Preliminary studies on the involvement of BcCHC1 in the regulation of TuMV infection in non-heading Chinese cabbage

Abstract

Abstract [Objective] To enhance the resistance of non-heading Chinese cabbage to turnip mosaic virus (TuMV), this study preliminarily explored the interaction mechanism between BcCHC1 protein of nonheading Chinese cabbage and TuMV. [Methods] The study firstly identified members of the heavy chain clathrin (CHC) gene family, BcCHCs, from non-heading Chinese cabbage, and then cloned CHC1 gene. BcCHC1 subcellular localization was analyzed, and bimolecular fluorescence complementation (BiFC) assay was used to verify the interaction between TuMV and BcCHC1. Finally, virus-induced gene silencing (VIGS) was employed to silence BcCHC1 expression and its function was assessed. [Results] BcCHC1 gene from non-heading Chinese cabbage was cloned, with a coding sequence length of 5 124 bp, encoding 1 708 amino acids. qRT-PCR results showed a significant decrease in the relative expression level of Bc- CHC1 gene in non-heading Chinese cabbage infected with TuMV after 30 days. Subcellular localization revealed that BcCHC1 protein was located in the cell membrane and nucleus of tobacco epidermal cells. BiFC assay analysis confirmed that BcCHC1 interacted with CI and 6K2: Interaction with CI was mainly occurring in the nucleus, while interaction with 6K2 mainly on the cell membrane. Observations of BcCHC1-silenced lines showed that silencing BcCHC1 gene led to plant death. [Conclusion] BcCHC1 regulates the infection of non-heading Chinese cabbage by TuMV through interaction with CI and 6K2, thereby affecting the clathrin-dependent endocytosis pathway and virus replication. However, the mechanism by which Bc- CHC1 regulates TuMV infection in non-heading Chinese cabbage requires further study.

Keywords

• non-heading chinese cabbage [brassica campestris (syn. brassica rapa) ssp. chinensis]
• tumv
• bcchc1
• protein interaction
• vigs