Transgenic tobacco plants expressing the Drosophila Polycomb (Pc) chromodomain show developmental alterations: possible role of Pc chromodomain proteins in chromatin-mediated gene regulation in plants.

The chromodomain of the Drosophila Polycomb (Pc) protein has been introduced into tobacco nuclei to determine its location in the nucleus and its effect on plant development. Pc is a repressor of homeotic Drosophila genes that shares a well-conserved, although not identical, chromodomain with a structural heterochromatin component, Heterochromatin Protein 1. The chromodomains might therefore play a common role in chromatin repression. An analysis of transgenic plants expressing the Pc chromodomain, which was linked to the green fluorescent protein, suggested that the Pc chromodomain has distinct target regions in the plant genome. Transgenic plants expressing the Pc chromodomain had phenotypic abnormalities in their leaves and flowers, indicating a disruption in development. In axillary shoot buds of plants displaying altered leaf phenotypes, enhanced expression of a homeodomain gene, which is downregulated in wild-type leaves, was found. In Drosophila, Pc has been shown to possess distinct chromosome binding activity and to be involved in the regulation of development-specific genes. Our results support the assumptions that the heterologous chromodomain affects related functions in Drosophila and in plants, and that chromatin modification mechanisms are involved in the regulation of certain plant genes, in a manner similar to chromatin-mediated gene regulation in Drosophila.

Developmental abnormalities associated with deoxyadenosine methylation in transgenic tobacco.

As in other higher eukaryotes, DNA methylation in plants is predominantly found at deoxycytosine residues, while deoxyadenosine residues are not methylated at significant levels. 6mdA methylation has been successfully introduced into yeast and Drosophila via expression of a heterologous methyltransferase, but similar attempts in tobacco had, up until now, proved unsuccessful despite the correct expression of a methyltransferase construct. It was unclear whether this result reflected the failure of heterologous methyltransferases to enter the nucleus, or whether 6mdA methylation, which has been shown to interfere with promoter activity, was toxic for plants. Here we show that 6mdA methylation can be successfully introduced into transgenic tobacco plants via expression of the bacterial dam enzyme. The efficiency of 6mdA methylation was directly proportional to expression levels of the dam construct, and methylation of all GATC sites was observed in a highly expressing line. Increasing expression levels of the enzyme in different plants correlated with increasingly abnormal phenotypes affecting leaf pigmentation, apical dominance, and leaf and floral structure. Whilst introduction of dam-specific methylation does not cause any developmental abnormalities in yeast or Drosophila, our data suggest that methylation of deoxyadenine residues in plants interferes with the expression of genes involved in leaf and floral development.

Gene expression in nematode-infected plant roots.

A major pathogen of potato plants (Solanum tuberosum) is the potato cyst nematode (Globodera spp.), which induces localized redifferentiation of a limited number of host cells to form a specialized feeding-site termed the syncytium. A novel strategy utilizing the polymerase chain reaction (PCR) was employed to construct a cDNA library from dissected potato roots highly enriched in syncytial material. The library was differentially screened with cDNA probes derived from the infected root tissue from a compatible interaction and from healthy root tissue. Characterization of one gene identified by the library screen indicated an expression pattern that correlated with events in the immediate vicinity of the pathogen after syncytial establishment. The strategy for library construction and screening could be applicable to the study of gene expression in any plant-pathogen interaction in which the limited supply of cells at the interface of the two organisms precludes a more traditional approach.