Skip to main page content (VSports最新版本)
U.S. flag

An official website of the United States government

Dot gov

The VSports app下载. gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2019 Jan;31(1):189-209.
doi: 10.1105/tpc.18.00535. Epub 2018 Dec 18.

Iron- and Reactive Oxygen Species-Dependent Ferroptotic Cell Death in Rice- Magnaporthe oryzae Interactions

Sarmina Dangol  1 , Yafei Chen  1 , Byung Kook Hwang  2 , Nam-Soo Jwa  3
Affiliations

Iron- and Reactive Oxygen Species-Dependent Ferroptotic Cell Death in Rice- V体育ios版 - Magnaporthe oryzae Interactions

Sarmina Dangol et al. Plant Cell. 2019 Jan.

Abstract

Hypersensitive response (HR) cell death is the most effective plant immune response restricting fungal pathogen invasion. Here, we report that incompatible rice (Oryza sativa) Magnaporthe oryzae interactions induce iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death in rice cells. Ferric ions and ROS (i. e. , H2O2) accumulated in tissues undergoing HR cell death of rice leaf sheath tissues during avirulent M. oryzae infection. By contrast, iron did not accumulate in rice cells during virulent M. oryzae infection or treatment with the fungal elicitor chitin. Avirulent M. oryzae infection in ΔOs-nadp-me2-3 mutant rice did not trigger iron and ROS accumulation and suppressed HR cell death, suggesting that NADP-malic enzyme2 is required for ferroptotic cell death in rice. The small-molecule ferroptosis inhibitors deferoxamine, ferrostatin-1, and cytochalasin E and the NADPH oxidase inhibitor diphenyleneiodonium suppressed iron-dependent ROS accumulation and lipid peroxidation to completely attenuate HR cell death in rice sheaths during avirulent M VSports手机版. oryzae infection. By contrast, the small-molecule inducer erastin triggered iron-dependent ROS accumulation and glutathione depletion, which ultimately led to HR cell death in rice in response to virulent M. oryzae These combined results demonstrate that iron- and ROS-dependent signaling cascades are involved in the ferroptotic cell death pathway in rice to disrupt M. oryzae infection. .

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Images of the Accumulation of ROS and Ferric Ions (Fe3+) and HR Cell Death Response in Rice Leaf Sheaths in Compatible and Incompatible Rice-M. oryzae Interactions. (A) CM-H2DCFDA staining (GF) shows the accumulation of ROS (H2O2) in rice cells 30 h after inoculation with avirulent M. oryzae INA168. (B) Prussian blue staining (blue color) shows the accumulation of ferric ions (Fe3+) in rice cells 48 h after inoculation with avirulent M. oryzae INA168. (C) HR cell death response (dark brown color) 48 h after inoculation with avirulent M. oryzae INA168. Images of rice leaf sheath cells (cv HY) infected by M. oryzae PO6-6 (virulent) and INA168 (avirulent) strains were taken by a fluorescence microscope (Zeiss equipped with Axioplan 2) using a bright field (BF) as well as a combination of excitation (wavelengths, 450‒490 nm) and emission (515‒565 nm) GF filters. The images shown are representative of the different leaf sheath samples that were analyzed in three independent experiments. Bars = 20 µm.
Figure 2.
Figure 2.
Time-Course Images of the Accumulation of ROS and Ferric Ions (Fe3+) and HR Cell Death Response in Rice Leaf Sheaths during avirulent M. oryzae Infection. (A) and (B) CM-H2DCFDA (GF) (A) and DAB (dark brown color) (B) staining shows the accumulation of ROS (H2O2) in rice cells at different time points after inoculation with avirulent M. oryzae 007. (C) Prussian blue staining (blue color) shows the accumulation of ferric ions (Fe3+) in rice cells at different time points after inoculation with avirulent M. oryzae 007. (D) HR cell death responses (dark brown) 36 to 48 h after inoculation with avirulent M. oryzae 007. Images were taken by a fluorescence microscope (Zeiss equipped with Axioplan 2) using a bright field (BF) as well as a combination of excitation (wavelengths, 450‒490 nm) and emission (515‒565 nm) GF filters. The images shown are representative of the different leaf sheath samples that were analyzed in three independent experiments. Bars = 20 μm.
Figure 3.
Figure 3.
DFO Suppresses the Accumulation of Ferric Ions (Fe3+) and ROS and HR Cell Death in Incompatible Rice-M. oryzae:GFP Interaction. (A) DFO suppresses iron accumulation and HR cell death in rice. Rice leaf sheaths were treated with mock (water) and 3 mM DFO solutions 42 h after inoculation with M. oryzae:GFP. Prussian blue staining (blue color) shows the accumulation of ferric ions (Fe3+) in rice cells. GFP fluorescence shows successful colonization of M. oryzae:GFP IH in rice leaf sheath cells. The images shown are representative of the different leaf sheath samples that were analyzed in three independent experiments. Bars = 20 μm. (B) Quantification of infected cell phenotypes in leaf sheaths of mock (water)- and DFO-treated rice (cv HY) 48 h after inoculation with avirulent M. oryzae INA168:GFP. The results are presented as mean values ± sd; n = 4 leaf sheaths from different plants. Asterisks indicate statistically significant differences (Student’s t test, **, P < 0.01). Similar results were obtained in three independent experiments. (C) Quantification of ROS production in mock (water)- and DFO-treated rice leaf sheaths 48 h after inoculation with avirulent M. oryzae INA168:GFP. ROS was detected using a GloMax 96 Microplate Luminometer (Promega). Values are means ± sd of total relative luminescence units (RLU) (n = 10). Asterisks indicate statistically significant differences (Student’s t test, **, P < 0.01). The experiments were repeated three times with similar results. Images of untreated and DFO-treated rice sheath cells were taken by a fluorescence microscope (Zeiss equipped with Axioplan 2) using a bright field (BF) as well as a combination of excitation (wavelengths, 450‒490 nm) and emission (515‒565 nm) GF filters.
Figure 4.
Figure 4.
Fer-1 Suppresses Ferric Ion (Fe3+) and ROS Accumulation, Lipid Peroxidation, and HR Cell Death during Incompatible Rice-M. oryzae Interactions. (A) Avirulent M. oryzae INA168:GFP colonizes the leaf sheaths of the resistant rice (cv HY) treated with 10 μM Fer-1. GFP fluorescence shows M. oryzae INA168:GFP IH growing in Fer-1-treated rice sheath cells. The images shown are representative of the different leaf sheath samples that were analyzed in three independent experiments. Fer-1 treatment suppressed the HR cell death 48 h after inoculation with avirulent M. oryzae INA168:GFP. (B) Prussian blue staining (blue color) shows the accumulation of ferric ions (Fe3+) in the HR cell death response of rice leaf sheaths (cv DJ) infected with avirulent M. oryzae 007. The images shown are representative of the different leaf sheath samples that were analyzed in three independent experiments. Fer-1 treatment (10 μM) suppressed HR cell death 48 h after inoculation with avirulent M. oryzae 007. (C) Quantification of ROS production in rice leaf sheath tissues using a chemiluminescence assay. Fer-1 treatment (10 μM) suppressed ROS (H2O2) accumulation 48 h after inoculation with avirulent M. oryzae 007. Values are means ± sd of total relative luminescence units (RLU) (n = 10). (D) Determination of lipid peroxidation levels by MDA assay. Fer-1 treatment (10 μM) inhibited lipid peroxidation 48 h after inoculation with avirulent M. oryzae 007. Images of untreated and treated rice sheath cells were taken by a fluorescence microscope (Zeiss equipped with Axioplan 2) using a bright field (BF) as well as a combination of excitation (wavelengths, 450‒490 nm) and emission (515‒565 nm) GF filters. The results are presented as mean values ± sd; n = 4 leaf sheaths from different plants. Asterisks indicate statistically significant differences (Student’s t test, *, P < 0.05 and **, P < 0.01). The experiments were repeated three times with similar results. Mock, water treated. Bars = 20 μm.
Figure 5.
Figure 5.
The Actin Filament Inhibitor Cyt E Suppresses the Accumulation of ROS and Ferric Ions (Fe3+) in the Incompatible Rice-M. oryzae Interaction. CM-H2DCFDA (A), DAB (B), and Prussian blue (C) staining shows the effect of Cyt E (10 µg/mL) on the accumulation of ROS (H2O2) and ferric ions (Fe3+) in rice leaf sheaths (cv DJ) 30 and 48 h after inoculation with avirulent M. oryzae 007. Images were taken by a fluorescence microscope (Zeiss equipped with Axioplan 2) using a bright field (BF) as well as a combination of excitation (wavelengths, 450‒490 nm) and emission (515‒565 nm) GF filters. The images shown are representative of the different leaf sheath samples that were analyzed in three independent experiments. Bars = 20 μm.
Figure 6.
Figure 6.
The NADPH Oxidase Inhibitor DPI Suppresses ROS and Ferric Ion (Fe3+) Accumulation and HR Cell Death during Incompatible Rice-M. oryzae Interaction. (A) CM-H2DCFDA (GF), DAB, and Prussian blue (blue color) staining shows the effect of DPI treatment (5 μM) on ROS (H2O2) and ferric ion (Fe3+) accumulation and HR cell death in rice leaf sheath cells (cv DJ) infected with avirulent M. oryzae 007. The images shown are representative of the different leaf sheath samples that were analyzed in three independent experiments. Bars = 20 μm. (B) Quantification of ROS production in rice leaf sheath tissues of mock (water)- and DPI-treated rice (cv DJ) 48 h after inoculation with avirulent M. oryzae 007. ROS was detected using a GloMax 96 Microplate Luminometer (Promega). Values are means ± sd of total relative luminescence units (RLU; n = 10). Asterisks indicate statistically significant differences (Student’s t test, **, P < 0.01). (C) Quantification of infected cell phenotypes in leaf sheaths of mock (water)- and DPI-treated rice (cv DJ) 48 h after inoculation with avirulent M. oryzae 007. The results are presented as mean values ± sd; n = 4 leaf sheaths from different plants. Asterisks indicate statistically significant differences (Student’s t test, **, P < 0.01). Images were taken by a fluorescence microscope (Zeiss equipped with Axioplan 2) using a bright field (BF) as well as a combination of excitation (wavelengths, 450‒490 nm) and emission (515‒565 nm) GF filters. The experiments were repeated three times with similar results.
Figure 7.
Figure 7.
Rice NADP-ME2 Is Involved in the Accumulation of ROS and Ferric Ions (Fe3+) in HR Cell Death during Avirulent M. oryzae Infection. (A) and (B) CM-H2DCFDA (GF) (A) and Prussian blue (blue color) (B) staining shows the accumulation of H2O2 and ferric ion (Fe3+) around IH in wild-type rice (cv HY) during avirulent M. oryzae INA168 infection. By contrast, the focal accumulation of H2O2 and ferric ion (Fe3+) was not detected in ΔOs-nadp-me2-3 mutant rice. (C) Avirulent M. oryzae INA168:GFP induces HR cell death in wild-type rice but successfully colonizes ΔOs-nadp-me2-3 mutant rice. Images in (A) and (C) were taken by a fluorescence microscope (Zeiss equipped with Axioplan 2) using a bright field (BF) as well as a combination of excitation (wavelengths, 450‒490 nm) and emission (515‒565 nm) GF filters. Images in (B) were taken by a fluorescence microscope using a bright field. The images shown are representative of the different leaf sheath samples that were analyzed in three independent experiments. Bars = 20 μm.
Figure 8.
Figure 8.
Erastin Triggers the Accumulation of ROS and Ferric Ions (Fe3+), Glutathione Depletion, and HR Cell Death in Compatible Rice-M. oryzae Interaction. Treatment with 10 µM erastin added to conidial suspensions induces HR cell death in susceptible rice sheath cells (cv HY) 48 h after inoculation with virulent M. oryzae PO6-6. (A) Effects of erastin on the focal accumulation of ROS (H2O2) and ferric ion (Fe3+) and HR cell death during virulent M. oryzae PO6-6 infection. CM-H2DCFDA (GF) and Prussian blue (blue color) staining shows the accumulation of H2O2 and ferric ion (Fe3+) in rice cells. The images shown are representative of the different leaf sheath samples that were analyzed in three independent experiments. Bars = 20 μm. (B) and (C) Quantification of HR cell death and ROS production in mock (water)- and erastin-treated rice leaf sheaths infected with virulent M. oryzae PO6-6. ROS production was detected using a GloMax 96 Microplate Luminometer (Promega). Values are means ± sd of total relative luminescence units (RLU) (n = 10). (D) Quantification of erastin effects on glutathione levels in rice leaf sheaths infected with virulent M. oryzae PO6-6. GSH and total glutathione (GSH+GSSG) contents were measured at 412 nm using a WKSP-2000UV Smart Plus UV/VIS spectrophotometer (Woongki Science). FW, fresh weight. Images were taken by a fluorescence microscope (Zeiss equipped with Axioplan 2) using a bright field (BF) as well as a combination of excitation (wavelengths, 450‒490 nm) and emission (515‒565 nm) GF filters. The results in (B) and (D) are presented as mean values ± sd; n = 4 leaf sheaths from different plants. Asterisks indicate statistically significant differences (Student’s t test, *, P < 0.05 and **, P < 0.01). The experiments were repeated three times with similar results.
Figure 9.
Figure 9.
Erastin Triggers the Accumulation of ROS and Ferric Ions (Fe3+) and HR Cell Death in ΔOs-nadp-me2-3 Mutant Rice in Compatible Rice-M. oryzae Interaction. (A) Effects of erastin on focal ROS (H2O2) and ferric ion (Fe3+) accumulation and HR cell death during virulent M. oryzae PO6-6 infection. CM-H2DCFDA (GF) and Prussian blue (blue color) staining shows the accumulation of H2O2 and ferric ion (Fe3+) in leaf sheath cells 48 h after inoculation with the conidial suspension/erastin, respectively. The images shown are representative of the different leaf sheath samples that were analyzed in three independent experiments. Bars = 20 μm. (B) Quantification of HR cell death in mock (water)- and erastin-treated rice leaf sheaths infected with virulent M. oryzae PO6-6. The results are presented as mean values ± sd; n = 4 leaf sheaths from different plants. (C) Quantification of ROS production in mock (water)- and erastin-treated rice leaf sheaths infected with virulent M. oryzae PO6-6. ROS production was detected using a GloMax 96 Microplate Luminometer (Promega). Values are means ± sd of total relative luminescence units (RLU) (n = 10). Images were taken by a fluorescence microscope (Zeiss equipped with Axioplan 2) using a bright field (BF) as well as a combination of excitation (wavelengths, 450‒490 nm) and emission (515‒565 nm) GF filters. Asterisks indicate statistically significant differences (Student’s t test, *, P < 0.05 and **, P < 0.01). The experiments were repeated three times with similar results.
Figure 10.
Figure 10.
The Fungal Elicitor Chitin Triggers the Accumulation of ROS, but Not Ferric Ions (Fe3+), in Rice Leaf Sheath Cells. The virulent M. oryzae PO6-6 and avirulent M. oryzae 007 (4 × 105 conidia/mL) were inoculated onto rice leaf sheaths (cv DJ). Chitin (10 µM) was applied onto rice leaf sheaths, followed by incubation for 48 h in the dark. (A) and (B) CM-H2DCFDA (GF) (A) and DAB (dark brown color) (B) staining shows the accumulation of ROS (H2O2) in rice leaf sheath cells. (C) Prussian blue (blue color) staining shows the accumulation of ferric ion (Fe3+) in rice leaf sheath cells. (D) Quantification of ROS production in rice leaf sheath cells 48 h after inoculation with M. oryzae or treatment with mock (water) and chitin (10 µM). ROS production was detected using a GloMax 96 Microplate Luminometer (Promega). Values are means ± sd of total relative luminescence units (RLU) (n = 10). Different letters above the bars indicate significantly different means (P < 0.05), as analyzed by Fisher’s protected LSD test. Images were taken by a fluorescence microscope (Zeiss equipped with Axioplan 2) using a bright field (BF) as well as a combination of excitation (wavelengths, 450‒490 nm) and emission (515‒565 nm) GF filters. The images shown are representative of the different leaf sheath samples that were analyzed in three independent experiments. Bars = 20 μm.
Figure 11.
Figure 11.
Proposed Model of Iron- and ROS-Dependent Ferroptotic Cell Death in Rice-M. oryzae Interactions. NADP-ME supplies NADPH as an electron (e–) donor to NADPH oxidase (Rboh). NADP-MEs and Rbohs are required for robust ROS generation. Aquaporin channels mediate H2O2 transport across biological membranes. The highly reactive Fe2+ present in the cell reacts with H2O2 to produce Fe3+ and hydroxyl radicals (∙OH). The small-molecule inhibitors DFO, Cyt E, Fer-1, and DPI are in red. The small-molecule inducer erastin is in blue. Erastin inhibits cystine uptake by the cystine antiporter (system Xc–) to induce glutathione depletion and possible GPX4 inactivation, leading to overwhelming lipid peroxidation that ultimately causes iron- and ROS-dependent ferroptotic cell death. SOD, superoxide dismutase.
None
See this image and copyright information in PMC

VSports app下载 - Comment in

"VSports" References

    1. Airaki M., Sánchez-Moreno L., Leterrier M., Barroso J.B., Palma J.M., Corpas F.J. (2011). Detection and quantification of S-nitrosoglutathione (GSNO) in pepper (Capsicum annuum L.) plant organs by LC-ES/MS. Plant Cell Physiol. 52: 2006–2015. - "VSports最新版本" PubMed
    1. An Q., Hückelhoven R., Kogel K.H., van Bel A.J. (2006). Multivesicular bodies participate in a cell wall-associated defence response in barley leaves attacked by the pathogenic powdery mildew fungus. Cell. Microbiol. 8: 1009–1019. - "VSports手机版" PubMed
    1. Apostol I., Heinstein P.F., Low P.S. (1989). Rapid stimulation of an oxidative burst during elicitation of cultured plant cells: Role in defense and signal transduction. Plant Physiol. 90: 109–116. - PMC - PubMed
    1. Bienert G.P., Chaumont F. (2014). Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide. Biochim. Biophys. Acta 1840: 1596–1604. - PubMed
    1. Blanch M., Rosales R., Goya L., Sanchez-Ballesta M.T., Escribano M.I., Merodio C. (2013). NADP-malic enzyme and glutathione reductase contribute to glutathione regeneration in Fragaria vesca fruit treated with protective high CO2 concentrations. Postharvest Biol. Technol. 86: 431–436.

"V体育官网" MeSH terms

LinkOut - more resources