High frequency, cell type-specific visualization of fluorescent-tagged genomic sites in interphase and mitotic cells of living Arabidopsis plants.

Background: Interphase chromosome organization and dynamics can be studied in living cells using fluorescent tagging techniques that exploit bacterial operator/repressor systems and auto-fluorescent proteins. A nuclear-localized Repressor Protein-Fluorescent Protein (RP-FP) fusion protein binds to operator repeats integrated as transgene arrays at defined locations in the genome. Under a fluorescence microscope, the tagged sites appear as bright fluorescent dots in living cells.

A stepwise pathway for biogenesis of 24-nt secondary siRNAs and spreading of DNA methylation.

We used a transgene system to study spreading of RNA-directed DNA methylation (RdDM) during transcriptional gene silencing in Arabidopsis thaliana. Forward and reverse genetics approaches using this system delineated a stepwise pathway for the biogenesis of secondary siRNAs and unidirectional spreading of methylation from an upstream enhancer element into downstream sequences. Trans-acting, hairpin-derived primary siRNAs induce primary RdDM, independently of an enhancer-associated 'nascent' RNA, at the target enhancer region.

Fluorescent transgenes to study interphase chromosomes in living plants.

Fluorescence tagging of genomic sites through the use of bacterial operator/repressor systems combined with fluorescent proteins permits high-resolution analysis of interphase chromosomes in living cells. This technique has been used to study interphase chromosome arrangement and dynamics in yeast, Drosophila, and mammalian cells, but is only beginning to be exploited in plant systems. In this chapter, we describe methods for producing and identifying Arabidopsis thaliana plants harbouring fluorescence-tagged transgenes.

RNA-directed DNA methylation mediated by DRD1 and Pol IVb: a versatile pathway for transcriptional gene silencing in plants.

RNA-directed DNA methylation, which is one of several RNAi-mediated pathways in the nucleus, has been highly elaborated in the plant kingdom. RNA-directed DNA methylation requires for the most part conventional DNA methyltransferases, histone modifying enzymes and RNAi proteins; however, several novel, plant-specific proteins that are essential for this process have been identified recently. DRD1 (defective in RNA-directed DNA methylation) is a putative SWI2/SNF2-like chromatin remodelling protein; DRD2 and DRD3 (renamed NRPD2a and NRPD1b, respectively) are subunits of Pol IVb, a putative RNA polymerase found only in plants.

Use of two-color fluorescence-tagged transgenes to study interphase chromosomes in living plants.

Sixteen distinct sites distributed on all five Arabidopsis (Arabidopsis thaliana) chromosomes have been tagged using different fluorescent proteins and one of two different bacterial operator-repressor systems: (1) a yellow fluorescent protein-Tet repressor fusion protein bound to tet operator sequences, or (2) a green or red fluorescent protein-Lac repressor fusion protein bound to lac operator sequences. Individual homozygous lines and progeny of intercrosses between lines have been used to study various aspects of interphase chromosome organization in root cells of living, untreated seedlings. Features reported here include distances between transgene alleles, distances between transgene inserts on different chromosomes, distances between transgene inserts on the same chromatin fiber, alignment of homologous chromosomes, and chromatin movement.

Evidence for nuclear processing of plant micro RNA and short interfering RNA precursors.

The Arabidopsis genome encodes four Dicer-like (DCL) proteins, two of which contain putative nuclear localization signals. This suggests one or more nuclear pathways for processing double-stranded (ds) RNA in plants. To study the subcellular location of processing of nuclear-encoded dsRNA involved in transcriptional silencing, we examined short interfering (si) RNA and micro (mi) RNA accumulation in transgenic Arabidopsis expressing nuclear and cytoplasmic variants of P19, a viral protein that suppresses posttranscriptional gene silencing.

Studies on the effects of a flanking repetitive sequence on the expression of single-copy transgenes in Nicotiana sylvestris and in N. sylvestris-N. tomentosiformis hybrids.

To test the influence of a Nicotiana tomentosiformis repetitive sequence (R8.3) on transgene expression in N. sylvestris and in N.

HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by double-stranded RNA.

To analyze relationships between RNA signals, DNA methylation and chromatin modifications, we performed a genetic screen to recover Arabidopsis mutants defective in RNA-directed transcriptional silencing and methylation of a nopaline synthase promoter-neomycinphosphotransferase II (NOSpro- NPTII) target gene. Mutants were identified by screening for recovery of kanamycin resistance in the presence of an unlinked silencing complex encoding NOSpro double-stranded RNA. One mutant, rts1 (RNA-mediated transcriptional silencing), displayed moderate recovery of NPTII gene expression and partial loss of methylation in the target NOSpro, predominantly at symmetrical C(N)Gs.

RNA-directed DNA methylation in Arabidopsis.

In plants, double-stranded RNA that is processed to short RNAs approximately 21-24 nt in length can trigger two types of epigenetic gene silencing. Posttranscriptional gene silencing, which is related to RNA interference in animals and quelling in fungi, involves targeted elimination of homologous mRNA in the cytoplasm. RNA-directed DNA methylation involves de novo methylation of almost all cytosine residues within a region of RNA-DNA sequence identity.