CLONING, EXPRESSION, AND CHARACTERIZATION OF (-DELTA-CADINENE SYNTHASE - A CATALYST FOR COTTON PHYTOALEXIN BIOSYNTHESIS())

In cotton, sesquiterpene phytoalexins are elicited in response to bact erial or fungal infection, A Gossypium arboreum cell suspension cultur e which produces the sesquiterpene phytoalexin gossypol showed a time- dependent 10-fold increase in a 1,9-kb mRNA in response to a challenge by a preparation from Verticillium dahliae, The mRNA prepared from th ese elicited cultures was used to isolate two cDNA clones that contain open frames coding for proteins of 554 amino acids with M(r) 64,096 a nd 64,118, The encoded protein shows a significant degree of sequence identity with the other known plant terpene cyclases, Western blot ana lyses with a cross-reactive monoclonal antibody from a related sesquit erpene synthase in Nicotiana tabacum showed a time-dependent increase of a 65-kDa protein which reached a maximal level 24 h post elicitor t reatment. The encoded protein from the pXC1 cDNA was produced in Esche richia coli and purified by affinity column chromatography. The enzyma tic properties of this protein were identified by a radiochemical assa y for cyclization of farnesyldiphosphate and a product structure was a ssigned by GC-MS, chiral phase GC, and NMR analyses as (+)-delta-cadin ene. The fungal-elicited production of a (+)-delta-cadinene synthase i s consistent with a role for this enzyme as the first committed step i n the pathways leading to the related phytoalexins gossypol and lacini lene C in cotton. (C) 1995 Academic Press, Inc.