Institute of Plant Biology, University, Zollikerstrasse 107, CH-8008 Zürich, Switzerland

Background and objectives
Stable transformation of wheat with putative antifungal transgenes is a tedious and time-consuming process. Therefore we have developed a transient assay system based on biolistic transformation of wheat leaves that allows the functional testing of transgenes in the interaction with the powdery mildew fungus Blumeria (syn. Erysiphe) graminis f. sp. tritici. Wheat oxalate oxidase (Oxox; E.C., also referred to as germin) is an extracellular germination- and pathogenesis-related protein producing hydrogen peroxide and releasing Ca2+ from Ca-oxalate [1].

Results and conclusions
Transient expression of Oxox under the control of the CaMV 35S promoter resulted in a pattern of cell-autonomous oxalate-dependent 1-chloro-4-naphthol oxidation in epidermal cells, indicating Oxox activity. Oxox-over-expressing cells were more resistant to fungal penetration than GUS-expressing control cells. However, no evidence was found for availability of endogenous Ca2+-oxalate and spontaneous production of hydrogen peroxide by cells transformed with Oxox. Instead, inoculated transformed as well as non-transformed cells exhibited a local burst of hydrogen peroxide and covalent protein crosslinking at the site of attempted penetration, similar to pathogen-attacked barley cells [2]. At the sites of attempted penetration, oxalate oxidase was resistant to washing in hot SDS, indicating covalent crosslinking. The hypothesis that oxalate oxidase is a novel crosslinking substrate is being addressed by over-expression of mutated Oxox constructs that have lost Oxox enzymatic activity. Although endogenous Oxox was not expressed in the epidermis, an oxox-like protein lacking Oxox activity was found to be induced exclusively in wheat and barley [3] epidermal cells attacked by Blumeria graminis. This protein might be the natural crosslinking substrate in epidermal cells of cereals, and the kinetics and extent of its accumulation might be critical for penetration resistance of papillae.

1. Lane BG, Dunwell JM, Ray JA et al., 1993. Journal of Biological Chemistry 268, 12239-12242.
2. Thordal-Christensen H, Zhang Z, Wei YD, Collinge D, 1997. Plant Journal 11, 1187-1194.
3. Wie Y, Zhang Z, Andersen CH et al., 1998. Plant Molecular Biology, in press.