Intra- and intercellular RNA interference in Arabidopsis thaliana requires components of the microRNA and heterochromatic silencing pathways

In RNA interference (RNAi), double-stranded RNA (dsRNA) is processed into short interfering RNA (siRNA) to mediate sequence-specific gene knockdown. The genetics of plant RNAi is not understood, nor are the bases for its spreading between cells. Here, we unravel the requirements for biogenesis and action of siRNAs directing RNAi in Arabidopsis thaliana and show how alternative routes redundantly mediate this process under extreme dsRNA dosages. We found that SMD1 and SMD2, required for intercellular but not intracellular RNAi, are allelic to RDR2 and NRPD1a, respectively, previously implicated in siRNA-directed heterochromatin formation through the action of DCL3 and AGO4. However, neither DCL3 nor AGO4 is required for non-cell autonomous RNAi, uncovering a new pathway for RNAi spreading or detection in recipient cells. Finally, we show that the genetics of RNAi is distinct from that of antiviral silencing and propose that this experimental silencing pathway has a direct endogenous plant counterpart.     

Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome

To gain insight into the function of DNA methylation at cis-regulatory regions and its impact on gene expression, we measured methylation, RNA polymerase occupancy and histone modifications at 16,000 promoters in primary human somatic and germline cells. We find CpG-poor promoters hypermethylated in somatic cells, which does not preclude their activity. This methylation is present in male gametes and results in evolutionary loss of CpG dinucleotides, as measured by divergence between humans and primates. In contrast, strong CpG island promoters are mostly unmethylated, even when inactive. Weak CpG island promoters are distinct, as they are preferential targets for de novo methylation in somatic cells. Notably, most germline-specific genes are methylated in somatic cells, suggesting additional functional selection. These results show that promoter sequence and gene function are major predictors of promoter methylation states. Moreover, we observe that inactive unmethylated CpG island promoters show elevated levels of dimethylation of Lys4 of histone H3, suggesting that this chromatin mark may protect DNA from methylation.     

Recombination and linkage disequilibrium in Arabidopsis thaliana

Linkage disequilibrium (LD) is a major aspect of the organization of genetic variation in natural populations. Here we describe the genome-wide pattern of LD in a sample of 19 Arabidopsis thaliana accessions using 341,602 non-singleton SNPs. LD decays within 10 kb on average, considerably faster than previously estimated. Tag SNP selection algorithms and 'hide-the-SNP' simulations suggest that genome-wide association mapping will require only 40%-50% of the observed SNPs, a reduction similar to estimates in a sample of African Americans. An Affymetrix genotyping array containing 250,000 SNPs has been designed based on these results; we demonstrate that it should have more than adequate coverage for genome-wide association mapping. The extent of LD is highly variable, and we find clear evidence of recombination hotspots, which seem to occur preferentially in intergenic regions. LD also reflects the action of selection, and it is more extensive between nonsynonymous polymorphisms than between synonymous polymorphisms.     

Gene silencing in cancer by histone H3 lysine 27 trimethylation independent of promoter DNA methylation

Epigenetic silencing in cancer cells is mediated by at least two distinct histone modifications, polycomb-based histone H3 lysine 27 trimethylation (H3K27triM) and H3K9 dimethylation. The relationship between DNA hypermethylation and these histone modifications is not completely understood. Using chromatin immunoprecipitation microarrays (ChIP-chip) in prostate cancer cells compared to normal prostate, we found that up to 5% of promoters (16% CpG islands and 84% non-CpG islands) were enriched with H3K27triM. These genes were silenced specifically in prostate cancer, and those CpG islands affected showed low levels of DNA methylation. Downregulation of the EZH2 histone methyltransferase restored expression of the H3K27triM target genes alone or in synergy with histone deacetylase inhibition, without affecting promoter DNA methylation, and with no effect on the expression of genes silenced by DNA hypermethylation. These data establish EZH2-mediated H3K27triM as a mechanism of tumor-suppressor gene silencing in cancer that is potentially independent of promoter DNA methylation.     

The PHYTOCHROME C photoreceptor gene mediates natural variation in flowering and growth responses of Arabidopsis thaliana

Light has an important role in modulating seedling growth and flowering time. We show that allelic variation at the PHYTOCHROME C (PHYC) photoreceptor locus affects both traits in natural populations of A. thaliana. Two functionally distinct PHYC haplotype groups are distributed in a latitudinal cline dependent on FRIGIDA, a locus that together with FLOWERING LOCUS C explains a large portion of the variation in A. thaliana flowering time. In a genome-wide scan for association of 65 loci with latitude, there was an excess of significant P values, indicative of population structure. Nevertheless, PHYC was the most strongly associated locus across 163 strains, suggesting that PHYC alleles are under diversifying selection in A. thaliana. Our work, together with previous findings, suggests that photoreceptor genes are major agents of natural variation in plant flowering and growth response.     

Flowering-time genes modulate meristem determinacy and growth form in Arabidopsis thaliana

Plants have evolved annual and perennial life forms as alternative strategies to adapt reproduction and survival to environmental constraints. In isolated situations, such as islands, woody perennials have evolved repeatedly from annual ancestors. Although the molecular basis of the rapid evolution of insular woodiness is unknown, the molecular difference between perennials and annuals might be rather small, and a change between these life strategies might not require major genetic innovations. Developmental regulators can strongly affect evolutionary variation and genes involved in meristem transitions are good candidates for a switch in growth habit. We found that the MADS box proteins SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FRUITFULL (FUL) not only control flowering time, but also affect determinacy of all meristems. In addition, downregulation of both proteins established phenotypes common to the lifestyle of perennial plants, suggesting their involvement in the prevention of secondary growth and longevity in annual life forms.     

Induction, suppression and requirement of RNA silencing pathways in virulent Agrobacterium tumefaciens infections

Regulation of gene expression through microRNAs (miRNAs) and antiviral defense through small interfering RNAs (siRNAs) are aspects of RNA silencing, a process originally discovered as an unintended consequence of plant transformation by disarmed Agrobacterium tumefaciens strains. Although RNA silencing protects cells against foreign genetic elements, its defensive role against virulent, tumor-inducing bacteria has remained unexplored. Here, we show that siRNAs corresponding to transferred-DNA oncogenes initially accumulate in virulent A. tumefaciens-infected tissues and that RNA interference-deficient plants are hypersusceptible to the pathogen. Successful infection relies on a potent antisilencing state established in tumors whereby siRNA synthesis is specifically inhibited. This inhibition has only modest side effects on the miRNA pathway, shown here to be essential for disease development. The similarities and specificities of the A. tumefaciens RNA silencing interaction are discussed and contrasted with the situation encountered with plant viruses.     

Formation of stable epialleles and their paramutation-like interaction in tetraploid Arabidopsis thaliana

Polyploidization is found frequently in plants, and species previously considered to be diploid may show remnants of earlier polyploidization events on closer inspection of their genomes. The success of polyploids may lie in increased genetic redundancy supporting subsequent genetic diversification. Although doubling the genome does not generate diversity per se, recent studies show that rapid genomic rearrangements and changes in DNA modification and gene expression patterns are associated with polyploid formation. But recessive modifications will not become phenotypically apparent in early polyploid generations. Here we show that epialleles in tetraploid plants (but not in diploids) interact in trans and lead to heritable gene silencing persisting after segregation from the inactivating allele. This mechanism, resembling paramutation, leads to the establishment of functional epigenetic homozygosity and, thus, to conversion of new recessive alleles into traits expressed in early polyploid generations. Such interactions probably contribute to rapid adaptation and evolution of polyploid plant species.     

Pathogen-induced, NADPH oxidase-derived reactive oxygen intermediates suppress spread of cell death in Arabidopsis thaliana

Plant immune responses are usually accompanied by the production of extracellular superoxide at and surrounding infection sites. Extracellular reactive oxygen intermediates (ROIs) in plants were proposed to drive programmed cell death correlated with disease resistance (the hypersensitive response). ROIs derived from this oxidative burst are generated by plasma membrane NADPH oxidases, anchored by gp91(phox) proteins related to those responsible for the respiratory oxidative burst activated in mammalian neutrophils during infection. Mutation of Arabidopsis thaliana respiratory burst oxidase (Atrboh) genes eliminated pathogen-induced ROI production but had only a modest effect on the hypersensitive response. We show that Atrboh function can be activated by exogenous ROIs. Unexpectedly, the subsequent oxidative burst can suppress cell death in cells surrounding sites of NADPH oxidase activation. This cell death requires salicylic acid, a plant immune system activator. Thus, ROIs generated by Atrboh proteins can antagonize salicylic acid-dependent pro-death signals. These results have implications for understanding how salicylic acid activates defense signaling in cells spatially removed from infection sites without causing cell death.