Isolation and characterization of a novel lectin gene, Allium triquetrum agglutin, conferring insecticidal properties against Myzus persicae

Abstract

Myzus persicae, more commonly known as green peach aphid, is an important pest in agriculture. Each year it causes great losses to agricultural and horticultural plants. It feeds on sap in the phloem, the main transportation route for plant nutrients. It also transmits more than 100 plant viruses, and as there are still no agrichemicals specifically against viruses, farmers often spray large amounts of pesticides to protect their crops. A group of widely-distributed plant proteins, termed lectins, have been recently studied because of their insecticidal properties against aphids. Snowdrop lectin, Galanthus nivalis agglutinin (GNA) was the first isolated monocot mannose-binding lectin, conferring properties of resistance to sap-sucking or homopteran pests. The aim of the project described in this thesis was to identify and express a novel lectin in Arabidopsis thaliana and to assess the performance of the green peach aphid, Myzus persicae, on both transgenic and wild type plants. A gene encoding a novel mannose-binding lectin was cloned from Allium triquetrum (wild garlic). The full-length cDNA of Allium triquetrum agglutinin (ATA) was 719 bp and contained a 522 bp open reading frame encoding a 173 amino acid polypepetide. Homology analysis showed that ATA has high similarity with other mannose-binding lectins and includes three putative mannose-binding subdomains, which suggests that ATA may also confer resistance against aphids. The pGreen 0029 vector and the 35S CaMV cassette were ligated to produce the expression construct. An expression vector was engineered using the pGreen vector together with CaMV 35S promoter and the novel ATA gene was inserted. Transgenic Arabidopsis thaliana plants were subsequently generated using the Agrobacterium-mediated floral dip method. Six homozygous ATA-transformed lines and one empty vector-transformed control line were obtained using kanamycin selection. Several key growth parameters of the transgenic plants were assessed to demonstrate that expressing ATA causes few III phenotypic changes to the host plant. The transgenic plants were subsequently used for aphid bioassays, including choice and non-choice behaviour tests, Mean Relative Growth Rate test, fecundity test, and survival test. The aphid bioassays revealed that (1) ATA does not change the behaviour of aphids to choose a host; (2) Adults are more sensitive to ATA than nymphs; (3) ATA has significant detrimental effects on aphid fecundity. The results described in the thesis show that the production of Allium triquetrum agglutinin in Arabidopsis confers partial resistance to the aphids, Myzus persicae. It is likely that this lectin in related Brassicaceae plants could be used, perhaps in conjunction with other pest-control measures in agriculture. Key words: Allium triquetrum agglutinin, aphid bioassay, Arabidopsis thaliana, choice test, fecundity test, lectin, non-choice test, mean relative growth rate, Myzus persicae, pleiotropic effects, resistance against aphids, survival test