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Summary Methylation at the MdMYB1 promoter in apple sports has been reported as a regulator of the anthocyanin pathway, but little is known about how the locus is recognized by the methylation machinery to regulate anthocyanin accumulation. In this study, we analysed three differently coloured ‘Fuji’ apples and found that differences in the transcript levels of MdMYB1, which encodes a key regulator of anthocyanin biosynthesis, control the anthocyanin content (and therefore colour) in fruit skin. The CHH methylation levels in the MR3 region (−1246 to −780) of the MdMYB1 promoter were found to be negatively correlated with MdMYB1 expression. Thus, they were ideal materials to study DNA methylation in apple sports. The protein of RNA‐directed DNA methylation (RdDM) pathway responsible for CHH methylation, MdAGO4, was found to interact with the MdMYB1 promoter. MdAGO4s can interact with MdRDM1 and MdDRM2s to form an effector complex, fulfilling CHH methylation. When MdAGO4s and MdDRM2s were overexpressed in apple calli and Arabidopsis mutants, those proteins increase the CHH methylation of AGO4‐binding sites. In electrophoretic mobility shift assays, MdAGO4s were found to specifically bind to sequence containing ATATCAGA. Knockdown of MdNRPE1 did not affect the binding of MdAGO4s to the c3 region of the MdMYB1 promoter in 35S::AGO4 calli. Taken together, our data show that the MdMYB1 locus is methylated through binding of MdAGO4s to the MdMYB1 promoter to regulate anthocyanin biosynthesis by the RdDM pathway.

We combined de novo mutation (DNM) data from 10,927 individuals with developmental delay and autism to identify 253 candidate neurodevelopmental disease genes with an excess of missense and/or likely gene-disruptive (LGD) mutations. Of these genes, 124 reach exome-wide significance (P

The Cucumber mosaic virus (CMV) suppressor 2b co-localizes with AGO4 in cytoplasmic and nuclear fractions of Arabidopsis thaliana. Biochemical fractionation of A. thaliana cellular extracts revealed that 2b and AGO4 coexist in multiple size exclusions. 2b transgenic A. thaliana exhibited an enhanced accumulation of 24nt siRNAs from flowering wageningen (FWA) and other heterochromatic loci. These plants also exhibited hypo-methylation of an endogenous- as well as transgene-FWA promoter at non-CG sites. In corroboration, both transgenic 2b and CMV infection affected the regulation of transposons which mimics the ago4 phenotype. In conclusion, 2b perturbs plant defense by interfering with AGO4-regulated transcriptional gene silencing., Summary: CMV suppressor 2b perturbs host defense by interrupting epigenetic signaling. 2b causes FWA siRNAs sequestration and hypo-methylation of FWA promoter region. It shines light on a hitherto little understood mechanism of viral suppression of host epigenetics.

Background DNA methylation ensures genome integrity and regulates gene expression indiverse eukaryotes. In Arabidopsis, methylation occurs in threesequence contexts: CG, CHG and CHH. The initial establishment of DNAmethylation at all three sequence contexts occurs through a process known asRNA-directed DNA methylation (RdDM), in which small RNAs bound by Argonaute4(AGO4) guide DNA methylation at homologous loci through the de novomethyltransferase DRM2. Once established, DNA methylation at each of thethree sequence contexts is maintained through different mechanisms. Althoughsome players involved in RdDM and maintenance methylation have beenidentified, the underlying molecular mechanisms are not fully understood. Toaid the comprehensive identification of players in DNA methylation, wegenerated a transgenic reporter system that permits genetic and chemicalgenetic screens in Arabidopsis. Results A dual 35S promoter (d35S) driven luciferase (LUC)reporter was introduced into Arabidopsis and LUCL, a linewith a low basal level of luciferase activity, was obtained. LUCLwas found to be a multi-copy, single-insertion transgene that containsmethylated cytosines in CG, CHG and CHH contexts, with the highestmethylation in the CG context. Methylation was present throughout thepromoter and LUC coding region. Treatment with an inhibitor ofcytosine methylation de-repressed luciferase activity. A mutation inMET1, which encodes the CG maintenance methyltransferase,drastically reduced CG methylation and de-repressed LUC expression.Mutations in AGO4 and DRM2 also de-repressed LUCexpression, albeit to a smaller extent than loss of MET1. UsingLUCL as a reporter line, we performed a chemical screen forcompounds that de-repress LUC expression, and identified achemical, methotrexate, known to be involved in biogenesis of the methyldonor. Conclusion We developed a luciferase-based reporter system, LUCL, which reportsboth RdDM and CG maintenance methylation in Arabidopsis. The lowbasal level of LUCL expression provides an easy readout in geneticand chemical genetic screens that will dissect the mechanisms of RdDM andmethylation maintenance.

As important components of small RNA (smRNA) pathways, Argonaute (AGO) proteins mediate the interaction of incorporated smRNAs with their targets. Arabidopsis contains 10 AGO proteins with specialized or redundant functions. Among them, AGO1 mainly acts in microRNA (miRNA) and small-interfering RNA (siRNA) pathways for post-transcriptional gene silencing (PTGS), whereas AGO4 regulates transcriptional gene silencing (TGS) via endogenous 24-nucleotide (nt) smRNAs. To fully characterize smRNAs associated with AGO1 and AGO4, we developed a two-step protocol to purify AGO/smRNA complexes from flowers, leaves, roots and seedlings with enhanced purity, and sequenced the smRNAs by Illumina technology. Besides recovering most previously annotated smRNAs, we also identified some additional miRNAs, phased smRNA clusters and small-interfering RNAs derived from the overlapping region of natural antisense transcript pairs (NAT) (nat-siRNAs). We also identified a smRNA distribution feature on miRNA precursors which may help to identify authentic miRNAs. Organ-specific sequencing provided digital expression profiles of all obtained smRNAs, especially miRNAs. The presence and conservation of collateral miRNAs on known miRNA precursors were also investigated. Intriguingly, about 30% of AGO1-associated smRNAs were 24-nt long and unrelated to the 21-nt species. Further analysis showed that DNA-dependent RNA polymerase IV (Pol IV)-dependent smRNAs were mainly 24 nt and associated with AGO4, whereas the majority of the potential Pol V-dependent ones were 21-nt smRNAs and bound to AGO1, suggesting the potential involvement of AGO1 in Pol V-related pathways.