The mpi gene encodes a maize proteinase inhibitor (MPI) protein whose mRNA accumulates in response to mechanical wounding. In this study, mpi gene expression in response to different types of damage was investigated. In mechanically damaged leaves of maize (Zea mays L.), mpi mRNA accumulation was affected by the degree of damage inflicted on the leaf. Consecutive wounds resulted in higher levels of mpi transcripts. The MPI protein was expressed in Escherichia coli and purified. Polyclonal antibodies were then produced and used to study MPI accumulation in insect-wounded and mechanically wounded maize leaves. When larvae of the lepidopteran insect Spodoptera littoralis were fed on maize leaves, MPI accumulated in tissues adjacent to the wound site. The level of inhibitor accumulation was higher in leaves chewed by larvae than in leaves that had been damaged mechanically. Longer feeding periods also resulted in higher levels of MPI accumulation. Additionally, the inhibitory properties of MPI toward mammalian and insect digestive serine proteinases were determined. Contrary to the majority of the plant proteinase inhibitors described, MPI is an inhibitor of mammalian elastase that only weakly inhibits mammalian chymotrypsin. However, both elastase and chymotrypsin-like activities from the larval midgut of S. littoralis were effectively inhibited by MPI. We discuss these results with regard to the function and evolution of plant proteinase inhibitors. The availability of a plant proteinase inhibitor which is able to inhibit the two types of insect digestive proteinase, elastase and chymotrypsin, might be useful for engineering protection against lepidopteran insect pests in transgenic plants.

Characterization of the insecticidal and hemolytic activity of solubilized crystal proteins of

A gene designated cyt1Ab1, encoding a 27,490-Da protein, was isolated from Bacillus thuringiensis subsp. medellin (H30 serotype) by using an oligonucleotide probe corresponding to the cyt1Aa1 gene. The sequence of the Cyt1Ab1 protein, as deduced from the sequence of the cyt1Ab1 gene, was 86% identical to that of the Cyt1Aa1 protein and 32% identical to that of the Cyt2Aa1 protein from B. thuringiensis subsp. kyushuensis. The cyt1Ab1 gene was flanked upstream by a p21 gene, in the same orientation, encoding a 21,370-Da protein that showed 84% similarity to the putative chaperone P20 protein from B. thuringiensis subsp. israelensis and downstream, on the opposite strand, by a sequence showing 85% identity to the IS240A insertion sequence. The cyt1Ab1 gene was expressed at a high level in a nontoxic strain of B. thuringiensis subsp. israelensis in which large inclusions of the Cyt1Ab1 protein were produced. Purified Cyt1Ab1 crystals were as hemolytic as those of the Cyt1Aa1 protein and were twice as hemolytic as those from the wild-type strain. Mosquitocidal activity toward Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae was assayed. The toxicity of the Cyt1Ab1 protein was slightly lower than that of the Cyt1Aa1 protein for all three mosquito species, and Cyt1Ab1 was 150, 300, and 800 times less active toward Culex, Anopheles, and Aedes larvae, respectively, than were the native crystals from B. thuringiensis subsp. medellin.