PUMILIO/Fem-3 Binding Factor (PUF) proteins are conserved eukaryotic mRNA-binding proteins. Higher plants encode an expanded family of PUF proteins with 26 members in Arabidopsis thaliana of which 6 (PUM1-PUM6) have a domain organization equivalent to metazoan PUF proteins implicated in mRNA regulation. Here, we show that the PUF proteins PUM2 and PUM6 are expressed in mitotically active cells of root and shoot meristems, while PUM5 is mostly expressed in the differentiation zone of root meristems exiting from active division. Increased PUM2 dosage in proliferating cells causes growth repression and developmental delay reminiscent of mutants with defects in cytoplasmic YTHDF proteins that bind to N6 -methyladenosine (m6A) in mRNA. Remarkably, even weak defects in the m6A-YTHDF system enhance the effect of increased PUM2 dosage. In line with this genetic interaction, mRNA targets overlap significantly between PUFs and the major YTHDF proteins ECT2 and ECT3. Furthermore, the enrichment of the core PUF recognition motif URUAY around m6A sites is largely explained by PUF targeting, not by direct URUAY methylation as previously suggested. Our results indicate that the balance between the reciprocal activities of the deeply conserved cytoplasmic RNA binding protein families PUF and YTHDF is an important element of growth control in plants. ### Competing Interest Statement The authors have declared no competing interest. Novo Nordisk Foundation, https://ror.org/04txyc737, NNF19OC0054973, NNF24SA0092867 Carlsberg Foundation, https://ror.org/01kpjmx04, CF20-0659

ARGONAUTE (AGO) proteins associate with small RNAs to form RNA-induced silencing complexes (RISCs). Arabidopsis AGO1 effects post-transcriptional silencing by microRNAs (miRNAs) and small interfering RNAs (siRNAs) and is necessary for siRNA amplification through conversion of RISC target RNAs into double-stranded RNA by the RNA-dependent RNA Polymerase RDR6 and its mandatory cofactors SGS3 and SDE5. Many AGO proteins harbor hydrophobic pockets that interact with tryptophan residues, often surrounded by glycine (GW/WG), in intrinsically disordered regions (IDRs) of RISC cofactors. Here, we show that GW/WG dipeptides in the IDR of SGS3 and the hydrophobic pockets in AGO1 are required for fully functional RDR6-dependent siRNA amplification. We also show that this mechanism requires AGO1-specific structural elements, including positively charged residues surrounding the binding pockets, and a conserved, negatively charged patch in the IDR of SGS3. Thus, the same, conserved protein-protein interaction site is used for different purposes in distinct eukaryotic AGO proteins: the GW/WG-mediated TNRC6-Ago2 interaction is crucial for miRNA-guided silencing in metazoans whereas the GW/WG-mediated SGS3-AGO1 interaction facilitates siRNA amplification via RDR6 in plants. ### Competing Interest Statement The authors have declared no competing interest. Novo Nordisk Foundation, https://ror.org/04txyc737, Hallas Møller 2010 European Research Council, https://ror.org/0472cxd90, ERC-2011-StG 282460, ERC-2016-CoG 726417 Lundbeck Foundation, https://ror.org/03hz8wd80, R83-A7859 Villum Fonden, https://ror.org/05nqkay65, 13397 Carlsberg Foundation, https://ror.org/01kpjmx04, CF18-1075, CF20-0659 Augustinus Foundation, https://ror.org/030y53541, 12-5126 Hartmann Fonden, https://ror.org/05t3dfy66, A35879

ARGONAUTE (AGO) proteins bind to small non-coding RNAs to form RNA-induced silencing complexes. In the RNA-bound state, AGO is stable while RNA-free AGO turns over rapidly. Molecular features unique to RNA-free AGO that allow its specific recognition and degradation remain unknown. Here, we identify a confined, linear region in Arabidopsis AGO1 and human Ago2, the N-coil, as a structural switch with preferential accessibility in the RNA-free state. RNA-free Arabidopsis AGO1 interacts with the autophagy cargo receptor ATI1 by direct contact with specific N-coil amino acid residues whose mutation reduces the degradation rate of RNA-free AGO1 in vivo. The N-coil of human Ago2 has similar degron activity dependent on residues in positions equivalent to those required for the Arabidopsis AGO1–ATI1 interaction. These results elucidate the molecular basis for specific recognition and degradation of the RNA-free state of eukaryotic AGO proteins. ARGONAUTE (AGO) is the core protein component of small RNA-guided silencing complexes. Free, but not RNA-bound, AGO turns over rapidly. The authors identify a structural AGO switch whose accessibility only in the free state confers rapid degradation.

Flowering plant genomes encode four or five DICER-LIKE (DCL) enzymes that produce small interfering RNAs (siRNAs) and microRNAs, which function in RNA interference (RNAi). Different RNAi pathways in plants effect transposon silencing, antiviral defense, and endogenous gene regulation. DCL2 acts genetically redundantly with DCL4 to confer basal antiviral defense. However, DCL2 may also counteract DCL4 since knockout of DCL4 causes growth defects that are suppressed by DCL2 inactivation. Current models maintain that RNAi via DCL2-dependent siRNAs is the biochemical basis of both effects. Here, we report that DCL2-mediated antiviral resistance and growth defects cannot be explained by the silencing effects of DCL2-dependent siRNAs. Both functions are defective in genetic backgrounds that maintain high levels of DCL2-dependent siRNAs, either with specific point mutations in DCL2 or with reduced DCL2 dosage because of heterozygosity for dcl2 knockout alleles. Intriguingly, all DCL2 functions require its catalytic activity, and the penetrance of DCL2-dependent growth phenotypes in dcl4 mutants correlates with DCL2 protein levels but not with levels of major DCL2-dependent siRNAs. We discuss this requirement and correlation with catalytic activity but not with resulting siRNAs, in light of other findings that reveal a DCL2 function in innate immunity activation triggered by cytoplasmic double-stranded RNA.

ABSTRACT In plants, RNA interference (RNAi) mediated by the endonucleolytic RNA-Induced Silencing Complex (RISC) defends against foreign RNA and regulates endogenous genes. Targeting of RISC to foreign RNA establishes amplification loops, wherein RNA-dependent RNA Polymerase 6 (RDR6) synthesizes double-stranded RNA (dsRNA) for secondary small interfering RNA (siRNA) biogenesis, using cleavage fragments of RNA targeted by RISC programmed with a primary siRNA as template. Secondary siRNA production from endogenous RISC targets requires a particular primary small RNA size or target site multiplicity. siRNA amplification in yeast and nematodes requires terminal nucleotidyl transferases (TNTases), but their roles in plants are unclear. Here, we demonstrate two functions of TNTases in siRNA amplification in Arabidopsis thaliana . URT1 prevents initiation of microRNA-induced secondary siRNA formation through uridylation of 5’-cleavage fragments, sometimes redundantly with the exosome and the TNTase HESO1. Once initiated via RDR6 recruitment, HESO1 and other TNTases stimulate secondary siRNA formation by producing 2-nt 3’overhangs on RDR6-synthesized dsRNA to yield substrates for processing into siRNAs by DICER-LIKE4. These results define molecular mechanisms by which TNTases control siRNA amplification in plants.

Plants use mRNA methylation to regulate gene expression. As in other eukaryotes, the only abundant methylated nucleotide in plant mRNA bodies is N6-methyladenosine (m6A). The conserved core components of m6A-based genetic control are a multi-subunit nuclear methyltransferase, and a set of nuclear and cytoplasmic RNA-binding proteins consisting of an m6A recognition module, the YT521-B homology (YTH) domain, and long intrinsically disordered regions (IDRs). In plants, this system is essential for growth during embryonic and post-embryonic development, but emerging evidence also points to key functions in plant-virus interactions and stimulus-dependent gene regulation. Cytoplasmic YTH-domain proteins are particularly important for these functions, and recent progress has identified two elements of the underlying molecular mechanisms: IDR-mediated phase separation and conserved short linear motifs mediating interactions with other key mRNA-binding proteins.

Abstract N6-methyladenosine (m6A) exerts many of its regulatory effects on eukaryotic mRNAs by recruiting cytoplasmic YT521-B homology-domain family (YTHDF) proteins. Here, we show that in Arabidopsis thaliana, the interaction between m6A and the major YTHDF protein ECT2 also involves the mRNA-binding ALBA protein family. ALBA and YTHDF proteins physically associate via a deeply conserved short linear motif in the intrinsically disordered region of YTHDF proteins and their mRNA target sets overlap, with ALBA4 binding sites being juxtaposed to m6A sites. These binding sites correspond to pyrimidine-rich elements previously found to be important for m6A binding to ECT2. Accordingly, both the biological functions of ECT2, and its binding to m6A targets in vivo, require ALBA association. Our results introduce the YTHDF-ALBA complex as the functional cytoplasmic m6A-reader in Arabidopsis, and define a molecular foundation for the concept of facilitated m6A reading, which increases the potential for combinatorial control of biological m6A effects.

ABSTRACT N6 -methyladenosine (m 6 A) in mRNA is key to eukaryotic gene regulation. Many m 6 A functions involve specialized RNA-binding proteins that recognize m 6 A via a YT521-B Homology (YTH) domain. YTH domain proteins contain long intrinsically disordered regions (IDRs) that may mediate phase separation and interaction with protein partners, but whose precise biochemical functions remain largely unknown. The Arabidopsis thaliana YTH domain proteins ECT2, ECT3 and ECT4 accelerate organogenesis through stimulation of cell division in organ primordia. Here, we focus on ECT2 to reveal molecular underpinnings of this function of ECT2/3/4. We show that stimulation of leaf formation requires the long N-terminal IDR, and we identify two short IDR-elements required for ECT2-mediated organogenesis. Of these two, a tyrosine-rich 19-amino acid region is necessary for binding to a small subset of proteins that includes the major cytoplasmic poly(A)-binding proteins PAB2, PAB4 and PAB8. Remarkably, overexpression of PAB4 in leaf primordia partially rescues the delayed leaf formation in ect2 ect3 ect4 mutants, suggesting that the ECT2-PAB2/4/8 interaction on target mRNAs of organogenesis-related genes may overcome limiting PAB concentrations in primordial cells.

ABSTRACTARGONAUTE (AGO) proteins bind to small non-coding RNAs to form RNA Induced Silencing Complexes (RISCs). In the RNA-bound state, AGO proteins are stable while RNA-free AGOs turn over rapidly. Molecular determinants unique to RNA-free AGO that allow its specific recognition and degradation remain unknown. Here, we show that a confined, linear region in Arabidopsis AGO1, the N-coil, is accessible to antibodies preferentially in the RNA-free state of AGO1. Reanalysis of hydrogen-deuterium exchange data on human Ago2 indicates similar structural flexibility of the N-coil depending on small RNA binding. Unloaded Arabidopsis AGO1 interacts with the autophagy cargo receptor ATI1 via direct contact to specific amino acid residues in the N-coil, and mutation of residues required for ATI1 interaction reduces the degradation rate of unloaded AGO1in vivo. These results provide insight into the molecular basis for specific recognition and degradation of the RNA-free state of eukaryotic AGO proteins.

The effector complex of RNA interference (RNAi) contains at its core an ARGONAUTE (AGO) protein bound to a small guide RNA. AGO proteins adopt a two-lobed structure in which the N-terminal (N) and Piwi-Argonaute-Zwille (PAZ) domains make up one lobe, while the middle (MID) and Piwi domains make up the other. Specific biochemical func-tions of PAZ, MID and Piwi domains of eukaryotic AGO proteins have been described, but the functions of the N domain remain less clear. Here, we use yeast two-hybrid screening with the N domain of the founding member of the AGO protein family, Arabidopsis AGO1, to reveal that it interacts with many factors involved in regulated pro-teolysis. Interaction with a large group of proteins, including the autophagy cargo recep-tors ATI1 and ATI2, requires residues in a short, linear region, the N-coil, that joins the MID-Piwi lobe in the three-dimensional structure of AGO. In contrast, the F-box protein AUF1 interacts with AGO1 independently of the N-coil and requires distinct residues in the globular N domain itself. Mutation of AGO1 residues necessary for interaction with protein degradation factors in yeast stabilizes reporters fused to the AGO1 N domain in plants, supporting their in vivo relevance. Our results define distinct regions of the N domain implicated in protein-protein interaction, and point to a particular importance of the AGO1 N-coil as a site of interaction with regulatory factors.

In virus-host interactions, nucleic acid-directed first lines of defense that allow viral clearance without compromising growth are of paramount importance. Plants use the RNA interference pathway as a basal antiviral immune system, but additional RNA-based mechanisms of defense also exist. The infectivity of a plant positive-strand RNA virus, alfalfa mosaic virus (AMV), relies on the demethylation of viral RNA by the recruitment of the cellular N6-methyladenosine (m(6)A) demethylase ALKBH9B, but how demethylation of viral RNA promotes AMV infection remains unknown. Here, we show that inactivation of the Arabidopsis cytoplasmic YT521-B homology domain (YTH)-containing m(6)A-binding proteins ECT2, ECT3, and ECT5 is sufficient to restore AMV infectivity in partially resistant alkbh9b mutants. We further show that the antiviral function of ECT2 is distinct from its previously demonstrated function in the promotion of primordial cell proliferation: an ect2 mutant carrying a small deletion in its intrinsically disordered region is partially compromised for antiviral defense but not for developmental functions. These results indicate that the m(6)A-YTHDF axis constitutes a novel branch of basal antiviral immunity in plants.

ABSTRACT Sensors of intracellular double-stranded RNA are central components of metazoan innate antiviral immunity, but such sensors have not been identified in plants. RNA interference (RNAi) constitutes a potent plant antiviral defense mechanism that relies on conversion of viral RNA into small interfering RNAs by two DICER-LIKE (DCL) ribonucleases, DCL4 and DCL2. Here, we show that while plant DCL4 is dedicated to RNAi, cytoplasmic dicing by DCL2 also triggers RNAi-independent defense gene expression via at least two intracellular nucleotide-binding domain/leucine-rich repeat (NLR) immune receptors. Combined DCL4/NLR inactivation abrogates basal resistance to a positive strand RNA virus. Our results redefine the basis of plant antiviral immunity, including autoimmunity as an explanation for DCL2-dependent growth arrest in dcl and RNA decay mutants in several plant species. One sentence summary The plant immune system uses Dicer-like ribonucleases for both antiviral RNA interference and double-stranded RNA sensing.

N6 ‐methyladenosine (m 6 A) in mRNA is key to eukaryotic gene regulation. Many m 6 A functions involve RNA‐binding proteins that recognize m 6 A via a YT521‐B Homology (YTH) domain. YTH domain proteins contain long intrinsically disordered regions (IDRs) that may mediate phase separation and interaction with protein partners, but whose precise biochemical functions remain largely unknown. The Arabidopsis thaliana YTH domain proteins ECT2, ECT3, and ECT4 accelerate organogenesis through stimulation of cell division in organ primordia. Here, we use ECT2 to reveal molecular underpinnings of this function. We show that stimulation of leaf formation requires the long N‐terminal IDR, and we identify two short IDR elements required for ECT2‐mediated organogenesis. Of these two, a 19‐amino acid region containing a tyrosine‐rich motif conserved in both plant and metazoan YTHDF proteins is necessary for binding to the major cytoplasmic poly(A)‐binding proteins PAB2, PAB4, and PAB8. Remarkably, overexpression of PAB4 in leaf primordia partially rescues the delayed leaf formation in ect2 ect3 ect4 mutants, suggesting that the ECT2‐PAB2/4/8 interaction on target mRNAs of organogenesis‐related genes may overcome limiting PAB concentrations in primordial cells.

YT521-B homology (YTH) domain proteins act as readers of N6-methyladenosine (m6A) in mRNA. Members of the YTHDF clade determine properties of m6A-containing mRNAs in the cytoplasm. Vertebrates encode three YTHDF proteins whose possible functional specialization is debated. In land plants, the YTHDF clade has expanded from one member in basal lineages to eleven so-called EVOLUTIONARILY CONSERVED C-TERMINAL REGION1-11 (ECT1-11) proteins in Arabidopsis thaliana, named after the conserved YTH domain placed behind a long N-terminal intrinsically disordered region (IDR). ECT2, ECT3 and ECT4 show genetic redundancy in stimulation of primed stem cell division, but the origin and implications of YTHDF expansion in higher plants are unknown, as it is unclear whether it involves acquisition of fundamentally different molecular properties, in particular of their divergent IDRs. Here, we use functional complementation of ect2/ect3/ect4 mutants to test whether different YTHDF proteins can perform the same function when similarly expressed in leaf primordia. We show that stimulation of primordial cell division relies on an ancestral molecular function of the m6A-YTHDF axis in land plants that is present in bryophytes and is conserved over YTHDF diversification, as it appears in all major clades of YTHDF proteins in flowering plants. Importantly, although our results indicate that the YTH domains of all arabidopsis ECT proteins have m6A-binding capacity, lineage-specific neo-functionalization of ECT1, ECT9 and ECT11 happened after late duplication events, and involves altered properties of both the YTH domains, and, especially, of the IDRs. We also identify two biophysical properties recurrent in IDRs of YTHDF proteins able to complement ect2 ect3 ect4 mutants, a clear phase separation propensity and a charge distribution that creates electric dipoles. Human and fly YTHDFs do not have IDRs with this combination of properties and cannot replace ECT2/3/4 function in arabidopsis, perhaps suggesting different molecular activities of YTHDF proteins between major taxa.

Plants use different receptors to detect potential pathogens: membrane-anchored pattern recognition receptors (PRRs) activated upon perception of pathogen-associated molecular patterns (PAMPs) that elicit pattern-triggered immunity (PTI); and intracellular nucleotide-binding leucine-rich repeat proteins (NLRs) activated by detection of pathogen-derived effectors, activating effector-triggered immunity (ETI). The interconnections between PTI and ETI responses have been increasingly reported. Elevated NLR levels may cause autoimmunity, with symptoms ranging from fitness cost to developmental arrest, sometimes combined with run-away cell death, making accurate control of NLR dosage key for plant survival. Small RNA-mediated gene regulation has emerged as a major mechanism of control of NLR dosage. Twenty-two nucleotide miRNAs with the unique ability to trigger secondary siRNA production from target transcripts are particularly prevalent in NLR regulation. They enhance repression of the primary NLR target, but also bring about repression of NLRs only complementary to secondary siRNAs. We summarize current knowledge on miRNAs and siRNAs in the regulation of NLR expression with an emphasis on 22 nt miRNAs and propose that miRNA and siRNA regulation of NLR levels provides additional links between PTI and NLR defense pathways to increase plant responsiveness against a broad spectrum of pathogens and control an efficient deployment of defenses.

Amplification of short interfering RNA (siRNAs) via RNA-dependent RNA polymerases (RdRPs) is of fundamental importance in RNA silencing. Plant microRNA (miRNA) action generally does not involve engagement of RdRPs, in part thanks to a poorly understood activity of the cytoplasmic exosome adaptor SKI2. Here, we show that inactivation of the exosome subunit RRP45B and SKI2 results in similar patterns of miRNA-induced siRNA production. Furthermore, loss of the nuclear exosome adaptor HEN2 leads to secondary siRNA production from miRNA targets largely distinct from those producing siRNAs in ski2. Importantly, mutation of the Release Factor paralogue PELOTA1 required for subunit dissociation of stalled ribosomes causes siRNA production from miRNA targets overlapping with, but distinct from, those affected in ski2 and rrp45b mutants. We also show that in exosome mutants, miRNA targets can be sorted into producers and non-producers of illicit secondary siRNAs based on trigger miRNA levels and miRNA:target affinity rather than on presence of 5'-cleavage fragments. We propose that stalled RNA-Induced Silencing Complex (RISC) and ribosomes, but not mRNA cleavage fragments released from RISC, trigger siRNA production, and that the exosome limits siRNA amplification by reducing RISC dwell time on miRNA target mRNAs while PELOTA1 does so by reducing ribosome stalling.

Immune responses triggered by pathogen-associated molecular patterns (PAMPs) are key to pathogen defense, but drivers of the genetic reprogramming required to reach the immune state remain incompletely understood in plants. Here, we report a time-course study of the establishment of PAMP-triggered immunity (PTI) using cap analysis of gene expression (CAGE). Our results show that as much as 15% of all PAMP response genes display alternative transcription initiation. In several cases, use of alternative TSSs may be regulatory as it determines inclusion of target peptides or protein domains, or occurrence of upstream open reading frames (uORFs) in mRNA leader sequences. We also find that 60% of PAMP-response genes respond much earlier than previously thought. In particular, a previously unnoticed cluster of rapidly and transiently PAMP-induced genes is enriched in transcription factors whose functions, previously associated with biological processes as diverse as abiotic stress adaptation and stem cell activity, appear to converge on growth restriction. Furthermore, some examples of known potentiators of PTI, in one case under direct MAP kinase control, support the notion that the rapidly induced transcription factors could constitute direct links to PTI signaling pathways and drive gene expression changes underlying establishment of the immune state.

Specific recognition of N6 -methyladenosine (m 6 A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The Arabidopsis YTH-domain protein ECT2 is thought to bind to mRNA at URU(m 6 A)Y sites, yet RR(m 6 A)CH is the canonical m 6 A consensus site in all eukaryotes and ECT2 functions require m 6 A binding activity. Here, we apply iCLIP (individual-nucleotide resolution cross-linking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) to define high-quality target sets of ECT2, and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites. Our analyses show that ECT2 does in fact bind to RR(m 6 A)CH. Pyrimidine-rich motifs are enriched around, but not at m 6 A-sites, reflecting a preference for N6 -adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of m 6 A sites, are also implicated in ECT2 binding via its intrinsically disordered region (IDR). Finally, URUAY-type motifs are enriched at ECT2 crosslink sites, but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins. Our study provides coherence between genetic and molecular studies of m 6 A-YTH function in plants, and reveals new insight into the mode of RNA recognition by YTH-domain-containing proteins.

Gene regulation via N6 -methyladenosine (m 6 A) in mRNA involves RNA-binding proteins that recognize m 6 A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2 , ECT3 and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.