Date Published: October 4, 2018
Publisher: Public Library of Science
Author(s): Kun Jiang, Xiao-yue Hou, Tong-tong Tan, Zhang-lei Cao, Si-qi Mei, Bing Yan, Jin Chang, Lu Han, Dan Zhao, Jun Cai, David S. Schneider.
The vegetative insecticidal proteins (Vip), secreted by many Bacillus thuringiensis strains during their vegetative growth stage, are genetically distinct from known insecticidal crystal proteins (ICPs) and represent the second-generation insecticidal toxins. Compared with ICPs, the insecticidal mechanisms of Vip toxins are poorly understood. In particular, there has been no report of a definite receptor of Vip toxins to date. In the present study, we identified the scavenger receptor class C like protein (Sf-SR-C) from the Spodoptera frugiperda (Sf9) cells membrane proteins that bind to the biotin labeled Vip3Aa, via the affinity magnetic bead method coupled with HPLC-MS/MS. We then certified Vip3Aa protoxin could interact with Sf-SR-C in vitro and ex vivo. In addition, downregulation of SR-C expression in Sf9 cells and Spodoptera exigua larvae midgut reduced the toxicity of Vip3Aa to them. Coincidently, heterologous expression of Sf-SR-C in transgenic Drosophila midgut significantly enhanced the virulence of Vip3Aa to the Drosophila larvae. Moreover, the complement control protein domain and MAM domain of Sf-SR-C are involved in the interaction with Vip3Aa protoxin. Furthermore, endocytosis of Vip3Aa mediated by Sf-SR-C correlates with its insecticidal activity. Our results confirmed for the first time that Sf-SR-C acts as a receptor for Vip3Aa protoxin and provides an insight into the mode of action of Vip3Aa that will significantly facilitate the study of its insecticidal mechanism and application.
Microbial insecticides, as substitutes for chemical pesticides, are alternatives for insect control in crops. Bacillus thuringiensis (Bt) is the most extensively used biopesticide worldwide due to its ability to produce insecticidal crystal proteins (Cry and Cyt toxins)[1–3]. The classical pore-forming model is the widely accepted mode of action of the three-domain crystal protein (3d-Cry) . A signaling pathway model of the Cry toxin’s action has also been reported [4, 5]. Recently, Fengjuan et al. showed Cry6Aa could trigger the Caenorhabditis elegans death by necrosis signaling pathway . In spite of differences, all three models agree that binding to host specific receptors is a key step in the process involved in cytotoxicity. Several types of receptors for Cry toxins have been reported, such as aminopeptidase N (APN), the cadherin-like proteins, alkaline phosphatases, and ABC transporter [1, 7, 8]. Bt has been used successfully to control many crop pests by transgenic plant or traditional spray approaches, however, many pests are not sensitive to Cry toxins and a number of cases of insect resistance to Cry toxins have been reported as a result of laboratory or field selections [1–3].
Vip3Aa proteins have been studied for more than 20 years since they were first found by Estruch et al. in 1996 . They are considered as novel insecticidal toxins secreted by Bt because they have different insecticidal properties compared with known Cry toxins and have a broad insecticidal spectrum within Lepidoptera . To date, more than 138 Vip proteins have been found and were divided into four categories according to the classification of Bt Toxin Nomenclature Committee . However, there has been no report of a definite receptor for Vip toxins up to now. In this paper, via HPLC-MS/MS, 70 potential binding proteins of Vip3Aa, including ribosomal protein S2 and actin, were identified (S1 Dataset). Singh et al. identified ribosomal protein S2 as a toxicity-mediating interacting partner protein of Vip3A in Sf21 cells . However, as an intracellular protein, S2 protein is not likely to be a receptor of Vip3A. That maybe why Singh et al named it interacting partner protein, not a receptor. Our results also showed Vip3Aa could not bind to the S2-Sf-S2 cells, which heterologously expressed Sf-S2 into the S2 cells, and had no obvious cytotoxicity to them (Fig 2D and 2E). It suggests that S2 protein is not a receptor for Vip3Aa. Actin was identified as a novel Cry1Ac binding protein in Manduca sexta midgut through proteomic analysis . For the same reason, it is unlikely that this protein is serving as a receptor for Vip3Aa. We speculate that the Vip3Aa may interfere with the function of the ribosome and actin after entering the cells.