Screening of proteins interacting with HbMYC26 which regulates laticifer differentiation in rubber tree by using yeast two-hybrid technology

Aims: MYC (myelocytomatosis) is a transcription factor belonging to the basic helix-loop-helix (bHLH) family and plays a key role in jasmonic acid mediated plant development and stress responses. Previous research has confirmed that HbMYC26 was involves in laticifer differentiation by activating the expression of the phytosulfokine encoding gene HbPSK5 in rubber tree. This study aimed to investigate the molecular mechanism of laticifer differentiation regulating by HbMYC26 and its interacting proteins. Methods: The bait vector pGBKT7-HbMYC26 was constructed in this study, and the interacting proteins of HbMYC26 were screened using a yeast two-hybrid (Y2H) assay with rubber tree bark yeast two-hybrid library. Results: The yeast containing the bait vector pGBKT7-HbMYC26 exhibited autoactivation on the SD/-Trp-Leu-His medium. The 3-AT inhibition experiment confirmed that SD/-Trp-Leu-His medium supplemented with 5 mmol·L-1 3-AT and SD/-Trp-Leu-His-Ade medium were suitable for Y2H screening. Using the co-transformation method, 43 proteins interacting with HbMYC26 were identified from the bark yeast nuclear library. These candidate interacting proteins include hormone-response related proteins such as DELLA and ARF8; development related proteins such as SPL, MADS-box and XTH; the aquaporin PIP1; 3 involved in water transport; metacaspase associated with programmed cell death; as well as E3 ligases and the 26S proteasome involved in protein degradation. The interaction between HbSPL3470 and HbMYC26 was further verified by point-to-point Y2H assay. Conclusion: The results lay a foundation for further revealing the functions and mechanism of HbMYC26 and its interacting proteins in regulating laticifer differentiation in rubber tree.