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. 2020 Dec 29;12(1):16. doi: 10.3390/insects12010016

Figure 4.

Figure 4

The interactions between NtWRKY6 and five tobacco MAPKs, wound-induced protein kinase (WIPK), salicylic acid-induced protein kinase (SIPK), NTF4-1, NTF4-2, and NRK1. (A) In vivo interaction between WRKY6 and NtMAPKs as shown by BiFC analysis. NtWRKY6-YFPN and NtMAPKs-YFPC were transiently co-expressed in N. benthamiana line H2B-RFP, of which the nuclei were marked with RFP fusion protein. Photos were imaged at 48 h using a Zeiss LSM710 confocal microscope. Columns from left to right represent YFP fluorescence, bright field, RFP fluorescence, and YFP/RFP/bright field overlay. Scale bars: 50 μm. (B) In vitro interaction between NtWRKY6 and NtMAPKs as shown by pull-down assay. Proteins GST, NtMAPKs-GST, MBP, and NtWRKY6-MBP were expressed via prokaryotic expression, and purified using glutathione agarose beads or Amylose resin. GST or NtMAPK-GST fusion proteins were used to pull down MBP or NtWRKY6-MBP fusion proteins. Binding proteins were analyzed via SDS-PAGE and Western blot assays using anti-MBP antibodies. At the start, samples (Input) were stained with Coomassie blue solution. GST and MBP proteins were used as negative controls.