Resistance gene-dependent activation of a calcium-dependent protein kinasein the plant defense response

In the Cf-9/Avr9 gene-for-gene interaction, the Cf-9 resistance gene from t omato confers resistance to the fungal pathogen Cladosporium fulvum, which expresses the corresponding pathogen-derived avirulence product Avr9. To un derstand R gene function and dissect the signaling mechanisms involved in t he induction of plant defenses, we studied Cf-9/Avr9-dependent activation o f protein kinases in transgenic Cf9 tobacco cell cultures. Using a modified in-get kinase assay with histone as substrate, we identified a membrane-bo und, calcium-dependent protein kinase (CDPK) that showed a shift in electro phoretic mobility from 68 to 70 kD within 5 min after Avr9 elicitor was add ed. This transition from the nonelicited to the elicited CDPK form was caus ed by a phosphorylation event and was verified when antibodies to CDPK were used for protein gel blot analysis. In addition, the interconversion of th e corresponding CDPK forms could be induced in vitro in both directions by treatment with either phosphatase or ATP. In vitro protein kinase activity toward syntide-2 or histone with membrane extracts or gel-purified enzyme w as dependent on Ca2+ content and was compromised by the calmodulin antagoni st N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) but not by its inactive isoform N-(6-aminohexyl)-1-naphthalenesulfonamide In these assays, the CDPK activity in elicited samples, reflecting predominantly the phosph orylated 70-kD CDPK form, was greater than in nonelicited samples. Thus, Av r9/Cf-9-dependent phosphorylation and subsequent transition from the noneli cited to the elicited form correlate with the activation of a CDPK isoform after in vivo stimulation. Because that transition was not inhibited by W-7 , the in vivo CDPK activation probably is not the result of autophosphoryla tion. Studies with pharmacological inhibitors indicated that the identified CDPK is independent of or is located upstream from a signaling pathway tha t is required for the Avr9-induced active oxygen species.