FIG 5.

Binding of HrcT to hrpX promoter in vivo and in vitro. (A) Agrobacterium-mediated transient expression assay of hrpX activated by HrcT. GUS reporter constructs are codelivered via A. tumefaciens into N. benthamiana with (+) and without (−) the construct HrcT. PthXo1 of X. oryzae pv. oryzae and its target Os8N3 (pOs8N3) were used as a positive control. The GUS activity was determined 2 dpi by stained leaf disks (0.8 cm in diameter) with X-Gluc (5-bromo-4-chloro-3-indolyl-β-d-glucuronide). Blue indicates a positive reaction. Error bars indicate standard deviations (n = 3 samples). 4-MU, 4-methyl-umbelliferone. (B) Binding of HrcTΔ41N to the hrpX promoter by EMSA. Twenty femtomoles of biotinylated probes was used to react with 1 μg of purified HrcTΔ41N (presence indicated by +, absence by −). Different sizes of the hrpX promoter upstream of the hrpX transcriptional start codon were used as probes, displayed on the right. Probe A is 149 bp long, probe B 78 bp, probe C 57 bp, all upstream of the hrcT transcription start code, and probe D is 70 bp long, upstream of the probe B as displayed on the left. (C) Competition of biotinylated probe C with unlabeled probe C bound to HrcTΔ41N by EMSA. The unlabeled probe C with higher concentration, at 0.5×, 1×, and 2× more than 20 fmol of biotinylated probe C, was used. +, present; −, absent. The above-described experiments were repeated three times, and results from one representative experiment are shown.