1.1.36
ISOLATION OF THE AVR9-SPECIFIC BINDING SITE FROM Cf9 TOMATO MEMBRANES

RAL VAN DER HOORN, G HONEE and PJGM DE WIT

Department of Phytopathology, Wageningen Agricultural University, Binnenhaven 9, 6709 PD Wageningen, The Netherlands

Background and objectives
The interaction between the pathogenic fungus Cladosporium fulvum and tomato conforms to the gene-for-gene model. The Avr9 gene is the only fungal genetic component responsible for induction of the hypersensitive response in tomato plants that carry the Cf-9 resistance gene. Biochemical interpretation of the gene-for-gene model predicts that the avirulence gene product AVR9 and the gene product of the Cf-9 resistance gene interact directly to elicit a resistance response. Binding studies using radiolabelled AVR9 peptide demonstrated the presence of a single class of binding sites that shows high affinity for AVR9 on membranes isolated from Cf9 tomato [1]. This high-affinity binding site is also present in tomato plants without the Cf-g gene, as well as in other plant species [1]. This indicates therefore that the binding site is not the CF9 protein. Furthermore, when single amino-acid changes were introduced in AVR9, a correlation between necrosis-inducing activity and affinity for the AVR9 binding site was shown in these mutants [2]. These results suggest that the AVR9 binding site is directly required for AVR9 induced HR, whereas the involvement of CF9 in AVR9 perception is probably indirect. To study AVR9 perception in more detail, biochemical isolation of the AVR9 binding site was initiated.

Results and conclusions
The AVR9-binding site could be solubilized efficiently without affecting AVR9 binding affinity. The binding site was absorbed from solubilized membrane fractions by affinity chromatography using immobilized AVR9. The amount of AVR9-binding sites that were bound to the column was calculated by determining remaining binding activity in the flow-through. Current experiments are focused on elution of the affinity column.

References
1. Kooman-Gersmann M, Honee G, De Wit PJGM, 1996. Plant Cell 8, 929-938.
2. Kooman-Gersmann M, Vogelsang R, Vossen P et al., 1998. Plant Physiology (in press).