1.2.38
EARLY DEFENCE RESPONSES INDUCED BY AVR9 IN TRANSGENIC TOBACCO CELL SUSPENSIONS CONTAINING THE CF-9 RESISTANCE GENE

CF DE JONG, G HONEE and PJGM DE WIT

Department of Phytopathology, Wageningen Agricultural University, PO Box 8025, 6700 EE Wageningen, The Netherlands

Background and objectives
The interaction of Cladosporium fulvum and its only host, tomato, follows the gene-for-gene model. The tomato Cf-9 gene confers resistance to races of C. fulvum that contain the Avr9 avirulence gene. Recognition of AVR9 by tomato plants that carry the Cf-9 resistance gene results in induction of a hypersensitive response (HR) and other defence-related responses. Single amino-acid changes in the AVR9 peptide resulted in mutant peptides that showed different necrosis-inducing activities. Injections of chemically synthesized mutant peptides AVR9 (R8K) and AVR9 (Fl0A) into leaves of Cf9 tomato plants have shown that AVR9 (R8K) is more active, while AVR9 (Fl0A) is less active compared to wild-type AVR9 elicitor peptide. The mutant AVR9 (F21A) only caused chlorosis and no necrosis, even upon injection of very high doses [1].

To investigate early defence responses induced by the AVR9 elicitor peptide, cell suspension cultures derived from plants that contain the Cf-9 resistance gene have been established. Cell cultures derived from Cfg tomato plants do not respond to AVR9 [2], whereas cell cultures derived from transgenic tobacco plants that express the Cf-g gene do respond to the AVR9 elicitor.

Materials and methods
AVR9-responsive cell suspension cultures derived from transgenic tobacco plants (cv. Petit Havana) that express the Cf-g resistance gene were established. Wild-type AVR9 and the mutants AVR9 (R8K), AVR9 (F10A) and AVR9 (F21A) were chemically synthesized. The oxidative burst was determined by recording decreasing fluorescence upon oxidation of the fluorescent dye pyranin. To quantify the magnitude of the oxidative burst, the slope of the linear part of the obtained curve was determined. From these data, dose-response curves and IC50 values for wt AVR9 and the AVR9 mutants R8K, F10A and F21A were determined.

Results and conclusions
The lowest AVR9 and AVR9 mutant peptide concentration at which an oxidative burst could be detected was 1000-fold lower than the lowest concentration needed to induce necrosis in planta. In addition, the magnitude of the oxidative burst proved to be dose-dependent. Results obtained from the oxidative burst measurements in Cfg transgenic tobacco cell suspensions showed similar differences between AVR9 and the AVR9 mutant peptides, as was observed upon injection in tomato leaves: AVR9 (R8K) is more than or equally as active as AVR9; both AVR9 (Fl0A) and AVR9 (F21A) are less active than AVR9. Other AVR9-specific responses, such as extracellular medium alkalinization, and potential inhibitors of AVR9 induced responses, will also be discussed.

References
1. Kooman-Gersmann M, Vogelsang R, Vossen P et al., 1998. Plant Physiology (in press).
2. Honee G, Buitink J, Jabs T et al., 1998. Plant Physiology (in press).