ABSTRACT
Currently, crop protection relies heavily on chemical treatments, which ultimately leads to environmental contamination and pest resistance. Societal and public policy considerations urge the need for new eco-friendly solutions. In this perspective, biopesticides are effective alternatives to chemical insecticides for the control of various insect pests. Legumes contain numerous insecticidal proteins aimed at protecting their high nitrogen content from animal/insect predation. Investigating one such protein family at genome scale, we discovered a unique diversity of the albumin 1 family in the (model) barrel medic genome. Only some members retained very high insecticidal activity. We uncovered that AG41 peptide from the alfalfa roots displays an outstanding insecticidal activity against several pests such as aphids and weevils. Here we report the 3D structure and activity of AG41 peptide. Significant insights into the structural/functional relationships explained AG41 high insecticidal activity. Such observations pave the way for the development of bio-insecticides, with AG41 peptide as the lead compound.
Subject(s)
Fabaceae , Insecticides , Animals , Insecticides/pharmacology , Insecticides/chemistry , Insecta , Peptides/pharmacology , AlbuminsABSTRACT
PA1b (Pea Albumin 1, subunit b) peptide is an entomotoxin, extracted from Legume seeds, with a lethal activity towards several insect pests, such as mosquitoes, some aphids and cereal weevils. This toxin acts by binding to the subunits c and e of the plasma membrane H+-ATPase (V-ATPase) in the insect midgut. In this study, two cereal weevils, the sensitive Sitophilus oryzae strain WAA42, the resistance Sitophilus oryzae strain ISOR3 and the insensitive red flour beetle Tribolium castaneum, were used in biochemical and histological experiments to demonstrate that a PA1b/V-ATPase interaction triggers the apoptosis mechanism, resulting in insect death. Upon intoxication with PA1b, apoptotic bodies are formed in the cells of the insect midgut. In addition, caspase-3 enzyme activity occurs in the midgut of sensitive weevils after intoxication with active PA1b, but not in the midgut of resistant weevils. These biochemical data were confirmed by immuno-histochemical detection of the caspase-3 active form in the midgut of sensitive weevils. Immuno-labelling experiments also revealed that the caspase-3 active form and V-ATPase are close-localized in the insect midgut. The results concerning this unique peptidic V-ATPase inhibitor pave the way for the utilization of PA1b as a promising, more selective and eco-friendly insecticide.
Subject(s)
Insect Proteins/genetics , Insecticides/toxicity , Peptides/toxicity , Pisum sativum/genetics , Plant Proteins/toxicity , Toxins, Biological/toxicity , Vacuolar Proton-Translocating ATPases/genetics , Animals , Apoptosis , Caspase 3/genetics , Caspase 3/metabolism , Gastrointestinal Tract/drug effects , Gastrointestinal Tract/metabolism , Gene Expression Regulation , Insect Proteins/antagonists & inhibitors , Insect Proteins/metabolism , Insecticides/isolation & purification , Insecticides/metabolism , Pisum sativum/chemistry , Pisum sativum/parasitology , Peptides/isolation & purification , Peptides/metabolism , Plant Proteins/isolation & purification , Plant Proteins/metabolism , Protein Binding , Protein Subunits/antagonists & inhibitors , Protein Subunits/genetics , Protein Subunits/metabolism , Seeds/chemistry , Seeds/genetics , Seeds/parasitology , Toxins, Biological/isolation & purification , Toxins, Biological/metabolism , Tribolium/drug effects , Tribolium/metabolism , Vacuolar Proton-Translocating ATPases/antagonists & inhibitors , Vacuolar Proton-Translocating ATPases/metabolism , Weevils/drug effects , Weevils/metabolismABSTRACT
The Pea Albumin 1 subunit b (PA1b) peptide is an entomotoxin extracted from legume seeds with lethal activity towards several insect pests. Its toxic activity occurs after the perception of PA1b by a plasmalemmic proton pump (V-ATPase) in the insects. Assays revealed that PA1b showed no activity towards mammalian cells displaying high V-ATPase activity. Similarly, PA1b displayed no binding activity and no biological activity towards other non-insect organisms. We demonstrate here that binding to labelled PA1b was found in all the insect families tested, regardless of the sensitivity or insensitivity of the individual species. The coleopteran Bruchidae, which are mainly legume seed pests, were found to be fully resistant. A number of insect species were seen to be insensitive to the toxin although they exhibited binding activity for the labelled PA1b. The fruit fly, Drosophila melanogaster (Diptera), was generally insensitive when maintained on an agar diet, but the fly appeared to be sensitive to PA1b in bioassays using a different diet. In conclusion, the PA1b toxin provides legumes with a major source of resistance to insects, and insects feeding on legume seeds need to overcome this plant resistance by disrupting the PA1b - V-ATPase interaction.
Subject(s)
Fabaceae/chemistry , Insecta/drug effects , Pesticides/toxicity , Plant Proteins/toxicity , Vacuolar Proton-Translocating ATPases/chemistry , Amino Acid Sequence , Animals , Cell Differentiation/drug effects , Cell Line, Tumor , Coleoptera/drug effects , Drosophila melanogaster/drug effects , Humans , Insect Proteins/chemistry , Insecticide Resistance , MCF-7 Cells , Mice , Molecular Sequence Data , Osteoclasts/drug effects , Pesticides/chemistry , Plant Proteins/chemistry , Seeds/chemistry , Toxicity TestsABSTRACT
The PA1b (Pea Albumin 1, subunit b) peptide is an entomotoxin extract from Legume seeds with lethal activity on several insect pests, such as mosquitoes, some aphids and cereal weevils. This 37 amino-acid cysteine-rich peptide has been, until now, obtained by biochemical purification or chemical synthesis. In this paper, we present our results for the transient production of the peptide in Nicotiana benthamiana by agro-infiltration, with a yield of about 35 µg/g of fresh leaves and maximum production 8 days after infiltration. PA1b is part of the PA1 gene which, after post-translational modifications, encodes two peptides (PA1b and PA1a). We show that transforming tobacco with the PA1b cDNA alone does not result in production of the toxin and, in fact, the entire cDNA is necessary, raising the question of the role of PA1a. We constructed a PA1-cassette, allowing for the quick "cut/paste" of different PA1b mutants within a conserved PA1 cDNA. This cassette enabled us to produce the six isoforms of PA1b which exist in pea seeds. Biological tests revealed that all the isoforms display similar activity, with the exception of one which is inactive. The lack of activity in this isoform led us to conclude that the amphiphilic nature of the peptide is necessary for activity. The possible applications of this expression system for other cysteine-rich biomolecules are discussed.
Subject(s)
Insecticides/chemistry , Nicotiana/genetics , Pisum sativum/chemistry , Plant Proteins/chemistry , Protein Subunits/chemistry , Toxins, Biological/chemistry , Amino Acid Sequence , Biological Control Agents , DNA, Complementary , Gene Expression , Hydrophobic and Hydrophilic Interactions , Insecticides/metabolism , Models, Molecular , Molecular Sequence Data , Pisum sativum/metabolism , Plant Proteins/biosynthesis , Plant Proteins/genetics , Protein Isoforms/biosynthesis , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Processing, Post-Translational , Protein Subunits/biosynthesis , Protein Subunits/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Nicotiana/metabolism , Toxins, Biological/biosynthesis , Toxins, Biological/geneticsABSTRACT
PA1b (Pea Albumin 1, subunit b) is a peptide extract from pea seeds showing significant insecticidal activity against certain insects, such as cereal weevils (genus Sitophilus), the mosquitoes Culex pipiens and Aedes aegyptii, and certain species of aphids. PA1b has great potential for use on an industrial scale and for use in organic farming: it is extracted from a common plant; it is a peptide (and therefore suitable for transgenic applications); it can withstand many steps of extraction and purification without losing its activity; and it is present in a seed regularly consumed by humans and mammals without any known toxicity or allergenicity. The potential of this peptide to limit pest damage has stimulated research concerning its host range, its mechanism of action, its three-dimensional structure, the natural diversity of PA1b and its structure-function relationships.
