Screening and Identification of Interacting Proteins of Sweet Potato WRKY Transcription Factor IbWRKY61
ZHANG Kai1,2,*, LIU Ying-Ying1,3,*, PENG De-Liang1, WU Zheng-Dan1,2, MAO Li-Min1,2, TANG Dao-Bin1,2, WANG Ji-Chun1,2,**
1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China; 2 Chongqing Key Laboratory of Biology and Genetic Breeding for Tuber and Root Crops, Chongqing 400715, China; 3 Institute of Health Inspection and Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100175, China
Abstract：WRKY transcription factors play key roles in the responses against biotic or abiotic stress in plants. To elucidate the biological function of sweet potato (Ipomoea batatas) WRKY transcription factor IbWRKY61 and to clarify its role in the protein interaction network, in this study, the bait construct pGBKT7-DEST-IbWRKY61 was constructed using Gateway method and used to screen the sweet potato cDNA library via the yeast (Saccharomyces cerevisiae) two-hybrid (Y2H) library screening system. The results showed that 241 positive clones were obtained through Y2H screening. These clones were cultured and detected using PCR, and a total of 121 positive monoclones were selected and the PCR products were recovered from agarose gel. High thought-out sequencing was performed with mixed PCR products, and the obtained reads were mapped to genes using STAR software package. According to transcripts per million (TPM) values, the top 100 gene sequences were selected and blasted against NCBI database for annotation, and the results showed that screened interacting proteins of IbWRKY61 were mainly involved in biotic stress response and pathogen defense. The interaction of IbWRKY61 and 12 of the screened interacting proteins were validated using Y2H and bimolecular fluorescence complementation (BiFC). Y2H test confirmed the interaction between IbWRKY61 and all the 12 proteins, and BiFC results confirmed that IbWRKY61 interacted with CCR4-associated factor 1 (CAF1) in the nucleus of Nicotiana benthamiana mesophyll cells, and interacted with ubiquitin-activating enzyme E1, Clp protease proteolytic subunit-related protein 2 (CLP2) and leucine-rich repeat receptor-like protein kinase BAK1-interacting receptor-like kinase 1 (BIR1), in the plasma membrane, chloroplast, plasma membrane or plasmodesma, respectively. The present study provides a reference for further research on the biological function of WRKY transcription factors in stress response and disease resistance of sweet potato.
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