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Selfish genetic elements contribute to hybrid incompatibility and bias or ‘drive’ their own transmission1,2. Chromosomal drive typically functions in asymmetric female meiosis, whereas gene drive is normally post-meiotic and typically found in males. Here, using single-molecule and single-pollen genome sequencing, we describe Teosinte Pollen Drive, an instance of gene drive in hybrids between maize (Zea mays ssp. mays) and teosinte mexicana (Z. mays ssp. mexicana) that depends on RNA interference (RNAi). 22-nucleotide small RNAs from a non-coding RNA hairpin in mexicana depend on Dicer-like 2 (Dcl2) and target Teosinte Drive Responder 1 (Tdr1), which encodes a lipase required for pollen viability. Dcl2, Tdr1 and the hairpin are in tight pseudolinkage on chromosome 5, but only when transmitted through the male. Introgression of mexicana into early cultivated maize is thought to have been critical to its geographical dispersal throughout the Americas3, and a tightly linked inversion in mexicana spans a major domestication sweep in modern maize4. A survey of maize traditional varieties and sympatric populations of teosinte mexicana reveals correlated patterns of admixture among unlinked genes required for RNAi on at least four chromosomes that are also subject to gene drive in pollen from synthetic hybrids. Teosinte Pollen Drive probably had a major role in maize domestication and diversification, and offers an explanation for the widespread abundance of ‘self’ small RNAs in the germ lines of plants and animals.An example of hybrid incompatibility between maize and teosinte reveals a selfish toxin–antidote system mediated by small RNAs that may have contributed to the origin of maize. [ABSTRACT FROM AUTHOR]

Active DNA demethylation is required for sexual reproduction in plants but the molecular determinants underlying this epigenetic control are not known. Here, we show in Arabidopsis thaliana that the DNA glycosylases DEMETER (DME) and REPRESSOR OF SILENCING 1 (ROS1) act semi-redundantly in the vegetative cell of pollen to demethylate DNA and ensure proper pollen tube progression. Moreover, we identify six pollen-specific genes with increased DNA methylation as well as reduced expression in dme and dme;ros1. We further show that for four of these genes, reinstalling their expression individually in mutant pollen is sufficient to improve male fertility. Our findings demonstrate an essential role of active DNA demethylation in regulating genes involved in pollen function. Active DNA demethylation is required for sexual reproduction in plants, but the underlying mechanisms are unknown. Here, the authors show that the DNA glycosylases DEMETER and REPRESSOR OF SILENCING 1 enable the DNA demethylation-dependent activation of genes involved in pollen tube progression. [ABSTRACT FROM AUTHOR]

Plant genomes encode various small RNAs that function in distinct, yet overlapping, genetic and epigenetic silencing pathways. However, the abundance and diversity of small-RNA classes varies among plant species, suggesting coevolution between environmental adaptations and gene-silencing mechanisms. Biogenesis of small RNAs in plants is well understood, but we are just beginning to uncover their intricate regulation and activity. Here, we discuss the biogenesis of plant small RNAs, such as microRNAs, secondary siRNAs and heterochromatic siRNAs, and their diverse cellular and developmental functions, including in reproductive transitions, genomic imprinting and paramutation. We also discuss the diversification of small-RNA-directed silencing pathways through the expansion of RNA-dependent RNA polymerases, DICER proteins and ARGONAUTE proteins. [ABSTRACT FROM AUTHOR]

In plants, post-transcriptional gene silencing (PTGS) is mediated by DICER-LIKE 1 (DCL1)-dependent microRNAs (miRNAs), which also trigger 21-nucleotide secondary short interfering RNAs (siRNAs) via RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), DCL4 and ARGONAUTE 1 (AGO1), whereas transcriptional gene silencing (TGS) of transposons is mediated by 24-nucleotide heterochromatic (het)siRNAs, RDR2, DCL3 and AGO4 (ref. 4). Transposons can also give rise to abundant 21-nucleotide 'epigenetically activated' small interfering RNAs (easiRNAs) in DECREASED DNA METHYLATION 1 (ddm1) and DNA METHYLTRANSFERASE 1 (met1) mutants, as well as in the vegetative nucleus of pollen grains and in dedifferentiated plant cell cultures. Here we show that easiRNAs in Arabidopsis thaliana resemble secondary siRNAs, in that thousands of transposon transcripts are specifically targeted by more than 50 miRNAs for cleavage and processing by RDR6. Loss of RDR6, DCL4 or DCL1 in a ddm1 background results in loss of 21-nucleotide easiRNAs and severe infertility, but 24-nucleotide hetsiRNAs are partially restored, supporting an antagonistic relationship between PTGS and TGS. Thus miRNA-directed easiRNA biogenesis is a latent mechanism that specifically targets transposon transcripts, but only when they are epigenetically reactivated during reprogramming of the germ line. This ancient recognition mechanism may have been retained both by transposons to evade long-term heterochromatic silencing and by their hosts for genome defence. [ABSTRACT FROM AUTHOR]

A growing number of functions are emerging for RNA interference (RNAi) in the nucleus, in addition to well-characterized roles in post-transcriptional gene silencing in the cytoplasm. Epigenetic modifications directed by small RNAs have been shown to cause transcriptional repression in plants, fungi and animals. Additionally, increasing evidence indicates that RNAi regulates transcription through interaction with transcriptional machinery. Nuclear small RNAs include small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) and are implicated in nuclear processes such as transposon regulation, heterochromatin formation, developmental gene regulation and genome stability. [ABSTRACT FROM AUTHOR]

Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. The inheritance of heterochromatin through mitosis requires RNA interference (RNAi), which guides histone modification during the DNA replication phase of the cell cycle. Here we show that the alternating arrangement of origins of replication and non-coding RNA in pericentromeric heterochromatin results in competition between transcription and replication in Schizosaccharomyces pombe. Co-transcriptional RNAi releases RNA polymerase II (Pol II), allowing completion of DNA replication by the leading strand DNA polymerase, and associated histone modifying enzymes that spread heterochromatin with the replication fork. In the absence of RNAi, stalled forks are repaired by homologous recombination without histone modification. [ABSTRACT FROM AUTHOR]

Centromere-binding protein B (CENP-B) is a widely conserved DNA binding factor associated with heterochromatin and centromeric satellite repeats. In fission yeast, CENP-B homologues have been shown to silence long terminal repeat (LTR) retrotransposons by recruiting histone deacetylases. However, CENP-B factors also have unexplained roles in DNA replication. Here we show that a molecular function of CENP-B is to promote replication-fork progression through the LTR. Mutants have increased genomic instability caused by replication-fork blockage that depends on the DNA binding factor switch-activating protein 1 (Sap1), which is directly recruited by the LTR. The loss of Sap1-dependent barrier activity allows the unhindered progression of the replication fork, but results in rearrangements deleterious to the retrotransposon. We conclude that retrotransposons influence replication polarity through recruitment of Sap1 and transposition near replication-fork blocks, whereas CENP-B counteracts this activity and promotes fork stability. Our results may account for the role of LTR in fragile sites, and for the association of CENP-B with pericentromeric heterochromatin and tandem satellite repeats. [ABSTRACT FROM AUTHOR]

In the ovules of most sexual flowering plants female gametogenesis is initiated from a single surviving gametic cell, the functional megaspore, formed after meiosis of the somatically derived megaspore mother cell (MMC). Because some mutants and certain sexual species exhibit more than one MMC, and many others are able to form gametes without meiosis (by apomixis), it has been suggested that somatic cells in the ovule are competent to respond to a local signal likely to have an important function in determination. Here we show that the Arabidopsis protein ARGONAUTE 9 (AGO9) controls female gamete formation by restricting the specification of gametophyte precursors in a dosage-dependent, non-cell-autonomous manner. Mutations in AGO9 lead to the differentiation of multiple gametic cells that are able to initiate gametogenesis. The AGO9 protein is not expressed in the gamete lineage; instead, it is expressed in cytoplasmic foci of somatic companion cells. Mutations in SUPPRESSOR OF GENE SILENCING 3 and RNA-DEPENDENT RNA POLYMERASE 6 exhibit an identical defect to ago9 mutants, indicating that the movement of small RNA (sRNAs) silencing out of somatic companion cells is necessary for controlling the specification of gametic cells. AGO9 preferentially interacts with 24-nucleotide sRNAs derived from transposable elements (TEs), and its activity is necessary to silence TEs in female gametes and their accessory cells. Our results show that AGO9-dependent sRNA silencing is crucial to specify cell fate in the Arabidopsis ovule, and that epigenetic reprogramming in companion cells is necessary for sRNA–dependent silencing in plant gametes. [ABSTRACT FROM AUTHOR]

Overlapping epigenetic mechanisms have evolved in eukaryotic cells to silence the expression and mobility of transposable elements (TEs). Owing to their ability to recruit the silencing machinery, TEs have served as building blocks for epigenetic phenomena, both at the level of single genes and across larger chromosomal regions. Important progress has been made recently in understanding these silencing mechanisms. In addition, new insights have been gained into how this silencing has been co-opted to serve essential functions in 'host' cells, highlighting the importance of TEs in the epigenetic regulation of the genome. [ABSTRACT FROM AUTHOR]

The external appearance of flowering plants is determined to a large extent by the forms of flower-bearing branch systems, known as inflorescences, and their position in the overall structure of the plant. Branches and branching patterns are produced by tissues called shoot apical meristems. Thus, inflorescence architecture reflects meristem number, arrangement and activity, and the duration of meristem activity correlates with branch length. The inflorescences of maize, unlike those of related grasses such as rice and sorghum, predominantly lack long branches, giving rise to the tassel and familiar corncob. Here we report the isolation of the maize ramosa1 gene and show that it controls inflorescence architecture. Through its expression in a boundary domain near the nascent meristem base, ramosa1 imposes short branch identity as branch meristems are initiated. A second gene, ramosa2, acts through ramosa1 by regulating ramosa1 gene expression levels. ramosa1 encodes a transcription factor that appears to be absent in rice, is heterochronically expressed in sorghum, and may have played an important role in maize domestication and grass evolution. [ABSTRACT FROM AUTHOR]

Gene regulation by RNA interference requires the functions of the PAZ domain protein Argonaute. In plants, mutations in ARGONAUTE1 (AGO1) are associated with distinctive developmental defects that suggest a role for microRNA (miRNA) in organ polarity. Potential targets of miRNA regulation are the homeodomain/leucine zipper genes PHABULOSA (PHB) and PHAVOLUTA (PHV). These genes are expressed in a polar fashion in leaf primordia and are required for adaxial cell fate. Here we show that a 21-nucleotide miRNA that directs cleavage of PHB/PHV messenger RNA accumulates first in the embryonic meristem, and then in the abaxial domain of the developing leaf. miRNA distribution is disrupted by mutations in AGO1, indicating that AGO1 affects the regulation of miRNA. In addition, interactions between homeodomain/leucine zipper genes and an allelic series of ago1 indicate that miRNA acts as a signal to specify leaf polarity. [ABSTRACT FROM AUTHOR]

To ensure flowering in favourable conditions, many plants flower only after an extended period of cold, namely winter. In Arabidopsis, the acceleration of flowering by prolonged cold, a process called vernalization, involves downregulation of the protein FLC, which would otherwise prevent flowering. This lowered FLC expression is maintained through subsequent development by the activity of VERNALIZATION (VRN) genes. VRN1 encodes a DNA-binding protein whereas VRN2 encodes a homologue of one of the Polycomb group proteins, which maintain the silencing of genes during animal development. Here we show that vernalization causes changes in histone methylation in discrete domains within the FLC locus, increasing dimethylation of lysines 9 and 27 on histone H3. Such modifications identify silenced chromatin states in Drosophila and human cells. Dimethylation of H3 K27 was lost only in vrn2 mutants, but dimethylation of H3 K9 was absent from both vrn1 and vrn2, consistent with VRN1 functioning downstream of VRN2. The epigenetic memory of winter is thus mediated by a ‘histone code’ that specifies a silent chromatin state conserved between animals and plants. [ABSTRACT FROM AUTHOR]

RISC, the RNA-induced silencing complex, uses short interfering RNAs (siRNAs) or micro RNAs (miRNAs) to select its targets in a sequence-dependent manner. Key RISC components are Argonaute proteins, which contain two characteristic domains, PAZ and PIWI. PAZ is highly conserved and is found only in Argonaute proteins and Dicer. We have solved the crystal structure of the PAZ domain of Drosophila Argonaute2. The PAZ domain contains a variant of the OB fold, a module that often binds single-stranded nucleic acids. PAZ domains show low-affinity nucleic acid binding, probably interacting with the 3' ends of single-stranded regions of RNA. PAZ can bind the characteristic two-base 3' overhangs of siRNAs, indicating that although PAZ may not be a primary nucleic acid binding site in Dicer or RISC, it may contribute to the specific and productive incorporation of siRNAs and miRNAs into the RNAi pathway. [ABSTRACT FROM AUTHOR]

In plants, animals and fungi, active centromeres are associated with arrays of repetitive DNA sequences. The outer repeats at fission yeast ( Schizosaccharomyces pombe) centromeres are heterochromatic and are required for the assembly of an active centromere. Components of the RNA interference (RNAi) machinery process transcripts derived from these repeats and mediate the formation of silent chromatin. A subfragment of the repeat ( dg) is known to induce silencing of marker genes at euchromatic sites and is required for centromere formation. We show that the RNAi components, Argonaute (Ago1), Dicer (Dcr1) and RNA-dependent RNA polymerase (Rdp1), are required to maintain silencing, lysine 9 methylation of histone H3 and association of Swi6 via this dg ectopic silencer. Deletion of Ago1, Dcr1 or Rdp1 disrupts chromosome segregation leading to a high incidence of lagging chromosomes on late anaphase spindles and sensitivity to a microtubule poison. Analysis of dg transcription indicates that csp mutants, previously shown to abrogate centromere silencing and chromosome segregation, are also defective in the regulation of non-coding centromeric RNAs. In addition, histone H3 lysine 9 methylation at, and recruitment of Swi6 and cohesin to, centromeric repeats is disrupted in these mutants. Thus the formation of silent chromatin on dg repeats and the development of a fully functional centromere is dependent on RNAi. [ABSTRACT FROM AUTHOR]

Shows that the Arabidopsis mutant asymmetric leaves1 (as1) disrupts the process of meristem function. Finding that AS1 encodes a myb domain protein; Role of negatively regulating the homeobox genes KNAT1 and KNAT2; Negative regulation of as1 by the meristematic homeobox gene SHOOT MERISTEMLESS; Implications for differentiation between stem cells and organ founder cells within the shoot apical meristem; Demonstration that genes expressed in organ primordia interact with meristematic genes to regulate shoot morphogenesis.

The Arabidopsis genome sequence is scheduled for completion at the end of this year (December 2000). It will be the first higher plant genome to be sequenced, and will allow a detailed comparison with bacterial, yeast and animal genomes. Already, two of the five chromosomes have been sequenced, and we have had our first glimpse of higher eukaryotic centromeres, and the structure of heterochromatin. The implications for understanding plant gene function, genome structure and genome organization are profound. In this review, the lessons learned for future genome projects are reviewed as well as a summary of the initial findings in Arabidopsis. [ABSTRACT FROM AUTHOR]

The genomes of higher plants and animals are highly differentiated, and are composed of a relatively small number of genes and a large fraction of repetitive DNA. The bulk of this repetitive DNA constitutes transposable, and especially retrotransposable, elements. It has been hypothesized that most of these elements are heavily methylated relative to genes, but the evidence for this is controversial. We show here that repeat sequences in maize are largely excluded from genomic shotgun libraries by the selection of an appropriate host strain because of their sensitivity to bacterial restriction-modification systems. In contrast, unmethylated genic regions are preserved in these genetically filtered libraries if the insert size is less than the average size of genes. The representation of unique maize sequences not found in plant reference genomes is also greatly enriched. This demonstrates that repeats, and not genes, are the primary targets of methylation in maize. The use of restrictive libraries in genome shotgun sequencing in plant genomes should allow significant representation of genes, reducing the number of reactions required. [ABSTRACT FROM AUTHOR]

Arabidopsis thaliana has emerged as a model system for studies of plant genetics and development, and its genome has been targeted for sequencing by an international consortium (the Arabidopsis Genome Initiative; http://genome-www. stanford.edu/Arabidopsis/agi.html). To support the genomesequencing effort, we fingerprinted more than 20,000 BACs (ref. 2) from two high-quality publicly available libraries, generating an estimated 17-fold redundant coverage of the genome, and used the fingerprints to nucleate assembly of the data by computer. Subsequent manual revision of the assemblies resulted in the incorporation of 19,661 fingerprinted BACs into 169 ordered sets of overlapping clones ('contigs'), each containing at least 3 clones. These contigs are ideal for parallel selection of BACs for large-scale sequencing and have supported the generation of more than 5.8 Mb of finished genome sequence submitted to GenBank; analysis of the sequence has confirmed the integrity of contigs constructed using this fingerprint data. Placement of contigs onto chromosomes can now be performed, and is being pursued by groups involved in both sequencing and positional cloning studies. To our knowledge, these data provide the first example of whole-genome random BAC fingerprint analysis of a eucaryote, and have provided a model essential to efforts aimed at generating similar databases of fingerprint contigs to support sequencing of other complex genomes, including that of human. [ABSTRACT FROM AUTHOR]

Arabidopsis thaliana is a small flowering plant that serves as the major model system in plant molecular genetics. The efforts of many scientists have produced genetic maps that provide extensive coverage of the genome (http://genome-www. stanford.edu/Arabidopsis/maps.html). Recently, detailed YAC, BAC, P1 and cosmid-based physical maps (that is, representations of genomic regions as sets of overlapping clones of corresponding libraries) have been established that extend over wide genomic areas ranging from several hundreds of kilobases to entire chromosomes. These maps provide an entry to gain deeper insight into the A. thaliana genome structure. A. thaliana has been chosen as the subject of the first large-scale project intended to determine the full genome sequence of a plant. This sequencing project, together with the increasing interest in map-based gene cloning, has highlighted the requirement for a complete and accurate physical map of this plant species. To supply the scientific community with a high-quality resource, we present here a complete physical map of A. thaliana using essentially the IGF BAC library. The map consists of 27 contigs that cover the entire genome, except for the presumptive centromeric regions, nucleolar organization regions (NOR) and telomeric areas. This is the first reported map of a complex organism based entirely on BAC clones and it represents the most homogeneous and complete physical map established to date for any plant genome. Furthermore, the analysis performed here serves as a model for an efficient physical mapping procedure using BAC clones that can be applied to other complex genomes. [ABSTRACT FROM AUTHOR]

The α- Amy1 and α- Amy2 genes of wheat produce distinct subsets of α-amylase isozymes which show different patterns of expression in wheat aleurone cells and in developing grain. In order to characterise the organisation and expression of these genes, clones of α- Amy1 and α- Amy2 cDNA have been isolated. The two types of cDNA clone were distinguished within a small library of α-amylase cDNA clones (Baulcombe and Buffard, Planta 157 493-501 [1983]) by restriction endonuclease mapping and by cross hybridisation. The identity of α- Amy1 or α- Amy2 type was assigned from the results of hybrid selected translation analysis in which small subfragments of the cDNA clones were used. These subfragments were derived from the 3′ ends of the cDNA and did not cross hybridise between the different types of cDNA. Hybridisation of α- Amy1 and α- Amy2 cDNA probes to restriction enzyme digests of wheat nuclear DNA revealed that these are multigene families located on the group 6 (α- Amy1) and group 7 (α- Amy2) chromosomes. Studies on the levels of α- Amy1 and α- Amy2 mRNA in developing grain and in aleurone tissue indicated that the differences in isozyme expression are due to the patterns of mRNA accumulation. In aleurone tissue the α- Amy1 transcripts accumulate in parallel with other genes which are regulated by gibberellic acid, while the accumulation of α- Amy2 genes is sustained for 36 h longer. [ABSTRACT FROM AUTHOR]

One of the most striking properties of RNA interference (RNAi) in Caenorhabditis elegans is its persistence in offspring after the triggering double-stranded RNA (dsRNA) has disappeared. A new study reveals that a heterochromatic silencing mark is deposited around the targets of RNAi and is transmitted through generations. These results show that RNAi can induce stable and heritable chromatin modifications in animals. [ABSTRACT FROM AUTHOR]

Tandem repeats are prone to epigenetic silencing regulated by RNA interference. This may be because siRNAs from tandem array transcripts are regenerated by RNA-dependent RNA polymerase (RdRP) and Dicer, but siRNAs from single-copy sequences are exhausted by sequential use of downstream primers by RdRP. This could account for the formation of heterochromatin from tandem repeats. [ABSTRACT FROM AUTHOR]

Reports that adenine methylation is directed to eukaryotic target sites in vivo when DNA-binding proteins are tethered to dam methylase from Escherichia coli. Key issues of interest; Analysis of pertinent topics and relevant issues; Implications on genetics.