LPS trigger weak early, but strong late MAMP-triggered immunity responses. A, Elevation of [Ca2+]cyt. [Ca2+]cyt was measured using 5-d-old transgenic seedlings expressing the calcium reporter protein, aequorin. Signals were recorded for 15 min after treatment. B, ROS burst. ROS were monitored using a chemiluminescence assay with luminol as a substrate. Leaf disks were punched from 4-week-old mature plants. Signals were recorded for 30 min after treatment. C, MAP kinase phosphorylation. Total protein was extracted from 10-d-old seedlings at different time points after treatment. Immunoblot analysis was performed using an antip42/p44-MAPK antibody. Ponceau S staining was used for protein loading control. D and E, WRKY53 (D) and WRKY33 (E) gene expression. Gene expression was quantified by RT-qPCR. RNA was extracted from 10-d-old seedlings 30 min after treatment. UBQ10 was used as the reference gene. F, Callose deposition. Ten-day-old seedlings were treated with water, flg22, or LPS for 24 h. Callose was stained with aniline blue. Representative images are shown. Bars = 100 μm. G, Quantification of the callose intensity in F. The percentages were quantified as the number of fluorescent callose-corresponding pixels relative to the total number of pixels covering the cotyledon surface using Image J software. H, Growth of P. syringae pv. tomato DC3000 (Pst DC3000). Five-week-old Arabidopsis leaves were primed with the indicated treatment before bacterial infection. The inoculation concentration of Pst DC3000 was 105 cfu/mL. Bacterial growth was determined 3 d after inoculation. I, Inhibition of seedling growth. Five-day-old seedlings were transferred to the indicated liquid media and images were taken 5 d after incubation. Bar = 1 cm. J, Primary root length in I. LPS (50 μg/mL) and flg22 (100 nm) were used in these assays. The data are shown as means ± se (n = 8 in A, B, G, and J; n = 3 in D, E, and H). Asterisks indicate significant differences from the control treatment (Student’s paired t test: *P ≤ 0.05, **P ≤ 0.01). All experiments were repeated twice with similar results.