1.14.7S
TRANSCRIPTIONAL REGULATION AND PROMOTER ELEMENTS IN NEMATODE FEEDING SITES

C FENOLL, S SANZ-ALFÉREZ, J URIBE, FA ARISTIZÁBAL and C ESCOBAR

Departamento de Biología, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Background and objectives
Root-knot nematodes are sedentary biotrophic endoparasites which feed from root cells of a large number of different plant species. Their successful parasitic strategy is based on the establishment of permanent feeding sites (NFSs) composed of one or more modified root cells. From these modified cells the nematodes withdraw nutrients with such subtlety that the cells remain metabolically active and fully competent during the whole nematode reproductive cycle, and may last for many weeks. NFS development seems to involve the action of signal molecules contained in nematode secretions. Such enigmatic components are believed to act somehow upon vascular parenchyma cells positioned at a particular root region, the root elongation zone [1]. These cells are probably poised at a particular developmental stage which makes them competent to respond to nematodes in a stereotyped way remarkably conserved across species: the nucleus enlarges and experiences repeated mitosis and endo-reduplication processes, the central vacuole disappears, internal membrane compartments increase and become active, and numerous ribosomes, mitochondria and plastids accumulate, as the cell grows and develops a highly folded cell wall which provides a large interface with the adjacent vascular tissue. These changes can be interpreted as the consequence of a very particular cell differentiation process triggered by the nematode. Recent evidence from several laboratories shows that changes in gene expression, as expected, accompany the ultrastructural and morphological transformations [2], but the molecular characterization of NFSs is still too preliminary to allow comparison of the NFS formation process with other plant cell differentiation processes in molecular terms, which is the objective of our work.

Results and conclusions
Extensive changes in gene expression during cell differentiation are often due to changes in transcriptional regulation which are generally brought about by signal transduction cascades triggered by a unique key event. Such an event, in the case of NFSs, may involve interactions of nematode secretions with specific cell components of a signal transduction cascade in which transcriptional control is involved. Therefore, analysis of promoters differentially regulated during NFSs formation should eventually lead us one step closer to the mechanisms that initiate the nematode-dependent developmental process.

We have approached NFS differentiation by tracking a number of nematode-responsive promoters and by identifying in them putative target sites for transcription factors. The identification of such putative promoter elements is being performed by: (i) mapping protein-binding sites in in vitro DNA-protein binding assays and in vivo footprinting; and (ii) circumscribing the functional nematode-responsive sequences by expression analysis of deletion series and of chimaeric promoters. Nematode-responsive sequences which bind nuclear proteins from NFSs can be used as molecular traps to clone the genes encoding such DNA-binding proteins, which are putative transcription factors. We are using a yeast one-hybrid system based on the GAL4 trans-activation domain to screen for NFS proteins which interact with specific DNA sequences.

In a parallel strategy, we are searching NFS cDNA libraries for the presence of selected transcription factors, on the basis of the presence of their DNA target sites in nematode-responsive promoters. By combining both approaches, we expect to gain insight into the complement of transcription factors which are present in NFSs. They must lie somewhere downstream in the cascade initiated by nematode signals, and may provide useful tools to proceed upstream towards the regulatory molecule(s) that control the differentiation of a root pro-vascular cell into an NFS.

References
1. Sijmons PC, Atkinson HJ, Wyss U, 1994. Annual Review of Phytopathology 32, 235-259.
2. Fenoll C, Aristizábal FA, Sanz-Alférez S, del Campo FF, 1997. In: Fenoll C, Grundler FWM, Ohl SA, eds, Cellular and Molecular Aspects of Plant-Nematode Interactions, (Developments in Plant Pathology, Vol. 10). Kluwer, Dordrecht, pp. 133-149.