Max-Planck-institut fur Zuchtungsforschung, Department of Biochemistry, Cari-von-Linne-Weg 10, D-50829 Koln, Germany

Background and objectives
To study in detail cell biological aspects of one of the major defence responses of plants against microbial infection, the hypersensitive reaction, we have recently established a model system of reduced complexity: cultured parsley (Petroselinum crispum) cells infected with the phytopathogenic fungus Phytophthora infestans. This model system closely reflected the non-host relationship between parsley plants and Phytophthora sojae and exhibited major features of the hypersensitive reaction that we observed in planta [1]. With this system we have in our hands a powerful tool to investigate in detail pivotal events in the plant defence response at the single-cell level.

Results and conclusions
By cytological methods we have monitored in infected cells rapid morphological and metabolic changes. Simultaneously with the translocation of cytoplasm and nucleus to the penetration site, we observed the increase in mitochondrial activity, the activation of defence-related genes and the intracellular accumulation of reactive oxygen species [2]. Studies on the cytoskeleton architecture of infected cells revealed the dissociation of microtubules and the formation of a new actin focus at the penetration site. In particular, the actin filament network appeared to be involved in the cytoplasmic rearrangements [1]. By cytochemical staining with the dye 2',7'-dichlorofluorescin diacetate (DCFH-DA), visualizing intracellular reactive oxygen species, and the vital dyes fluorescein diacetate or Neutral red, we have obtained evidence that the intracellular accumulation of reactive oxygen species (ROS) correlates with deterioration and leakage of membranes in infected parsley cells [2]. We suppose that unsaturated fatty acids may become more and more oxidized by ROS, resulting in increasing membrane damage and final collapse. The defence reactions of parsley cells can be grouped in two categories: morphological changes (translocation of cytoplasm and nucleus, wall apposition, hypersensitive cell death) and biochemical changes (increased ion fluxes, generation of reactive oxygen species, increase of mitochondrial activity, activation of defence-related genes and production of phytoalexins). Only the biochemical changes can be induced by treatment of cultured parsley cells with a defined peptide elicitor molecule from the non-host pathogen P. sojae. The question may be asked, which additional signals are needed for the induction of the complete defenve response? It is conceivable that attempted penetration by the fungus causes in addition to chemical, also mechanical signals and that, hence, a combination of signals has to be perceived by the plant cell to perform all steps of the hypersensitive reaction.

To test this hypothesis we studied by cytological methods the response of parsley cells upon local mechanical stimulation and local application of a defined elicitor molecule. The local mechanical stimulus alone was able to induce translocation of cytoplasm and nucleus to the site of triggering as well as the generation of intracellular reactive oxygen species (ROS). If a defined elicitor molecule of the oomycetous pathogen P. sojae was applied locally to the surface of cultured parsley cells, intracellular ROS rapidly accumulated but essentially no morphological changes were observed. Combining the local physical and chemical stimulus, we detected interference with respect to induction of the cytoplasmic rearrangements dependent on the elicitor concentration. Applying locally low doses of the elicitor simultaneously with gentle, local physical pressure, the 'natural' situation of early stages in fungal infection was largly simulated: the plant cells responded with cytoplasmic aggregation at the site of stimulation, nuclear migration to this site and oxidative burst.

1. Schmeizer E, Naton B, Freytag S et al., 1995. Canadian Journal of Botany 73 (Suppl. 1), S426-S434.
2. Naton B, Hahlbrock K, Schmeizer E, 1996. Plant Physiology 112, 433-444.