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Expression of Modified Snowdrop Lectin (Galanthus nivalis Agglutinin) Protein Confers Aphids and Plutella xylostella Resistance in Arabidopsis and Cotton.

ABSTRACT: Cotton is a major fiber crop in the world that can be severely infested by pests in agricultural fields. Identifying new insect-resistance genes and increasing the expression of known insect-resistance genes are imperative in cultivated cotton. Galanthus nivalis agglutinin (GNA), a lectin that is toxic to both chewing and sucking pests, is mainly expressed in monocotyledons. It is necessary to improve the expression of the GNA protein and to test whether the lectin confers insect resistance to dicotyledons plants. We report a modified GNA gene (ASGNA) via codon optimization, its insertion into Arabidopsis thaliana, and transient expression in cotton to test its efficacy as an insect-resistance gene against cotton aphids and Plutella xylostella. The amount of ASGNA in transgenic plants reached approximately 6.5 μg/g of fresh weight. A feeding bioassay showed that the survival rate of aphids feeding on the leaves of ASGNA transgenic plants was lower than those of aphids feeding on the leaves of non-optimized GNA (NOGNA) transgenic plants and wild-type plants. Meanwhile, the fertility rate was 36% when fed on the ASGNA transgenic plants, while the fertility was 70% and 95% in NOGNA transgenic plants and wild-type plants. Correspondingly, the highest mortality of 55% was found in ASGNA transgenic lines, while only 35% and 20% mortality was observed in NOGNA transgenic plants and wild-type plants, respectively. Similar results were recorded for aphids feeding on cotton cotyledons with transient expression of ASGNA. Taken together, the results show that ASGNA exhibited high insecticidal activity towards sap-sucking insects and thus is a promising candidate gene for improving insect resistance in cotton and other dicotyledonous plants.

SUBMITTER: He P

PROVIDER: S-EPMC9316576 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundNew bioinsecticides with novel modes of action are urgently needed to minimise the environmental and safety hazards associated with the use of synthetic chemical pesticides and to combat growing levels of pesticide resistance. The pea seed albumin PA1b knottin peptide is the only known proteinaceous inhibitor of insect vacuolar adenosine triphosphatase (V-ATPase) rotary proton pumps. Oral toxicity towards insect pests and an absence of activity towards mammals makes Pa1b an attractive candidate for development as a bioinsecticide. The purpose of this study was to investigate if Pichia pastoris could be used to express a functional PA1b peptide and if it's insecticidal activity could be enhanced via engineering to produce a fusion protein comprising the pea albumin protein fused to the mannose-specific snowdrop lectin (Galanthus nivalis agglutinin; GNA).ResultsWe report the production of a recombinant full-length pea albumin protein (designated PAF) and a fusion protein (PAF/GNA) comprised of PAF fused to the N-terminus of GNA in the yeast Pichia pastoris. PAF was orally toxic to pea (Acyrthosiphon pisum) and peach potato (Myzus persicae) aphids with respective, Day 5 LC50 values of 54 µM and 105 µM derived from dose-response assays. PAF/GNA was significantly more orally toxic as compared to PAF, with LC50 values tenfold (5 µM) and 3.3-fold (32 µM) lower for pea and peach potato aphids, respectively. By contrast, no phenotypic effects were observed for worker bumble bees (Bombus terristrus) fed PAF, GNA or PAF/GNA in acute toxicity assays. Confocal microscopy of pea aphid guts after pulse-chase feeding fluorescently labelled proteins provides evidence that enhanced efficacy of the fusion protein is attributable to localisation and retention of PAF/GNA to the gut epithelium. In contact assays the fusion protein was also found to be significantly more toxic towards A. pisum as compared to PAF, GNA or a combination of the two proteins.ConclusionsOur results suggest that GNA mediated binding to V-type ATPase pumps acts to potentiate the oral and contact aphicidal activity of PAF. This work highlights potential for the future commercial development of plant protein-based bioinsecticides that offer enhanced target specificity as compared to chemical pesticides, and compatibility with integrated pest management strategies.

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Expression of Modified Snowdrop Lectin (Galanthus nivalis Agglutinin) Protein Confers Aphids and Plutella xylostella Resistance in Arabidopsis and Cotton.

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