Several members of the Rubiaceae and Violaceae plant families produce a series of cyclotides or macrocyclic peptides of 28 -37 aa with an embedded cystine knot. The cyclic peptide backbone together with the knotted and strongly braced structure confers exceptional chemical and biological stability that has attracted attention for potential pharmaceutical applications. Cyclotides display a diverse range of biological activities, such as uterotonic action, anti-HIV activity, and neurotensin antagonism. In plants, their primary role is probably protection from insect attack. Ingestion of the cyclotide kalata B1 severely retards the growth of larvae from the Lepidopteran species Helicoverpa armigera. We examined the gut of these larvae after consumption of kalata B1 by light, scanning, and transmission electron microscopy. We established that kalata B1 induces disruption of the microvilli, blebbing, swelling, and ultimately rupture of the cells of the gut epithelium. The histology of this response is similar to the response of H. armigera larvae to the Bacillus thuringiensis delta-endotoxin, which is widely used to control these insect pests of crops such as cotton. circular peptides ͉ insecticidal ͉ kalata B1 ͉ Lepidoptera ͉ microscopy C yclotides are a series of cyclic minipeptides of 28-37 aa that are expressed at high levels in the leaves, stems, and roots of several plant species (1, 2). They have an unusual topology based on a cyclic peptide backbone together with a cystine knot in which two disulfide bonds are threaded by a third disulfide bond (2, 3). This structure forces hydrophobic amino acids onto the surface of the molecule, creating a hydrophobic face on the otherwise hydrophilic surface. The cyclotides are thus soluble in both organic and aqueous solvents and are very stable at extremes of pH and temperature (4). The absence of free N and C termini, together with the tightly packed cystine knot, also renders the cyclotides resistant to the activity of proteases (4). The cyclotides are the largest family of circular proteins, although others have been described in bacteria, plants, and animals (5).Cyclotides were first isolated from the African plant Oldenlandia affinis. Two peptides, kalata B1 and B2, were recognized as the active components in a traditional medicine used to accelerate childbirth (6). Additional members of the cyclotide family were subsequently identified in screening studies directed toward discovery of bioactive molecules such as neurotensin antagonists (7), inhibitors of HIV replication (8), and hemolytic agents (9). More than 100 cyclotides have since been isolated from various members of the Rubiaceae, Violaceae, and Cucurbitaceae plant families (10, 11). A single plant may have at least 12 cyclotide genes and produce dozens of different cyclotides (12)(13)(14). Although an earlier study reported antimicrobial activity (15), it appears that their predominant activity in plants is insecticidal (13, 16). Jennings et al. (13) demonstrated that Helicoverpa punctigera failed to devel...

The vacuolar location of cyclotides supports our hypothesis that the vacuolar processing enzyme, asparaginyl endoproteinase, has a pivotal role in excision and cyclization from cyclotide precursors.

BackgroundPlant defensins are small (45–54 amino acids), basic, cysteine-rich proteins that have a major role in innate immunity in plants. Many defensins are potent antifungal molecules and are being evaluated for their potential to create crop plants with sustainable disease resistance. Defensins are produced as precursor molecules which are directed into the secretory pathway and are divided into two classes based on the absence (class I) or presence (class II) of an acidic C-terminal propeptide (CTPP) of about 33 amino acids. The function of this CTPP had not been defined.ResultsBy transgenically expressing the class II plant defensin NaD1 with and without its cognate CTPP we have demonstrated that NaD1 is phytotoxic to cotton plants when expressed without its CTPP. Transgenic cotton plants expressing constructs encoding the NaD1 precursor with the CTPP had the same morphology as non-transgenic plants but expression of NaD1 without the CTPP led to plants that were stunted, had crinkled leaves and were less viable. Immunofluorescence microscopy and transient expression of a green fluorescent protein (GFP)-CTPP chimera were used to confirm that the CTPP is sufficient for vacuolar targeting. Finally circular dichroism and NMR spectroscopy were used to show that the CTPP adopts a helical confirmation.ConclusionsIn this report we have described the role of the CTPP on NaD1, a class II defensin from Nicotiana alata flowers. The CTPP of NaD1 is sufficient for vacuolar targeting and plays an important role in detoxification of the defensin as it moves through the plant secretory pathway. This work may have important implications for the use of defensins for disease protection in transgenic crops.

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