PURIFICATION OF A PROTEIN FROM CULTURE FILTRATES OF FUSARIUM-OXYSPORUM THAT INDUCES ETHYLENE AND NECROSIS IN LEAVES OF ERYTHROXYLUM-COCA

Culture filtrates of Fusarium oxysporum elicited ethylene production and necrosis when applied to leaves of Erythroxylum coca. A protein that induces ethylene production and necrosis in leaves of E. coca was purified from culture filtrates of an isolate of F. oxysporum pathogenic to E. coca. The protein was first concentrated using ultrafiltration where it was retained by a 1-kDa filter. The protein was purified by fast protein liquid chromatography using cation exchange chromatography followed by hydrophobic interaction chromatography. Gel filtration was used as the final purification step and to exchange salts. The protein migrated as a single band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular mass of 22.5 kDa. The exact mass was determined by mass spectroscopy to be 23,996.1 Da. The mobility of the protein was not affected by reducing agents, but the 24-kDa protein broke down and was inactivated by excessive heat. The protein constituted a major component of the extracellular proteins produced in cultures three or more days old and reached a maximum concentration between 6 and 12 days. Biological activity of the protein could be detected by induction of ethylene down to a concentration of 50 ng per leaf (approximately 500 ng/g fresh weight) when applied as a hanging drop to the petiole of an excised E. coca leaf. The 24-kDa protein induced ethylene biosynthesis and necrosis in a wide variety of Dicotyledoneae, but we were unable to demonstrate activity in members of the Monocotyledoneae tested. It remains to be determined if the 24-kDa protein plays a role in disease development in the F. oxysporum-E. coca interaction.