Proteomic analysis of Cry2Aa-binding proteins and their receptor function in Spodoptera exigua

Abstract

The bacterium Bacillus thuringiensis produces Crystal (Cry) proteins that are toxic to a diverse range of insects. Transgenic crops that produce Bt Cry proteins are grown worldwide because of their improved resistance to insect pests. Although Bt “pyramid” cotton that produces both Cry1A and Cry2A is predicted to be more resistant to several lepidopteran pests, including Spodoptera exigua, than plants that produce Cry1Ac alone, the mechanisms responsible for the toxicity of Cry2Aa in S. exigua are not well understood. We identified several proteins that bind Cry2Aa (polycalin, V-ATPase subunits A and B, actin, 4-hydroxybutyrate CoA-transferase [4-HB-CoAT]), and a receptor for activated protein kinase C (Rack), in S. exigua. Recombinant, expressed versions of these proteins were able to bind the Cry2Aa toxin in vitro assays. RNA interference gene knockdown of the Se-V-ATPase subunit B significantly decreased the susceptibility of S. exigua larvae to Cry2Aa, whereas knockdown of the other putative binding proteins did not. Moreover, an in vitro homologous competition assay demonstrated that the Se-V-ATPase subunit B binds specifically to the Cry2Aa toxin, suggesting that this protein acts as a functional receptor of Cry2Aa in S. exigua. This the first Cry2Aa toxin receptor identified in S. exigua brush-border membrane vesicles.

Polycalin is involved in the toxicity and resistance to Cry1Ac toxin in Helicoverpa armigera (Hübner)

“…Since polycalin was first reported it could strongly bind with Cry1A toxins (Hossain et al, 2004(Hossain et al, , 2005, later the result showed polycalin also could bind with Cry2Aa in Spodoptera exigua and H. armigera (Qiu et al, 2017;Wang et al, 2019).…”

“…Since polycalin was first reported it could strongly bind with Cry1A toxins (Hossain et al, 2004, 2005), later the result showed polycalin also could bind with Cry2Aa in Spodoptera exigua and H. armigera (Qiu et al, 2017; Wang et al, 2019). Overexpression or down expression of the receptors have always been used to study the function of binding proteins in the mode action of Bt toxins.…”

Docking‐based generation of antibodies mimicking Cry1A/1B protein binding sites as potential insecticidal agents against diamondback moth (Plutella xylostella)

“… 43 However, both Cry2Aa and Cry1Ca bind to V‐ATPase A from Chilo suppresalis and V‐ATPase A down‐regulation reduces susceptibility, 42 in agreement with a relevant role for binding to V‐ATPase A in toxicity. Orthologues of P. xylostella V‐ATPase A were proposed as binding proteins for Cry1Ac in Heliothis virescens , 23 for Cry2Aa in Spodoptera exigua 44 and for Cry8Ea3 in the scarab Holotrichia parallela 45 . Binding of Cry1A or GEAb‐dV L to V‐ATPase A may result in non‐functional V‐ATPase pumps, 46 altering gut homeostasis and probably resulting in toxicity.…”

Functional validation of DvABCB1 as a receptor of Cry3 toxins in western corn rootworm, Diabrotica virgifera virgifera

“…often coupled with tandem liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Examples include actin, vacuolar ATP synthase (V-ATPase) subunits A and B, heat shock cognate protein, polycalin, and prohibitin 25,[29][30][31][32][33][34] . Additional functional validation would provide a better understanding of the role that these interacting proteins might play as bona fide receptors in vivo resulting in toxicity.…”