 2020) combine genome editing, small RNA sequencing, and transcriptomics to investigate the functions of DCL2-dependent 22-nucleotide siRNAs in soybean (Jia et al., 2020). The authors used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing to simultaneously generate loss-of-function mutants of the two GmDLC2 genes in soybean, and performed small RNA sequencing on a selection of tissues. The analysis revealed a substantial decrease in 22-nucleotide siRNAs abundance throughout the genome in Gmdcl2, but also a drastic reduction of secondary 21-nucleotide siRNAs derived from the 22-nucleotide siRNAs. By investigating the distribution of these DCL2-dependent siRNAs in wild-type soybean, the authors identify that a large majority of 22-nucleotide siRNAs are overlapping with transposable elements (TEs), suggesting a possible role in silencing. In the Gmdcl2 mutant, the production of such TE-associated 22-nucleotide siRNAs is drastically reduced, but this does not correlate with local reduction in DNA methylation, nor accumulation of TE transcripts. Next, the authors examined the structural features of GmDCL2 substrates. The transcriptomic approach reveals that DCL2 preferentially processes PolII-transcribed long inverted repeats to generate 22-nucleotide siRNAs.
The most dramatic developmental phenotype resulting from GmDCL2 loss-of-function mutants is a darkening of the seed coat, from yellow in wild-type soybean to dark brown in Gmdlc2 (see figure). Seed color in soybean is dictated by the activity of Chalcone Synthase (CHS)-encoding genes that enable flavonoid biosynthesis that results in a dark brown seed coat. The yellow seed-coat phenotype is associated to the abundant production of siRNAs from a particular CHS gene cluster, with the siRNAs targeting and silencing other CHS genes in trans. With a higher genomic resolution provided by a de novo assembly of the CHS cluster, Jia et al. (2020) propose that the CHS cluster-generating siRNAs can form a long inverted repeat transcript that is possibly processed by DCL2. This hypothesis is further supported by the accumulation of DCL2-dependent 22-nucleotide siRNAs at this CHS cluster, which disappears in Gmdcl2. In the absence of CHS trans-targeting siRNAs, other CHS genes accumulate transcripts at a higher levels compared with wild type, and flavonoid accumulation results in a dark seed coat.
Loss-of-Function Mutations in GmDCL2 Results in the Darkening of the Seed Coat.
CRISPR-Cas9–engineered frame-shift mutations in GmDCL2a and GmDCL2b causes the overaccumulation of CHS transcripts and the darkening of the seed coats in the Tianlong1 cultivar.
(Adapted from Jia et al. [2020], Figure 1.)
Further from facilitating trait diversification, DCL2-dependent 22-nucleotide siRNAs are also involved in translational repression and stress adaptation (Wu et al., 2020). The high abundance of 22-nucleotide siRNAs in crops suggests more unexplored functions in regulating genome activity in plants.
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