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. 2021 Aug 2;12:710794. doi: 10.3389/fpls.2021.710794

FIGURE 9.

FIGURE 9

OsMPK1 overexpression induces ROS and ferric Ion (Fe3+) accumulation, lipid peroxidation and cell death in rice leaf sheaths during virulent Magnaporthe oryzae PO6-6 infection. Leaf sheaths of the susceptible wild-type (WT) cultivar NB and OsMPK1-overexpressed (35S:OsMPK1) plants were inoculated with the conidial suspension (4 × 105 conidia/mL) of virulent M. oryzae PO6-6 and then treated with 10 μM Fer-1. (A) Microscopic images of cell death and ROS and ferric Ion (Fe3+) accumulation in rice sheath cells untreated or treated with 10 μM Fer-1 at 48 hpi. ROS accumulation in the infected leaf sheath epidermal cells was detected by CM-H2DCFDA (green fluorescence) and DAB (dark brown color) staining. Prussian blue (blue color) staining shows ferric ion accumulation in rice cells. The images are representatives of different leaf sheath samples from three independent experiments. Scale bars = 20 μm. (B) Quantification of cell death (CD) and invasive hyphae (IH) in rice sheath cells at 48 hpi. (C) Quantification of Prussian blue-stained cells. Prussian blue-stained cells were divided into two phenotypes: Type I, cells that contain invasive hyphae (IH) but are weakly or not Prussian blue-stained; and Type II, strongly Prussian blue-stained with only a few poor hyphae. (D) DAB-stained cell phenotypes. DAB-stained cells were divided into two phenotypes: Type I, cells that contain invasive hyphae (IH) but are weakly or not DAB-stained; and Type II, strongly DAB-stained cells with only a few poor hyphae. (E) Quantification of ROS accumulation. ROS accumulation was monitored using a GloMax® 96 Microplate Luminometer (Promega). Values are means ± SD of total relative luminescent units (RLU) (n = 10). (F) Determination of lipid peroxidation by MDA (malondialdehyde) assay. Images were taken using a fluorescence microscope (Zeiss equipped with Axioplan 2) with bright field and green fluorescence (GF) filters. Experiments were repeated three times with similar results. Results are presented as mean values ± SD; n = 4 leaf sheaths from different plants. Different letters above the bars indicate significantly different means (P < 0.05) as analyzed by Fisher’s protected LSD test. Fer-1, ferrostatin-1, hpi, hours post-inoculation.