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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
A DnaJ protein that interacts with soybean mosaic virus coat protein serves as a key susceptibility factor for viral
Tingxuan Zong1, Jinlong Yin1, Tongtong Jin1
1National Center for Soybean Improvement, National Key Laboratory for Crop Genetics and Germplasm Enhancement, Key Laboratory of Biology and Genetic Improvement of Soybean, Ministry of Agriculture, Nanjing Agricultural University, Weigang 1, Nanjing, China.
Abstract:
Soybean mosaic virus (SMV)-disease is one of the most serious and widespread diseases in soybean (Glycine max). In the present study, a DnaJ protein in soybean designated GmCPIP (SMV coat protein-interacting protein) was screened by the QIS-Seq (quantitative interactor screening with next-generation sequencing) method, and the interaction between SMV CP and GmCPIP was confirmed by the yeast two-hybrid (Y2H) system and bimolecular fluorescence complementation (BiFC) assay. Subcellular localization analysis indicated that both proteins are localized in the cytoplasm, cytomembrane and nucleus. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that infection with SMV-SC4 temporarily increased the transcription of GmCPIP. Virus-induced gene silencing (VIGS) down-regulated the GmCPIP gene by 82%, and the accumulation of SMV was decreased by 88.6% in GmCPIP-silenced plants inoculated with SMV-SC4. The interaction of GmCPIP with SMV CP seems to contribute to SMV infection in soybean.
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