Abstract
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.
Highlights
Chemical pesticides are increasingly used around the world but they are increasingly stigmatized because of their persistence and their toxicity to non-target organisms
The mass spectrometry analysis corroborated the HPLC analysis: the only detectable peptide was PA1b in the pMDC32-(PA1cDNA) inoculated leaves extract, and the results confirmed that three disulfide bridges exist in the molecule
PA1b is an entomotoxin with considerable potential for applications on an industrial scale, the main advantage being that it is derived from an edible plant and, it can be used in organic farming
Summary
Chemical pesticides are increasingly used around the world but they are increasingly stigmatized because of their persistence and their toxicity to non-target organisms. Crop protection against two very important pests, namely cereal weevils and aphids, is currently carried out almost exclusively by chemical treatments. A few alternative methods do exist to combat these insects, but they are either much less effective or prohibitively expensive, compared with chemical control. The PA1b (Pea Albumin 1, subunit b) peptide is an entomotoxin extract from pea seeds and, more generally, from Legume seeds [1]. PA1b is one of the few orally active peptide toxins currently known. It displays outstanding insecticidal activity against certain insects, such as grain weevils (genus Sitophilus) and the mosquitoes Culex pipiens and Aedes aegyptii
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