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 (m6A) 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 m6A‐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 m6A‐YTHDF axis constitutes a novel branch of basal antiviral immunity in plants.
N6-methyladenosine (m6A) in mRNA is key to eukaryotic gene regulation. Many m6A functions involve specialized RNA-binding proteins that recognize m6A 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.
YT521-B homology (YTH) domain proteins act as readers of N6-methyladenosine (m6A), the most common internal covalent modification in eukaryotic mRNA. Members of the YTHDF subclade can determine properties of m6A-containing mRNAs in the cytoplasm of animal and plant cells. Vertebrates encode three YTHDF proteins, and whether they perform specialized or redundant molecular functions is currently debated. In land plants, the YTHDF clade has expanded from just 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 found at the C-terminus following a long intrinsically disordered region (IDR) at the N-terminus. The origin and implications of YTHDF expansion in higher plants are not known, as it is unclear whether it involves acquisition of fundamentally different properties, in particular of their divergent IDRs. Here, we used the leaf formation defects in ect2/ect3/ect4 mutants to test whether the many Arabidopsis YTHDF proteins can perform the same function if expressed at similar levels in leaf primordia. We show that the ancestral molecular function of the m6A-YTHDF axis in land plants is conserved over YTHDF diversification, and currently present in all major clades of YTHDF proteins in flowering plants. Nevertheless, lineage-specific neo-functionalization of a few members also happened after late duplication events. ECT1, the closest homolog of ECT2/3/4, is one such divergent YTHDF protein. Accordingly, mutation of ECT1 does not aggravate the defective organogenesis of ect2/ect3/ect4 mutants, even though the four proteins are naturally expressed in the same population of primordial cells.
SUMMARYIn 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 such a basal antiviral immune system, but additional RNA-based mechanisms of defense also exist. The infectivity of the plant positive strand RNA virus alfalfa mosaic virus (AMV) relies on demethylation of viral RNA by recruitment of the cellularN6-methyladenosine (m6A) demethylase ALKBH9B, but how demethylation of viral RNA promotes AMV replication remains unknown. Here, we show that inactivation of the cytoplasmic YT521-B homology domain (YTH)-containing m6A-binding proteins, ECT2, ECT3, and ECT5 is sufficient to restore AMV infectivity in partially resistantalkbh9bmutants. We also show that the antiviral function of ECT2 is distinct from its previously demonstrated function in promotion of proliferation of primordial cells, because 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 m6A-YTH axis constitutes a novel branch of basal antiviral immunity in plants.
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