Arabidopsis histone deacetylase 6: a green link to RNA silencing.

Epigenetic reprogramming is at the base of cancer initiation and progression. Generally, genome-wide reduction in cytosine methylation contrasts with the hypermethylation of control regions of functionally well-established tumor suppressor genes and many other genes whose role in cancer biology is not yet clear. While insight into mechanisms that induce aberrant cytosine methylation in cancer cells is just beginning to emerge, the initiating signals for analogous promoter methylation in plants are well documented. In Arabidopsis, the silencing of promoters requires components of the RNA interference machinery and promoter double-stranded RNA (dsRNA) to induce a repressive chromatin state that is characterized by cytosine methylation and histone deacetylation catalysed by the RPD3-type histone deacetylase AtHDA6. Similar mechanisms have been shown to occur in fission yeast and mammals. This review focuses on the connections between cytosine methylation, dsRNA and AtHDA6-controlled histone deacetylation during promoter silencing in Arabidopsis and discusses potential mechanistic similarities of these silencing events in cancer and plant cells.

Endogenous viral sequences and their potential contribution to heritable virus resistance in plants.

Tobacco endogenous pararetroviruses (TEPRVs) represent the first virus-derived repetitive sequence family found in plants. The sequence conservation of TEPRVs and the lack of an exogenous form of the virus suggest that TEPRVs serve a beneficial function, perhaps by furnishing virus resistance via homologous sequence interactions. This hypothesis is supported by the observation that TEPRVs are methylated and negligibly transcribed. Moreover, transgenes driven by the TEPRV enhancer are silenced and methylated when introduced into tobacco, but remain active and unmethylated in non-host species devoid of sequences homologous to TEPRVs. In transgenic Arabidopsis, the TEPRV enhancer is active primarily in shoot meristems. This suggests that the virus giving rise to TEPRVs could infect germ cell precursors, a prerequisite for meiotically heritable insertions into host chromosomes. The copy number, organization and methylation of TEPRVs in tetraploid tobacco and one of its diploid ancestors, Nicotiana sylvestris, the presumed original host for the virus, have remained constant since polyploid formation. The remarkable conservation of these features in two independently evolving species further supports a role for TEPRVs in viral immunity.

Transcriptional silencing and promoter methylation triggered by double-stranded RNA.

Double-stranded RNA induces a post-transcriptional gene silencing process, termed RNAi, in diverse organisms. It is shown here that transcriptional gene silencing accompanied by de novo methylation of a target promoter in plants can be triggered by a double-stranded RNA containing promoter sequences. Similar to the double-stranded RNA involved in RNAi, this promoter double-stranded RNA, which is synthesized in the nucleus, is partially cleaved into small RNAs approximately 23 nucleotides in length. Both transcriptional and post-transcriptional gene silencing can thus be initiated by double-stranded RNAs that enter the same degradation pathway. The results also implicate double-stranded RNA in directing DNA methylation. Different constructs designed to produce double-stranded promoter RNA in various ways were evaluated for their ability to induce gene silencing in tobacco and Arabidopsis. RNA hairpins transcribed from inverted DNA repeats were the most effective trans-acting silencing signals. This strategy could be useful for transcriptionally downregulating genes in a variety of plants.

Host defenses to parasitic sequences and the evolution of epigenetic control mechanisms.

The analysis of transgene silencing effects in plants and other eukaryotic organisms has revealed novel mechanisms of epigenetic regulation that are based on recognition of nucleic acid sequence homology. These homology-dependent gene silencing phenomena are characterized by an inverse relationship between copy number of a particular sequence and expression levels. Depending on whether promoter regions or transcribed sequences are repeated, silencing occurs at the transcriptional or post-transcriptional level, respectively. Different silencing effects involving DNA-DNA or RNA-DNA associations in the nucleus, and RNA-RNA interactions in the cytoplasm appear to reflect distinct host defense responses to parasitic sequences, including transposable elements (TEs), viroids and RNA viruses. Natural epigenetic phenomena that resemble transgene silencing effects often involve endogenous genes comprising recognizable TE sequences or rearrangements generated by TEs and can thus be interpreted in terms of host defense systems. A genome defense that inactivates TEs by methylation might have been recruited during evolution to regulate the transcription of plant and vertebrate genes that contain remnants of TE insertions in promoter regions.

The ECS1 gene of Arabidopsis encodes a plant cell wall-associated protein and is potentially linked to a locus influencing resistance to Xanthomonas campestris.

The interaction between Arabidopsis and Xanthomonas campestris pv. campestris (Xcc) provides a useful model system to identify components that are involved in incompatible interactions of this phytopathogen and cruciferous plants. We have previously described a new gene from Arabidopsis thaliana, ECS1 (formerly CXC750), which showed an ecotype-specific expression pattern, although homologous sequences are present in the genomes of all ecotypes tested. Interestingly, ECS1 mRNA transcripts were only detected in ecotypes which showed the resistant phenotype against Xcc race 750. Subsequent genetic studies with F3 progeny of a cross between a Xcc750 resistant ecotype (Col-0) and a Xcc750 sensitive ecotype (Oy-0) revealed segregation of the ECS1RNA-phenotype away from the Xcc750 susceptible phenotype in one out of 16 F3 families. In addition, transformation of Xcc750 sensitive plants that did not express ECS1, with a constitutively transcribed ECS1 gene, did not lead to the resistant phenotype in the transgenic plants. From these results we conclude that ECS1 is not a Xcc750 resistance gene, but the genetic data indicate that ECS1 is linked to a locus influencing resistance to Xcc750. ECS1 was localized to YACs 3H12 and 12F3 of the CIC library, which map to chromosome 1. Subcellular localization of the gene product indicated that the ECS1 protein is associated with the plant cell wall. Further molecular investigation of 10 ecotypes revealed the utility of ECS1 as a new marker for chromosome 1, which is detectable by RFLP, PCR or expression analysis.

The hrpRS locus of Pseudomonas syringae pv. phaseolicola constitutes a complex regulatory unit.

The right part of the hrp cluster of Pseudomonas syringae pv. phaseolicola contains two regulatory genes, the previously described hrpS gene and an adjacent locus, hrpR. In this study we determined the sequence of hrpR and analysed the functional organization of the two genes. HrpR and HrpS show high sequence similarities to each other and to other response regulators of the two-component regulatory system. This has recently also been described for the hrpRS system of the closely related pathogen Pseudomonas syringae pv. syringae. The results of our genetic analyses strongly indicate that hrpS expression is regulated by the hrpR gene product. DNA-protein binding studies and site-directed mutagenesis of the hrpR sequence provided further evidence that HrpR activates hrpS transcription by binding to an activator site. This HrpR binding site was mapped in a fragment which is located 378-609 nucleotides upstream of the hrpS transcription start site. The hrpS transcription start site maps 179 nucleotides upstream of the initiation codon ATG, as determined by primer extension analysis, and is preceded by a typical -12/-24 promoter motif.

A new, pathogen-inducible gene of Arabidopsis is expressed in an ecotype-specific manner.

In this study we describe a novel gene, which was isolated in an attempt to search for specific plant resistance genes of Arabidopsis against isolates of the phytopathogenic bacterium Xanthomonas campestris pv. campestris. The gene was cloned by differential screening of a genomic library of the Xcc 750-resistant ecotype Col-0, using cDNA populations derived from ecotype Col-0 and the Xcc 750-susceptible ecotype Oy-0. The isolated gene, CXc750, is differentially expressed in ecotypes of Arabidopsis thaliana. In addition, although highly expressed in uninfected plants, gene expression increases in response to pathogen attack. CXc750 potentially codes for a small, basic protein of about 10 kDa. The predicted protein product contains a potential signal leader peptide at the amino-terminal end but no ER retention sequence and no further transmembrane domain. This indicates that the gene product is transported to other compartments or out of the cell. The possible function of CXc750 as a member of the plant defense response system is discussed.