PHB2 affects the virulence of Vip3Aa to Sf9 cells through internalization and mitochondrial stability

Abstract

The vegetative insecticidal proteins (Vip3A) secreted by some Bacillus thuringiensis (Bt) strains during vegetative growth are regarded as a new generation of insecticidal toxins. Like insecticidal crystal proteins, they are also used in transgenic crops to control pests. However, their insecticidal mechanisms are far less defined than those of insecticidal crystal protein. Prohibitin 2 (PHB2) is a potential Vip3Aa binding receptor identified from the membrane of Sf9 cells in our previous work. In this paper, we demonstrated the interaction between Vip3Aa and PHB2 using pull-down, dot blotting, microscale thermophoresis, and co-immunoprecipitation assays. PHB2 is distributed on the cell membrane and in the cytoplasm, and the co-localization of PHB2 and Vip3Aa was observed in Sf9 cells using a confocal laser scanning microscope. Moreover, PHB2 could interact with scavenger receptor-C via its SPFH (stomatin, prohibitin, flotillin, and HflK/C) domain. Downregulation of phb2 expression reduced the degree of internalization of Vip3Aa, exacerbated Vip3Aa-mediated mitochondrial damage, and increased Vip3Aa toxicity to Sf9 cells. This suggested that PHB2 performs two different functions: Acting as an interacting partner to facilitate the internalization of Vip3Aa into Sf9 cells and maintaining the stability of mitochondria. The latter has a more important influence on the virulence of Vip3Aa.

RNA interference of PHB1 enhances virulence of Vip3Aa to Sf9 cells and Spodoptera frugiperda larvae

“…In our previous study, we found the dual functions of PHB2 in the Vip3Aa action process: (i) PHB2 mediates the internalization of Vip3Aa by interacting with it and (ii) PHB2 is an essential structural protein for mitochondrial stability and helps cells resist Vip3Aa virulence. The latter was more important than the former for Vip3Aa toxicity, suggesting a critical role of PHB2 in mitochondria for cellular resistance 12 . PHB1 and PHB2 form ring‐like structures with high molecular weights in yeast, nematodes and mammals, and this complex performs multiple functions to maintain the structure and regulate the function of mitochondria 19 .…”

“…In our previous research, 48 h was a suitable time point to detect the virulence of Vip3Aa to Sf9 cells. 12 Based on this, we tested the virulence of Vip3Aa to Sf9 cells, and the LC 50 of Vip3Aa to Sf9 cells at 48 h was 82.1 ± 10.6 μg mL −1 . Then, we chose a sublethal concentration (60 μg mL −1 ) for the following experiments.…”

“…The PHB1 protein was identified from the PHB2 potential interacting proteins in our previous research. 12 Then, the amplified fragment of phb1 (NCBI accession number: XM_035589856.2) was inserted into the pET28a and pGEX-4 T-1 vectors and the reconstructed vectors were transformed into BL21 to produce PHB1-His, PHB1-Flag, and GST-PHB1 proteins. All the reconstructed BL21 strains were induced with 0.5 mM IPTG at 16 °C overnight.…”

“…indicated that sublethal concentrations of Vip3Aa protein activated the signal pathway of apoptotic cell death in S. exigua larvae 7 . With an increasing number of receptors being discovered, including S2, 9 SR‐C, 10 FGFR 11 and PHB2, 12 the mode of action of Vip3Aa has gradually been understood. Vip3Aa can be endocytosed into lysosomes under the mediation of receptors to exert its virulence, and thus the internalization of Vip3Aa is closely related to its virulence 10,13 …”

Vegetative Insecticidal Protein Vip3Aa Is Transported via Membrane Vesicles in Bacillus thuringiensis BMB171

“…Binding to the receptor is a crucial step in the virulence process of Vip3A. Recently, several Vip3A receptors have been identified, including ribosome S2 protein from sf21 cells of S. frugiperda [ 12 ], scavenger receptor class C protein (Sf-SR-C) [ 13 ], fibroblast growth factor receptor protein (Sf-FGFR) [ 14 ] and prohibitin 2 (PHB2) from the Sf9 cell lines of S.frugiperda [ 15 ], and a tenascin-like glycoprotein from A. ipsilon [ 16 ]. After binding to its receptor, Vip3A may exert its potency through pore formation and induce apoptosis [ 1 , 17 , 18 ].…”