1.1.31
AVR9 AND Cf-9-DEPENDENT ACTIVATION OF A MAP KINASE: A LINK BETWEEN THE WOUNDING AND PATHOGEN DEFENCE RESPONSE?

T ROMEIS1, P PIEDRAS 1, KE HAMMOND-KOSACK1, M SMOKER 1, H HIRT2 and JDG JONES1

1Sainsbury Laboratory, John lnnes Centre, Colney Lane, Norwich NR4 7UH, Norfolk, UK; 2lnsitute of Microbiology and Genetics, Vienna Biocenter, Dr Bohrgasse 9, 1030 Vienna, AT

Background and objectives
The Cf-9 gene of tomato confers resistance the fungal pathogen Cladosporium fulvum through recognition of the corresponding pathogen-encoded avirulence gene product (Avr9). Upon recognition, Cf-9 protein initiates downstream signalling processes that activate the plant defence response. In transgenic tobacco which expresses Cf-9 the specific responsiveness to the Avr9 elicitor is retained. Treatment with Avr9 causes grey necrosis and results in a rapid synthesis of active oxygen species (AOS) in tobacco leaves and suspension cultures, respectively. In order to investigate early signalling events mediating the induction of the specific defence response, we analysed Avr9/Cf-9-dependent changes in protein phosphorylation.

Materials and methods
Leaves of Cf-9 expressing tobacco plants or suspension culture cells were treated with intercellular fluid (IF) with or without the Avr9 peptide. Leaf discs and cell samples were harvested at different time points. Crude cell extracts were either analysed immediately for kinase activity, using an in-gel kinase assay with MBP as a substrate, or subjected to a prior immunoprecipitation with various antibodies. RNA was also isolated, blotted and probed with WIPK cDNA.

Results and conclusions
48-kDa protein kinase, which can utilize MBP as a substrate, became activated in both leaves and suspension culture cells in an Avr9/Cf-9-dependent manner. The kinase activity was detectable as soon as 2-5 min after elicitation, showed a maximum at 15 min and decreased during the following 2 h. The inhibitors staurosporin and lanthanum chloride added 5 min prior to elicitation with Avr9 and compromised the kinase activation, suggesting that the 48-kDa MBP kinase requires a phosphorylation step for activation and is located downstream of a Ca2+ channel. Since DPI had no inhibitory effect, the 48-kDa kinase seems to function upstream or in a signalling pathway distinct from the induction of AOS. The Avr9-induced activation of the 48-kDa MBP kinase was associated with tyrosine phosphorylation, indicating that the kinase might be a MAP kinase. Furthermore, the activated kinase could be immunoprecipitated with the M7 antibody directed against a wound- and stress-induced MAP kinase [1]. To investigate whether the Avr9-induced kinase is related to WIPK from tobacco [2], elicited leaf and suspension culture samples were subjected to RNA blot analysis. WIPK transcript accumulated within 1 h in samples treated with the Avr9 elicitor but not in untreated ones, suggesting that the Avr9-induced 48-kDa MBP kinase might be identical with WIPK. We conclude that the signalling pathways of the race-specific pathogen defence and wound responses, although triggered by distinct stimuli and resulting in a different set of induced responses, appear to be interconnected through a conserved MAP kinase.

References
1. Bogre L, Ligterink, W, Heberle-Bors E, Hirt H, 1997. Nature 383, 489-490.
2. Seo S, Okamoto M, Seto H et al., 1995. Science 270, 1988-1992.