To analyze the subcellular localization of the OpWRKY2 protein, the open reading frame (ORF) was amplified by PCR from the O. pumila hairy-root cDNA library with OpWRKY2 gene-specific primers (Table S2) and inserted into the modified plant expression vector pHB-YFP (yellow fluorescent protein) to generate the pHB-OpWRKY2-YFP construct. The pHB-YFP construct without OpWRKY2 was used as the negative control. The plasmids pHB-OpWRKY2-YFP and pHB-YFP were introduced into the A. tumefaciens strain GV3101 and transiently infected the epidermal cells of 5-week-old N. benthamiana leaves. YFP signals were analyzed using an LSM880 confocal laser microscope (Carl Zeiss, Germany) 48 h post infection, and three biological replicates were performed to confirm the results as reported previously23,28.

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