BackgroundAlbumin 1b peptides (A1b) are small disulfide-knotted insecticidal peptides produced by Fabaceae (also called Leguminosae). To date, their diversity among this plant family has been essentially investigated through biochemical and PCR-based approaches. The availability of high-quality genomic resources for several fabaceae species, among which the model species Medicago truncatula (Mtr), allowed for a genomic analysis of this protein family aimed at i) deciphering the evolutionary history of A1b proteins and their links with A1b-nodulins that are short non-insecticidal disulfide-bonded peptides involved in root nodule signaling and ii) exploring the functional diversity of A1b for novel bioactive molecules.ResultsInvestigating the Mtr genome revealed a remarkable expansion, mainly through tandem duplications, of albumin1 (A1) genes, retaining nearly all of the same canonical structure at both gene and protein levels. Phylogenetic analysis revealed that the ancestral molecule was most probably insecticidal giving rise to, among others, A1b-nodulins. Expression meta-analysis revealed that many A1b coding genes are silent and a wide tissue distribution of the A1 transcripts/peptides within plant organs. Evolutionary rate analyses highlighted branches and sites with positive selection signatures, including two sites shown to be critical for insecticidal activity. Seven peptides were chemically synthesized and folded in vitro, then assayed for their biological activity. Among these, AG41 (aka MtrA1013 isoform, encoded by the orphan TA24778 contig.), showed an unexpectedly high insecticidal activity. The study highlights the unique burst of diversity of A1 peptides within the Medicago genus compared to the other taxa for which full-genomes are available: no A1 member in Lotus, or in red clover to date, while only a few are present in chick pea, soybean or pigeon pea genomes.ConclusionThe expansion of the A1 family in the Medicago genus is reminiscent of the situation described for another disulfide-rich peptide family, the “Nodule-specific Cysteine-Rich” (NCR), discovered within the same species. The oldest insecticidal A1b toxin was described from the Sophorae, dating the birth of this seed-defense function to more than 58 million years, and making this model of plant/insect toxin/receptor (A1b/insect v-ATPase) one of the oldest known.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0745-0) contains supplementary material, which is available to authorized users.
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.
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