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. 2020 Dec;32(12):3662-3673.
doi: 10.1105/tpc.20.00562. Epub 2020 Oct 19.

Soybean DICER-LIKE2 Regulates Seed Coat Color via Production of Primary 22-Nucleotide Small Interfering RNAs from Long Inverted Repeats

Jinbu Jia  1   2   3 , Ronghuan Ji  4 , Zhuowen Li  5   2   3 , Yiming Yu  1   2   3 , Mayumi Nakano  6 , Yanping Long  1   2   3 , Li Feng  1   2   3 , Chao Qin  4 , Dongdong Lu  1   2   3 , Junpeng Zhan  1   2   3   6 , Rui Xia  7 , Blake C Meyers  6   8 , Bin Liu  9 , Jixian Zhai  5   2   3
Affiliations

Soybean DICER-LIKE2 Regulates Seed Coat Color via Production of Primary 22-Nucleotide Small Interfering RNAs from Long Inverted Repeats

Jinbu Jia et al. Plant Cell. 2020 Dec.

Abstract

In plants, 22-nucleotide small RNAs trigger the production of secondary small interfering RNAs (siRNAs) and enhance silencing. DICER-LIKE2 (DCL2)-dependent 22-nucleotide siRNAs are rare in Arabidopsis (Arabidopsis thaliana) and are thought to function mainly during viral infection; by contrast, these siRNAs are abundant in many crops such as soybean (Glycine max) and maize (Zea mays). Here, we studied soybean 22-nucleotide siRNAs by applying CRISPR-Cas9 to simultaneously knock out the two copies of soybean DCL2, GmDCL2a and GmDCL2b, in the Tianlong1 cultivar. Small RNA sequencing revealed that most 22-nucleotide siRNAs are derived from long inverted repeats (LIRs) and disappeared in the Gmdcl2a/2b double mutant. De novo assembly of a Tianlong1 reference genome and transcriptome profiling identified an intronic LIR formed by the chalcone synthase (CHS) genes CHS1 and CHS3 This LIR is the source of primary 22-nucleotide siRNAs that target other CHS genes and trigger the production of secondary 21-nucleotide siRNAs. Disruption of this process in Gmdcl2a/2b mutants substantially increased CHS mRNA levels in the seed coat, thus changing the coat color from yellow to brown. Our results demonstrated that endogenous LIR-derived transcripts in soybean are predominantly processed by GmDCL2 into 22-nucleotide siRNAs and uncovered a role for DCL2 in regulating natural traits.

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Figures

Figure 1.
Figure 1.
CRISPR/Cas9-Engineered Mutations in GmDCL2a/2b Result in a Brown Seed Coat. (A) Deletion mutations at target sites in the Gmdcl2a/2b mutants. The gRNA target and PAM sequences are highlighted with boldface in black and blue, respectively. The gene structures of GmDCL2a and GmDCL2b are shown above. The coding sequence is shown as a dark box, the untranslated region is shown as a gray box, and the intron region is shown as a line. (B) Seed coat color phenotype of the Gmdcl2a/2b mutants. Bar = 1 cm. WT, wild type.
Figure 2.
Figure 2.
The Accumulation Levels of 22-Nucleotide siRNAs Are Sharply Decreased in the Gmdcl2a/2b Mutant. (A) The length distribution of siRNAs in the wild type (WT) and the Gmdcl2a/2b mutant. nt, nucleotide; TPM, transcripts per million. (B) Proportions of total locus count and siRNA accumulation of different kinds of loci. The numbers above each column represent the number of siRNA loci identified in different samples. nt, nucleotide; WT, wild type. (C) The length distribution of sRNAs from loci predominantly generating 21-, 22-, or 24-nucleotide (nt) siRNAs. TPM, transcripts per million; WT, wild type. (D) The comparison of sRNA accumulation levels of 22-nucleotide (nt) siRNA loci in seed coat between the wild type (WT) and the Gmdcl2a/2b mutant. The arrow and circle mark a miRNA-like locus shown in (F). The dashed lines indicate an sRNA fold change of 10. TPM, transcripts per million. (E) The comparison of sRNA accumulation levels of 21-nucleotide siRNA loci in seed coat between the wild type and the Gmdcl2a/2b mutant. The dashed lines indicate an sRNA fold change of 10. (F) A miRNA-like locus. The potential miRNA/miRNA* pairs and their expression levels in the wild type (WT) and the Gmdcl2a/2b (dcl2) mutant are shown in the left panel. The depth of uniquely mapped mRNA reads and the accumulation levels of uniquely mapped sRNA reads in seed coat are shown in the right panel. nt, nucleotide.
Figure 3.
Figure 3.
TE-Derived 22-Nucleotide siRNAs in Seed Coat. (A) TE enrichment analysis of 21-nucleotide, 22-, and 24-nucleotide (nt) siRNA loci. (B) Length distribution of sRNAs derived from different TE families. TPM, transcripts per million; WT, wild type. (C) Examples of 22-nucleotide (nt) TE siRNA loci (siRNA loci overlapping with TE) and 21-nucleotide TE siRNA loci. Only uniquely mapped RNA and sRNA reads are shown. TPM, transcripts per million; WT, wild type. (D) Log2 fold change of sRNA and mRNA accumulation levels of TE siRNA loci in the Gmdcl2a/2b mutant compared with the wild type (WT). The locus numbers (N) are indicated. nt, nucleotide; TPM, transcripts per million.
Figure 4.
Figure 4.
IR-Derived 22-Nucleotide siRNAs. (A) The IR enrichment analysis of 21-, 22-, and 24-nucleotide (nt) siRNA loci in seed coat samples. TPM, transcripts per million. (B) and (C) The RNA expression levels (B) in fragments per kilobase of exon model per million mapped reads (FPKM) and repeat lengths (C) of the repeat region of IRs overlapped with 21-, 22-, and 24-nucleotide (nt) siRNA loci in seed coat samples. TPM, transcripts per million. (D) Examples of 22- and 21-nucleotide (nt) siRNA loci in seed coat samples. Only uniquely mapped mRNA and sRNA reads are shown. TPM, transcripts per million; WT, wild type.
Figure 5.
Figure 5.
The 22-Nucleotide siRNAs Suppress the Expression of CHS Genes That Control the Color of the Seed Coat. (A) The gene structures of the I locus in the wild soybean W05 genome as well as cultivated soybean ZH13 and Tianlong1 genomes. The mRNA-seq and sRNA-seq results of the Tianlong1 seed coat are shown below the gene structures. *, Due to the duplication of CHS1 and CHS3 genes, the reads specifically mapped to CHS1 or CHS3, but not to other CHS genes, are also shown as “Uniquely mapped.” The junction read counts (n) are indicated. nt, nucleotide; WT, wild type. (B) The length distribution of CHS siRNAs in the wild type (WT). nt, nucleotide; TPM, transcripts per million. (C) The sRNA and mRNA accumulation levels of CHS genes in the Gmdcl2a/2b mutant and the wild type (WT). FPKM, fragments per kilobase of exon model per million mapped reads. TPM, transcripts per million. (D) A model of DCL2-dependent 22-nucleotide (nt) siRNAs regulating the expression levels of CHS genes.
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