A novel tetratricopeptide repeat-containing J-protein localized in a plasma membrane-bound protein complex of the phytopathogenic oomycete Phytophthora megasperma

Abstract

Phytoalexins originating from plant tissues may cause within cells of fungi or oomycetes a change in the localization of actin, tubulin and chaperones. To test the hypothesis in a filamentously growing oomycete, we compared the distribution of cellular markers in the presence and absence of hydroxystilbene phytoalexins. Using cDNA from the phytopathogenic organism Phytophthora megasperma, the causal agent of root rot on soybean and many other plants, and including probes for Hsp70 and Hsp40, we cloned a DnaJ-protein (Jcp) with the capacity of interacting with both a particular Hsp70 isoform via its J-domain and with other proteins via its tetratricopeptide repeat (TPR) domain. Antisera raised against the bacterially expressed protein Jcp allowed the analysis of its intracellular localization during hyphal growth. Following the subfractionation of cell homogenates, we detected virtually all immunoreactive Jcp in the plasma membrane-enriched fraction and as constituent of a membrane-associated protein complex. In agreement with the biochemical findings, immunocytochemical stains of hyphae showed Jcp as part of cortical patches positioned along the plasma membrane similar to the distribution of actin patches. Confocal microscopy, however, revealed that the Jcp-containing patches did not generally co-localize with the patches visualized by the actin stain. The 59-kDa Jcp, characterized by a large 8-fold TPR domain at the N-terminal region and a J-domain close to the C-terminus, is a good candidate for bridging the gap between Hsp70 and Hsp90 by protein-protein interactions. By administration of plant-derived phytoalexins it was shown that the presence of resveratrol or piceatannol significantly reduces the amount of the Jcp-containing patches, but does not lead to a relocalization of intracellular Jcp.