Split luciferase (LUC) complementation
The full-lengthMdHSFA1dand MdHSFA9b coding sequences were cloned independently into the
CLUC vector, while MdNup62 was cloned into the NLUC vector. The
split-LUC complementation assay was performed with tobacco leaves. The
reconstituted LUC activity was detected in the dark
using
a Princeton Lumazone Pylon 2048B cooling camera (Princeton, USA). The
LUC activity was quantified using the Dual-Luciferase Reporter Assay
System (Promega, USA). The primers used are listed in Table S5.
Pull-down assays
The ORFs of MdNup62 and MdHSFA9b were cloned into the
pET-28a and pGEX-6p-1 vectors, respectively, and subsequently
overexpressed independently in Escherichia coli BL21(DE3)
(Transgene). The pull-down assays were conducted using the His-Tagged
Protein Purification Kit (Clontech) in accordance with the
manufacturer’s instructions. The primers used are listed in Table S5.