A silencing signal in plants with an RNA specificity determinant moves through plasmodesmata and the phloem. To identify the mobile RNA, we grafted Arabidopsis thaliana shoots to roots that would be a recipient for the silencing signal. Using mutants that block small RNA (sRNA) biogenesis in either source or recipient tissue, we found that transgene-derived sRNA as well as a substantial proportion of the endogenous sRNA had moved across the graft union, and we provide evidence that 24-nucleotide mobile sRNAs direct epigenetic modifications in the genome of the recipient cells. Mobile sRNA thus represents a mechanism for transmitting the specification of epigenetic modification and could affect genome defense and responses to external stimuli that have persistent effects in plants.

Argonaute (AGO) effectors of RNA silencing bind small RNA (sRNA) molecules and mediate mRNA cleavage, translational repression, or epigenetic DNA modification. In many organisms, these targeting mechanisms are devolved to different products of AGO multigene families. To investigate the basis of AGO functional diversification, we characterized three closely related Arabidopsis thaliana AGOs (AGO4, AGO6, and AGO9) implicated in RNA-directed DNA methylation. All three AGOs bound 59 adenosine 24-nucleotide sRNAs, but each exhibited different preferences for sRNAs from different heterochromatin-associated loci. This difference was reduced when AGO6 and AGO9 were expressed from the AGO4 promoter, indicating that the functional diversification was partially due to differential expression of the corresponding genes. However, the AGO4-directed pattern of sRNA accumulation and DNA methylation was not fully recapitulated with AGO6 or AGO9 expressed from the AGO4 promoter. Here, we show that sRNA length and 59 nucleotide do not account for the observed functional diversification of these AGOs. Instead, the selectivity of sRNA binding is determined by the coincident expression of the AGO and sRNA-generating loci, and epigenetic modification is influenced by interactions between the AGO protein and the different target loci. These findings highlight the importance of tissue specificity and AGOassociated proteins in influencing epigenetic modifications.

Most eukaryotes produce small RNA (sRNA) mediators of gene silencing that bind to Argonaute proteins and guide them, by base pairing, to an RNA target. MicroRNAs (miRNAs) that normally target messenger RNAs for degradation or translational arrest are the best-understood class of sRNAs. However, in Arabidopsis thaliana flowers, miRNAs account for only 5% of the sRNA mass and less than 0.1% of the sequence complexity. The remaining sRNAs form a complex population of more than 100,000 different small interfering RNAs (siRNAs) transcribed from thousands of loci. The biogenesis of most of the siRNAs in Arabidopsis are dependent on RNA polymerase IV (PolIV), a homologue of DNA-dependent RNA polymerase II. A subset of these PolIV-dependent (p4)-siRNAs are involved in stress responses, and others are associated with epigenetic modifications to DNA or chromatin; however, the biological role is not known for most of them. Here we show that the predominant phase of p4-siRNA accumulation is initiated in the maternal gametophyte and continues during seed development. Expression of p4-siRNAs in developing endosperm is specifically from maternal chromosomes. Our results provide the first evidence for a link between genomic imprinting and RNA silencing in plants.

Hybrid organisms may fail to develop, be sterile or they may be more vigorous than either of the parents. Examples of hybrid vigour or hybrid necrosis in the F1 are often not inherited stably in subsequent generations if they are associated with overdominance. There can also be transgressive phenotypes that are inherited stably in these later generations, but the underlying mechanisms are not well understood. Here we have investigated the possibility that stable transgressive phenotypes in the progeny of crosses between cultivated tomato (Solanum lycopersicum cv. M82) and a wild relative (Solanum pennellii, accession LA716) are associated with micro or small interfering(si) RNAs. We identified loci from which these small(s)RNAs were more abundant in hybrids than in either parent and we show that accumulation of such transgressive sRNAs correlated with suppression of the corresponding target genes. In one instance this effect was associated with hypermethylation of the corresponding genomic DNA. Our results illustrate a potential role of transgressive sRNAs in plant breeding and in natural evolution with wild plants.