Mitogen-Activated Protein Kinase Kinase OsMEK2 Positively Regulates Ca²⁺ Influx and Ferroptotic Cell Death during Rice Immune Responses

Abstract

Mitogen-activated protein (MAP) kinase (MAPK) signaling pathway is important in plant immune responses, involved in iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death mediated by Ca²⁺. High Ca²⁺ influx triggered iron-dependent ROS accumulation, lipid peroxidation, and subsequent hypersensitive response (HR) cell death in rice (Oryza sativa). Apoplastic Ca²⁺ chelation by EGTA during avirulent Magnaporthe oryzae infection altered Ca²⁺, ROS, and Fe²⁺ accumulation, increasing rice susceptibility to infection. By contrast, acibenzolar-S-methyl (ASM), a plant defense activator, significantly enhanced Ca²⁺ influx, and H₂O₂ accumulation, triggering rice ferroptotic cell death during virulent Magnaporthe oryzae infection. Here, we report a novel role of the MAPK signaling pathway in regulating cytoplasmic Ca²⁺ increase during ferroptotic cell death in rice immunity, using the ΔOsmek2 knockout mutant rice. The knockout of rice OsMEK2 impaired the ROS accumulation, lipid peroxidation, and iron accumulation during avirulent M. oryzae infection. This study has shown that OsMEK2 could positively regulate iron- and ROS-dependent ferroptotic cell death in rice by modulating the expression of OsNADP-ME, OsRBOHB, OsPLC, and OsCNGC. This modulation indicates a possible mechanism for how OsMEK2 participates in Ca²⁺ regulation in rice ferroptotic cell death, suggesting its broader role in plant immune responses in response to M. oryzae infection.

1. Introduction

Plants have evolved numerous defense mechanisms to protect themselves from pathogen attacks [1]. The defense response starts with the successful recognition of pathogens. Pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) to activate pattern-triggered immunity (PTI), and intracellular or extracellular receptors which detect effectors from the pathogens to activate effector-triggered immunity (ETI) [2,3]. Mitogen-activated protein (MAP) kinase (MAPK) signaling pathway is one of the major signal pathways involved in intracellular events, such as developmental changes and immune responses. Activation of MAPKs by PRRs induces a series of defense responses, including expression of pathogenesis-related (PR) genes, and plant cell death during plant immune responses [4,5]. One well-studied Arabidopsis MAPK cascade in plant immunity is the MEKK1-MKK1/2-MPK4 cascade [6]. MEKK1 interacts with MKK1/2 on the plasma membrane, and MKK1/2 interacts with MPK4 on both the plasma membrane and the nucleus, indicating that the MEKK1-MKK1/2-MPK4 cascade transmits the signal from the PRR to the nucleus [6]. MKK1 and MKK2 function redundantly with MPK4 and MEKK1 to regulate plant immunity by modulating ROS accumulation and plant cell death [6]. Similarly, the Pseudomonas syringae effector HopAI1 is an effector that can directly target Arabidopsis MPK3 and MAPK6 to inactivate MAPKs to promote pathogenesis [7]. The MAPK cascade plays an essential role in plant innate immunity, involved in both PTI and ETI.

Plant MAPK signaling proceeds through a kinase cascade: MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK, or MEK), and MAP kinase (MAPK) [8]. One structural non-canonical MAPKK, MPKK10.2, was found to enhance rice resistance against Xanthomonas oryzae pv. oryzicola infection and drought stress, by phosphorylating MPK6 and MPK3, indicating a role of MAPKK in plant immunity [9]. We previously performed Y2H screening of the rice MAPK interactors and found that OsMEK2 could interact with OsMPK1 and OsMPK6 [10]. This was confirmed by mapping the MAPK interactome network to underline the cellular and physiological responses of the MAPK signaling pathway in rice [11]. When signals are perceived on the plasma membrane by PRRs or NLRs, the MAPK cascade can be activated. MAP kinase kinases (MEKs) regulate transcriptional reprograming by activating MAP kinase (MAPK) [12]. Rice OsMEK2 interacts with OsMPK1, and then OsMPK1 interacts with the downstream transcription factor OsWRKY90 to activate plant immune responses [10,11,13].

The hypersensitive response (HR) in rice has been identified to share characteristics of animal ferroptotic cell death, including iron accumulation, GSH depletion, and lipid peroxidation during avirulent Magnaporthe oryzae (M. oryzae) infection [14,15]. Ca²⁺ channels and Ca²⁺ signals are well known to be extensively involved in plant immunity [16]; however, the discovery of the HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) resistosome has shown that the role of Ca²⁺ in plant immune responses may be more complex than previously thought [17]. The knockout of rice OsMEK2 impaired the ROS accumulation, lipid peroxidation, and iron accumulation during avirulent M. oryzae infection, indicating that OsMEK2 participates in rice ferroptosis during incompatible rice–M. oryzae interaction [13]. Further study has elucidated that OsMEK2 could positively regulate ROS- and iron-dependent ferroptotic cell death in rice, by modulating the expression of rice NADP-malic enzyme (OsNADP-ME), and respiratory burst oxidative homologs (OsRbohs), which are plant nicotinamide adenine dinucleotide phosphate (NADPH) oxidases [13]. In rice, Rbohs are localized in the plasma membrane with six transmembrane domains, playing an important role in ROS production during defense signaling [18].

In addition to ROS accumulation, recent studies have found that resistosome-mediated Ca²⁺ influx is an essential feature during plant HR cell death [19,20]. In contrast to the active research on the role of MAPKs in regulating ROS generation [21], MAPK studies on intracellular Ca²⁺ regulation are still limited. In this study, we investigated the role of OsMEK2 in association with Ca²⁺ influx during avirulent M. oryzae-induced hypersensitive cell death. We applied the Ca²⁺ chelator ethylene glycol-bis (2-aminoethylether)-N, N, N′, N′-tetra-acetic acid (EGTA), and systemic acquired resistance (SAR) inducer acibenzolar-S-methyl (ASM) to analyze the role of OsMEK2 in Ca²⁺ regulation during M. oryzae–rice interaction. In the present study, we further confirmed that the expression levels of OsNADP-ME and OsRBOHB, which are important for ROS production, were severely reduced in ΔOsmek2 rice during avirulent M. oryzae infection. Here, we suggest a possible mechanism for how OsMEK2 participates in Ca²⁺ regulation in rice ferroptotic cell death and immune responses.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Seeds of rice (Oryza sativa L.) cultivar Donjin (DJ) and Kitaake were obtained from the National Institute of Crop Science (http://www.nics.go.kr, accessed on 1 September 2010), and ΔOsmek2 mutant seeds were provided by the Rice Functional Genomic Express Database (RiceGE) managed by the Salk Institute (http://signal.salk.edu./cgi-bin/RiceGE, accessed on 8 March 2017). The plants were grown in Baroker soil (Seoul Bio, Seoul, Korea) in a growth chamber and the settings were as follows: 28 °C, 60% humidity, white fluorescent light (150 µmol photons m⁻² s⁻¹), and a 16 h day/8 h night photoperiod.

2.2. Fungal Cultures and Growth Conditions

Virulent M. oryzae strain PO6-6 and RO1-1 and avirulent strain 007 were obtained from the Center for Fungal Genetic Resources, Seoul National University, Seoul, Korea (http://genebank.snu.ac.kr, accessed on 5 January 2010). M. oryzae strains were grown on rice bran agar media (20 g rice bran, 20 g sucrose, and 20 g agar in 1 L water) in the dark at 25 °C for 10 days, followed by sporulation under continuous fluorescent light (80 µmol photons m⁻² s⁻¹) for another 3–4 days. Conidial suspensions were adjusted to 4 × 10⁵ spore mL⁻¹ in 0.025% (v/v) Tween 20 before use [14].

2.3. Fungal Inoculation and Infection Evolution

M. oryzae conidial suspensions were inoculated onto 6-week-old rice leaf sheaths, and the samples were kept in a dark moistened chamber, at 28 °C, for 48 h. The epidermal 2–3 cell layers were monitored and investigated under a microscope (Zeiss equipped with Axioplan 2, Bentonville, AR, USA). Infected epidermal cells were classified into three infection types: IH (invasive hyphae colonized inside the cell), type Ⅰ HR (severe hypersensitive response), and type Ⅱ HR (mild hypersensitive response). The experiment was independently repeated three times.

2.4. Treatment of Pharmacological Compounds

Ethylene glycol-bis (2-aminoethylether)-N, N, N′, N′-tetra-acetic acid (EGTA; Sigma-Aldrich, St. Louis, MO, USA) was applied during avirulent M. oryzae infection to chelate apoplastic Ca²⁺, and acibenzolar-S-methyl (ASM) was applied during virulent M. oryzae infection to enhance Ca²⁺ influx [15]. EGTA (3 mM) was treated to rice sheath cells, together with M. oryzae 007; ASM (500 µM) was treated to rice sheath cells, 2 h before virulent M. oryzae PO6-6 treatment.

2.5. Detection and Quantification of Ca²⁺ and H₂O₂

Fluo-5F AM is a Ca²⁺ indicator, which monitors intracellular Ca²⁺ [22]. Peroxy orange 1 (PO1) is an H₂O₂ probe, which emits orange intracellular fluorescence after incubation with H₂O₂ [23]. We applied Fluo-5F AM and PO1 to observe intracellular Ca²⁺ and H₂O₂, respectively. Briefly, the leaf sheath epidermal cells were incubated in 50 µM of Fluo-5F AM and 5 µM of PO1 at 37 °C for 1 h, followed by a three-time washing using 3DW. The samples were monitored and investigated under a fluorescence microscope (Zeiss equipped with Axioplan 2). The settings for filters were as follows: green fluorescence: Ex/Em: 450–490/515–565 nm, and red fluorescence: Ex/Em: 546/590 nm.

At least three regions of interest (ROIs) were chosen to quantify the green fluorescence intensities to monitor changes in Ca²⁺ levels at different time points using ImageJ software, https://imagej.net/ij/ (Classic, Wayne Rasband, Bethesda, MD, USA) [24]. To properly present the images which were corresponded with the generated plots, an image enhancement process was carried out when necessary. Especially, in figure in below, the brightness of the green field (GF) and red field (RF) images were enhanced by 50% to compare the changes in cytoplasmic Ca²⁺ and H₂O₂ accumulations at different time points. Corrected total cell fluorescence (CTCF) values were calculated as previously described [25,26]: CTCF = integrated fluorescence density − (ROI area × mean fluorescence of background readings). H₂O₂ levels were quantified by measuring red fluorescence intensities in a similar way using the ImageJ software (Classic, Wayne Rasband, Bethesda, MD, USA).

2.6. Prussian Blue Staining

Prussian blue staining was performed to visualize ferric ion (Fe³⁺) accumulation in rice leaf sheath cells infected with M. oryzae [14]. Epidermal layers of rice sheath cells were submerged in the Prussian blue solution (7% potassium ferrocyanide and 2% hydrochloric acid) overnight at room temperature (RT), and the stained samples were washed several times using 3DW to remove excess background. The samples were monitored and observed under a microscope (Zeiss equipped with Axioplan 2).

2.7. Glutathione Measurement

Total glutathione (oxidized form and reduced form) and reduced glutathione (GSH) were measured spectrophotometrically [14]. Avirulent M. oryzae 007 spores were inoculated onto rice leaf sheaths, and the samples were incubated at 25 °C for 48 h in the dark condition. The samples were ground in liquid nitrogen. An equal amount of the sample powder was mixed with 5% (w/v) meta-phosphoric acid (Sigma Aldrich) and the samples were centrifuged at 21,000× g for 20 min at 4 °C. The supernatants were used immediately to quantify glutathione contents [14]. Reduced glutathione (GSH) can be directly measured by observing OD₄₁₂ after reacting with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), and total glutathione (GSSG and GSH) can be measured by reducing GSSG to GSH first, and then measuring OD₄₁₂ after reacting with DTNB [27].

2.8. MDA Quantification

Malondialdehyde (MDA) is a product of unsaturated fatty acid peroxidation and can indicate the level of lipid peroxidation in rice leaf sheath samples, using thiobarbituric acid (TBA). Rice leaf sheaths were ground in liquid nitrogen, and the sample powder was mixed with the reaction solution (0.5% [w/v] TBA [Sigma-Aldrich], 20% [v/v] trichloroacetic acid [TCA; Sigma-Aldrich], and 0.25 mL of 175 mM NaCl in a total of 2 mL of 50 mM Tris-Cl [pH 8.0]). The samples were kept in a water bath (100 °C) for 5 min and the supernatant was collected by centrifuging at 14,000× g for 5 min at 4 °C. The absorbance was measured at 450, 532, and 600 nm with a spectrophotometer (Woongki Science, Seoul, Republic of Korea) [14]. MDA concentration was calculated based on the equation as follows [14].

$$C_{MDA}=\left[6.45\times \left({OD}_{532}-{OD}_{600}\right)\right]-\left(0.56\times {OD}_{450}\right)$$

where C_MDA is the concentration of MDA, and OD₄₅₀, OD₅₃₂, and OD₆₀₀ represent the optical density (OD) at 450, 532, and 600 nm, respectively.

2.9. RNA Extraction and Gene Expression Analysis

Total RNA was extracted from rice plants using the TRIzol Reagent (Invitrogen) and used for cDNA synthesis. Transcript levels of OsNADP-ME, OsRBOHB, OsPLC2, OsPLC4, OsCNGC2, OsCNGC13, and OsUbiquitin (OsUbi) genes were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (real-time qRT-PCR) (Supplementary Table S1). Transcript levels of genes were normalized relative to that of OsUbi and presented as mean ± standard deviation (SD) of three biological replicates.

3. Results

3.1. OsMEK2 Positively Regulates Cytoplasmic Ca²⁺ and H₂O₂ Levels during Avirulent M. oryzae Infection

OsMEK2 is required for ROS, iron accumulation, and lipid peroxidation to cause rice ferroptotic cell death [13]. We investigated cytoplasmic Ca²⁺ and H₂O₂ accumulation in rice DJ and ΔOsmek2 mutant rice during avirulent M. oryzae infection at different time points, and the results showed that cytoplasmic Ca²⁺ and H₂O₂ were highly accumulated in WT rice DJ during avirulent M. oryzae 007 infection at 36 hpi, compared to that in the ΔOsmek2 mutant rice (Figure 1). Changes in cytoplasmic Ca²⁺ and H₂O₂ were analyzed at 12, 24, 36, and 48 hpi in rice DJ and ΔOsmek2 mutant rice during avirulent M. oryzae infection (Figure 1A). In WT rice DJ, cytoplasmic Ca²⁺ and H₂O₂ accumulation started from 12–24 hpi and peaked at 36 hpi, however, cytoplasmic Ca²⁺ and H₂O₂ accumulation in ΔOsmek2 mutant rice was significantly impaired from 12–36 hpi (Figure 1). The ΔOsmek2 mutant rice showed no expression of the OsMEK2 (Supplementary Figure S1), and had a different disease phenotype during avirulent M. oryzae infection, compared to that in the WT rice DJ (Supplementary Figure S2). We classified the disease phenotypes into IH (invasive hyphae grew inside the rice cell), Type I HR (typical hypersensitive cell death with brown granules filled inside the infected rice cell), and Type II HR (mild hypersensitive cell death with light brown color inside the infected rice cell) (Supplementary Figure S2A). The ΔOsmek2 mutant rice showed a significantly higher HR cell death than WT rice DJ, with the majority of HR cell death being mild HR cell death (Supplementary Figure S2B). To investigate the effects of virulent M. oryzae on cytoplasmic Ca²⁺ and H₂O₂ accumulation, we inoculated virulent M. oryzae PO6-6 onto WT rice DJ and ΔOsmek2 mutant rice. Virulent M. oryzae infection induced minor cytoplasmic Ca²⁺ and H₂O₂ accumulation in both WT rice DJ and ΔOsmek2 mutant rice, with no significant difference between WT rice DJ and ΔOsmek2 mutant rice (figure in below).

3.2. EGTA Abrogates Ca²⁺, ROS Increase, Iron Accumulation, and HR Cell Death in WT and ΔOsmek2 Mutant Rice

During avirulent M. oryzae infection, cytoplasmic Ca²⁺ elevation, ROS accumulation, and iron accumulation induced rice ferroptotic cell death in WT rice DJ (Figure 2). Avirulent M. oryzae infection induced high levels of cytoplasmic Ca²⁺ elevation, and ROS and iron accumulation in rice leaf sheath cells, as visualized by the microscopy (Figure 2A). We compared cytoplasmic Ca²⁺, ROS accumulation, iron accumulation, and HR cell death in rice DJ and ΔOsmek2 mutant rice during avirulent M. oryzae inoculation and EGTA treatment to investigate whether Ca²⁺ influx is required for rice ferroptotic cell death. To determine the proper concentration of EGTA, we applied 1 and 3 mM EGTA to investigate the effects on HR cell death, based on our primary experimental data. It was shown that 3 mM EGTA greatly suppressed HR cell death, with a better effect on reducing HR cell death than 1 mM EGTA treatment (Supplementary Figure S3). During avirulent M. oryzae infection, WT rice DJ showed a typical HR cell death (48 hpi), featured by a high cytoplasmatic Ca²⁺ concentration (36 hpi), ROS accumulation (36 hpi), and iron accumulation (48 hpi) (Figure 2). However, the ΔOsmek2 mutant rice showed less HR cell death but more cell infections; especially, the HR cell death that occurred in ΔOsmek2 mutant rice was less dense, compared with that which occurred in the WT rice DJ (Figure 2). Also, less cytoplasmatic Ca²⁺ and less ROS accumulation (36 hpi) were detected in the ΔOsmek2 mutant rice. In addition, the ΔOsmek2 mutant rice had less iron accumulation compared to that in the WT rice DJ during avirulent M. oryzae infection (Figure 2). The treatment of the Ca²⁺ chelator EGTA blocked the Ca²⁺ influx, ROS accumulation, iron accumulation, and eventual cell death (Figure 2).

3.3. ASM Enhanced Ca²⁺, ROS Increase, Iron Accumulation, and HR Cell Death in WT and ΔOsmek2 Mutant Rice

During virulent M. oryzae infection, cytoplasmic Ca²⁺ and ROS levels were at baseline in WT rice DJ, with invasive hyphae growing inside the rice sheath cells, as visualized by the microscopy (Figure 3A). We compared cytoplasmic Ca²⁺, ROS accumulation, iron accumulation, and HR cell death in rice DJ and ΔOsmek2 mutant rice during virulent M. oryzae inoculation and ASM treatment to investigate whether Ca²⁺ influx is required for rice ferroptotic cell death. To determine the proper concentration of ASM, we applied 125 and 500 µM ASM to investigate the effects on HR cell death, based on our primary experimental data. Consequently, 500 µM ASM significantly increased HR cell death, with a better effect on inducing HR cell death than 125 µM ASM treatment (Supplementary Figure S4). During virulent M. oryzae infection, both WT rice DJ and the ΔOsmek2 mutant rice showed a susceptible phenotype, with fungal hyphae growing inside the rice epidermal cells (Figure 4A). The treatment of the SAR inducer ASM enhanced the Ca²⁺ influx (36 hpi), ROS accumulation (36 hpi), iron accumulation (48 hpi), and HR cell death (48 hpi) in both WT rice DJ and the ΔOsmek2 mutant rice (Figure 4). However, the ΔOsmek2 mutant rice showed less HR cell death after ASM treatment, compared to that in ASM-treated WT rice DJ (Figure 4B).

3.4. OsMEK2 Increased Glutathione Content and Impaired Lipid Peroxidation during Avirulent M. oryzae Infection

Rice ferroptotic cell death is featured with glutathione depletion and lipid peroxidation [14]. The glutathione content in the cell reflects the redox level of the cell and represents the ROS scavenging capacity of the cell [28]. To compare the glutathione content and ROS scavenging capacity in WT rice DJ and the ΔOsmek2 mutant rice during avirulent M. oryzae 007 infection, we investigated reduced glutathione (GSH) and total glutathione (GSSG + GSH), and the results showed that ΔOsmek2 mutant rice had higher GSH, as well as total glutathione content, representing a higher capacity for ROS scavenging (Figure 5A,B).

ROS accumulation always leads to oxidative stress that finally results in MDA formation, and the reaction of ROS and lipids can cause lipid peroxidation [29]. To compare the MDA content and the lipid peroxidation level in WT rice DJ and the ΔOsmek2 mutant rice during avirulent M. oryzae 007 infection, we measured the MDA content, and the results showed that the ΔOsmek2 mutant rice showed less MDA content, compared to WT rice DJ, suggesting a lower lipid peroxidation level (Figure 5C).

3.5. Defense-Related Genes Were Downregulated in the ΔOsmek2 Mutant Rice during Avirulent M. oryzae Infection

To analyze the ROS regulation and cellular oxidative condition, we analyzed the expression levels of OsNADP-ME and OsRBOHB at different time points after avirulent M. oryzae inoculation to rice sheath cells of WT and ΔOsmek2 mutant rice, and the results showed that the ΔOsmek2 mutant rice had lower expressions of OsNADP-ME and OsRBOHB, compared to that in the WT rice DJ. The lower expressions of OsNADP-ME and OsRBOHB corresponded to the lower level of cellular ROS in the ΔOsmek2 mutant rice (Figure 6).

We analyzed the expressions of OsPLC2 and OsPLC4, OsCNGC2 and OsCNGC13, and the results showed that the expressions of OsPLC2 and OsPLC4, OsCNGC2 and OsCNGC13 were significantly suppressed in ΔOsmek2 mutant rice during avirulent M. oryzae infection, compared to those in the WT rice DJ, suggesting that the external and internal Ca²⁺ release were less active in the ΔOsmek2 mutant rice, where less HR cell death occurred (Figure 6).

4. Discussion

4.1. OsMEK2 Regulated the Accumulation of Cytoplasmic Ca²⁺ and H₂O₂ Induced by Avirulent M. oryzae

Avirulent M. oryzae induces cytoplasmic Ca²⁺ increase and ROS accumulation to trigger immune responses [14,30]. Increased cytoplasmic Ca²⁺ can come from the apoplast or the internal stores [31]. Apoplastic Ca²⁺ acts as the main supplier of pathogen-induced cytoplasmic Ca²⁺ influx [32]. When plant cells were treated with elicitors, radioactive Ca²⁺ uptake from the apoplast could be observed, suggesting that apoplastic Ca²⁺ could be transported to the cytoplasm and participate in cellular activities during plant immunity [33,34]. The inhibition of Ca²⁺ influx compromised MAPK activation, suggesting that Ca²⁺ influx is necessary for the MAPK signaling pathway [35]. A sustained cytoplasmic Ca²⁺ elevation could activate sustained MAPK signaling, which could ultimately lead to HR cell death [36]. The constitutive expression of tobacco MEK2 or Arabidopsis MEK4/5 could induce H₂O₂ production, indicating the importance of the MAPK cascades during ROS production and plant immune responses [37]. However, MAPK cascades are not the sole signaling pathway to regulate plant HR cell death. This study investigated cytoplasmic Ca²⁺ and H₂O₂ accumulation in both WT rice DJ and the ΔOsmek2 mutant rice during avirulent M. oryzae infection. The ΔOsmek2 mutant rice showed impaired cytoplasmic Ca²⁺ and H₂O₂ accumulation (Figure 1), suggesting that OsMEK2 is required for cytoplasmic Ca²⁺ increase and ROS accumulation for inducing rice ferroptotic cell death. During plant immunity, there is extensive interplay between Ca²⁺ and ROS [38]. Ca²⁺ is extensively involved in plant immune responses for signaling transduction [39]. Recently, the discovery of resistosomes has suggested a potential new role for Ca²⁺ in plant HR cell death [19]. Specifically, the elevation of cytoplasmic Ca²⁺ has emerged as a determinant of cell death during the plant immune responses [19]. Cytoplasmic Ca²⁺ increase can be due to the influx from apoplast through cyclic-nucleotide-gated channels (CNGC) or from the internal stores through the phospholipase C (PLC) pathway [40,41]. It is worth investigating the Ca²⁺ and ROS homeostasis in rice DJ and ΔOsmek2 mutant rice to elucidate the interplay between Ca²⁺, ROS, and the MAPK signaling pathway during rice immune responses.

4.2. EGTA Blocked Ca²⁺ Influx, ROS Accumulation, Iron Accumulation, and HR Cell Death in WT and ΔOsmek2 Mutant Rice

Ethylene glycol-bis (2-aminoethylether)-N, N, N′, N′-tetra-acetic acid (EGTA) could effectively chelate Ca²⁺ influx in tobacco during the bacteria-induced hypersensitive response [42]. Moreover, EGTA also significantly decreased cell death induced by INF1, suggesting that Ca²⁺ is an essential component during elicitin-induced cell death [43]. The recent discovery of resisitosomes has highlighted the importance of apoplastic Ca²⁺ during plant ferroptotic cell death [44]. In this study, we used EGTA as an apoplastic Ca²⁺ chelator to investigate the role of apoplastic Ca²⁺ during avirulent M. oryzae-induced rice ferroptotic cell death. EGTA treatment completely blocked Ca²⁺ influx, ROS accumulation, iron accumulation, and HR cell death in both WT and ΔOsmek2 mutant rice (Figure 2), suggesting that the influx of apoplastic Ca²⁺ into the cytosol might be an early essential event to trigger downstream rice immune responses against M. oryzae infection. Rice cells infected with avirulent M. oryzae accumulate numerous dark brown granules during hypersensitive cell death response while simultaneously restricting fungal hyphae growth [14]. However, cheating the apoplastic Ca²⁺ using EGTA completely prohibited the accumulation of such granules, with the invasive hyphae extensively growing inside the rice cell (Figure 2A). Glutathione depletion, ROS accumulation, lipid peroxidation, and iron accumulation are marker events during rice ferroptotic cell death [14], and this study has suggested that Ca²⁺ increase might be an earlier essential event during rice hypersensitive response. EGTA could block Ca²⁺ influx from the apoplast to the cytoplasm, and this prevented the subsequent ROS accumulation and lipid peroxidation, suggesting that Ca²⁺ plays a vital role in ROS generation and downstream defense signaling.

4.3. ASM Enhanced Ca²⁺ Influx, ROS Accumulation, Iron Accumulation, and HR Cell Death in WT and ΔOsmek2 Mutant Rice

Acibenzolar-S-methyl (ASM) is one of the salicylic acid (SA) analogs that protects rice against various pathogens [45]. Pretreatment of ASM on apple seedlings could induce resistance against fire blight caused by Erwinia amylovora, by inducing the accumulation of defense-related enzymes such as peroxidases and β-1,3-glucanases [46]. In tomato seedlings, the application of ASM effectively reduced disease severity and induced plant resistance by increasing the activity of peroxidase (POX) and chitinase [47]. Cheng et al. [48] applied both ASM and MAKP inhibitor PD98059 to investigate the effects of ASM on the role of the MAPK cascade on apple resistance against Penicillium ex pansum. ASM treatment inhibited lesion expansion, promoted ROS accumulation, and regulated the expression of the genes involved in the MAPK cascade, including MdMAPK2, MdMAPK4, MdMAPKK1, and MdMAPK3 [48]. In this study, we investigated the effects of the plant activator ASM on rice ferroptotic cell death. ASM induced Ca²⁺ influx, ROS accumulation and iron accumulation, and the consequent HR cell death in WT and ΔOsmek2 mutant rice, indicating a role of ASM in cytoplasmic Ca²⁺ increase, and the subsequent immune responses during rice ferroptotic cell death (Figure 4). Surprisingly, ASM induced a significant increase in intracellular Ca²⁺ levels even in ΔOsmek2 mutant rice. Although ASM induced slightly less HR cell death in ΔOsmek2 mutant rice compared to WT rice DJ (Figure 4B), the maintenance of ferroptotic cell death in ΔOsmek2 mutant rice suggests the possibility that ASM primarily regulates Ca²⁺ through mechanisms other than MAPK signaling. To investigate whether ASM triggers external or internal Ca²⁺ influx to the cytosol, we used ASM/EGTA co-treatment. Compared to ASM treatment, ASM/EGTA co-treatment resulted in less cytoplasmic Ca²⁺ accumulation and HR cell death (Supplementary Figure S5). This suggests that ASM-induced cytoplasmic Ca²⁺ elevation may have primarily originated from the apoplastic Ca²⁺ influx. However, further research is still needed to elucidate the detail mechanism by which ASM mediates the increase in intracellular Ca²⁺ in rice cells.

4.4. OsMEK2 Regulated Glutathione Depletion and Impaired Lipid Peroxidation during Avirulent M. oryzae Infection

Glutathione acts as a crucial antioxidant to control reactive oxygen species (ROS) during plant immune responses [49]. GSH depletion could directly cause ROS accumulation due to its antioxidant function [50]. Glutathione depletion has been identified as a hallmark event during ferroptosis and other forms of cell death [51]. At normal conditions, around 90% of glutathione exists in the reduced form (GSH), and 10% exists in the oxidized form (GSSG) [52]. When the plants encounter biotic or abiotic stresses, cellular oxidative stress changes the ratio of glutathione into the oxidized form [52,53]. We compared the levels of reduced glutathione (GSH) and total glutathione (GSH + GSSG) in WT rice DJ and the ΔOsmek2 mutant rice during avirulent M. oryzae 007 infection, and the higher glutathione contents in the ΔOsmek2 mutant rice support the lower H₂O₂ levels and the reduced HR cell death (Figure 1 and Figure 2). Glutathione depletion during avirulent M. oryzae infection can induce H₂O₂ accumulation, which might lead to iron accumulation and lipid peroxidation, ultimately causing ferroptotic cell death [54].

Lipid peroxides (ROOH) are an important class of reactive oxygen species, and lipid peroxidation has been identified as a key downstream feature of ferroptotic cell death [55]. Lipids are essential for maintaining the integrity of cell membranes, and the peroxidation of lipids could alter the structure, properties, or dynamics of the cell membranes [56]. Malondialdehyde (MDA), a biomarker for lipid peroxidation in biological samples, is one well-described lipid peroxide degradation product, and the reaction of MDA with thiobarbituric acid could yield a product that can be detected by the spectrophotometer [14,57]. The MDA content in the ΔOsmek2 mutant rice was less compared to that in the WT rice DJ (Figure 5C), suggesting less peroxidized lipids of the cellular membrane.

4.5. Time-Course Expression of Defense-Related Genes in WT and ΔOsmek2 Mutant Rice during M. oryzae Infection

Respiratory burst oxidase homologue (RBOH), the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, could generate superoxide ions in a Ca²⁺-dependent manner, which are later converted into H₂O₂ by superoxide dismutase (SOD), inducing a subsequent Ca²⁺ influx and cytoplasmic Ca²⁺ elevation [36]. During pathogen infections, OsRBOHB plays a vital role in ROS burst and rice immune responses [58]. The NADPH conversation contributes to the acidification in the cytoplasm, modulating plant immune responses [59]. MAPK cascades regulate transcriptional reprograming via the WRKY transcription factors [60]. Our earlier research has discovered that OsMEK2 could interact with OsMPK1, which moves from the cytoplasm to the nucleus and interacts with the transcription factor OsWRKY90 [10,11,13]. The NADP-malic enzyme (NADP-ME) functions to catalyze the oxidative decarboxylation of malate under stress conditions [61]. In the current research, we found that the NADP-malic enzyme (OsNADP-ME) and NADP-oxidase respiratory burst oxidase homolog protein B (OsRBOHB) were downregulated in the ΔOsmek2 mutant rice during avirulent M. oryzae 007 infection (Figure 6), suggesting an impaired ability of the mutant rice for ROS accumulation. Cytoplasmic Ca²⁺ could activate the RBOHB, both directly and indirectly [62,63]. In the ΔOsmek2 mutant rice, less cytoplasmic Ca²⁺ was detected (Figure 1), which may lead to the downregulation of OsRBOHB (Figure 6). The phospholipase C (PLC) can cleave the membrane phospholipid phosphoatidylinositol-4,5-bisphopshate (PIP₂) to produce inositol trisphosphate (IP₃), which enables the Ca²⁺ release from the internal stores [41]. While the membrane-bound cyclic nucleotide-gated channels (CNGCs) facilitate Ca²⁺ influx to the cytoplasm from the apoplast, increasing the cytoplasmic Ca²⁺ concentration [40]. The current data showed that OsPLC2 and OsPLC4, and OsCNGC2 and OsCNGC13 were downregulated in the ΔOsmek2 mutant rice during avirulent M. oryzae 007 infection, suggesting an impaired ability of the mutant rice for cytoplasmic Ca²⁺ increase from both internal and external Ca²⁺ release (Figure 6). The qRT-PCR data suggest that OsMEK2 might contribute to rice ferroptotic cell death by regulating ROS- and Ca²⁺-related genes to modulate ROS accumulation and cytoplasmic Ca²⁺ elevation.

4.6. Model of OsMEK2-Mediated Rice Ferroptotic Cell Death and Plant Immune Response

Based on the previous publications and the current data, we proposed a model of the OsMEK2-mediated signaling pathway and its involvement in rice ferroptotic cell death (Figure 7). The mitogen-activated protein kinase (MAPK) pathway is activated through the recognition of the M. oryzae effector by PRRs. In the MAPK signaling cascade, OsMEK2 triggers the OsMPK1-OsWRKY90 pathway in the nucleus, upregulating OsNADP-ME, NADP-oxidase (OsRBOHB), phospholipase C (OsPLC), and cyclic nucleotide-gated channels (OsCNGC). NADP-ME converts L-malate to pyruvate, with NADP+ being converted to NADPH. NADPH gives electrons to RBOHB to promote ROS production. Ca²⁺ release from the internal stores and Ca²⁺ influx through the cell membrane are regulated by the PLC signaling pathway and the CNGC channels. The produced ROS and elevated cytoplasmic Ca²⁺ regulate the downstream immune responses, including iron accumulation and rice ferroptotic cell death. The recently discovered resistosomes are localized on the plasma membrane to facilitate apoplastic Ca²⁺ influx to mediate rice ferroptotic cell death, while EGTA can chelate apoplastic Ca²⁺ to prevent Ca²⁺ influx, as well as the subsequent ROS accumulation, iron accumulation, and the eventual cell death. The plant activator ASM used in this the study stimulates cytoplasmic Ca²⁺ increase and ROS accumulation through an unelucidated mechanism, contributing to ferroptotic cell death. Further research on the detail mechanism of ASM in the increase of intracellular Ca²⁺ is necessary to understand ferroptotic cell death and plant immune responses.