Cloning, expression, and characterization of (+)-delta-cadinene synthase: a catalyst for cotton phytoalexin biosynthesis.
In cotton, sesquiterpene phytoalexins are elicited in response to bacterial or fungal infection. A Gossypium arboreum cell 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 from Verticillium dahliae. The mRNA prepared from these elicited cultures was used to isolated two cDNA clones that contain open frames coding for proteins of 554 amino acids with M(r) 64,096 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 in Nicotiana tabacum showed 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 in Escherichia coli and 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 (+)-delta-cadinene. The fungal-elicited production of a (+)-delta-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.
Chen
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Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907-1333, USA.
Chen
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Heinstein
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Davisson
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VJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D003368:Gossypium / Q000201:enzymology / Q000235:genetics*; D015151:Immunoblotting; D007535:Isomerases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D010936:Plant Extracts / Q000096:biosynthesis*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012717:Sesquiterpenes; D013045:Species Specificity; D013379:Substrate Specificity; D013729:Terpenes / Q000378:metabolism*; D000094162:Phytoalexins
D018076:DNA, Complementary; D010936:Plant Extracts; D011106:Polyisoprenyl Phosphates; D011993:Recombinant Fusion Proteins; D012717:Sesquiterpenes; D013729:Terpenes; C004808:farnesyl pyrophosphate; D007535:Isomerases; C097608:delta-cadinene synthase; D000094162:Phytoalexins