Fig. 2.

pri-miR825 has a negative impact on PTI. (A) Sequence of pri-miR825 indicating the target for amiR anti825. (B) Plants expressing amiR anti825 display significantly reduced precursor levels, as well as significantly reduced levels of the mature forms of miR825-5p and miR825-3p compared with wild-type Col-0 plants (WT). Asterisks indicate that the results are significantly different from WT plants, as established by a Student’s t-test (P<0.05). Numbers above the bars indicate P-values. Error bars correspond to the SE. Similar results were obtained in two biological replicates. (C) Bacterial multiplication assay in WT or anti825 lines: leaves were inoculated by infiltration with a solution of 5×10⁴ cfu ml^–1 of P. syringae DC3000. Samples were taken 4 days post-inoculation and plated. Bacterial counts are shown. Mean values are shown for each plant genotype, although individual values are also represented. Mean values marked with the same letter were not significantly different from each other as established by Student’s t-test (P<0.05). Similar results were obtained in two biological replicates. (D) ROS production at different time points after treatment with 100 nM flg22 of WT or anti825 lines. Similar results were obtained in two biological replicates. (E) Western blot analysis showing levels of phosphorylated mitogen-activated protein kinases (MPK3, MPK4, MPK6, and MPK11) after treatment with 100 nM flg22 of wild-type (WT) or anti825 lines at three different time points (0, 10, or 15 min post-flg22 treatment). Anti-tubulin was used for normalization. Numbers below the blot indicate fold differences between MPK/tubulin signal ratios calculated using ImageJ (http://imagej.nih.gov/ij/) in anti825 lines and the ratio obtained for WT plants at each time point. Similar results were obtained in two biological replicates. (F) Western blot analysis showing PR1 protein levels in WT or anti825 plants after inoculation with 5×10⁷ cfu ml^–1 of P. syringae DC3000 expressing effector AvrRpt2 from a plasmid under a constitutive nptII promoter. Samples were taken at 0 or 12 h post-inoculation. Anti-tubulin was used as loading control. Similar results were obtained in two biological replicates.