Significance Small RNAs (sRNAs) of 24 nt are associated with transcriptional gene silencing by targeting DNA methylation to complementary sequences. We demonstrated previously that sRNAs move from shoot to root, where they regulate DNA methylation of three endogenous transposable elements (TEs). However, the full extent of root DNA methylation dependent on mobile sRNAs was unknown. We demonstrate that DNA methylation at thousands of sites depends upon mobile sRNAs. These sites are associated with TE superfamilies found in gene-rich regions of the genome, which lose methylation selectively in an sRNA-deficient mutant. If the TEs were able to reactivate, they could cause genome instability and altered gene expression patterns, with negative effects on the plant. Consequently, mobile sRNAs may defend against these TEs.

Abstract RNA silencing at the transcriptional and posttranscriptional levels regulates endogenous gene expression, controls invading transposable elements (TEs), and protects the cell against viruses. Key components of the mechanism are small RNAs (sRNAs) of 21–24 nt that guide the silencing machinery to their nucleic acid targets in a nucleotide sequence-specific manner. Transcriptional gene silencing is associated with 24-nt sRNAs and RNA-directed DNA methylation (RdDM) at cytosine residues in three DNA sequence contexts (CG, CHG, and CHH). We previously demonstrated that 24-nt sRNAs are mobile from shoot to root in Arabidopsis thaliana and confirmed that they mediate DNA methylation at three sites in recipient cells. In this study, we extend this finding by demonstrating that RdDM of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot and that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. Mobile sRNA-dependent non-CG methylation is largely dependent on the DOMAINS REARRANGED METHYLTRANSFERASES 1/2 (DRM1/DRM2) RdDM pathway but is independent of the CHROMOMETHYLASE (CMT)2/3 DNA methyltransferases. Specific superfamilies of TEs, including those typically found in gene-rich euchromatic regions, lose DNA methylation in a mutant lacking 22- to 24-nt sRNAs (dicer-like 2, 3, 4 triple mutant). Transcriptome analyses identified a small number of genes whose expression in roots is associated with mobile sRNAs and connected to DNA methylation directly or indirectly. Finally, we demonstrate that sRNAs from shoots of one accession move across a graft union and target DNA methylation de novo at normally unmethylated sites in the genomes of root cells from a different accession.

RNA silencing in plants and animals is a process that controls gene expression at both the transcriptional and posttranscriptional levels (1). In plants, small RNAs (sRNAs), 21–24 nt in length (2, 3), direct the RNA silencing machinery to target nucleic acids in a sequence-specific manner (4). The 21/22-nt sRNAs are primarily associated with mRNA cleavage and are involved in posttranscriptional gene silencing (PTGS) (3). The 24-nt sRNAs are primarily associated with RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (2, 5). However, a recent study suggests that both 21- and 24-nt sRNAs are involved in deposition of DNA methylation (6). It is proposed that the 21-nt sRNAs may establish DNA methylation, whereas the 24-nt species are involved in its amplification and maintenance (7, 8).

RdDM involves methylation of cytosine residues in CG, CHG, and CHH sequence contexts (where H denotes any base except G) (9, 10). It is closely associated with repressive chromatin marks at target loci (11) and blocks gene transcription when present in promoter regions (12). RdDM maintains genome integrity by repression of transposable element (TE) activity, as well as contributing to environmental and developmental regulation of gene expression (4, 8, 13⇓⇓⇓–17). The methylation status of DNA is heritable through both meiosis and mitosis, allowing it to persistently alter gene expression (18⇓–20).

Initial establishment of RdDM involves cleavage of double-stranded RNA by DICER-LIKE (DCL) proteins to form 21- to 24-nt sRNAs, which load into ARGONAUTE (AGO) proteins (4, 21). These nucleoprotein complexes target chromatin-associated scaffold transcripts in a sequence-specific manner (22⇓–24). The chromatin-bound complexes then recruit DOMAINS REARRANGED METHYLTRANSFERASES 1 and 2 (DRM1 and DRM2), which methylate DNA in CG, CHG, and CHH sequence contexts (8, 25). A complex set of maintenance mechanisms ensures persistence of established DNA methylation through cell division and even between generations. Most of these mechanisms are independent of RNA, and involve epigenetic histone marks. The VARIANT IN METHYLATION (VIM) family proteins 1, 2, and 3 and DNA METHYLTRANSFERASE 1 (MET1) efficiently maintain CG context methylation, resulting in near-complete methylation of target sequences (26⇓–28). Non-CG context methylation (i.e., CHG, CHH) is maintained by a self-reinforcing loop involving KRYPTONITE family enzymes (29). These proteins recognize non-CG context methylated DNA and methylate lysine 9 of adjacent histone H3 (H3K9me2). CHROMOMETHYLASE (CMT) proteins CMT2 and CMT3 bind H3K9me2 and methylate adjacent non-CG sites (30) of the newly replicated DNA. Redundancy exists between target sites of CMT2 and CMT3, but their predominant functions are to maintain CHH and CHG context methylation, respectively (31). The activity of CMT2 is substantially less efficient than that of MET1, so that CMT2 target sites exhibit variable levels of DNA methylation. CMT3 efficiency is intermediate between MET1 and CMT2. Both CMT2 and CMT3 typically target long TEs and gene-distal TEs (31).

There are also RNA-dependent mechanisms to maintain DNA methylation that involve plant-specific DNA-dependent RNA polymerases IV and V (POL IV and POL V). These polymerases are recruited to chromatin by methyl-DNA–binding proteins Sawadee homeodomain homolog 1/DNA-binding transcription factor 1 (SHH1) SU(VAR)3–9 homolog 2 (SUVH2) and 9 (32). POL IV produces precursor RNAs that are processed into 24-nt sRNAs, whereas POL V produces chromatin-bound scaffold transcripts at sites of DNA methylation (33). Together, they ensure maintenance of CG, CHG, and CHH context DNA methylation through an AGO-dependent mechanism similar to the mechanism that establishes methylation, in which an AGO–sRNA recruits DRM1 and DRM2 to maintain non-CG DNA methylation (8, 25). The target sites of the DRM1/DRM2 DNA methylation maintenance pathway are largely nonoverlapping with those of CMT2 and CMT3, and tend to be short, gene-proximal TEs and the edges of long TEs (31, 34).

RdDM can operate cell-to-cell and systemically due to translocation of 23- to 24-nt sRNAs from shoots to roots (35). In our previous studies, we confirmed that these mobile 23- to 24-nt sRNAs target RdDM and TGS at one transgene and RdDM at three endogenous TEs (35⇓–37). Depletion of shoot sRNAs corresponded to reduction of 23- to 24-nt sRNAs in wild-type roots, indicating that shoot-derived sRNAs contribute to the total root sRNA population (36). In this study, we investigate the extent to which mobile sRNAs mediate genome-wide RdDM. Our approach, as before, is to analyze sRNA and DNA methylation in roots of grafted plants that are defective for the production of the 24-nt sRNA species associated with RdDM. The sRNAs in these grafted plants move predominantly from shoot to root following source–sink gradients (36) and, by grafting different genotypes as shoots, we identify changes in DNA methylation and gene expression in the roots that are dependent on mobile sRNAs.

We show that mobile sRNAs influence genomic DNA methylation at thousands of loci, and that the affected loci are predominantly associated with transposons of specific classes. A very small number of protein-coding genes were influenced by this mobile RdDM. The mobile sRNA-dependent DNA methylation is associated with the DRM1/DRM2 RdDM pathway but not the CMT2/3 DNA methyltransferase pathway. Furthermore, we demonstrate that mobile sRNAs unique to one accession established DNA methylation de novo in unmethylated regions of the genome of a second accession.

Discussion In this study, we have characterized the genome-wide distribution of 23- to 24-nt shoot/root mobile sRNAs and identified regions of DNA methylation that they target directly (class A loci). We also identified regions of DNA methylation dependent indirectly upon mobile sRNAs (class B loci). We found that loss of mobile sRNAs generated in the shoots disrupts DNA methylation at thousands of sites in the roots. The class B (indirect) loci were most numerous, but the two classes were otherwise indistinguishable. Both were almost entirely in the non-CG (CHG/CHH) DNA methylation context. The class A and B loci were also significantly associated with the same TE superfamilies. We found that these TE superfamilies also lost DNA methylation in the dcl234 mutant, which is deficient in 22- to 24-nt sRNAs, indicating that the feature association of class A and B loci was driven by the dependency of mobile sRNAs on DCL234. Our results suggest that the function of mobile sRNAs is to reinforce silencing of these TEs. Furthermore, we have provided mechanistic insights into the specific RdDM pathways regulating mobile sRNA-dependent methylation. Data can be visualized on our interactive genome browser (neomorph.salk.edu/mobile_methylome.php). Our data confirm that mobile sRNA-dependent DNA methylation requires DRM1 and DRM2, key components of the 24-nt RdDM pathway. There was only limited dependence upon CMT3 and CMT2, both of which belong to a distinct DNA methylation maintenance pathway. Furthermore, class A and B loci were associated with the superfamilies containing the shortest (on average) TEs (Figs. 2 and 4 and SI Appendix, Table S1), including type 1 retroelements, such as RAth elements (E1, E2, E3), SINEs, and LINEs, as well as certain type 2 DNA elements (Mariner, Pogo, RC Helitron). The DNA methylation pathways containing DRM1/2 and CMT2/3 are distinct but overlapping, and both deposit non-CG methylation (8, 34). The DRM1/2 pathway methylates small TEs and TE edges, whereas the CMT2/3 pathway is responsible for methylation of long TEs (31, 34). Our data are in concordance with this pattern, and establish a relationship of mobile sRNA-dependent methylation with a specific RNA silencing mechanism. The direct and indirect (class A and B) loci are essentially identical according to our data, except that no mobile sRNAs were identified as being associated with indirect class B loci. These findings lead to three hypotheses for further investigation. The first is that the mobile sRNAs from the shoot regulate an unidentified secondary signal in the roots. This secondary signal would then directly regulate DNA methylation at the B loci. The second is that mobile sRNAs can direct the RNA silencing machinery to sites that they match imperfectly (i.e., with which they have mismatches in sequence homology). We required perfect matching between sRNAs and genomic sequence when identifying sRNA target loci, but it is possible that a certain degree of mismatch between mobile sRNAs and their targets is either tolerated or required in certain circumstances. However, allowing mismatches during sRNA mapping (and therefore in targeting) permits individual sRNAs to target multiple sites due to their inherently short sequence, many of which are presumably spurious (42). Therefore, detailed analysis of individual methylation regions and thorough experimental validation are required to test this hypothesis. Moreover, it appears unlikely that the conclusions of our study would be altered by the outcome of such experiments, because we find the characteristics of class A (direct) and class B (indirect) loci to be essentially identical. The third hypothesis is that these loci are targeted by mobile sRNAs of very low abundance, which could not be detected in our sRNA-seq libraries. Class A and B loci were substantially more numerous than the other classes (Fig. 1, Table 1, and SI Appendix, Fig. S1 and Table S2). The class D–F loci described loci not methylated by mobile sRNAs, not remethylated by mobile sRNAs, and whose methylation was independent of DCL234-derived sRNAs, respectively. Class F loci were the least numerous. This observation suggests there may be few mobile sRNA-targeted loci at which that sRNA does not regulate DNA methylation. sRNAs of 24-nt length were proportionally the most common sRNA species detected in our sRNA-seq experiments and demonstrated the clearest shoot–root transmission (Fig. 2) (36). This study and another indicate that 21- to 22-nt sRNAs are also mobile and associated with DNA methylation (37). These data are consistent with previous proposals of a role for the 21- to 22-nt size class in RdDM (6, 7, 37). Furthermore, the type 1 retroelements that we find to be the predominant targets of mobile sRNA-dependent DNA methylation produce RNA intermediate replication stages, making it possible that if transcriptionally active they are also targets of PTGS via mobile 21-nt sRNAs (43). However, fewer 21- to 22-nt sRNAs were mobile than 24-nt sRNAs, suggesting 24-nt sRNAs are the primary shoot–root signal. The design of our experiment did not permit us to completely eliminate the possibility that “mobile” 21-nt sRNAs are produced in the roots. This is because the dcl234 mutant contains functional DCL1, which generates 21-nt sRNAs, and the dcl234 mutant eliminates their production only partially (23, 44⇓–46). Detailed investigation of their contribution would involve generation of an additional series of Arabidopsis mutant grafts and is complicated by the lethality of a dcl1 null plant (47). Although mobile sRNAs have minimal effects on gene expression in Arabidopsis, we predict that their influence would be much greater in TE-rich genomes. In such genomes, including those of many common crops, mobile sRNAs may be an important mechanism of genome defense. The TE superfamilies targeted by mobile sRNAs are enriched in euchromatic regions, typically within c. 500 nt of the nearest gene (48, 49). TEs inserted near genes can have dramatic effects on gene expression (15, 49). Expression of a small number of transcripts was differentially regulated in our study, consistent with previous observations (39). However, the A. thaliana genome contains substantially fewer TEs than related outcrossing species, such as Arabidopsis lyrata (50, 51). Furthermore, TEs in the A. thaliana genome show a lower rate of active transposition (50). This suggests that TEs in the A. thaliana genome may have relatively less influence on gene expression than TEs in outcrossing species, which would result in fewer mobile sRNA-dependent regulated genes. Nonetheless, those genes that did exhibit mobile sRNA-dependent regulation had diverse functions, indicating they may have significant influence in the correct conditions. Moreover, mobile sRNAs are able to move into both meristematic and meiotically active tissues, where they can alter DNA methylation and gene expression (37, 52). In these tissues it is essential to protect genome stability by repression of TEs, so that gametes and developing organs are not harmed (53, 54). Grafting is routinely used in agriculture to combine rootstocks and shoots (scions) with desirable characteristics, such as for grapevine, apples, and tomatoes (55, 56). We have demonstrated that mobile sRNAs regulate patterns of DNA methylation genome-wide, and that expression of specific genes exhibits mobile regulation. Base-resolution methylomes are being actively generated in multiple species, including crops and nonmodel plants (38, 57⇓–59). Clear diversity in the epigenomes of closely related subspecies and accessions has been observed from these data (38, 57). With our demonstration that site-specific transmission of epiallelic states from one accession to another can be achieved by grafting and by de novo methylation of unmethylated DNA, it is likely that at least some effects of grafting are due to the movement of RNA. Our findings also indicate that DNA methylomes may provide a potential new resource to growers who use grafting. They could consider potential modification of gene expression patterns in sink tissues via sRNA transmission from source tissues. The mobile sRNAs can alter DNA methylation in germ-line tissue (37), so that DNA methylation patterns altered by grafting may be heritable by progeny plants (37, 60). In summary, we have shown that transmission of sRNAs from shoots to roots of Arabidopsis regulates DNA methylation at thousands of sites genome-wide. Mobile sRNA-dependent methylation is predominantly in the non-CG context (CHG and CHH), and is associated with short type 1 retroelements found in gene-rich regions of the genome. We confirm that deposition of mobile sRNA-dependent methylation is dependent upon the DRM1 and DRM2 RdDM pathway and largely independent of the CMT2 and CMT3 methylation pathway. Our conclusions underpin future research into why plants possess a system for communicating methylation status from shoot to root tissues.

Acknowledgments We thank Matthew D. Schultz and Yupeng He for assistance with analyses; Huaming Chen for assistance with web browser development; and Roberto Solano, Robert J. Schmitz, Taiji Kawakatsu, Chongyuan Luo, Ryan Lister, Ronan C. O’Malley, and Lindsay Robinson for helpful discussions. M.G.L. was funded by an EU Marie Curie FP7 International Outgoing Fellowship (252475). Work in J.R.E.’s laboratory is funded by the Gordon and Betty Moore Foundation (3034) and the National Science Foundation (MCB-1024999). J.R.E. is an investigator of the Howard Hughes Medical Institute. C.W.M. was funded by a Clare College (Cambridge, United Kingdom) Junior Research Fellowship. Work in the D.C.B. laboratory was supported by the Gatsby Charitable Foundation, European Union FP7 Collaborative Project Grant AENEAS, and European Research Council Advanced Investigator Grant ERC-2013-AdG 340642. D.C.B. is the Royal Society Edward Penley Abraham Research Professor.

Footnotes Author contributions: M.G.L., T.J.H., C.W.M., A.M., D.C.B., and J.R.E. designed research; M.G.L., T.J.H., C.W.M., A.M., A.V., M.A.U., and J.R.N. performed research; T.J.H. contributed new analytic tools; M.G.L., T.J.H., C.W.M., A.M., D.C.B., and J.R.E. analyzed data; and M.G.L., T.J.H., C.W.M., A.M., D.C.B., and J.R.E. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The data reported in this paper have been deposited in the European Nucleotide Archive (accession no. E-MTAB-3473).

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