FIG 4.
Analysis of the relationships between immune function induced by V. dahliae VdR3e and the peripheral extracellular localization and interactions with the N. benthamiana proteins BAK1 and SOBIR1. (A) ROS accumulation after transient expression of VdR3e, VdR3eΔSP, and GFP control for 2 days. Scale bars, 500 μm. ROS accumulation levels in N. benthamiana leaves were measured after the transient expression of VdR3e, VdR3eΔSP, and the GFP control for 2 days. ImageJ was used to analyze the gray value. Values represent the means ± the SE of three independent samples. Bars not sharing letters represent significant differences of the mean at P < 0.05, based on unpaired Student t tests. (B) Detection of electrolyte leakage induced by VdR3e and VdR3eΔSP. N. benthamiana leaves that transiently expressed VdR3e, VdR3eΔSP, and the GFP control were tested for electrolyte leakage after 2 days. Values represent the averages of three independent measurements with three replicates each. Error bars represent standard errors of the mean. Asterisks (*), double asterisks (**), and triple asterisks (***) represent statistical significance at 0.01 < P < 0.05, 0.001 < P < 0.01, and P < 0.001, respectively, based on unpaired Student t tests. (C) Detection of defense gene expression induced by VdR3e or VdR3eΔSP. N. benthamiana leaves were harvested 2 days after transient expression of VdR3e, VdR3eΔSP, and the GFP control. RT-qPCR was used to quantify the transcription levels of defense genes NbPR1, NbPR2, NbLOX, and NbPR4. Values represent the means ± the SE of three independent samples. Bars not sharing letters represent significant mean differences at P < 0.05, based on unpaired Student t tests. (D) Verification of the relationship between N. benthamiana proteins BAK1 and SOBIR1 and the cell death-inducing function of VdR3e. Virus-induced gene silencing was performed on 3-week-old N. benthamiana. VdR3erec (50 μg/mL) and PBS were infiltrated in N. benthamiana leaves silenced with BAK1 and SOBIR1. (E) The expression levels of BAK1 and SOBIR1 in silenced plants were quantified by RT-qPCR. Values represent the means ± the SE of three independent samples. Asterisks (*), double asterisks (**), and triple asterisks (***) represent statistical significance at 0.01 < P < 0.05, 0.001 < P < 0.01, and P < 0.001, respectively, based on unpaired Student t tests. (F) Detection of transcription levels of VdR3e induced defense genes in silenced plants. Transient transformation of VdR3e was performed using Agrobacterium infiltration in N. benthamiana silenced leaves of BAK1 and SOBIR1. The expression levels of HSR203, NbPR1, NbPR5, and NbPR4 were detected by RT-qPCR. Values represent the means ± the SE of three independent samples. Asterisks (*), double asterisks (**), and triple asterisks (***) represent statistical significance at 0.01 < P < 0.05, 0.001 < P < 0.01, and P < 0.001, respectively, based on unpaired Student t tests.