INSECT CELL EXPRESSION OF A PLANT DISEASE RESISTANCE PROTEIN Cf-9 FOR STRUCTURAL STUDIES

T. Teh,^a S. Lang,^b D. A. Jones,^b and B. Kobe ^a

^aDepartment of Biochemistry and Molecular Biology, University of Queensland, St. Lucia, Qld 4072, Australia; ^bPlant Cell Biology, Research School of Biological Sciences, Australian National University, Canberra, ACT 2601, Australia (t.teh@mailbox.uq.edu.au).

Plants have evolved specific mechanisms to recognise individual pathogens and activate defence systems. The induction of plant defence responses, which often include a hypersensitive response leading to rapid localised cell death at the site of infection, requires the interaction of a plant-derived resistance protein (R protein) with a specific pathogen-derived avirulence protein (Avr protein) [1]. The molecular and structural basis of the recognition events involving most R and Avr protein combinations remains unknown. It has been postulated that R proteins are receptors for Avr components [2,3] and that R and Avr proteins participate in a complex with other proteins to initiate signal transmission and activate host defence responses [4,5].

One well established model in which to study molecular aspects of host resistance and pathogen avirulence is that of the tomato Cf-9 protein which confers resistance to the leaf mould fungus Cladosporium fulvum through recognition of the fungus encoded Avr9 peptide [6]. Cf-9 is a mainly extracytoplasmic membrane-anchored glycoprotein, composed predominantly of leucine rich repeats (LRRs). LRRs are sequence motifs present in a variety of proteins involved in protein-protein interactions, including many plant R proteins; however, their role in disease resistance is unknown. In an effort to provide the first structural view of a plant R protein and to extend the present knowledge of the molecular basis of plant disease resistance, the Cf-9 protein is being expressed in insect cells. Through optimisation of expression conditions, it is hoped to produce adequate quantities of protein for crystallisation trials.

References

1 Flor, H. (1971) Annu. Rev.Phytopthol. 9, 275-296.

2 Staskawicz, B.J., Ausubel, F.M., Baker, B.J., Ellis, J.G., and Jones, J.D.G. (1995) Science 268, 661-667.

3 Ellis, J., Dodds, P. and Pryor, T. (2000) Curr. Opin.Plant Biol 3, 278-284.

4 Dixon, M.S., Golstein, C., Thomas, C.M., van Der Biezen, E.A., and Jones, J.D.G. (2000) Proc. Natl. Acad. Sci. USA 97, 8807-8814.

5 Dangl, J.L., and Jones, J.D.G. (2001) Nature 411, 826-833.

6 Joosten, M.H.A.J., and De Wit, P.J.G.M. (1999) Annu. Rev.Phytopthol. 37, 335-367.