RNA: guiding gene silencing.

In diverse organisms, small RNAs derived from cleavage of double-stranded RNA can trigger epigenetic gene silencing in the cytoplasm and at the genome level. Small RNAs can guide posttranscriptional degradation of complementary messenger RNAs and, in plants, transcriptional gene silencing by methylation of homologous DNA sequences. RNA silencing is a potent means to counteract foreign sequences and could play an important role in plant and animal development.

Localization of Agrobacterium rhizogenes T-DNA in plant chromosomes by in situ hybridization.

We have used in situ hybridization to determine the sites of insertion of Agrobacterium rhizogenes Ri T-DNA in the chromosomes of Crepis capillaris (2n = 6) transformed roots. Four transformed root lines were obtained by infecting Crepis stem segments with A. rhizogenes. Southern hybridization analysis indicated that each root line was the result of one or more independent T-DNA insertion events. In two root lines, one copy of T-DNA was present; the other two root lines each contained two copies of T-DNA. To localize these T-DNA inserts on Crepis chromosomes, metaphase spreads were perpared from each root line, and hybridized in situ to a biotinlabeled T-DNA probe. The results indicated that T-DNA was present in a different chromosomal location in each root line, and that each chromosome had been a target for T-DNA insertion at least once. In the root lines containing two T-DNA inserts, two patterns of integration were observed: in one case the T-DNAs were present on separate chromosomes; in the other case the two T-DNAs were close together (but not tandemly arranged) on a single chromosome. A comparison of these results and those obtained previously for a fifth Crepis-transformed root line demostrated that Ri T-DNA does not insert preferentially into a particlar chromosomal location.

RNA-based silencing strategies in plants.

In plants, double-stranded RNA can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling RNA-silencing mechanisms, which require common proteins in diverse organisms, and are assessing the role of methylation in transcriptional and posttranscriptional gene silencing.

Resistance of RNA-mediated TGS to HC-Pro, a viral suppressor of PTGS, suggests alternative pathways for dsRNA processing.

In plants, double-stranded (ds) RNA that is degraded to small (sm) RNAs that are approximately 23 nucleotides in length can trigger the degradation of homologous RNAs in the cytoplasm (posttranscriptional gene silencing or PTGS) and de novo methylation of homologous DNA in the nucleus [1]. PTGS is similar to quelling in fungi [2] and RNAi in animals [3]. RNA-directed DNA methylation (RdDM) can lead to transcriptional gene silencing (TGS) and the methylation of homologous target promoters if dsRNAs containing promoter sequences are involved [4]. HC-Pro is a plant viral suppressor of PTGS that acts by preventing the accumulation of smRNAs [5, 6] that provide the specificity determinant for homologous RNA degradation [7-10]. Here, we show that HC-Pro does not suppress TGS induced by promoter dsRNA. Moreover, the amount of promoter smRNAs is elevated 5-fold in the presence of HC-Pro, and target promoter methylation is slightly increased without a concomitant rise in the level of promoter dsRNA. The promoter dsRNA, which is not polyadenylated, failed to trigger substantial degradation of polyadenylated, single-stranded promoter RNA. The differential effects of HC-Pro on smRNA accumulation associated with dsRNA-mediated TGS and at least some cases of PTGS suggest that dsRNA processing can occur by alternative pathways, and they support the idea that RdDM is triggered by smRNAs.

Position effects and epigenetic silencing of plant transgenes.

Nuclear processes that silence plant transgenes are being revealed by analyses of natural triggers of epigenetic modifications, particularly cytosine methylation, and by comparisons of the genomic environments of differentially expressed transgene loci. It is increasingly apparent that plant genomes can sense and respond to the presence of foreign DNA in certain sequence contexts and at multiple dispersed sites. Determining the basis of this sensitivity and how nuclear defense systems are activated poses major challenges for the future.

Host defenses to transposable elements and the evolution of genomic imprinting.

Genomic imprinting is the differential expression of maternally and paternally inherited alleles of specific genes. Several organismic level hypotheses have been offered to explain the evolution of genomic imprinting. We argue that evolutionary explanations of the origin of imprinting that focus exclusively on the organismic level are incomplete. We propose that the complex molecular mechanisms that underlie genomic imprinting originally evolved as an adaptive response to the mutagenic potential of transposable elements (TEs). We also present a model of how these mechanisms may have been co-opted by natural selection to evolve molecular features characteristic of genomic imprinting.

A test for transvection in plants: DNA pairing may lead to trans-activation or silencing of complex heteroalleles in tobacco.

To study whether DNA pairing that influences gene expression can take place in somatic plant cells, a system designed to mimic transvection was established in transgenic tobacco. Pairing was evaluated by testing whether an enhancerless GUS gene on one allele could be activated in trans by an enhancer on the second allele. The required heteroalleles were obtained at four genomic locations using Cre-lox-mediated recombination. In one transgenic line, elevated GUS activity was observed with the heteroallelic combination, suggesting that trans-activation occurred. Conversely, when the unaltered allele was homozygous, GUS activity dropped to hemizygous levels in a silencing phenomenon resembling dosage compensation. Double-stranded GUS RNAs or small GUS RNAs indicative of RNA-based silencing mechanisms were not detected in plants displaying reduced GUS activity. These results suggested that a transgene locus capable of pairing, as revealed by trans-activation, could also become silenced in an RNA-independent manner, thus linking DNA pairing and gene silencing. The transgene locus was complex and comprised an inverted repeat, which possibly potentiated allelic interactions. The locus was unable to trans-activate transgenes at ectopic sites, further implicating allelic pairing in the transvection effects.

A large conductance ion channel in the nuclear envelope of a higher plant cell.

To detect and characterize ion channel activity in the nuclear envelope of a higher plant cell, we performed patch clamp experiments on nuclei isolated from coconut endosperm cells and on giant liposomes containing nuclear envelope fragments prepared from the same cells. An ion channel exhibiting a number of conductance substates, with a maximum of ca. 1,000 pS, was observed. Above an applied potential of +/- 100 mV, the behavior of the channel was similar in isolated nuclei and liposomes, indicating that both patch clamp modes were detecting the same channel. That such a channel has now been identified in members of both the animal and plant kingdoms reinforces the notion that the nuclear pores are not always open to ions.

Detection of a large cation-selective channel in nuclear envelopes of avian erythrocytes.

To determine whether the nuclear envelope of eukaryotic cells has the capability to regulate ion fluxes, we have used the patch-clamp technique to detect ion channels in this membrane system. Since possible sites for ion channels in the nuclear envelope include not only the nuclear pores, but also both the inner and outer nuclear membranes, we have patched giant liposomes composed of phosphatidylcholine and nuclear envelope fragments isolated from mature avian erythrocytes. A large, cation-selective channel with a maximum conductance of approximately 800 pS in symmetrical 100 mM KCl was detected. This channel is a possible candidate for a nuclear pore.

Gene silencing in plants: relevance for genome evolution and the acquisition of genomic methylation patterns.

Transgenes often become silenced in plants because of repressive influences exerted by flanking plant DNA and/or because of interactions among multiple copies of closely linked transgenes. Repeated transgenes on different chromosomes can also interact in a way that leads to silencing and methylation, suggesting a previously unrecognized ability of unlinked homologous sequences to cross-talk in complex genomes. Non-Mendelian inheritance is a frequent consequence of these interactions because the silenced genes do not fully reactivate or lose methylation after segregating in progeny. Several examples of gene silencing in plants appear to reflect the action of genome defence system that methylates and inactivates foreign or invasive sequences such as transgenes and transposable elements. Because certain types of transposable elements are embedded in regulatory regions of plant genes and have become greatly amplified in plant genomes, they could contribute substantially to normal gene expression and to the generation of genomic methylation patterns. Polyploidy, which has been a major force in plant and vertebrate evolution, might encourage proliferation of transposable elements because genes in polyploids are duplicated and hence less susceptible to the consequences of insertional mutagenesis. Accordingly, the appearance of genome-wide methylation has often coincided with episodes of polyploidization.

Use of forward genetic screens to identify genes required for RNA-directed DNA methylation in Arabidopsis thaliana.

RNA-directed DNA methylation is a small RNA-mediated epigenetic modification that contributes to transcriptional silencing of transposons and repetitive sequences in plants. We have conducted several forward genetic screens to identify factors required for RNA-directed DNA methylation and transcriptional gene silencing in Arabidopsis thaliana. Here, we review the findings from these screens and report on two new mutants, dms12 and dms13, that are defective in Pol V-specific subunits NRPE5 and NRPE9b. Cumulative results from genetic screens performed in our laboratory and those of other investigators have revealed that RNA-directed DNA methylation requires a complex transcriptional machinery comprising a number of plant-specific factors, many of which were functionally uncharacterized before being implicated in this pathway. Future challenges include unraveling the detailed mechanism and full range of functions of RNA-directed DNA methylation.

RNA-directed DNA methylation and Pol IVb in Arabidopsis.

Recent work in Arabidopsis has revealed a plant-specific RNA polymerase, pol IV, that is specialized for RNA interference (RNAi)-mediated, chromatin-based gene silencing. Two functionally diversified pol IV complexes have been identified: pol IVa is required to produce or amplify the small RNA trigger, whereas pol IVb, together with the plant-specific SWI/SNF-like chromatin remodeling factor DRD1, acts downstream from small RNA formation to induce de novo cytosine methylation of homologous DNA by an unknown mechanism. Retrotransposon long terminal repeats (LTRs) and other unannotated sequences that encode small RNAs are prime targets for DRD1/pol IVb-mediated cytosine methylation. In drd1 and pol IVb mutants, silent LTRs in euchromatin can be derepressed, resulting in enhanced transcription of adjacent genes or intergenic regions. In addition to mediating de novo methylation, some evidence suggests that DRD1 and pol IVb are also involved in a reciprocal process of active demethylation, perhaps in conjunction with DNA glycosylase domain-containing proteins such as ROS1. We speculate that DRD1/pol IV-dependent methylation/demethylation evolved in the plant kingdom as a means to facilitate rapid, reversible changes in gene expression, which might have adaptive significance for immobile plants growing in unpredictable environments.

A set of novel Ti plasmid-derived vectors for the production of transgenic plants.

We describe in this paper the construction and use of a set of novel Ti plasmid-derived vectors that can be used to produce transgenic plants. These vectors are based on one of two strategies: 1) double recombination into the wild-type Ti plasmid of genetic information flanked by two T-DNA fragments on a wide-host range plasmid; 2) the binary vector strategy. The vector based on the double recombination principle contains a kanamycin resistance gene for use as a plant selectable marker, a polylinker for the insertion of foreign genes, and a nopaline synthase gene. The vector was constructed such that a disarmed T-DNA results from the double recombination event. The binary vector combines several advantageous features including an origin of replication that is stable in Agrobacterium in the absence of selection, six unique sites for insertion of foreign genes, an intact nopaline synthase gene, and a kanamycin resistance marker for selection of transformed plant cells. All of these vectors have been used to produce tobacco plants transformed with a variety of foreign genes.

Epigenetic silencing of plant transgenes as a consequence of diverse cellular defence responses.

Linked and unlinked copies of transgenes and related endogenous genes in plants can be epigenetically silenced by homology-based mechanisms that operate at either the transcriptional or post-transcriptional level. Transcriptional inactivation is associated with promoter homology and meiotically heritable methylation. Post-transcriptional silencing requires homology in protein-coding regions and is fully reversed during meiosis. Recently, the notion that both of these processes reflect the action of different host defence systems has been strengthened: (i) Obvious parallels have emerged between promoter homology-dependent silencing/methylation of transgenes and paramutation of endogenous genes that contain transposable elements in their promoters: (ii) remarkable similarities have been observed between post-transcriptional silencing involving transgenes and natural forms of virus resistance in nontransgenic plants. These results and others implicate two distinct cellular defence responses in transgene silencing. One is active in the nucleus and is manifested by transgene methylation, a reaction that might have originated as a means to oppose the spread of transposable elements. A second line of defence resides in the cytoplasm and operates through enhanced RNA turnover, a process that might help plants overcome viral infection.