Gene pulserxcell electroporation systems

Vectors for subcellular localization assay were constructed by sub-cloning the coding regions of MaWRKY26 and MaVQ5 into the pEAQ-HT-GFP vector (kindly supplied by Dr. George P. Lomonossoff), resulting in MaWRKY26-GFP and MaVQ5-GFP, respectively. Primers used are shown in Supplementary Table S1. The fusion constructs and control vector were electroporated into Agrobacterium tumefaciens strain GV3101 using Gene PulserXcell^TM Electroporation Systems (Bio-Rad, CA). Tobacco (Nicotiana benthamiana) leaf infiltration assay for sub-cellular localization was performed as described previously66 (link). After infiltration, plants were incubated at 22 °C with 16 h photoperiod for at least 2 days before analysis. GFP fluorescence signals were observed with a fluorescence microscope (Zeiss Axioskop 2 Plus). All transient expression assays were repeated at least three times.

Dual-luciferase assay was carried out to investigate the transactivation activity of transcription factors to target promoters. Full-length EjODO1 was amplified and inserted into NotI and SpeI sites of pGreen II 0029 62-SK vector, while pGreen II 0800-LUC carrying promoters of lignin biosynthesis were constructed by Xu et al. (2014) (link). All constructs were electroporated into Agrobacterium tumefaciens GV3101, using Gene PulserXcell^TM Electroporation Systems (Bio-Rad). Dual luciferase assays were performed according to our previous reports (Zeng et al., 2015 (link)). The empty vector mixed with the promoters was set as 1 and the analysis was carried out with at least three replicates.

The coding sequences of MaDEAR1 were amplified and cloned into the pEAQ-GFP vectors (kindly gifted by Dr. George P. Lomonossoff), then the fusion construct and positive control GFP vector were electroporated into the Agrobacterium tumefaciens strain GV3101 using Gene PulserXcell^TM Electroporation Systems (Bio-Rad, Hercules, CA, USA). The primers for construct development are listed in Supplementary Table S1. The Agrobacterium harboring MaDEAR1-GFP or the positive control was inoculated for 16 h at 28°C. Cells were pelleted, resuspended at OD600 = 0.1 in infiltration buffer [10 mM MgCl2, 10 mM MES (pH 5.6), 100 μM acetosyringone], incubated for 4 h at room temperature, then was infiltrated into the abaxial side of 4- to 6-week-old tobacco leaves using a 1-mL needleless syringe as described previously by Sainsbury et al. (2009) (link). Two days after infiltration, GFP fluorescence signals were observed by a fluorescence microscope (Zeiss Axioskop 2 Plus) with a beam splitter for excitation at 500 nm. All assays were repeated at least three times.