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Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro
Published on: August 14, 2011
Cucumber Green Mottle Mosaic Virus Coat Protein Hijacks Mitochondrial ATPδ to Promote Viral Infection
Xue Yang1,2, Xing-Lin Jiang1, Han Fu1
1College of Plant Protection, Henan Agricultural University, Zhengzhou, China.
Abstract:
The production and scavenging of reactive oxygen species (ROS) are critical for plants to adapt to biotic and abiotic stresses. In this study, we investigated the interaction between the coat protein (CP) of cucumber green mottle mosaic virus (CGMMV) and ATP synthase subunit δ (ATPδ) in mitochondria. Silencing of ATPδ by tobacco rattle virus-based virus-induced gene silencing impeded CGMMV accumulation in Nicotiana benthamiana leaves. Both the overexpression of ATPδ in transgenic plants and transient expression promoted CGMMV infection. Nitro blue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB) staining revealed that ATPδ inhibited O2 - production but not H2O2 production. The treatment of CGMMV-infected leaves with the ROS inhibitor diphenylene iodonium (DPI) induced a ROS burst that inhibited CGMMV infection. Reverse transcription-quantitative PCR and superoxide dismutase (SOD) activity assays showed that ATPδ, CGMMV infection, and CP expression specifically induced NbFeSOD3/4 expression and SOD activity, and silencing NbFeSOD3/4 inhibited CGMMV infection. We speculate that CGMMV CP interacts with ATPδ and hijacks it, thereby enhancing O2 - quenching by upregulating NbFeSOD expression and, in turn, SOD activity.
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