Figure 5.

Induction of cell death by transient expression of CaRING1 in pepper leaves. A, Cell death phenotypes in pepper leaves transiently expressing 35S:CaRING1 at 7 d after Agrobacterium-mediated transformation with the concentrations indicated (OD₆₀₀ = 1.0, 0.5, and 0.1). UV-fluorescing phenolic compounds associated with cell death are indicated in the same leaves. B, Site-directed CaRING1 mutations cause reduced cell death. Reduced-cell-death phenotypes are shown in pepper leaves infiltrated with Agrobacterium strains (OD₆₀₀ = 1.0) carrying the 35S:CaRING1C110S or 35S:CaRING1H131Y mutant. The marked regions on each leaf indicate the area infiltrated with Agrobacterium strains (OD₆₀₀ = 1.0). Photographs were taken 3 d after infiltration. UV-fluorescing phenolic compounds on the same leaf are shown in the right panels. C, Quantification of electrolyte leakage from pepper leaf discs transiently expressing empty vector control (35S:00), 35S:CaRING1, 35S:CaRING1C110S, or 35S:CaRING1H131Y at various time points after infiltration (OD₆₀₀ = 1.0). The data represent means ± sd from three independent experiments. D, Transient expression of empty vector control (35S:00) and 35S:CaRING1, 35S:CaRING1C110S, or 35S:CaRING1H131Y in pepper leaves 24 and 48 h after infiltration (OD₆₀₀ = 1.0). Gene expression was analyzed by quantitative RT-PCR. E, Immunoblot analysis of 35S:CaRING1 and 35S:CaRING1C110S expression in pepper leaves 24 and 48 h after infiltration with Agrobacterium carrying 35S:CaRING1:GFP, 35S:CaRING1C110S:GFP, or 35S:CaRING1H131Y:GFP. Total protein was extracted from mature leaves and used to detect GFP-tagged protein. Coomassie blue (CBB) staining confirmed equal protein loading. H, Healthy leaves.