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

"VSports注册入口" Avirulent M. oryzae Induces ROS and Fe³⁺ Accumulation and HR Cell Death in Rice

The rice blast disease and HR cell death responses caused by M. oryzae include some cellular infection processes in rice plants (Howard et al. , 1991; Howard and Valent, 1996; Kankanala et al. , 2007). Upon coming into contact with the rice plant surface, M. oryzae conidia germinate and form dome-shaped appressoria that develop at the end of germ tubes (12 h post inoculation [hpi]). The appressoria generate enormous turgor pressure to penetrate into host cells (Howard and Valent, 1996). The penetrated specialized hypha, called a penetration peg, develops a filamentous primary hypha that differentiates into bulbous IH at the infection sites in rice cells (24 hpi; Heath et al. , 1990; Kankanala et al. , 2007) VSports手机版. During the infection processes (24 to 48 hpi), various interactive reactions between rice and M. oryzae result in either disease or HR cell death in rice plants (Figures 1 and 2).

An early response of plants against pathogen attack is characterized by the accumulation of ROS (H₂O₂) and ferric ions (Fe³⁺) at infection sites (Apostol et al., 1989; Liu et al., 2007). We investigated whether H₂O₂ and Fe³⁺ are involved in HR cell death during the defense response of rice plants to M. oryzae (Figures 1 and 2). In the compatible interaction of rice (cv Hwayeong [HY]) with virulent M. oryzae PO6-6, primary hyphae grew from the appressorium, differentiated into thicker, bulbous IH in the invaded rice cell, and spread into neighboring cells (Figure 1). Virulent (compatible) M. oryzae PO6-6 grew well with plentiful IH inside rice leaf sheath epidermal cells during infection. However, avirulent (incompatible) M. oryzae INA168 and 007 grew poorly, with only a few IH leading to the HR cell death in rice (cv HY and Dongjin [DJ]) leaf sheath cells, respectively (Figures 1 and 2). In this study, 5-(and-6) chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H₂DCFDA) and 3,3′-diaminobenzidine (DAB) staining methods were used to determine the localization and accumulation of H₂O₂ in M. oryzae-infected rice leaf sheaths (Figures 1 and 2). The ROS-sensitive dye CM-H₂DCFDA produces a highly fluorescent product, 2′,7′-dichlorofluorescein, in the presence of ROS in living plant cells (Gomes et al., 2005); however, DAB reacts with H₂O₂ in the presence of peroxidase to form a reddish-brown polymer (Thordal-Christensen et al., 1997). CM-H₂DCFDA staining (green fluorescence [GF]) revealed significant ROS (H₂O₂) accumulation inside and around IH as well as inside the invaded and neighboring cells of rice leaf sheaths (cv HY) at 30 hpi with avirulent M. oryzae INA168 (Figure 1A). By contrast, H₂O₂ accumulation was not detected in the rice sheath cells infected by virulent M. oryzae PO6-6 in compatible M. oryzae interactions with rice.

To identify the role of Fe³⁺ in HR cell death during avirulent M. oryzae infection, we stained rice leaf sheath tissues with Prussian blue solution to detect Fe³⁺ (Figure 1B). The Prussian blue staining method is one of the most sensitive histochemical tests to detect Fe³⁺ in tissues (Liu et al., 2007). Avirulent M. oryzae INA168, but not virulent M. oryzae PO6-6, induced Fe³⁺ accumulation inside and around the IH in rice sheath cells (cv HY) at 48 h after inoculation (Figure 1B). Fe³⁺ was observed (blue color) inside IH and at hyphal invasion sites in the HR cell death response of rice leaf sheath cells where ROS accumulated. HR cell death appeared with cellular aggregates (dark brown color) including vesicles inside avirulent M. oryzae-infected sheath cells at 48 h after inoculation (Figure 1C). These results indicate that H₂O₂ and Fe³⁺ accumulated in HR cell death in response to IH in rice cells infected by avirulent rice blast fungus. Vesicles are known to contain ROS, phytoalexins, or phenolics involved in defense signaling and antimicrobial functions (Lamb and Dixon, 1997; Hückelhoven et al., 1999; An et al., 2006; Jwa and Hwang, 2017). Vesicle trafficking may increase the abundance of NADPH oxidase, a ROS-generating enzyme, at the plasma membrane in rice cells (Jwa and Hwang, 2017).

We further investigated a time course change in ROS (H₂O₂) and Fe³⁺ accumulation and the HR cell death response in rice cells during infection in the incompatible interactions of rice (cv DJ) with avirulent M. oryzae 007 (Figure 2). The ROS-sensitive CM-H₂DCFDA fluorescent signals showed the localization and spread of active ROS accumulation inside and around the living IH of avirulent M. oryzae 007 at 12 to 36 hpi; however, active ROS did not accumulate inside the HR cell but were distributed evenly throughout the neighboring rice cells at 48 hpi (Figure 2A). In addition, DAB staining showed that ROS accumulated gradually inside and around IH during infection for 48 h. Notably, the ROS burst peaked at 48 hpi and generated a typical HR cell death (Figures 2B and 2D). As detected by the Prussian blue staining (Figure 2C), Fe³⁺ accumulation was induced distinctly inside and around the IH in rice sheath cells during avirulent M. oryzae 007 infection for 36 to 48 h. Avirulent M. oryzae 007 infection started to induce HR cell death with cellular aggregates (dark brown color) 36 h after inoculation, which subsequently resulted in the severe HR death response in the rice cell, full of dark brownish vesicles 48 h after inoculation (Figure 2D). Collectively, these results indicate that avirulent M. oryzae INA168 and 007 induced ROS and Fe³⁺ accumulation in the HR cell death response in the leaf sheaths of resistant rice cv HY and DJ (Figures 1 and 2). Thus, the iron-dependent ferroptotic cell death likely requires continued iron-dependent ROS formation over an extended time period of infection to trigger HR cell death in incompatible rice-M. oryzae interactions.

DFO Is an Iron Chelator that Suppresses Fe³⁺ and ROS Accumulation and HR Cell Death

The iron chelator DFO (Dixon et al., 2012) was applied onto rice leaf sheaths (cv HY) 42 h after inoculation with virulent M. oryzae PO6-6:GFP (green fluorescent protein) and avirulent M. oryzae INA168:GFP. DFO treatment inhibited Fe³⁺ and ROS accumulation and HR cell death in rice leaf sheaths during avirulent M. oryzae INA168:GFP infection (Figure 3). Virulent M. oryzae PO6-6:GFP successfully colonized the leaves with IH in the leaf sheath epidermal cells; however, avirulent M. oryzae INA168:GFP induced Fe³⁺ accumulation and HR cell death in the infected rice sheath epidermal cells (Figure 3A). In the incompatible interaction, the fluorescence of GFP-tagged M. oryzae INA168 was not detected in the rice sheath epidermal cells. Treatment with DFO (3 mM) suppressed Fe³⁺ accumulation and the induction of HR cell death by the avirulent M. oryzae INA168:GFP and 007:GFP infection, which led to the successful colonization of IH in rice leaf sheath cells (Figure 3A; Supplemental Figure 1A). Increasing concentrations (1to 3 mM) of DFO drastically suppressed HR cell death in rice leaf sheaths (cv HY) infected with avirulent M. oryzae INA168 (Supplemental Figures 2A and 2B).

We quantified the infected cell phenotypes (IH and HR) and ROS production in mock (water)- and DFO-treated rice (cv HY) 48 h after inoculation with avirulent M. oryzae INA168:GFP (Figures 3B and 3C). DFO-treated rice leaf sheaths had more cells with IH but fewer HR cells than the mock (water)-treated leaf sheaths during avirulent M. oryzae INA168:GFP and 007:GFP infection (Figure 3B; Supplemental Figure 1B). Treatment with 3 mM DFO suppressed ROS production in rice sheath epidermal cells infected with avirulent M. oryzae INA168:GFP (Figure 3C). These combined data indicate that DFO suppresses the iron-dependent accumulation of ROS to restrict HR cell death in rice during avirulent M. oryzae infection.

Fer-1 Is a Potent Ferroptosis Inhibitor That Suppresses Fe³⁺ and ROS Accumulation, Lipid Peroxidation, and HR Cell Death

Treatment with the potent ferroptosis inhibitor Fer-1 (Dixon et al., 2012) suppressed Fe³⁺ and ROS accumulation, lipid peroxidation, and HR cell death, leading to an increase in the number of IH inside rice leaf sheath epidermal cells (cv HY and DJ) infected with the avirulent M. oryzae INA168:GFP and 007, respectively (Figure 4). Avirulent M. oryzae INA168:GFP and 007 successfully colonized the incompatible rice leaf sheath tissue pretreated with Fer-1 (Figures 4A and 4B). The fluorescence (green) of GFP-tagged M. oryzae INA168 IH was detected in rice leaf sheath cells treated with Fer-1 but not in mock-treated cells (Figure 4A). Treatment with high concentrations of Fer-1 (5–10 μM) triggered the formation of normal hyphal structures of M. oryzae 007 inside rice sheath cells (Supplemental Figure 3A).

Fer-1 treatment suppressed HR cell death in rice leaf sheath cells infected with avirulent M. oryzae (Figures 4A and 4B; Supplemental Figure 3B). The suppression of HR cell death in resistant (incompatible) rice sheaths was dependent on the concentration of pretreated Fer-1 (Supplemental Figure 3). Fer-1 treatment significantly inhibited ROS accumulation in leaf sheaf cells infected with avirulent rice blast (Figure 4C). We evaluated lipid peroxidation levels in mock- and Fer-1-treated rice leaf sheath cells during the avirulent M. oryzae 007 infection using the malondialdehyde (MDA) assay (Figure 4D). Avirulent M. oryzae 007 infection enhanced lipid peroxidation in rice leaf sheath cells that displayed HR cell death. Treatment with 10 μM Fer-1 suppressed lipid peroxidation in rice leaf sheaths infected with avirulent M. oryzae 007. These combined results indicate that Fe³⁺ and ROS accumulation and lipid peroxidation in rice are involved in the iron-and ROS-dependent ferroptotic cell death response in incompatible rice-M. oryzae interactions.

The Actin Microfilament Inhibitor Cyt E Suppresses ROS and Fe³⁺ Accumulation in HR Cell Death (V体育官网入口)

Cyt E is an inhibitor of actin microfilament polymerization in both animals and plants (Cooper, 1987; Yun et al., 2003; Shimada et al., 2006). We investigated whether Cyt E affects ROS (H₂O₂) and Fe³⁺ accumulation in rice leaf sheaths (cv DJ) during avirulent M. oryzae 007 infection. Tissues were stained with CM-H₂DCFDA and DAB for ROS detection and with Prussian blue for Fe³⁺ detection (Figure 5), and this revealed the accumulation of H₂O₂ and Fe³⁺ in avirulent M. oryzae-infected rice leaf sheath tissues at 30 and 48 h after inoculation.

CM-H₂DCFDA staining (GF) detects H₂O₂ inside living cells (Shin et al., 2005) and identified a focal H₂O₂ accumulation inside and around IH in rice leaf sheath cells 30 h after inoculation with avirulent M. oryzae 007 (Figure 5A). DAB staining showed that H₂O₂ accumulated inside the avirulent M. oryzae-infected necrotic rice cells to cause serious damage to the fungal IH at 48 h after inoculation (Figure 5B). Numerous vesicles containing H₂O₂ were observed inside rice cells including IH (Figure 5B), similar to those observed in HR cell death in plant-powdery mildew interactions (Hückelhoven et al., 1999; Collins et al., 2003). Plant NADPH oxidases (Respiratory burst oxidase homologs [Rbohs]) localize to the plasma membrane and endomembranes (Jwa and Hwang, 2017), and vesicle trafficking may increase Rboh abundance at the plasma membrane, ultimately leading to the ROS burst in rice cells.

Prussian blue staining showed that avirulent M. oryzae 007 induced the accumulation of Fe³⁺ inside and around IH in rice leaf sheath cells 48 h after inoculation (Figure 5C). However, pretreatment with 10 µg/mL Cyt E suppressed the M. oryzae 007-induced accumulation of H₂O₂ and Fe³⁺ inside and around IH in rice leaf sheath cells (Figures 5A to 5C). These results indicate that actin microfilament reorganization is involved in the focal accumulation of H₂O₂ and Fe³⁺ in rice cells during avirulent M. oryzae infection.

The NADPH Oxidase Inhibitor DPI Suppresses ROS and Fe³⁺ Accumulation and HR Cell Death

DPI preferentially inhibits the plasma membrane NADPH oxidase activity that is required to generate extracellular ROS in plant and mammalian cells (Li and Trush, 1998; Morré, 2002; Kadota et al., 2015). In this study, the NADPH oxidase inhibitor DPI was used to determine whether NADPH oxidases are involved in ROS and iron accumulation in incompatible rice-M. oryzae interactions (Figure 6). Avirulent M. oryzae 007 infection induced a strong accumulation of ROS (H₂O₂) and ferric ions (Fe³⁺) inside and around IH as well as HR cell death with cellular aggregates (dark brown color) in the invaded and neighboring cells of rice leaf sheaths (cv DJ). However, the DPI treatment (5 μM) distinctly inhibited H₂O₂ and Fe³⁺ accumulation and HR cell death, leading to the successful colonization of IH inside rice leaf sheath cells during avirulent M. oryzae 007 infection (Figure 6A). A chemiluminescence assay of ROS production shows that the DPI treatment significantly suppressed ROS production in rice sheath epidermal cells infected with avirulent M. oryzae 007 (Figure 6B). When observed using a microscope, DPI-treated rice leaf sheaths had more cells with IH but fewer HR death cells than did the mock (water)-treated leaf sheaths 48 h after inoculation with avirulent M. oryzae 007 (Figure 6C). These combined data suggest that a possible inhibition of NADPH oxidases by DPI suppresses the iron-dependent accumulation of ROS to restrict HR cell death in rice during avirulent M. oryzae infection.

"V体育2025版" Rice NADP-ME2 Is Required for ROS and Fe³⁺ Accumulation in HR Cell Death

In previous studies, we showed that rice NADP-MEs are required for robust ROS generation in incompatible rice-M. oryzae interactions (Singh et al., 2016; Jwa and Hwang, 2017). To investigate whether rice NADP-ME2 is involved in ferroptotic cell death, the accumulation of ROS and Fe³⁺ and the HR cell death response were detected histochemically in the leaf sheath cells of wild-type rice (cv HY) and ΔOs-nadp-me2-3 mutant rice during avirulent M. oryzae INA168 infection (Figures 7A and 7B). CM-H₂DCFDA (GF) and Prussian blue staining revealed that H₂O₂ and Fe³⁺ accumulated along with cellular aggregates inside and around IH in the infected leaf sheaths of wild-type rice 48 h after inoculation (Figures 7A and 7B). However, the focal accumulations of H₂O₂ and Fe³⁺ were not detected in leaf sheath cells of the ΔOs-nadp-me2-3 mutant rice (harboring the deletion of OsNADP-ME2) during infection. Avirulent M. oryzae INA168:GFP induced typically necrotic HR cell death in wild-type rice but successfully colonized (with extended IH) ΔOs-nadp-me2-3 mutant rice 48 h after inoculation (Figure 7C). GFP-tagged IH of M. oryzae INA168:GFP were visualized (green) in ΔOs-nadp-me2-3 mutant rice but not in wild-type rice. These combined results suggest that rice NADP-MEs have crucial roles in the accumulation of ROS and Fe³⁺ in the ferroptotic HR cell death response in rice cells.

Erastin Triggers ROS and Fe³⁺ Accumulation and Glutathione Depletion in HR Cell Death

Erastin is a small-molecule inducer that can trigger iron-dependent ferroptotic cell death in mammalian cells (Dixon et al., 2012). Inoculation with conidial suspensions of virulent M. oryzae PO6-6 that were supplemented with erastin induced ROS burst, Fe³⁺ accumulation, glutathione depletion, and HR cell death in susceptible (compatible) rice leaf sheath cells (cv HY) (Figure 8). Virulent M. oryzae PO6-6 causes disease but not HR cell death without erastin treatment. Combined with erastin treatment, virulent M. oryzae PO6-6 significantly induced HR cell death in rice leaf sheaths. Notably, CM-H₂DCFDA-specific fluorescence derived by ROS signals was clearly visible at living neighbor cells in the erastin-treated rice leaf sheath (Figure 8A). Prussian blue staining (blue color) showed Fe³⁺ accumulation inside and around IH in the infected leaf sheath cell treated with erastin 48 h after inoculation (Figure 8A). Erastin treatment significantly reduced the number of cells with IH, but increased the number of cells with HR, in rice leaf sheaths infected with virulent M. oryzae PO6-6 (Figure 8B). Treatment with erastin at higher concentrations (5–10 μM) also increased the number of HR cell deaths in susceptible rice leaf sheaths (Supplemental Figure 4). These results indicate that erastin triggers susceptible rice cells to express iron-dependent HR cell death responses against virulent M. oryzae PO6-6 at 48 h after inoculation.

A chemiluminescent assay with a luminometer showed that erastin treatment induced significant accumulation of H₂O₂ in rice leaf sheath cells at 48 h after inoculation with virulent M. oryzae PO6-6 (Figure 8C). To investigate the effect of erastin on glutathione depletion in rice, we monitored the glutathione levels in M. oryzae-infected rice leaf sheaths treated with erastin (Figure 8D). Glutathione (γ-l-glutamyl-l-cysteinylglycine) is one of the major soluble, low molecular weight antioxidants in plant cells (Airaki et al., 2011). The intracellular concentration of glutathione is an indicator of oxidative stress in cells (Pastore et al., 2001). Within cells, glutathione exists in reduced (GSH) and oxidized (GSSG) states. Erastin treatment inhibited the accumulation of GSH and total (GSH+GSSG) glutathione in rice leaf sheath cells at 48 h after inoculation with virulent M. oryzae PO6-6 (Figure 8D). This indicates that erastin triggered glutathione depletion by inhibiting the accumulation of both GSH and GSSG in M. oryzae-infected rice cells. Collectively, these results suggest that ROS and Fe³⁺ accumulation and glutathione depletion are required for erastin-induced HR cell death in rice-M. oryzae interactions.

Erastin Triggers ROS and Fe³⁺ Accumulation and HR Cell Death in ΔOs-nadp-me2-3 Mutant Rice

Erastin treatment coupled with virulent M. oryzae PO6-6 infection triggered the accumulation of ROS (H₂O₂) and Fe³⁺ in HR cell death in leaf sheath cells of both wild-type rice (cv HY) and ΔOs-nadp-me2-3 mutant rice (Figure 9; Supplemental Figure 5). CM-H₂DCFDA (GF) and Prussian blue staining showed the accumulation of H₂O₂ and Fe³⁺ in leaf sheath cells 48 h after inoculation with the M. oryzae conidial suspension/erastin, respectively (Figure 9A). Erastin treatment significantly induced HR cell death in ΔOs-nadp-me2-3 mutant rice compared with the mock-treated mutant rice during infection (Figure 9B). During M. oryzae PO6-6 infection, erastin treatment led to H₂O₂ accumulation in the ΔOs-nadp-me2-3 mutant, as detected with a luminometer (Figure 9C). Fe³⁺ accumulation and HR cell death were strongly induced in erastin-treated rice leaf sheaths of the ΔOs-nadp-me2-3 mutant 72 to 96 h after virulent M. oryzae PO6-6 infection (Supplemental Figure 5). These results suggest that erastin triggers iron- and ROS-dependent, but NADP-ME-independent, ferroptotic HR cell death in rice.

"VSports app下载" The Fungal Elicitor Chitin Triggers ROS Accumulation, but Not Fe³⁺ Accumulation, in Rice

To investigate whether the fungal elicitor chitin triggers iron- and ROS-dependent ferroptotic cell death in rice, we treated rice leaf sheaths with chitin (hexa-N-acetyl-chitohexaose; Figure 10; Supplemental Figure 6). Chitin is a primary component of cell walls in M. oryzae, and chitin fragments act as elicitors in rice (Kishimoto et al., 2010). Chitin oligosaccharides recognize the rice plasma membrane glycoprotein CEBiP, which contributes to disease resistance to the rice blast fungus M. oryzae (Kishimoto et al., 2010).

Treatment with 10 µM chitin induced a strong ROS (H₂O₂) generation in rice leaf sheath cells (cv DJ) (Figures 10A to 10C). CM-H₂DCFDA (GF) staining indicated that CM-H₂DCFDA-specific fluorescence derived by ROS was distributed uniformly throughout the cytoplasm in chitin-treated rice leaf sheath cells (Figure 10A). However, DAB staining revealed that H₂O₂ accumulation was much stronger in avirulent M. oryzae 007-infected rice leaf sheath cells than in the chitin-treated rice cells (Figure 10B). The chemiluminescent assay with a luminometer showed that chitin treatment induced ROS accumulation in rice leaf sheath cells as much as avirulent M. oryzae 007 infection (Figure 10D). Notably, chitin treatment produced 2 times more ROS in leaf sheath cells of wild-type rice (cv HY) than in those of ΔOs-nadp-me2-3 mutant rice (Supplemental Figures 6A and 6B).

Chitin did not trigger Fe³⁺ accumulation in rice leaf sheath cells (Figure 10C; Supplemental Figure 6A). By contrast, avirulent M. oryzae 007 infection triggered Fe³⁺ and H₂O₂ accumulation in rice leaf sheath cells (Figure 10), similar to that observed in other incompatible rice-M. oryzae interactions (Figures 1 and 2). Fe³⁺ accumulated inside and around IH in rice leaf sheath cells infected with avirulent M. oryzae 007 (Figure 10C). However, chitin treatments did not trigger Fe³⁺ accumulation in the rice cv DJ and HY and the ΔOs-nadp-me2-3 mutant rice (Figure 10C; Supplemental Figure 6A). These combined data indicate that the fungal elicitor chitin is involved in ROS-induced HR cell death signaling, but not in iron-dependent ferroptotic cell death, in rice.

Fe³⁺ and ROS Accumulation in HR Cell Death in Incompatible Rice-M. oryzae Interactions

Plant pathogens have evolved to overcome host ROS defense and obtain virulence and/or pathogenicity (Huang et al., 2011; Hemetsberger et al., 2012; Doehlemann et al., 2014; Jwa and Hwang, 2017). By contrast, avirulent plant pathogen infection induces strong ROS bursts leading to HR cell death in host plants (Lamb and Dixon, 1997; Torres, 2010; Jwa and Hwang, 2017). In this study, we found that ferric iron (Fe³⁺) and ROS are involved in HR cell death in resistant rice in response to the avirulent M. oryzae. Irrespective of resistant rice and avirulent M. oryzae genotypes, avirulent M. oryzae infection accumulates Fe³⁺ and ROS (H₂O₂) in HR cell death of resistant rice, suggesting that an iron-dependent ROS burst may mediate HR cell death in rice. In the presence of H₂O₂, highly reactive Fe²⁺ causes the Fenton reaction to produce Fe³⁺ with a highly toxic·OH (Fenton, 1894; Pierre and Fontecave, 1999). The resulting ·OH is destructive, oxidizing lipids, proteins, and DNA and disturbing normal cell function (Zaho et al., 1994; Henle and Linn, 1997; Yang et al., 2016). Fe³⁺ accumulation in infected areas of rice may enhance ROS toxicity to destroy the components of host cells and ultimately inhibit M. oryzae growth, as suggested previously (Zaho et al., 1994; Henle and Linn, 1997; Yang et al., 2016). A previous study showed that iron (Fe³⁺) accumulated at pathogen attack sites to mediate the ROS burst in epidermal cells of wheat (Triticum aestivum) leaves infected with powdery mildew (Liu et al., 2007).

DFO, Fer-1, and Cyt E Inhibit Iron- and ROS-Dependent Ferroptotic Cell Death

In mammals, intracellular iron reacts with lipid ROS in oncogenic RAS mutant cell lines to induce an iron-dependent form of nonapoptotic cell death called ferroptosis (Dixon et al., 2012; Stockwell et al., 2017). The iron-dependent ferroptotic cell death is characterized by the accumulation of lipid-based ROS such as lipid peroxides (Yang and Stockwell, 2016). Iron is required for the accumulation of lipid peroxides to execute ferroptotic cell death (Stockwell et al., 2017). Recently, Distéfano et al. (2017) demonstrated that heat stress induced ferroptosis-like cell death in plants.

The iron chelator DFO prevents iron-dependent lipid peroxidation in mammalian cells (Dixon et al., 2012; Stockwell et al., 2017). DFO treatment effectively inhibits HR cell death in rice, a hallmark of rice innate immunity against avirulent M. oryzae, which may enable rice cells to become susceptible to the blast fungus. DFO suppresses Fe³⁺ and ROS accumulation to completely suppress HR cell death of rice. Treatment with DFO at a high concentration (3 mM) did not inhibit M. oryzae growth in rice cells, suggesting that M. oryzae could not take up DFO-chelated iron from the host rice tissues to colonize them. The lipid ROS scavenger Fer-1 prevents the onset of ferroptosis by inhibiting the production of toxic lipid hydroperoxides by blocking lipid peroxidation in mammalian cells (Dixon et al., 2012; Stockwell et al., 2017). Fer-1 inhibits iron-dependent ROS accumulation, lipid peroxidation, and HR cell death, which subsequently increases the growth of IH inside rice cells during avirulent M. oryzae infection. These combined results suggest that iron and ROS are responsible for lipid peroxidation to trigger ferroptotic HR cell death.

Plant-pathogen interactions are associated with dynamic actin microfilament rearrangement at plant cell infection sites (Kobayashi et al., 1997; Opalski et al., 2005; Takemoto et al., 2006; Hückelhoven and Panstruga, 2011). Cyt E interferes with actin microfilament polymerization (Yun et al., 2003; Shimada et al., 2006) and inhibits Fe³⁺ and H₂O₂ accumulation inside and around IH of avirulent M. oryzae. It has been demonstrated that the actin microfilament disruptor cytochalasin A blocks Fe³⁺ accumulation at pathogen attack sites to mediate the ROS burst in response to powdery mildew in wheat leaves (Liu et al., 2007). In wheat leaf cells, cytosolic Fe³⁺ is transported to cell wall appositions in vesicle-like bodies guided by actin microfilament polymerization. These vesicles contain ROS (H₂O₂), which has antimicrobial activity in cell wall cross-linking and HR cell death signaling pathways (Lamb and Dixon, 1997). ROS-containing vesicles aggregate at the site of pathogen invasion in plant cells (Hückelhoven et al., 1999; Collins et al., 2003). Numerous cellular aggregates that may include vesicles containing H₂O₂ were observed in rice cells beneath IH of avirulent M. oryzae. Actin microfilaments have a crucial role in vesicle trafficking and multivesicular endosomes in plant cells (An et al., 2006).

Rice NADP-ME2 and NADPH Oxidase Are Involved in Fe³⁺ and ROS Accumulation in HR Cell Death

NADP-MEs catalyze the oxidative decarboxylation of l-malate to yield pyruvate, CO₂, and NADPH in the presence of Mg²⁺ (Figure 11; Blanch et al., 2013). They provide NADPH for the biosynthesis of defense-related compounds such as lignin and flavonoids (Drincovich et al., 2001). ROS generation by Rbohs (NADPH oxidases) requires a supply of electrons to plant cell membranes (Figure 11; Singh et al., 2016). NADPH oxidase contributes significantly to ROS accumulation in ferroptotic cell death (Dixon et al., 2012). Avirulent M. oryzae infection in ΔOs-nadp-me2-3 mutant rice (NADP-ME deletion mutant) does not induce Fe³⁺ and ROS accumulation, which inhibits HR cell death and immune responses at the early infection stage. The NADPH oxidase inhibitor DPI suppresses ROS and Fe³⁺ accumulation in HR cell death in avirulent M. oryzae-infected rice cells. Together, these results suggest that rice NADP-ME and NADPH oxidase have crucial roles in Fe³⁺ and ROS accumulation in ferroptosis-like HR cell death in rice cells.

Erastin Triggers Iron- and ROS-Dependent, but NADP-ME2-Independent, Ferroptotic Cell Death

In mammalian cells, the RAS-selective lethal small molecule erastin inhibits the intracellular entry of cystine from the cystine/Glu antiporter (system X_c^–), leading to glutathione depletion and the inactivation of GPX4, which ultimately results in lethal lipid peroxidation and cell death (Yang and Stockwell, 2016; Stockwell et al., 2017). Cystine is introduced into cells and converted to Cys, a substrate for the synthesis of glutathione. Cys is required for cell growth and to prevent cell death (Stockwell et al., 2017). However, cystine antiporters with the same role as system X_c^– have not been identified in plant cells. Glutathione, an important cellular antioxidant, is involved in the regulation of intracellular ROS homeostasis and is crucial for disease resistance in plants (Hwang et al., 1992; Parisy et al., 2007).

Erastin induced Fe³⁺ and ROS accumulation and inhibited the production of cellular glutathione, which ultimately resulted in iron- and ROS-dependent ferroptotic cell death in rice cells during virulent M. oryzae infection. Erastin may induce intracellular conditions such as iron and ROS accumulation and glutathione depletion that block plant cell colonization by virulent M. oryzae. The activation of electron supply to NADPH oxidases by NADP-MEs may induce an apoplastic ROS burst, leading to HR cell death (Singh et al., 2016). Erastin treatment of the ΔOs-nadp-me2-3 rice mutant restored Fe³⁺ and ROS accumulation, leading to HR cell death. However, the erastin-mediated induction of ferroptotic cell death likely does not require NADP-MEs to stimulate ROS accumulation. These combined results suggest that erastin triggers NADP-ME2-independent ferroptotic cell death in rice, similar to the interaction of rice with avirulent M. oryzae. However, how erastin triggers NADP-ME2-independent ferroptotic cell death in rice remains to be clarified.

The Fungal Elicitor Chitin Triggers ROS Burst without Fe³⁺ Accumulation in Rice

Chitin is a component of fungal cell walls, and chitin fragments act as pathogen-associated molecular patterns that trigger cell defense and cell death responses in many plants (Shibuya and Minami, 2001; Tanabe et al., 2006). Chitin oligosaccharides of blast fungal cell walls perceive OsCEBiP and trigger various defense responses such as ROS generation and defense-related gene expression, but not cell death, which ultimately contributes to basal resistance to M. oryzae infection in rice (Kishimoto et al., 2010). Chitin treatment induced focal H₂O₂ accumulation, but not Fe³⁺ accumulation and HR cell death, in rice leaf sheaths. Chitin oligosaccharides inside M. oryzae cell walls may trigger ROS burst, but not HR cell death, in rice, possibly due to the absence of Fe³⁺ accumulation. A focal ROS accumulation occurred around the avirulent M. oryzae IH; however, chitin treatment induced uniform ROS accumulation throughout rice cells. These combined results suggest that focal iron accumulation is required for HR cell death triggered by avirulent M. oryzae, which creates the physiological conditions for the Fenton reaction inside rice cells (Pierre and Fontecave, 1999).

Proposed Model of Iron- and ROS-Dependent Ferroptotic Cell Death in Rice-M. oryzae Interactions (V体育官网入口)

Based on the data presented in this study, we propose a working model of iron- and ROS-dependent signaling cascades in the ferroptotic HR cell death pathway in rice during interactions with the rice blast fungus M. oryzae (Figure 11). Fe³⁺ and ROS accumulate specifically in the HR cell death in rice leaf sheath cells during avirulent M. oryzae infection in differently incompatible rice-M. oryzae interactions. Iron does not accumulate in rice cells in response to infection with virulent M. oryzae or chitin treatment alone. NADP-ME coenzyme catalyzes the oxidative decarboxylation of l-malate and NADP⁺ and provides NADPH as an electron donor for plasma membrane-bound Rbohs (NADPH oxidases) that are required to generate extracellular ROS in rice cells (Figure 11; Bienert and Chaumont, 2014; Jwa and Hwang, 2017). The NADPH oxidase inhibitor DPI suppresses ROS and Fe³⁺accumulation in HR cell death in rice cells. Aquaporin channels mediate H₂O₂ transport inside the plant cell across plasma membranes (Jwa and Hwang, 2017). The coaccumulation of iron and ROS at pathogen attack sites suggests that highly reactive Fe²⁺ reacts with H₂O₂ to produce Fe³⁺ and hydroxyl radicals (Fenton, 1894; Pierre and Fontecave, 1999), which ultimately results in the iron-dependent accumulation of toxic lipid ROS by overwhelming lipid peroxidation, which causes cell death (Dixon et al., 2012; Stockwell et al., 2017).

The small-molecule ferroptosis inhibitors DFO, Cyt E, and Fer-1 suppress the accumulation of intracellular iron, which prevents M. oryzae growth in the HR cell death response in rice cells (Figure 11). The iron chelator DFO inhibits iron-dependent ROS accumulation and disturbs lipid peroxidation. Cyt E inhibits actin microfilament formation and interferes with iron transport into the infection site. Fer-1 suppresses the lipid peroxidation caused by the focal accumulation of iron and H₂O₂, subsequently significantly suppressing HR cell death. In mammalian cells, GSH is synthesized by Cys influx through the cystine transporter to eliminate lipid peroxides (Dixon et al., 2012; Stockwell et al., 2017). The RAS-selective lethal small-molecule inducer erastin inhibits cystine uptake by the cystine/Glu antiporter (system X_c^–) to induce glutathione depletion and GPX4 inactivation, ultimately leading to iron-dependent oxidative cell death (Dixon et al., 2012). However, the cystine transporter has not been identified in rice. Erastin treatment triggers iron-dependent ROS accumulation and glutathione depletion, which ultimately leads to HR cell death in rice to restrict the growth of virulent M. oryzae. These combined results suggest that the iron-dependent accumulation of lipid peroxides is required for HR cell death in rice during avirulent M. oryzae infection.

The strong ROS burst is a common signaling event occurring in HR cell death (Van Breusegem and Dat, 2006). Plant pathogens have undergone convergent evolution to suppress the plant immune system, including the ROS burst (Jwa and Hwang, 2017). By contrast, the recognition of nucleotide binding Leu-rich repeat receptors of host plants by specific pathogen effectors leads to robust resistance responses, such as the strong ROS burst and HR cell death (Jones and Dangl, 2006; Dangl et al., 2013; Han and Hwang, 2017). The experimental evidence of iron- and ROS-dependent ferroptotic cell death in incompatible rice-M. oryzae interactions may provide important clues to understand the fundamental functions of HR cell death in the plant immune system. The current hypothesis that plants share iron- and ROS-dependent ferroptotic cell death with mammals will improve our understanding of how HR cell death evolved in multicellular organisms.

Plant Materials and Growth Conditions

Seeds of rice (Oryza sativa) cv DJ and HY were provided by the National Institute of Crop Science, Jeonju, Korea (http://www.nics.go.kr). Seeds of the T-DNA insertion mutant rice, ΔOs-nadp-me2-3, were obtained from the Rice Functional Genomic Express Database managed by the Salk Institute (http://signal.salk.edu./cgi-bin/RiceGE; Jeon et al., 2000). This mutant is described in detail by Singh et al. (2016). Rice seeds were soaked in water under continuous light conditions (80 μmol photons m⁻² s⁻¹) for 5 d during seed germination. The germinated rice seeds were planted into pots containing Baroker soil for rice (Seoul Bio) and grown at 28°C and 60% relative humidity, using white fluorescent light (150 μmol photons m⁻² s⁻¹) with a 16-h photoperiod in growth chambers.

Fungal Cultures and Growth Conditions

The rice blast fungal strains, Magnaporthe oryzae INA168, 007, and PO6-6, were obtained from the Center for Fungal Genetic Resources, Seoul National University (http://genebank.snu.ac.kr). M. oryzae INA168 was avirulent (incompatible) to the rice cv HY. M. oryzae 007 was avirulent to the rice cv DJ. By contrast, M. oryzae PO6-6 was virulent (compatible) to both rice cv HY and DJ. All M. oryzae strains were stored at –20°C and cultured on rice bran agar medium (Singh et al., 2016). M. oryzae INA168 and INA168:GFP (Singh et al., 2016) were grown under continuous light conditions (80 μmol photons m⁻² s⁻¹) for 12 d at 25°C for conidial production, whereas M. oryzae 007 and PO6-6 were grown in the dark for 2 weeks at 25°C. M. oryzae cultures were sporulated by removing all aerial mycelia from the culture plates, followed by incubation under continuous fluorescent light (80 μmol photons m⁻² s⁻¹) for 2 to 3 d at 25°C.

Fungal Transformation

The GFP-tagged M. oryzae INA168, 007, and PO6-6, resistant to hygromycin B, were generated by polyethylene glycol-mediated protoplast transformation as described previously (Singh et al., 2016). Briefly, M. oryzae mycelia were cultured in broth medium (1 g of Suc, 0.6 g of yeast extract, and 0.6 g of casamino acid in 100 mL) for 3 d and then incubated with 5 mg/mL Lysing Enzymes (Sigma-Aldrich, L1412) for 3 to 4 h using a shaker. The binary vector pSK1044 used for the transformation contains the hygromycin B phosphotransferase gene as a selection marker and the enhanced GFP gene for labeling transformants (Park et al., 2013). Ten micrograms of plasmid pSK1044 was transformed into freshly harvested M. oryzae protoplasts (1 × 10⁷/mL) as described previously (Park et al., 2013; Singh et al., 2016). Positive (hygromycin-resistant) transformants were detected on TB3 agar medium supplemented with hygromycin B (20 g of sucrose, 0.3 g of yeast extract, 0.3 g of casamino acid, 1 g of glucose, 0.8 g of agar, and 0.2 mg/mL hygromycin B in 100 mL) and confirmed by PCR using hygromycin-specific primers.

V体育官网 - Fungal Inoculation and Evaluation of Infection

We used the rice leaf sheath-M. oryzae inoculation method as described previously (Kankanala et al., 2007; Singh et al., 2016). Conidia of GFP-tagged M. oryzae INA168, 007, and PO6-6 were harvested from the sporulated culture plates using Milli-Q water with 0.025% (v/v) Tween 20 (Sigma-Aldrich). The conidial suspensions were adjusted to 4 × 10⁵ conidia/mL using a hemacytometer. Intermediate-aged leaf sheaths from 4- or 5-week-old rice seedlings were cut into 5- to 7-cm lengths and inoculated with freshly prepared conidial suspensions of M. oryzae strains. Inoculated rice leaf sheaths were incubated in a moistened box with 100% relative humidity under dark conditions at 25°C for 24 to 48 h.

The middle thin epidermal layers excised from the inoculated rice leaf sheaths were cut into 1.5-cm lengths and fixed on glass slides to be used for microscopy analyses, as described previously by Kankanala et al. (2007). Using the light and fluorescence microscopes, all infected sheath epidermal cells were observed from each of the rice leaf sheath tissues inoculated with GFP-tagged M. oryzae strains. The infected cells were counted and divided into two infection phenotypes: cells with viable IH and cells with HR cell death. Counting was performed with three to five replicates from one of the three independent experiments.

Treatment with DFO, Fer-1, Cyt E, DPI, and Erastin

The inhibition of oxidative stress, such as ROS burst and HR in rice leaf sheath cells, was investigated by treatment with DFO and Fer-1. DFO and Fer-1 were purchased from Sigma-Aldrich. DFO treatment was performed as described previously with slight modification (Liu et al., 2007). The rice leaf sheaths infected with GFP-tagged M. oryzae strains were incubated in water (mock) and 3 mM DFO for 6 h at 42 h after inoculation. For Fer-1 treatment, rice leaf sheaths infected with GFP-tagged M. oryzae strains were incubated in the dark for 24 h at 25°C and vacuum infiltrated in water (mock) and 10 µM Fer-1 solution for 10 min, followed by incubation in the same solution for 24 h.

For Cyt E (Cayman Chemical) treatment, the rice leaf sheaths (5–7 cm in length) were inoculated with a conidial suspension (4 × 10⁵ conidia/mL) of avirulent M. oryzae 007, followed by incubation for 24 h in the dark at 25°C. The infected rice leaf sheaths were incubated in water (mock) and 10 µg/mL Cyt E solution for 24 h at room temperature. For DPI (Sigma-Aldrich) treatment, the rice leaf sheaths (5–7 cm in length) were inoculated with a conidial suspension (4 × 10⁵ conidia/mL) of avirulent M. oryzae 007 in water (mock) and DPI (5 µM) solution, followed by incubation in the dark at 25°C for 48 h (Chi et al., 2009).

For erastin (Sigma-Aldrich) treatment, conidia of virulent GFP-tagged M. oryzae PO6-6 were freshly harvested from sporulated fungal culture plates. The harvested M. oryzae conidia (4 × 10⁵ conidia/mL) were placed in 10 µM erastin solution (Skouta et al., 2014) and inoculated on rice leaf sheaths, followed by incubation in the dark at 25°C for 48 h.

After incubation in DFO, Fer-1, Cyt E, DPI, and erastin solutions, the middle thin epidermal layers of the infected rice leaf sheaths were mounted on microscope slides and observed with a fluorescence microscope (Zeiss equipped with Axioplan 2).

Treatment with the Fungal Elicitor Chitin

The fungal elicitor chitin (hexa-N-acetyl-chitohexaose; Santa Cruz Biotechnology) was used to treat rice leaf sheaths, which were then incubated in a moistened box at 25°C for 48 h in the dark. The chlorophyll-free upper epidermal layers were isolated from the infected rice sheaths, followed by CM-H₂DCFDA and Prussian blue staining.

CM-H₂DCFDA Assay and DAB Staining for ROS Detection

The localization of ROS in the rice leaf sheath tissue was visualized using the CM-H₂DCFDA assay and DAB staining. The ROS-sensitive dye CM-H₂DCFDA was used to monitor ROS localization in living plant cells (Kristiansen et al., 2009). CM-H₂DCFDA staining was performed as described previously with slight modification (Shin et al., 2005). Briefly, thin epidermal layers of rice leaf sheaths inoculated with M. oryzae strains were excised and immersed in Milli-Q water for 5 min at room temperature to minimize wound-induced ROS production, as described previously by Smith and Heese (2014). Their incubation in 2 µM CM-H₂DCFDA (Molecular Probes Life Technologies) in 1× PBS was then performed in the dark for 30 min on a horizontal shaker. The rice leaf sheath samples were then washed twice with 1× PBS for 5 min in the dark. ROS localization inside leaf sheath cells was observed with a fluorescence microscope (Zeiss equipped with Axioplan 2).

DAB staining was performed according to the method of Rustérucci et al. (2001) with slight modification. M. oryzae-inoculated rice leaf sheath cells were vacuum infiltrated with 1 mg/mL DAB (Sigma-Aldrich) for 5 min, followed by destaining with a mixture of ethanol:acetic acid:glycerol (3:1:1, v/v/v) overnight. ROS localization was observed with a microscope.

ROS Quantification

ROS production in rice leaf sheath tissues was measured using the chemiluminescence assay as described previously with minor modification (Singh et al., 2016). The thin epidermal layer of rice sheaths infected with virulent and avirulent M. oryzae strains was cut into small pieces (0.5 cm length), followed by treatment with DFO, Fer-1, DPI, or erastin. Each treated leaf sheath sample was incubated with 30 μL of luminol (Bio-Rad), 1 μL of horseradish peroxidase (Jackson Immunoresearch), and 69 μL of Milli-Q water on 96-well plates for 5 min, followed by ROS detection using a GloMax 96 Microplate Luminometer (Promega).

Prussian Blue Staining for Fe³⁺ Detection

Histochemical staining of Fe³⁺ was performed as described previously with slight modification (Liu et al., 2007). Prussian blue reaction involves treating rice leaf sheath tissues with acid solutions of ferrocyanides. Thin epidermal layers of rice sheaths inoculated with M. oryzae strains were incubated in 7% (w/v) potassium ferrocyanide and 2% (v/v) hydrochloric acid (1:1, v/v) for 15 h at room temperature. Fe³⁺ inside the leaf sheath epidermal cells combines with the ferrocyanides, which results in the formation of bright blue pigments called Prussian blue, or ferric ferrocyanides.

Lipid Peroxidation Assay (VSports在线直播)

Lipid peroxidation was determined by quantifying MDA, a product of unsaturated fatty acid peroxidation present in the leaf sheath sample, by its reaction with thiobarbituric acid as described previously with slight modification (Zhang et al., 2009). Avirulent M. oryzae 007 was inoculated on the leaf sheaths, followed by Fer-1 treatment. The leaf sheath tissues were harvested and ground using liquid nitrogen. The fine tissue powder then was mixed with the reaction solution with 0.5% (w/v) thiobarbituric acid, 20% (v/v) trichloroacetic acid, and 0. 25 mL of 175 mM NaCl in a total of 2 mL of 50 mM Tris-Cl, pH 8.0. The mixture was incubated in boiling water for 5 min, cooled in an ice bath for 5 min, and centrifuged at 14,000g for 5 min at 4°C. The absorbances of the resultant supernatant were measured at 450, 532, and 600 nm. MDA concentration (C) was calculated according to the equation C = 6.45 (OD₅₃₂ − OD₆₀₀) − (0.56 OD₄₅₀), where OD₅₃₂ is the OD at 532 nm.

VSports app下载 - Measuring Glutathione Content

Glutathione, which is present in GSH and GSSG states in rice leaf sheath tissues, was assayed spectrophotometrically. The freshly harvested conidial suspension (4 × 10⁵ conidia/mL) of virulent M. oryzae PO6-6, which was supplemented or not with 10 µM erastin, was inoculated on rice leaf sheaths followed by incubation in a moistened box at 25°C in the dark for 48 h. The leaf sheaths were sampled and ground using a prechilled mortar and pestle. The ground sheath tissue sample was first weighed and made an equal volume and then mixed with 5% (w/v) meta-phosphoric acid (Sigma-Aldrich). The homogenates were centrifuged at 21,000g for 20 min at 4°C. The supernatants were collected and filtered through 0.45-µm nylon filters (Sigma-Aldrich) and used immediately to quantify glutathione content as described previously (Airaki et al., 2011).

To measure the GSH content in rice leaf sheath tissues, the rice sheath extract sample (10 µL) was added to 600 μL of reaction buffer (100 mM sodium phosphate buffer, pH 7.5, and 1 mM EDTA), 40 μL of 0.4% (w/v) 5,5′-dithiobis (2-nitrobenzoic acid) (Ellman’s reagent; Sigma-Aldrich), and 350 μL of Milli-Q water to a final volume of 1000 µL, followed by incubation at room temperature for 5 min. Total glutathione (GSH+GSSG) content in rice leaf sheath tissues also was measured in 1 mL of assay mixture containing 600 μL of the reaction buffer, 40 μL of 0.4% (w/v) 5,5′-dithiobis (2-nitrobenzoic acid), 50 μL of 0.4% (v/v) NADPH, 1 μL of 0.5 units of glutathione reductase, and Milli-Q water, followed by incubation for 5 min at room temperature. The absorbance of glutathione in the reaction mixture was measured at 412 nm using a UV-visible spectrophotometer (Woongki Science; Salbitani et al., 2017). The calibration curve of glutathione concentrations was prepared using 0 to 25 µM standard glutathione (GSH; Sigma-Aldrich) solutions.

Microscopy

The microscopy imaging was done using a fluorescence microscope (Zeiss equipped with Axioplan 2). The images were captured by 40× oil-immersion objective lenses using a bright field. Both CM-H₂DCFDA-specific and IH:GFP fluorescence signals were visualized with the Zeiss fluorescence microscope using a combination of excitation (wavelengths, 450‒490 nm) and emission (515‒565 nm) GF filters.

Statistical Analysis of Data

Three to 10 independent biological replicates were sampled for each determination. Each biological replicate consisted of an independent leaf sheath sample that was taken from a different rice plant. The number of biological replicates is given in the figure legends. The experiments were repeated independently three times. Statistical analyses of the data obtained were performed using Prism 7 software (GraphPad). The data are represented as means ± sd. Statistical significances were analyzed by Student’s t test or Fisher’s LSD test.

Accession Numbers

Sequence data from this article can be found at the Rice Genome Project website (http://rice.plantbiology.msu.edu/) and the GenBank/EMBL data libraries under accession number LOC_Os01g52500.3 (Os-NADP-ME2-3).

Supplemental Data

• Supplemental Figure 1. DFO suppresses HR cell death in incompatible rice DJ-M. oryzae 007:GFP Interactions.

• Supplemental Figure 2. DFO concentration-dependent suppression of HR cell death in incompatible rice HY-M. oryzae INA168 interactions.

• V体育官网 - Supplemental Figure 3. Fer-1 concentration-dependent suppression of HR cell death in incompatible rice DJ-M. oryzae 007 interactions.

• Supplemental Figure 4. Erastin concentration-dependent induction of HR cell death in compatible rice DJ-M. oryzae PO6-6 interactions.

• Supplemental Figure 5. Erastin treatment induces ferric ion (Fe³⁺) accumulation and HR cell death in leaf sheath cells of wild-type (cv HY) and ΔOs-nadp-me2-3 mutant rice during virulent M. oryzae infection.

• Supplemental Figure 6. The fungal elicitor chitin induces ROS accumulation, but not ferric ion accumulation, in leaf sheath cells of wild-type (cv HY) and ΔOs-nadp-me2-3 mutant rice.