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Plant 22-nt siRNAs mediate translational repression and stress adaptation

Abstract

Small interfering RNAs (siRNAs) are essential for proper development and immunity in eukaryotes1. Plants produce siRNAs with lengths of 21, 22 or 24 nucleotides. The 21- and 24-nucleotide species mediate cleavage of messenger RNAs and DNA methylation2,3, respectively, but the biological functions of the 22-nucleotide siRNAs remain unknown. Here we report the identification and characterization of a group of endogenous 22-nucleotide siRNAs that are generated by the DICER-LIKE 2 (DCL2) protein in plants. When cytoplasmic RNA decay and DCL4 are deficient, the resulting massive accumulation of 22-nucleotide siRNAs causes pleiotropic growth disorders, including severe dwarfism, meristem defects and pigmentation. Notably, two genes that encode nitrate reductases—NIA1 and NIA2—produce nearly half of the 22-nucleotide siRNAs. Production of 22-nucleotide siRNAs triggers the amplification of gene silencing and induces translational repression both gene specifically and globally. Moreover, these 22-nucleotide siRNAs preferentially accumulate upon environmental stress, especially those siRNAs derived from NIA1/2, which act to restrain translation, inhibit plant growth and enhance stress responses. Thus, our research uncovers the unique properties of 22-nucleotide siRNAs, and reveals their importance in plant adaptation to environmental stresses.

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Fig. 1: Disruption of cytoplasmic RNA decay and DCL4 triggers massive production of 22-nt siRNAs and causes growth disorders.
Fig. 2: 22-nt siRNAs repress mRNA translation.
Fig. 3: 22-nt siRNAs mediate translational repression and siRNA amplification in an AGO1-dependent manner.
Fig. 4: Functional analysis and environmental induction of 22-nt siRNAs.

Data availability

Sequencing data are available at the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE136164. Source gel data for immunoblots and radiograms (Figs. 24 and Extended Data Figs. 2, 4, 6, 10) are provided in Supplementary Fig. 1; source data for all graphs (Figs. 14 and Extended Data Figs. 13, 5, 710) are also provided and are available with the online version of the paper.

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Acknowledgements

We thank X. Chen for assistance with AGO1-immunoprecipitation experiments; J. Jia for help with sRNA phase analysis; K. Kiyokawa for assistance with plasmid constructions and mRNA preparation; and Y. Tomari, A. Hutchins and P. Pimpl for critical comments on the manuscript and language editing. This work was supported by the National Natural Science Foundation of China (grant 91740203 to H.G.), the National Key Research and Development Program of China (grant 2018YFA0507101 to H.G.), the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (grants 2016ZT06S172 to J.Z. and B.L.), the Shenzhen Sci-Tech Fund (grant KYTDPT 20181011 104005 to J.Z. and B.L.), Grants-in-Aid for Scientific Research on Innovative Areas (‘Nascent-chain Biology’; grant 26116003 to H.-o.I.) and JST, PRESTO (grant JPMJPR 18K2 to H.-o.I.).

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Authors

Contributions

H.G., H.W. and B.L. conceived the project and designed the experiments; H.W. and X.Z. prepared the genetic materials; H.W. collected genetic phenotypes and carried out western blot assays, qRT–PCR assays and AGO1-immunoprecipitation sRNA sequencing with contributions from X.T., X.X. and S.S.; B.L. and Y.P. carried out sRNA-seq and mRNA-seq bioinformatics analyses; L.F., H.Z. and J.Z. performed sRNA phase analysis; Y.P., Z.T. and Q.L.-h. conducted polysome profiling; Y.L. carried out cytoplasmic and nuclear fraction isolation assays; H.-o.I. performed in vitro RNA silencing assays; H.G., H.W. and B.L. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Hongwei Guo.

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Peer review information Nature thanks Peter Waterhouse and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Differential expression analysis of siRNAs in ein5-1 dcl4-2 and ski2-2 dcl4-2 plants.

a, Normalized tasiRNA abundance in each genotype of 20-day-old plants. TPM, tags per million. Data are presented as means ± s.d.; n = 4 biologically independent samples. b, IGV of 21- and 22-nt siRNA abundance at eight TAS gene loci. c, 21- and 22-nt sRNA phasing score of tasiRNAs from representative TAS1a and TAS1c loci in the indicated genotypes. The y-axis shows the sRNA phasing score. See Methods for calling of sRNA phasing scores. d, Venn diagram depicting 22-nt-siRNA-generating genes that overlap between ein5-1 dcl4-2 (1,182 genes) and ski2-2 dcl4-2 (182 genes) plants. e, The top 20 gene loci and their FDRs in ein5-1 dcl4-2 (left, ed) and ski2-2 dcl4-2 (right, sd) plants, ranked by 22-nt siRNA abundance in each double mutant. Genes in red produce 22-nt siRNAs in both ein5-1 dcl4-2 and ski2-2 dcl4-2 plants. c, Col-0; ed, ein5-1 dcl4-2; sd, ski2-2 dcl4-2. FDR values are from Benjamini–Hochberg analysis; n = 4 biologically independent samples. Source Data

Extended Data Fig. 2 NIA1 and NIA2 mRNA and protein levels in ein5-1 dcl4-2 and ski2-2 dcl4-2 plants, and translational states of GTE2/7 in ein5-1 dcl4-2 plants.

a, mRNA-seq read abundance of NIA1/2 and SMXL4/5 genes in the indicated genotypes. b, Relative expression levels of NIA1/2 detected by qRT–PCR at 15:00 and 18:00 in 20-day-old Col-0, ein5-1 dcl4-2, ski2-2 dcl4-2 and ein5-1 ski2-3 plants. c, Separation of cytoplasmic and nuclear fractions of Col-0, ein5-1 dcl4-2 (ed) and ein5-1 dcl4-2 dcl2-1 (edd) plants. Tubulin and histone H3 proteins were used as cytoplasmic and nuclear markers, respectively. Cyto, cytoplasm; Nuc, nucleus. d, Relative expression level of NIA1 and NIA2 genes detected by qRT–PCR at 18:00 in the total and cytoplasmic fractions of 20-day-old plants. e, Verification of anti-NIA1 and anti-NR antibodies (the latter recognizing both NIA1 and NIA2) using nia1-3 and nia2-1 null alleles. HSP90 was used as a loading control. f, Protein levels of NIA1/2 detected by western blot at 15:00 and 18:00 in 20-day-old plants; sd, ski2-2 dcl4-2; sdd, ski2-2 dcl4-2 dcl2-1; ed, ein5-1 dcl4-2; edd, ein5-1 dcl4-2 dcl2-1; es, ein5-1 ski2-3; esdd, ein5-1 ski2-3 dcl4-2 dcl2-1. HSP90 was used as a loading control. g, GTE2/7 mRNA levels were normalized against PP2AA3 levels (locus AT1G13320). GTE2/7 expression in each polysomal fraction was calculated as the percentage of its expression in total RNA. Data are shown as means ± s.d. (b, d) or means with individual data points (technical replicates) (g). Numbers to the right of gels in cf indicate the molecular mass (in kDa) of proteins. Numbers of individual biological experiments are: a, n = 4; bd, n = 3; eg, n = 2. For gel source data, see Supplementary Fig. 1. Source Data

Extended Data Fig. 3 Accumulation of siRNAs and polysome profiles of the ein5-1 ski2-3 mutant.

a, Representative images of 20-day-old Col-0, ein5-1 ski2-3 and ein5-1 ski2-3 dcl4-2 dcl2-1 plants. Scale bar, 2 cm. Experiments were repeated three times with similar results. b, Normalized abundance of 21- and 22-nt siRNAs from all genes, excluding miRNAs and tasiRNAs, in each genotype. Data shown as means ± s.d.; n = 4 biological independent samples. c, IGV of 21- and 22-nt siRNAs from representative NIA1/2 gene loci in 20-day-old plants of each genotype. d, Polysome profiling of global translation efficiency in 20-day-old ein5-1 ski2-3 plants; 10–50% sucrose gradient absorbance (at 260 nm) was monitored in different fractions. Experiments were repeated twice with similar results. eg, Polysome distribution of NIA1/2 transcripts, with numbers on x-axis corresponding to fractions shown in d. NIA1/2 mRNA levels were normalized against PP2AA3 (AT1G13320). In e, f, NIA1/2 expression in each polysomal fraction was calculated as the percentage of its expression in total RNA. In g, TUB2 was used as a control. Data in eg are presented as means and individual data points (technical replicates). Experiments were repeated twice with similar results. Source Data

Extended Data Fig. 4 Genetic combinations of ein5-1 dcl4-2, ski2-2 dcl4-2 and sRNA-pathway-component mutants.

a, Western blot showing NIA1/2 protein levels at 15:00 in 20-day-old plants of the indicated genotypes. HSP90 was used as a control. bg, Genetic interaction of ein5-1 dcl4-2 and ski2-2 dcl4-2 with ago2-1 (b, c), ago4-1 (b, c), hen1-8 (d, e) and amp1-30 (f, g). Representative images of 20-day-old plants of the indicated genotypes. Scale bars, 2 cm. h, NR protein levels (anti-NR antibody recognizes both NIA1 and NIA2) in 20-day-old plants of the indicated genotypes. HSP90 was used as a control. a, h, Numbers to the right of gels indicate the molecular mass (in kDa) of proteins. The numbers of individual biological experiments are as follows: a, h, n = 2; bg, n = 3. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 5 AGO1 associates with 22-nt sRNAs in ein5-1 dcl4-2 plants.

a, Length distribution (18–26 nt) of AGO1-associated sRNAs in Col-0, ein5-1 dcl4-2 (ed) and ein5-1 dcl4-2 dcl2-1 (edd) plants. b, Classification (miRNAs, tasiRNAs and protein-coding-gene-derived siRNAs) and abundance of AGO1-associated sRNAs in Col-0 (Col-0_IP), ein5-1 dcl4-2 (ed_IP) and ein5-1 dcl4-2 dcl2-1 (edd_IP). The abundance of 21- and 22-nt sRNAs only was calculated. c, d, Sequence conservation analysis of AGO1-associated 21-nt (c) or 22-nt (d) sRNA using Weblogo 3 software. e, Venn diagram shows the overlap between 1,182 22-nt-siRNA-generating genes in ein5-1 dcl4-2 (ed_22DEG) and 642 AGO1-associated 22-nt-siRNA-generating genes in ein5-1 dcl4-2 (AGO1_IP_22). f, Heat map depicting the sRNA abundance (log2(TPM + 1)) of 1,182 22-nt-siRNA-generating genes from ein5-1 dcl4-2 plants in total (Input) and AGO1-immunoprecipitated (AGO1_IP) sRNA sequencing experiments. Source Data

Extended Data Fig. 6 Scheme for RNA silencing by 21- and 22-nt siRNAs in vitro.

a, Scheme underlying the experimental procedure of Fig. 3d. See Methods for further details. b, Structures of 21- and 22-nt siNIA2 siRNAs. The guide strands shown in red were radiolabelled. The grey strands show the passenger strand. The 5′-ends of siRNAs were phosphorylated and the 3′-ends were methylated. c, Schematic of 3× FLAG NIA2 mRNA and siNIA2 siRNA target site. d, We incubated 1.5 μM radiolabelled NIA2-siRNA duplexes (21- and 22-nt) with in vitro translated 3× FLAG-AGO1 in BY-2 lysate at 25 °C for 90 min. AGO1–RISC was then isolated using anti-FLAG antibody. Input (1/20) and AGO1-associated (IP (FLAG)) 21- and 22-nt siRNAs were then separated on denaturing gels and analysed by autoradiograph. Experiments were repeated twice with similar results. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 7 The ago1 mutant rescues the growth defects and eliminates the accumulation of 22-nt siRNAs in ein5-1 dcl4-2 and ski2-2 dcl4-2 plants.

a, b, Representative images of 20-day-old plants. Scale bars, 2 cm. Experiments were repeated three times with similar results. c, IGV of 22-nt siRNAs derived from NIA1/2 and SMXL4/5 gene loci in each genotype of 20-day-old plants. d, e, Heat map showing the abundance of 22-nt siRNAs (log2(TPM + 1)) from 1,182 (from ein5-1 dcl4-2 plants) and 182 (from ski2-2 dcl4-2 plants) genes that produce 22-nt siRNAs in the indicated genotypes. Source Data

Extended Data Fig. 8 Root growth and phloem formation are impaired in ein5-1 dcl4-2 and ski2-2 dcl4-2 plants.

a, Root length of 8-day-old plants of indicated genotypes. Data are means ± s.d.; n = 20 biologically independent roots. b, Number of meristematic cortex cells in 9-day-old plants. Data are means ± s.d.; n = 14 (for Col-0 and ski2-2) and 15 (for dcl4-2, ski2-2 dcl4-2 and ski2-2 dcl4-2 dcl2-1) biologically independent roots. c, Cotyledons from 18-day-old plants shown under bright field or with aniline-based callose visualization. Scale bars, 500 μm. n = 5 biologically independent cotyledons with similar results. See Methods for further details. d, Relative expression of phloem-related genes (CALS7 and NEN4) in the indicated genotypes of 20-day-old plants. Data are means ± s.d.; n = 3 biologically independent experiments. e, Measurement of relative anthocyanin content (ΔA/g.FW: absorbance at 525 nm minus absorbance at 650 nm per gram of fresh weight) in 20-day-old plants of the indicated genotypes. Data are means ± s.d.; n = 5 biologically independent samples. P-values in a, b, d are from two-tailed Student’s t-tests. Source Data

Extended Data Fig. 9 Transcriptome profiles and gene-ontology analysis of differentially expressed genes in ein5-1 dcl4-2 and ski2-2 dcl4-2 plants.

a, c, Venn diagrams depicting numbers of DCL2-dependent differentially expressed genes (at least twofold changes, FDR < 0.01) in ein5-1 dcl4-2 (a) and ski2-2 dcl4-2 (c) plants. b, d, Pie charts representing the numbers of upregulated and downregulated genes in ein5-1 dcl4-2 (b) and ski2-2 dcl4-2 (d) plants. eh, Representative gene-ontology enrichment analysis of upregulated (red) and downregulated (blue) genes in ein5-1 dcl4-2 (e, f) and ski2-2 dcl4-2 (g, h) plants. Circle sizes represent gene numbers and colour gradients indicate enrichment significance. Numbers at the bottom are the ratios of genes for each indicated biological process in the total differentially expressed genes. i, Heatmap depicting the log2(fold change) of representative genes involved in ABA biogenesis and response in 20-day-old Col-0 (c) versus ein5-1 dcl4-2 (ed), ein5-1 dcl4-2 dcl2-1 (edd), ski2-2 dcl4-2 (sd) and ski2-2 dcl4-2 (sdd) plants. j, qRT–PCR analysis of an ABA-responsive gene (RD29B) in 20-day-old plants of the indicated genotypes; data shown are means ± s.d. FDR values used to filter the differentially expressed genes in ah were obtained by Benjamini–Hochberg analysis; n = 3 biologically independent samples. Source Data

Extended Data Fig. 10 ABA and salt treatments induce the production of 22-nt siRNAs from NIA1 and NIA2 loci.

a, Normalized abundance of 21- and 22-nt siRNAs from all genes, excluding miRNAs and tasiRNAs, in each condition. b, Accumulation of 22-nt siRNAs from NIA1/2 gene loci in 14-day-old plants under 1 μM ABA treatment. c, Pie chart showing percentages of loci generating 22-nt siRNAs under 1 μM ABA treatment. d, Relative NIA1/2 mRNA expression level at 15:00 in 14-day-old plants on MS medium or 1 μM ABA medium. e, NIA1/2 protein levels at 15:00 in 14-day-old plants on MS medium or 1 μM ABA medium. HSP90 was used as a control to normalize the protein level. f, Representative images of 14-day-old seedlings grown on the indicated medium. Scale bar, 5 mm. g, Normalized abundance of 21- and 22-nt siRNAs from all genes, excluding miRNAs and tasiRNAs, in 15-day-old Col-0 plants under 250 mM NaCl treatment (data retrieved from GEO, accession number GSE66599). All P-values are from two-tailed Student’s t-tests. h, Normalized abundance of 22-nt siRNAs from NIA1 and NIA2 genes under the same conditions as in g). i, NIA1/2 protein level detected by western blot in 8-day-old wild-type seedlings upon 250 mM NaCl treatment for 24 h. HSP90 was used as a control. e, i, Numbers to the right of gels indicate the molecular mass (in kDa) of proteins. Data are presented as means ± s.d. in a, d, g, h. The numbers of individual biological experiments are as follows: a, d, f, n = 3; e, n = 2; i, n = 4. For gel source data, see Supplementary Fig. 1. Source Data

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Wu, H., Li, B., Iwakawa, Ho. et al. Plant 22-nt siRNAs mediate translational repression and stress adaptation. Nature 581, 89–93 (2020). https://doi.org/10.1038/s41586-020-2231-y

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