Identification of Insect Proteins Interacting with P6 of SRBSDV in the Midgut of White-backed Planthopper (Sogatella furcifera)
ZHAO Zhong-Hao1, 2, PAN Hui1, 2, DU Jiao2, CHEN Jian-Bin2, LIU Yong1, 2, ZHANG Song-Bai1, *, ZHENG Li-Min2, *
1 College of Agricultural, Yangtze University, Jingzhou 434025, China; 2 Institute of Plant Protection, Hunan Academy of Agricultural Sciences, Changsha 410125, China
Abstract：Southern rice black-streaked dwarf virus (SRBSDV) is transmitted by white-backed planthopper (Sogatella furcifera) in a persistent-propagative manner. During SRBSDV infection in its insect vector, viroplasm, putative sites of viral replication, contained the nonstructural viral protein P5, P6, P9-1 and insect vector proteins. The P6 protein is initially expressed, and interacts with P5, P9-1 and insect proteins in insect vector, respectively, which demonstrate that P6 protein is essential for viroplasm formation and viral replication. In order to investigate which insect proteins play an important role during viral replication, yeast two-hybrid experiments was be used to identify insect proteins interacting with SRBSDV P6 protein in the white-backed planthopper midgut. Firstly, SRBSDV P6 gene was constructed into the expression vector (pGBKT7-P6) and transformed into Y2HGold cell. Western blot showed P6 protein successfully expressed in yeast cell, which was non-toxicity and non-autonomous activation to cells. Then cDNA library plasmids were transformed into Y2HGold containing bait plasmid pGBKT7-P6. Transformants were selected from high stringency medium SD/-Leu/-Trp/-His/-Ade, and then replicated to SD/-Leu/-Trp/-His/-Ade/X-α-gal medium for further analysis. Colony growth and blue color indicated an interaction between the two-hybrid proteins. Plasmid DNA was isolated from yeast and then transformed into Escherichia coli DH5α. Lastly, cDNA inserts were sequenced to verify the preference of an open reading frame (ORF) and compared the sequence in Blast of NCBI.. The results showed 5 insect proteins, interacting with P6, which were involved in gene transcription, translation, post-translational modification and protein systhesis. Results of this study will be useful in revealing the replication mechanisms of SRBSDV in its insect vector, which will facilitate the development of effective control strategies against SRBSDV disease.
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