Transient expression assays
For ProPAL1:GUS vector construction, an approximately 2000-bp
promoter fragment of PAL1 was amplified by gene-specific primers
(Table S1) and cloned into the pCAMBIA-1301 vector. To generatePro35S:WRKY75 , the WRKY75 cDNA fragment was amplified and
inserted into the pCAMBIA-1301 vector. The 35S:PtrWRKY75construct was used as an effector. Leaves of N. benthamiana were
infiltrated with A. tumefaciens cells containing the effector and
reporter using the agroinfiltration method (Yang et al. , 2000).
Activity of GUS was measured using a previously described method
(Jefferson et al., 1987), which monitored the cleavage of the
β-glucuronidase substrate 4-methylumbelliferyl β-D-glucuronide by
hydrolysis to produce the fluorescent 4-methylumbelliferone. Protein
concentrations were measured as described by Bradford (1976).