Purification, characterization and rôle in infection of an extracellular cutinolytic enzyme from Colletotrichum gloeosporioides Penz. on Carica papaya L

Abstract

Colletrichum gloeosporioides was grown in still culture in a mineral salts medium contaning purified papaya cutin as the sole source of carbon. After 2 weeks of incubation, the cutinase from culture filtrates was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The purified enzyme, which hydrolyzes tritiated cutin, is a cationic glycoprotein (16% carbohydrate) with a molecular weight of about 24000 as determined by SDS-polyacrylamide gel electrophoresis. The enzyme hydrolyzed all p-nitrophenyl esters (model substrates) of C 4 to C 16 acids thus differing from previously reported fungal cutinases which could not hydrolyze the longer chain esters. Diisopropylflurophosphate inhibits the enzyme indicating that catalysis involves an active serine. Immunological studies with rabbit antibodies prepared against the cutinase showed a single sharp precipitin band when Ouchterlony double diffusion analysis was performed. Rabbit antibodies prepared against the cutinase isolated from Fusarium solani f. sp. pisi neither cross reacted nor inhibited the present enzyme. The antiserum prepared against C. gloeosporioides cutinase completely inhibited the enzyme in vitro and suppressed lesion formation when papaya fruits were inoculated with spore suspensions containing the anticutinase. Similarly, the cutinase inhibitor diisopropylfluorophosphate also suppressed lesion formation; however, neither anticutinase nor diisopropylfluorophosphate suppressed lesion formation when the fruit's cutin barrier was breached by a needle prick prior to inoculation. These findings show that C. gloeosporioides penetrates the cuticular layer of papaya by secreting a cutinase. Additionally, when papayas are pretreated with the purified cutinase of C. gloeosporioides and subsequently inoculated with spores of Mycosphaerella sp. a wound pathogen of papaya, infection and lesion formation result.