Expression and chloroplast targeting of cholesterol oxidase in transgenic tobacco plants.

Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism.

Cloning of an insecticidal cholesterol oxidase gene and its expression in bacteria and in plant protoplasts.

We cloned and sequenced structural gene choM, which encodes an insecticidally active cholesterol oxidase in Streptomyces sp. strain A19249. The primary translation product was predicted to be a 547-amino-acid protein whose first 43 amino acids constitute a secretory signal peptide. Expression of the gene with the signal sequence in Escherichia coli resulted in production of a protein that had enzymatic and insecticidal properties which were indistinguishable from those of the cholesterol oxidase secreted by Streptomyces sp. strain A19249. Expression of the gene with or without the signal sequence in tobacco protoplasts resulted in production of an enzymatically active cholesterol oxidase.

Structure and formation of the peritrophic membrane in the larva of the southern corn rootworm, Diabrotica undecimpunctata.

The peritrophic membrane (PM) in larvae of the southern corn rootworm Diabrotica undecimpunctata (Coleoptera:Chrysomelidae) forms along the full length of the midgut epithelium, defining D. undecimpunctata as a Type I insect with respect to PM formation. PM formation occurs in three phases: organization of a continuous lamella of matrix from material secreted into the interstices between the microvilli, maturation and apical movement of the lamella along the microvilli, and shedding of the lamella from the tips of the microvilli into the midgut lumen. Subsequent cycles of synthesis and shedding give rise to multiple, concentric lamellae which surround the food in the gut lumen. PM lamellae are 0.2 mum in profile width and consist of a core of bundles of 5 nm-diameter microfibers encased in a finely-granular homogeneous material. The microfiber bundles are arranged in an orthogonal grid-like array with dimensions consistent with formation around the microvilli. The homogeneous material separates from the PM lamellae to enclose food particles suggesting it may contain digestive enzymes. The PM, microvilli and intracellular vesicles in the midgut epithelium stain intensely with wheat germ agglutinin reflecting the presence and sites of secretion and synthesis of chitin.

Cholesterol oxidase: a potent insecticidal protein active against boll weevil larvae.

The discovery of proteins that control insects is critical for the continued growth of the agricultural biotechnology industry. A highly efficacious protein that killed boll weevil (Anthonomus grandis grandis Boheman) larvae was discovered in Streptomyces culture filtrates. The protein was identified as cholesterol oxidase (E.C. 1.1.3.6). Purified cholesterol oxidase was active against boll weevil larvae at a concentration (LC50 = 20.9 micrograms/ml) comparable to the bioactivity of Bacillus thuringiensis proteins against other insect pests. Histological studies demonstrated that cholesterol oxidase lysed the boll weevil midgut epithelium, suggesting that this is the primary mechanism of lethality.

Peritrophic membrane structure and formation in the larva of a moth, Heliothis.

The peritrophic membrane (PM) in tobacco budworm larvae (Heliothis virescens, Lepidoptera: Noctuidae), is a continuous sac which encloses the food bolus in the midgut and hindgut. The PM is a single-walled structure 3-5 mum thick which is comprised of two main layers or laminae. The laminae may be fused into a single structure or remain separated by a space which may contain additional thin strands of matrix. Staining with an anti-PM antibody and wheat germ agglutinin (WGA) illustrate the laminar nature of the PM and suggest that protein and chitin have co-incident spatial distributions within the matrix. By transmission electron microscopy, the PM is composed of a loose network of fibrils and small granules, the only structural difference among laminae being a compaction of the matrix along the edges of the two limiting laminae facing the endoperitrophic and ectoperitrophic spaces. By scanning electron microscopy, the PM surface has a wrinkled, felt-like texture without pores or slits. Contrary to the classical view that lepidopterans are Type I insects with respect to PM formation in which the PM forms along the full length of the midgut, the PM in the tobacco budworm forms primarily from secretions of specialized midgut epithelial cells at the junction of the foregut and midgut. The secretory cells, their secretions and the nascent PM stain intensely with the anti-PM antibody but not with WGA suggesting that chitin is added more posteriorly. The PM may be supplemented by the addition of minor amounts of matrix material along the length of the midgut. PM synthesis begins during embryogenesis prior to the initiation of feeding. The PM in neonates is only about 0.1 mum thick but otherwise is structurally similar to that in older larvae.

Scanning electron microscopy of the sheathed infective larva and parasitic third-stage larva of Haemonchus contortus (Nematoda: Trichostrongyloidea).

Scanning electron microscopy was used to describe the infective and parasitic third-stage larvae of Haemonchus contortus, the large stomach worm of ruminants. Infective larvae are ensheathed in the cuticle of the second stage, so the descriptions are of the second- and third-stage cuticles. Both larval stages had an inner circle of 6 labial papillae, an outer circle of 6 labial papillae and 4 somatic papillae, and lateral amphidial pits. Infective larvae (cuticle of the second stage) had the 6 internal labial papillae on prominent bluntly rectangular lappets in a star-shaped arrangement around a large triradiate mouth, small triangular or round amphidial pits, flattened ribbonlike lateral alae, and phasmidial apertures opening on the ventral surface of the lateral alae. Parasitic third-stage larvae had the 6 internal labial papillae on small elevations without lappets around a small mouth; large, oval amphidial pits; ribbonlike lateral alae for most of their length, but with the anterior 30-40 microns of the alae cordlike; and phasmidial apertures on the body cuticle ventral to the lateral alae.

Biochemical characterization of cuticle polypeptides from the infective larvae of Haemonchus contortus.

1. Ecdysis of infective Haemonchus contortus larvae is effected by the enzymatic degradation of a specialized region of the second molt cuticle containing a biochemically unique polypeptide (mol. wt = 160,000). 2. The 160,000 mol. wt polypeptide and related polypeptides are synthesized at approximately 6 days of larval development. Antigenically similar polypeptides occur in other ruminant trichostrongyles. 3. Cuticle polypeptides digested during ecdysis differ from second molt cuticle collagens in amino acid composition and collagenase sensitivity. However, some antigenic homology between the 160,000 mol. wt polypeptide and cuticle collagens suggests structurally similar regions.

Light and scanning electron microscopy of the ecdysis of Haemonchus contortus infective larvae.

During the second ecdysis of ruminant trichostrongyles, a region of the second molt cuticle is digested by a 44-kDa Zn-metalloprotease. We have examined this digestion process by light and scanning electron microscopy (SEM). The substrate region of the cuticle appeared, during the ecdysis process, as an indented ring at the 20th cuticular annulus coincident with the anterior terminus of the lateral alae. Continued digestion of the cuticle resulted in holes in the ring region that expanded until they became continuous and separation occurred between the anterior and posterior portions of the cuticle. Mechanical movements of the L3 forced aside the cuticle cap that generally remained attached on one side to the posterior portion as the larva escaped from the sheath. The site of secretion of the 44-kDa ecdysing enzyme causing cuticle digestion was not clear from morphological observations; however, existing evidence strongly points to the release of enzyme from the esophageal (pharyngeal) glands through the mouth.

Purification of a 44 kilodalton protease which mediates the ecdysis of infective Haemonchus contortus larvae.

We have characterized and purified a parasite protease which mediates the ecdysis of Haemonchus contortus, and at least several other ruminant trichostrongyles. The protease, with an apparent approximate molecular weight of 44,000, is a zinc metalloprotein which hydrolyzes several large protein substrates in in vitro assays. In vivo and in biological assays on isolated second molt cuticles this protease hydrolyzes a specific circular region of the second stage cuticle which results in removal of a cuticular cap, providing a rapid and synchronous method for the escape of infective larvae during the transition from free-living to parasitic environments.