Cloning, Expression, and Characterization of (+)-δ-Cadinene Synthase: A Catalyst for Cotton Phytoalexin Biosynthesis

In cotton, sesquiterpene phytoalexins are elicited in response to bacterial or fungal infection. AGossypium arboreumcell suspension culture 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 fromVerticillium dahliae.The mRNA prepared from these elicited cultures was used to isolate two cDNA clones that contain open frames coding for proteins of 554 amino acids withMr64,096 and 64,118. The encoded protein shows a significant degree of sequence identity with the other known plant terpene cyclases. Western blot analyses with a cross-reactive monoclonal antibody from a related sesquiterpene synthase inNicotiana tabacumshowed a time-dependent increase of a 65-kDa protein which reached a maximal level 24 h post elicitor treatment. The encoded protein from the pXC1 cDNA was produced inEscherichia coliand purified by affinity column chromatography. The enzymatic properties of this protein were identified by a radiochemical assay for cyclization of farnesyldiphosphate and a product structure was assigned by GC–MS, chiral phase GC, and NMR analyses as (+)-δ-cadinene. The fungal-elicited production of a (+)-δ-cadinene synthase is consistent with a role for this enzyme as the first committed step in the pathways leading to the related phytoalexins gossypol and lacinilene C in cotton.