VIGS silencing of SlWAK1 in Nicotiana benthamiana results in compromised PTI and increased susceptibility to Pst . (A) Cell death suppression assay. Silenced plants were induced by syringe infiltration of Pseudomonas fluorescens and challenged 8 h later with DC3000 in an overlapping area. Cell death symptoms were scored in the overlapping area, on four independently silenced plants with four infiltration sites on two different leaves. Results shown are from 7 days after infiltration (dai). Asterisks indicate significant differences based on Dunnett’s method, using EC1 as a control (P < 0.01). (B) Pst growth in leaves. Silenced plants were vacuum-infiltrated with P. fluorescens, inoculated 6 h later with DC3000ΔhopQ1-1 and sampled to measure bacterial populations. Results shown are the average of six plants per construct with the standard error. Asterisks indicate significant differences based on Dunnett’s method, using EC1 as a control (P < 0.01). (C) Disease symptoms of plants treated as described in (B) 7 dai. (D) Silencing efficiencies in EC1 and SlWAK1 silenced plants assessed by qRT-PCR. Silencing efficiency is shown as relative expression compared to the EC1 control. Data were generated using EF1α as reference gene and similar results were obtained using PP2A. Data from putative targets: (a) NbS00011055g0005.1, (b) NbS00011055g0014.1 and (c) NbS00011055g0002.1, and a predicted non-target (d) NbS00016938g0011.1 are shown. PP2A (e) was used as a cDNA integrity control. Asterisks indicate significant differences using a Student’s t-test (P < 0.01). dai, days after infiltration; qRT-PCR, quantitative reverse transcription-PCR; Pst, Pseudomonas syringae pv. tomato; PTI, pattern-triggered immunity; VIGS, virus-induced gene silencing.