Fig. 4: PdCDIE1-triggered plant cell death is dependent on ROS and Hsp70 is antagonistic to plant cell death.

a The production of H₂O₂ (brown) in N. benthamiana leaves expressing PdCDIE1 or GFP (control) at 3 dpi was detected by staining with 3,3-diaminobenzidine (DAB). b H₂O₂ content in N. benthamiana leaves expressing PdCDIE1 or GFP at a series of time points. Spectrophotometric determination of H₂O₂ content was performed based on the reaction between H₂O₂ and titanium. c The effect of dimethylthiourea (DMTU) treatment on PdCDIE1-triggered cell death in N. benthamiana. “-12 DMTU” indicates N. benthamiana leaves were treated with DMTU 12 h before Agrobacterium-mediated transient expression of PdCDIE1. Photos were taken at 4 dpi. “0 h DMTU” and “12 h DMTU” indicate N. benthamiana leaves were treated with DMTU 0 h or 12 h after Agrobacterium-mediated transient expression of PdCDIE1. d–f Electrolyte leakage (d), H₂DCFDA staining (e), and H₂O₂ content (f) assays in N. benthamiana leaves expressing PdCDIE1 upon DMTU treatment. Bars (in e), 50 μm. g Phenotypes of TRV:NbHsp70-, TRV:PDS-, TRV:00 (empty vector)-treated N. benthamiana leaves at 3 weeks post agroinfiltration. h, i Electrolyte leakage (h) and H₂O₂ content (i) assays in TRV:00- (control) and TRV:NbHsp70-treated N. benthamiana leaves. j Agrobacterium-mediated transient expression of NbHsp70 or CsHsp70 suppressed PdCDIE1-triggered cell death in N. benthamiana. N. benthamiana leaves were infiltrated with A. tumefaciens carrying NbHsp70, CsHsp70, or GFP (control), followed 24 h later with A. tumefaciens carrying PdCDIE1. Photos were taken at 4 dpi. In a, c, e, g, and j, these experiments were repeated three times with similar results and representative photos were shown. In b, d, f, h, i, data were shown as mean ± SD of three biological replicates. Differences were assessed using two-tailed t-test (***, p < 0.001; **, p < 0.01; *, p < 0.05) in b, h, i, and different letters in d and f indicate significant differences at p < 0.05 according to one-way ANOVA.