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Review
. 2024 Jan 30;36(2):246-275.
doi: 10.1093/plcell/koad253.

The plant siRNA landscape

Hervé Vaucheret  1 , Olivier Voinnet  2
Affiliations
Review

The plant siRNA landscape

Hervé Vaucheret et al. Plant Cell. .

Abstract

Whereas micro (mi)RNAs are considered the clean, noble side of the small RNA world, small interfering (si)RNAs are often seen as a noisy set of molecules whose barbarian acronyms reflect a large diversity of often elusive origins and functions. Twenty-five years after their discovery in plants, however, new classes of siRNAs are still being identified, sometimes in discrete tissues or at particular developmental stages, making the plant siRNA world substantially more complex and subtle than originally anticipated. Focusing primarily on the model Arabidopsis, we review here the plant siRNA landscape, including transposable elements (TE)-derived siRNAs, a vast array of non-TE-derived endogenous siRNAs, as well as exogenous siRNAs produced in response to invading nucleic acids such as viruses or transgenes. We primarily emphasize the extraordinary sophistication and diversity of their biogenesis and, secondarily, the variety of their known or presumed functions, including via non-cell autonomous activities, in the sporophyte, gametophyte, and shortly after fertilization.

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Conflict of interest statement

Conflict of interest statement. None declared.

Figures

Figure 1.
Figure 1.
RNAi sources and associated machineries. A) Sources of miRNAs. miRNAs derive from the of POLII-transcribed RNAs with imperfect foldback stem-loop structures. 1. Most MIRs produce a single miRNA via DCL1, but 2. some MIRs can produce up to 3 miRNAs. 3. Young MIRs produce multiple miRNAs via DCL4, of which one is considered the major product. B) Sources of primary siRNAs. siRNAs derive from perfectly paired dsRNA with 4 possible origins: 1. POLII-transcribed long inverted repeats produce RNAs with foldback stem-loop structures similar to, but longer than, those derive from young MIR genes. 2. POLII-transcribed genes arranged in convergent orientation on opposite DNA strands produce mRNAs complementary on the overlapping region 3. POLII-transcribed genes produce a fraction of abRNAs lacking a cap or a polyA tail, which can be converted to dsRNA by RDRs (mostly RDR1 and RDR6) when they evade RNA quality control. 4. POLIV transcribes short RNAs converted to dsRNA by RDR2. When produced in the nucleus, dsRNA are processed into 24-nt siRNAs by DCL3 or, alternatively, into 22-nt siRNAs by DCL2, whereas cytosolic dsRNAs are processed into 21-nt siRNAs by DCL4 or, alternatively, into 22-nt siRNAs by DCL2. C) Argonaute machineries. Depending on their size and 5′-terminal-nucleotide identities, siRNAs are loaded into different AGO proteins, which execute either PTGS or RdDM/TGS. Of note, AGO7 and AGO10 are not depicted in the figure as they have only been reported to load particular miRNAs.
Figure 2.
Figure 2.
Biogenesis of 21-22-nt siRNAs from non-TE loci. A) AGO1-dependent ta-siRNAs. Targeting TAS1a/b/c/or TAS2/4 RNAs by the 22-nt miR173/AGO1 complex attracts SGS3 and RDR6 to the cleavage products to spawn a first round of ta-siRNAs, including TAS1c3′D6(−). This ta-siRNA in complex with AGO1 also targets TAS1a/1b/1c/2 RNAs, resulting in double-cleaved, uncapped, and nonpolyadenylated TAS RNAs that are better substrates for RDR6. Successive dicing by DCL4 generates phased ta-siRNAs from both ends of the dsRNA. B) AGO7-dependent ta-siRNAs. Dual targeting of TAS3 RNAs by the 21-nt miR390/AGO7 complex attracts SGS3 and RDR6 to the cleavage products to produce phased ta-siRNAs from both dsRNA ends. In Arabidopsis, only 1 miR390 site is cleavable, resulting in phased ta-siRNAs from only 1 end of the dsRNA. C) AGO-dependent pha-siRNAs. 1. Targeting Arabidopsis protein-coding RNAs with the 22-nt miRNA/AGO1 complex attracts SGS3 and RDR6 to the cleavage products to spawn pha-siRNAs similarly to ta-siRNAs from TAS RNAs targeted by a single miRNA. 2. In some monocots, noncoding PHAS genes are targeted by miR2118 to produce 21-nt premeiotic pha-siRNAs. 3. In the same species, other noncoding PHAS genes are targeted by miR2275 to produce 24-nt meiotic pha-siRNAs. D) DCL-dependent nat-siRNAs. Protein-coding genes arranged as convergent units often involves a constitutively expressed gene and a stress-inducible gene. Dual expression produces bimolecular dsRNA, which upon DCL cleavage generates nat-siRNAs and 2 cleavage products lacking a polyA tail, which serves as substrates for RDR6. Subsequent processing of RDR6-derived dsRNA generates siRNAs from the 2 RNAs outside of the overlapping regions. E) RQC-dependent siRNAs. RQC normally eliminates abRNAs produced from protein-coding genes. When RQC is dysfunctional or impaired during virus infection, abRNAs become substrates for RDR1 or RDR6, resulting in the production of siRNAs from thousands of protein-coding mRNAs. F) UV-dependent siRNAs. UV irradiation induces DNA damages prevalently in intergenic regions, resulting in the production of 21-nt siRNA through the action of POLIV, RDR2 and a likely nuclear form of DCL4 (DCL4NLS, see also Fig. 6E). The DNA DAMAGE-BINDING PROTEIN 2 (DDB2), AGO1 and 21-nt uv-siRNAs form a chromatin-bound complex possibly facilitating sequence-specific recruitment of DNA repair-recognition factors at damaged sites.
Figure 3.
Figure 3.
Biogenesis of 24-nt siRNAs RdDM establishment and maintenance. A) RdDM pathway. Euchromatin exhibits H3K4me3 marks and is transcribed by POLII, whereas heterochromatin exhibits H3K9me2 marks and cytosine methylation. The former attracts SHH1, which, together with CLSY, likely recruits POLIV. The POLIV-interacting ZMP protein is enriched at hetero/eu- chromatic “junction” regions by presumably monitoring local changes in H3K4 methylation, thus promoting POLIV activity on H3K4-poor chromatin and impeding it in the H3K4-rich regions. POLIV produce short transcripts called P4-RNAs. Converted into dsRNA by RDR2, P4-RNAs are diced into 24-nt siRNAs by DCL3. The ensuing 24-nt siRNA/AGO4 complex interacts with POLV's carboxy-terminal domain enabling its annealing to nascent POLV transcripts. This attracts DRM2 to further methylate DNA, while HISTONE DEACETYLASE 6, JMJ14, and SUPPRESSOR OF VARIEGATION 4,5,6 reinforce the heterochromatic state. B) Detailed biogenesis of POLIV-dependent DCL3 substrates. 1. POLIV initiates transcription at POLII-like TSSs and produce 26- to 45-nt P4-RNAs often displaying a 5′ adenosine. 2. Complementary RNA synthesis by RDR2 preferentially starts from the third nucleotide. 3. RDR2's terminal-transferase activity adds an untemplated 3′-nucleotide to the P4-RNA. 4. DCL3 preferentially dices 5′A dsRNA explaining the bias for 5′ A in AGO4 associated 24-nt siRNAs. C) Targeting of POLV RNA by AGO4:siRNA complexes. Reiterated slicing events by AGO4 simultaneously enables AGO4-POLV dissociation and tethering of AGO4 to cleaved POLV RNA fragments. Ensuing AGO4:siRNA:ncRNA complexes might extend DRM2 local recruitment and RdDM without impeding POLV progression along the chromatin. D) Methylation maintenance. Methylation at CHH sites requires the constant action of DRM2 guided by 24-nt siRNAs. Methylation at CG and CHG sites, while established de novo by 24-nt siRNAs and DRM2, is maintained in a DNA-replication-dependent manner by MET1 at CG sites, and by the self-reinforcing action of CMT3 (among other factors) at CHG sites. All processes require the (hetero)-chromatin remodeler DMM1 presumably relaxing chromatin during DNA replication.
Figure 4.
Figure 4.
Epigenetic activation of TEs in mutant sporophytes and RdDM onset on EVD. A) EasiRNA biogenesis in ddm1 mutant sporophytes. Loss of DDM1 causes chromatin decompaction and gene expression at many TE and some MIRNA loci. Certain epigenetically activated TEs are targets of these epigenetically-activated miRNAs (eamiRNAs) in an AGO1-dependent manner. Their size (22 nt) or dual-hit mode of action (not shown) promotes RDR6 recruitment to produce TE-derived dsRNA in a DCL4-dependent manner. Presumably loaded into AGO1, these trigger PTGS of sequence-homologous TEs. B) PTGS-to-RdDM transitions underpin EVD de novo silencing. Upon reactivation in ddm1 or met1 epigenetic recombinant-inbred lines, EVD transposes despite production of RDR6-DCL4–dependent 21-nt siRNAs derived specifically from the EVD shGAG subgenomic RNA upon ribosome stalling and 5′OH RNA production. Its seclusion within VLPs likely protects the full-length EVD (flGAG-POL) against AGO1-mediated PTGS. When the EVD copy number reaches 40 to 50, the large amount of dsRNAs accrued over inbred generations likely saturates the capacity of DCL4, leading to its processing into 24-nt siRNAs by DCL3. Loaded into AGO4, these initiate RdDM, first on the shGAG-matching region of the GAG open Reading frame, and then onto the LTR on possible antisense transcript by POLV. Self-enforcing POLIV/V-dependent cytosine methylation and chromatin compaction on the LTRs eventually leads to TGS of EVD.
Figure 5.
Figure 5.
TE-derived siRNAs in gametophytes and seeds. A)TE methylation reprogramming in the Arabidopsis male germline. Left: at the meiocyte stage, polyploid somatic nurse cells forming the nourishing tapetum produce mobile P4-siRNAs (tap-siRNAs) from a subset of silent “HyperTEs” selectively enriched in CLASSY 3. Upon presumed CLASSY 3-mediated POLIV recruitment, up to 1,000 folds more P4-siRNAs accumulate at these compared with other TE loci, mediating cis-RDRM and silencing of sequence-related TEs within the tapetum. Upon movement into adjacent meiocytes, HyperTE-derived P4-siRNAs also mediate AGO4-dependent trans-RdDM by imperfectly pairing to target DNA/RNA produced at a subclass of MetGenes whose expression defines a paternal identity that persists through meiosis and mitosis into the mature pollen. For unexplained reasons, trans-methylation of MetGenes does not occur in tapetal nurse cells. Right: in the mature pollen grain, where a DDM1-deficient and DME-proficient vegetative cell encases 2 sperm cells (SCs), POLIV-dependent TE-derived 21-nt pollen-siRNAs are thought to move from the vegetative cell's nucleus (VN) into the SCs. Pollen-siRNA biogenesis occurs via an as yet-undefined pathway (scenario 1) that might involve longer-than-normal P4-RNAs spawned from hypomethylated DNA, subsequently targeted by TE-derived miRNAs such as the 22-nt-long miR845. Targeting promotes recruitment of RDR2 and/or possibly RDR6 to synthesize dsRNA processed into 21-nt pollen-siRNAs by the nuclear DCL4NLS isoform. Note that this pathway is entirely speculative. Alternatively (Scenario 2), POLIV requirement might be indirect and occur earlier during meiocyte differentiation, with POLIV andRDR2-dependent processes (e.g. tap biogenesis and action) ultimately delineating a paternal lineage-specific gene expression landscape of which a product might be inherited and amplified in the VN or VC where it would specifically activate pollen-siRNA biogenesis. See main text for details. As for scenario 1, scenario 2 is entirely speculative. B)TE methylation reprogramming in the Arabidopsis female germline. In a manner conceptually analogous to tap-siRNA biogenesis and action in the male germline, 24-nt siren-RNA are produced in the female sporophyte from a discrete number of TE loci enriched in CLASSY 3. In the sporophyte, siren-RNA mediate cis- and trans-methylation influencing gene expression and TE silencing. siren-RNA are also thought to move into the female gametophyte composed of a large and bi-nucleate central cell encasing an egg cell, among other cell types. As established for tap-siRNAs, siren-RNA might mediate trans-methylation and TE silencing therein, though this still requires experimental validation. Additionally, indirect evidence based on methylome comparisons suggests movement of TE-derived 24-nt siRNAs from the central cell's nucleus (which undergoes active demethylation via DME, resulting in TE activation) to the egg. C) Model for TB in paternal excess endosperm. Left: seeds consist in a diploid coat of maternal origin, a triploid endosperm with a 2:1 matrigenic/patrigenic ratio, and a diploid zygote with a 1:1 matrigenic/patrigenic ratio. Right: 1. Fertilization of a diploid central cell by haploid pollen brings 21-nt pollen-siRNAs in amounts that are insufficient to offset RdDM and TGS of PEGs by female 24-nt (siren?)-siRNAs. The ensuing adequate PEG expression levels allow normal endosperm and seed development. 2. Fertilization of a diploid central cell by diploid pollen provides an excess of 21-nt pollen-siRNAs, which overcomes RdDM and TGS of PEGs by female 24-nt (siren?)-siRNAs. This results in PEG overexpression, abnormal endosperm development and, ultimately, seed abortion.
Figure 6.
Figure 6.
PTGS and RdDM are systemic processes. A–D)NITRATE REDUCTASE (NIA) PTGS initiates locally and spreads throughout transgenic tobacco plants. 35S:NIA2 tobacco lines spontaneously trigger PTGS of both the endogenous NIA1/NIA2 and 35S:NIA2 loci, visible as small chlorotic spots indicated by arrow heads (A–B). PTGS subsequently spreads through the veins and progressively invades the entire plants (C–D). Age of the plants: A and B: 35 days, C: 50 days, D: 70 days. E–F) Transgene GFP PTGS artificially initiated locally spreads throughout transgenic N.benthamiana plants. 35S:GFP lines that do not spontaneously initiate PTGS can be induced to do so by local, transient introduction of extra 35S:GFP copies in a few leaves. The induced PTGS spreads through the veins (E) and progressively invades the entire plants (F). Adapted from Voinnet (2005)https://doi.org/10.1016/j.febslet.2005.09.039G) Possible mechanisms for spontaneous initiation and spread. A local stress-induced burst of aberrant (ab)RNAs that saturate RQC or a deficiency thereof allows abRNAs to be converted into dsRNA by RDR6/SGS3 to initiate PTGS via DCL2/4-dependent siRNAs. Movement of siRNA and/or dsRNA precursors thereof from cell-to-cell and over long distances allows PTGS re-initiation in recipient cells. This occurs independently of RQC saturation/deficiency, likely because siRNAs induce epigenetic changes at the transgene loci through the as-yet-understood action of JMJ14, NAC52, NRPD1, RDR2 and DCL3-AGO4. H) A PTGS signal moves through graft unions. Grafting non-silenced transgenic scions onto silenced transgenic rootstocks provokes systemic silencing of the homologous transgene in the grafted scion. I) Mobile endogenous siRNAs can mediate RdDM at distance. Grafting wildtype scions onto dcl234 rootstocks triggers mainly CHH methylation in dcl234 rootstocks at loci targeted by siRNAs (TEs depicted here) produced in the scions.
Figure 7.
Figure 7.
Anti-viral RNAi. A) dsRNA sources from diverse plant viruses and viroids. 1. Geminiviruses with a DNA genome can spawn dsRNA via read-through transcription of convergent overlapping ORFs (e.g. C3/V1 or C2/V1) located on opposite DNA strands. 2. Single-stranded RNA from RNA viruses can fold locally into dsRNA structures. dsRNA may also form during replication or transcription of sub-genomic (sg)RNA. Both RNA and DNA viruses combat antiviral RNAi by producing VSRs translated from their genomes, indicated here in red. 3. Viroids produce siRNAs but evade targeting by siRNA/AGO complexes, likely due to the rod-like complementary nature of their RNA. B) The affects of antiviral RNAi are mostly evident with VSR-deficient viruses. An illustrative framework for the hierarchical surrogacy linking DCL4 to DCL2 during RNA virus infections. Both 21- (DCL4-dependent) and 22-nt (DCL2-dependent) v-siRNAs promote PTGS upon their loading into antiviral AGOs. DCL4 action usually dominates but may be directly or indirectly inhibited by VSR activities, a circumstance mimicked by use of the dcl4 mutant background. In either situation, DCL2 action takes over to rescue antiviral PTGS via 22-nt v-siRNAs. Genetically, this translates into VSR-proficient (VSR+) viral titres remaining mostly unaffected in either the dcl4 single- or dcl2dcl4 double- mutant backgrounds due to the genetically-redundant VSR action. The VSR-deficient (VSR−) viral titres are, by contrast, strongly enhanced yet mostly in the dcl2dcl4 double mutants due the DCL4-DCL2 surrogacy. Neither the VSR+ nor VSR− viral titres are further enhanced in the dcl2dcl3dcl4 triple mutant background because, unlike DNA viruses (see F), most RNA viruses are insensitive to RdDM mediated by 24-nt v-siRNAs, should they accumulate at all. C) P19-mediated sequestration of tombusvirus-derived v-siRNAs prevents sequence-specific immunization of virus-proximal tissues. The images (adapted from Havelda et al. 2003) depict in situ hybridizations of the tombusviral (−)RNA strand (attesting replication). The P19-proficient virus unloads from the vasculature to the adjacent leaf laminal cells. By contrast, the P19-deficient virus only accumulates in the vasculature whereas the adjacent tissues exhibit nucleotide-sequence-specific immunity to secondary tombusvirus challenge, suggesting vascular-to-laminal movement of a virus-derived silencing signal. D) DNA viruses activate an RdDM-like response. Additionally to activating antiviral PTGS via 21-22-nt v-siRNAs, DNA viruses spawn DCL3-dependent 24-nt siRNAs in the nucleus, which trigger cytosine methylation of viral episomes/mini-chromosomes. E) Possible indirect effects of RdDM-deficient conditions on antiviral PTGS. DCL4 promoter demethylation in RdDM mutants allows alternative transcription start sites usage (TSSS#1 versus TSS#2 in WT, i.e. RdDM-proficient conditions). This enables production of a longer, NLS-containing DCL4 isoform that is more retained in the nucleus, with henceforth reduced activity in the cytosol where RNA viruses replicate.
Figure 8.
Figure 8.
RNAi homeostasis. A) Multiple RNAi pathways compete for the same component. HEN1 2′O-methylates miRNAs as well as siRNAs involved in the TGS and PTGS pathways, making its availability for one pathway limited by the others. VSRs can target HEN1, reducing further its availability for these sRNA pathways. B) AGO1 homoeostasis involves multiple levels of control. (1) AGO1 mRNA levels are feedback-regulated by a miRNA, miR168, which forms a complex with the AGO1 protein. (2) AGO1 is stabilized by the loading of 21-22-nt miRNAs and siRNAs. As a result, transgenic plants undergoing PTGS or virus-infected plants accumulate more AGO1 protein to accommodate the excess of 21-22-nt molecules. Transgene siRNAs and v-siRNAs may represent up to 30% and 70% of total siRNAs, respectively. (3) Lack of cellular 21-22-nt si/miRNAs (for example in miRNA-deficient mutants) provokes AGO1 degradation, thereby preventing its spurious loading with inappropriate RNA molecules. C) DCLs and virus-induced RTL1 compete for long dsRNA. (1) Endogenous long dsRNAs are normally processed by DCL4, DCL2 and DCL3 into signature siRNA products. (2) Upon virus infection, RTL1 is induced and compete with DCL2/3/4 for substrates, including viral-derived dsRNAs, which it degrades.
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