The CDS of ZmWAKLY, ZmWAKLQ, ZmBLK1, ZmWIK, ZmRBOH4 and their gene segments were cloned into the JW771-35S-CLuc vector (cLUC) and JW772-35S-NLuc vector (nLUC), respectively, to generate fusion proteins with the C-terminal or N-terminal fragment of the luciferase gene. ZmWIK and ZmWAKL were divided into their ECD/TM and ICD, marked as ZmWIKECD,TM (1-269 aa) and ZmWIKICD (270-594 aa), ZmWAKLY/ECD,TM (1-324 aa) and ZmWAKLY/ICD (325-665 aa), ZmWAKLQ/ECD,TM (1-363 aa) and ZmWAKLQ/ICD (364-704 aa). The N-terminal domain was marked as N-ZmRBOH4 (1-377 aa). The pairs of constructs were co-infiltrated into N. benthamiana leaves using the previously reported method55 (link). Two days after inoculation, 1 mM luciferin (Promega, E1601) was sprayed onto the inoculated leaves, and the luminescence signal was measured using the Chemiluminescent Imaging System (Tanon).
The luminescence signals were analyzed using ImageJ Launcher software (National Institutes of Health) to determine their intensities, so-called the mean gray value. This assay was immediately followed by protein extraction and immunoblotting with α-LUC antibody (Abcam, ab181640), and the immunoblot bands were measured using ImageJ Launcher as well. Total protein of the infiltrated leaf tissues was extracted and detected as described above.
The luminescence signals were analyzed using ImageJ Launcher software (National Institutes of Health) to determine their intensities, so-called the mean gray value. This assay was immediately followed by protein extraction and immunoblotting with α-LUC antibody (Abcam, ab181640), and the immunoblot bands were measured using ImageJ Launcher as well. Total protein of the infiltrated leaf tissues was extracted and detected as described above.
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