cDNA cloning, biochemical characterization and inhibition by plant inhibitors of the alpha-amylases of the Western corn rootworm, Diabrotica virgifera virgifera

We report the characterization and cDNA cloning of two a-amylase isozymes f rom larvae of the Western corn rootworm (Diabrotica virgifera virgifera LeC onte). Larvae raised on artificial media have very low levels of amylase ac tivity, and much higher levels are found in larvae raised on maize seedling s. At pH 5.7, the optimum pH for enzyme activity, the a-amylases are substa ntially but not completely inhibited by amylase inhibitors from the common bean (Phaseolus vulgaris) and from wheat (Triticum aestivum). Using the rev erse transcriptase polymerase chain reaction (RT-PCR), we cloned two cDNAs with 83% amino acid identity that encode a-amylase-like polypeptides. Expre ssion of one of the two cDNAs in insect cells with a baculovirus vector sho ws that this cDNA encodes an active amylase with a mobility that correspond s to that of one of the two isozymes present in larval extracts. The expres sed enzyme is substantially inhibited by the same two inhibitors. We also s how that expression in Arabidipsis of the cDNA that encodes the amylase inh ibitor AI-1 of the common bean results in the accumulation of active inhibi tor in the roots, and the results are discussed with reference to the possi bility of using amylase inhibitors as a strategy to genetically engineer ma ize plants that are resistant to Western corn rootworm larvae. (C) 2000 Els evier Science Ltd. All rights reserved.