Dokument

Summary:
P. indica is a symbiont with a biphasic colonization strategy. It colonizes the roots of a broad range of plant species, including the monocot plant H. vulgare (barley) where it has a variety of beneficial effects. The interaction is marked by characteristics of the plant innate immune response, including the localized production of reactive oxygen species (ROS) and formation of cell wall appositions (CWA). In barley, the colonization is furthermore accompanied by a localized accumulation of reactive Fe3+ in CWAs, which in turn mediates the production of ROS. In the plant immune response, ROS, among other functions, are responsible for the fortification and maturation of CWAs by polymerization of ferulic acid and crosslinking of defensive proteins and phenolic compounds with cell wall polymers. During the early stages of P. indica / barley interaction, when the fungus tries to penetrate the wall of root cells, resulting in the formation of CWAs, the expression of P. indica gene DLD1 is upregulated. The encoded protein Dld1, belongs to a family of small secreted proteins unique to P. indica, which exhibit a large number of regularly distributed histidine and alanine residues, as well as a conserved motif with the consensus sequence RSIDELD located at the C-terminus. In this study, the localization of Dld1, as well as its biophysical and biochemical characteristics were investigated. Although the secretion of Dld1 by P. indica remains unclear, it was demonstrated that Dld1 is secreted by Ustilago maydis Dld1-expression strains both in vitro and in planta, and during transient expression in barley. In this host, Dld1 co-localizes with Fe3+ in CWAs, formed in response to fungal infection. Dld1 was heterologously produced in E. coli. The purified protein was used for circular dichroism spectroscopy and protein x-ray crystallography in cooperation with the group of Prof. Lupas, Tübingen. The crystal structure demonstrates that Dld1 adapts a coiled-coil structure with two antiparallel helices. Its folding was shown to be pH-sensitive. While the histidines protrude from the face of the two helices and interdigitate like teeth of a zipper, alanines occupy most of the helix-inward positions to facilitate a very tight structural assembly, previously termed alacoil. In qualitative and quantitative metal ion binding assays, Dld1 bound several metal ions. The dissociation constants for the binding of Fe3+ and Zn2+ were determined in the low micromolar range. Originally, a function for Dld1 as metal ion scavenger was postulated, but the protein is unable to prevent the Fe3+-catalyzed oxidation of the chemical substrate diaminobenzidine. Instead, Dld1 interferes with the radical-induced polymerization of diaminobenzidine. This observation indicates that Dld1 might interfere with analogous reactions at the plant cell wall, e.g. the polymerization of ferulic acid in maturing CWAs. This might contribute to the ability of P. indica to overcome barley CWAs in order to establish a compatible interaction.

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