Isolation of a Protein Bound to Canker-forming Factor from Citrus Plant

Interaction analysis using affinity analysis (Affinity Sensors, Cambridge, UK) indicated the presence of proteins bound to Apl1, a virulence factor of Xanthomonas campestris pv. citri required for the formation of canker symptom on citrus, in the fraction 25–50% ammonium sulfate of citrus crude extracts. Three proteins of 25, 50 and 110-kD were eluted from an Apl1-affinity column. Western analysis revealed that Apl1 binds specifically to the 25-kD and 110-kD but not to the 50-kD protein. When crude extracts of soybean and of tobacco were applied to the Apl1-affinity column, only faint bands were detected. This result suggests that Apl1 targets exist specifically in citrus plants. The amino acid sequence of the N-terminal of the 25-kD protein was determined, and homology search analysis revealed that this sequence was almost identical to those commonly present in S-adenosyl-l-methionine : trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAMT) from several plants. This enzyme is specific to the substrate trans -caffeoyl-CoA and catalyzes the synthesis of trans-feruloyl-CoA for lignin formation. Received 4 November 1999/ Accepted in revised form 26 November 1999