The WT rice cultivars Dongjin (DJ) and Nipponbarre (NB) and the ΔOsmek2 knock-out, 35S:OsMEK2 and 35S:OsMPK1 overexpression lines were used in this study. ΔOsmek2 T-DNA insertion knock-out mutant seeds were provided by the Rice Functional Genomic Express Database (RiceGE) managed by the Salk Institute Genomic Analysis Laboratory¹ (Jeon et al., 2000). DJ and NB rice seeds were obtained from the National Institute of Crop Science, South Korea². Rice seeds were germinated in water for 5 days and then planted in plastic pots containing Baroker soil (Seoul Bio, South Korea). Rice plants were grown in growth chambers at 28°C under white fluorescent light (150 μmol photons m^–2 s^–1) with a 16 h photoperiod and 60% relative humidity, as described previously (Dangol et al., 2019).

The rice blast fungal strains Magnaporthe oryzae 007 and PO6-6 were provided by the Center for Fungal Genetic Resources, Seoul National University, Seoul, South Korea³. M. oryzae 007 was avirulent (incompatible) and M. oryzae PO6-6 was virulent (compatible) to the rice cultivar DJ. The rice cultivar NB was susceptible to M. oryzae PO6-6 infection. The fungal cultures were stored at –20°C and cultured on rice bran agar media (20 g rice bran, 20 g sucrose, and 20 g agar in 1 L Milli-Q water). M. oryzae strains were grown at 25°C in the dark for 2 weeks. M. oryzae sporulation was induced by removing aerial mycelia from the fungal culture plates, followed by their incubation under a continuous fluorescent light (80 μmol photons m^–2 s^–1) for 2–3 days at 25°C.

Conidial suspensions of M. oryzae strains were inoculated on rice leaves and leaf sheaths as described previously (Singh et al., 2016; Dangol et al., 2019). M. oryzae conidia were harvested from the sporulated culture plates using a 0.025% Tween 20 (Sigma-Aldrich) solution. The conidial concentration was adjusted to 4 × 10⁵ conidia mL^–1. The conidial suspension was spray-inoculated over the surface of 2-week-old rice seedlings. The inoculated seedlings were incubated at 25–28°C for 24 h under dark and moist conditions, and then were moved to normal conditions (16 h light/8 h dark). Disease phenotypes were observed at 5 days after inoculation and classified with respect to susceptible (large, elliptical, grayish, and expanded lesions) and resistant (slightly elongated, necrotic brownish spots) reactions.

Middle-aged leaf sheaths (5–7 cm lengths) of 4- or 5-week-old rice plants were inoculated with M. oryzae conidial suspensions (4 × 10⁵ conidia mL^–1). Inoculated leaf sheaths were incubated in a moistened box with 100% relative humidity at 25°C under dark conditions. After incubation for different times, epidermal layers were excised from the leaf sheaths, cut into 1.5 cm lengths, and fixed on glass microscope slides. The infected epidermal cells from each of three epidermal sheaths were observed under the microscope and divided into two infection phenotypes: cells with viable IH and cells with HR cell death. For the quantification of invasive hyphae (IH) and HR cell death, approximately 500 infected cells in each of the leaf sheaths were counted at least three times from one representative of three independent experiments.

ΔOsmek2 T-DNA insertion mutant seeds from RiceGE (Jeon et al., 2000) were screened by PCR using the left gene-specific primer (LP), the right gene-specific primer (RP), and the T-DNA right border primer (RB). The gene-specific primers are listed in Supplementary Table 1. To verify homozygosity in the knock-out mutant plants, PCR was performed with LP and RP primers of the gene, and homozygous plants were identified by the lack of specific PCR products. The LP and RB primers were used for PCR analysis to confirm the presence of the T-DNA insertion in ΔOsmek2 knock-out mutant plants. Quantitative real-time RT-PCR and immunoblotting analyses were performed to verify whether transcriptional and translational expression of OsMEK2 were blocked in ΔOsmek2 knock-out mutants.

The full length cDNAs of OsMEK2 and OsMPK1 were amplified from rice cDNA library and inserted into plant expression vector pCAMLA under the control of CaMV 35S promoter, followed by selection of hygromycin gene (hph). The constructed CaMV 35S:OsMEK2 and CaMV 35S:OsMPK1 were introduced in the rice cultivars DJ and NB, respectively, by Agrobacterium tumefaciens-mediated transformation, as described previously with slight modification (Hiei et al., 1994).

Briefly, 35S:OsMEK2 and 35S:OsMPK1 were delivered into rice calli using A. tumefaciens strain LBA4404 (Hoekema et al., 1983; Lee et al., 2005). The transformed calli were selected on the selection media containing gradually increasing concentrations of hygromycin (30 mg/L and 60 mg/L). After rooting and shooting, rice seedlings were transferred in water for 4 days. After adaption in water, rice seedlings were raised in soil in a growth chamber. The positive transformants from T₀ generation were selected by PCR using hygromycin primers. Next, seeds of T₁ generation were analyzed on the hygromycin-containing media and T₂ generation seeds were used for homozygote selection. The functional analysis was performed from T₃ generation. The levels of gene expression were determined by immunoblot analysis and qRT-PCR. The primers used for the experiments are listed in Supplementary Table 1.

Total RNA was isolated from rice tissue using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The gene expression levels were analyzed by reverse-transcription PCR (RT-PCR) and real-time quantitative PCR (qRT-PCR). First-strand cDNA was synthesized from 2 μg total RNA in 20 μL reaction mixture using a cDNA synthesis kit (Invitrogen) according to the manufacturer’s instructions. Prepared cDNA (1 μL) was used as a template for both RT-PCR and qRT-PCR. The qRT-PCR was performed using TOPreal^TM qPCR 2 × PreMIX (SYBR Green with low ROX; Enzynomics, Daejeon, South Korea) according to the manufacturer’s instructions. Relative gene expression levels were determined using rice 18S ribosomal RNA or rice ubiquitin as an internal standard gene. Gene-specific primers used for the real-time RT-PCR analysis are listed in Supplementary Table 1.

Rice proteins were extracted using trichloroacetic acid (TCA)/acetone extraction buffer [TCAAEB; 10% (w/v) trichloroacetic acid and 0.07% β-mercaptoethanol in 100 mL acetone] as described previously (Cho et al., 2006). Rice leaves were ground to a fine powder using liquid nitrogen in a mortar and pestle. Then, proteins were precipitated with TCAAEB and washed three times with wash buffer [0.07% β-mercaptoethanol, 2 mM ethylenediaminetetraacetic acid (EDTA), and EDTA-free protease inhibitor cocktail tablet (Roche) in a final volume of 100 mL acetone]. Protein precipitates were air-dried at room temperature, stored at –80°C for at least 24 h, and solubilized in lysis buffer containing thiourea and Tris (LB-TT) {7 M urea, 2 M thiourea, 4% (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-l-propanesulfonate (CHAPS), 18 mM Tris-HCI (pH 8.0), 14 mM Trizma base, two EDTA-free protease inhibitor cocktail tablets (Roche), 0.2% (v/v) Triton X-100, and 50 mM dithiothreitol (DTT) in a final volume of 100 mL}. After centrifuging at 15,000 × g for 15 min at 4°C, the supernatants were precipitated using pre-chilled acetone and solubilized in LB-TT buffer.

The OsMEK2 protein expression levels in DJ and ΔOsmek2 mutant plants were determined by 10% SDS-polyacrylamide gel electrophoresis (PAGE) followed by immunoblot analysis using rabbit polyclonal anti-MEK2 antibody (EnoGene^® E580135-A-SE). Immuno-reactive target bands were detected by Odyssey^® CLx Imaging System (LI-COR Biosciences). Equal gel loading was checked by Ponceau S staining.

The small molecule cell death inducer erastin was used to investigate whether erastin treatment triggered ferroptotic cell death in rice leaf sheath cells as described previously (Dangol et al., 2019). M. oryzae conidia (4 × 10⁵ conidia mL^–1) were mixed with 10 μM erastin (Sigma-Aldrich, St. Louis, MO, United States) and inoculated on leaf sheaths. The erastin-treated and M. oryzae-inoculated leaf sheath tissues were incubated in the dark at 25°C. The ferroptosis inhibitor, ferrostatin-1 (Fer-1, Sigma-Aldrich), was treated as described previously (Dangol et al., 2019). Rice epidermal layers were excised from the M. oryzae-infected leaf sheaths and then vacuum-infiltrated in 10 μM Fer-1 solution for 10 min, followed by their incubation for 24 h.

Cellular ROS (H₂O₂) localization in rice leaf sheath cells was visualized using 5- (and 6-) chloromethyl-2′,7′-dichlorofluorescin diacetate acetyl ester (CM-H₂DCFDA) and 3,3′-diaminobenzidine (DAB) staining methods as described previously (Shin et al., 2005; Dangol et al., 2019). Briefly, thin epidermal layers of rice leaf sheaths were excised and cut into equal pieces, followed by incubation in 1 mL water for 5 min to remove wound-induced ROS. Epidermal sheath samples were incubated in 2 μM CM-H₂DCFDA (Molecular Probes Life Technologies, Eugene, OH, United States) in 1 × phosphate-buffered saline (PBS) buffer in the dark for 30 min on a horizontal shaker. The incubated sheath samples were washed twice with 1 × PBS buffer for 5 min in the dark. ROS localization inside the epidermal sheath cells was observed immediately under a fluorescence microscope.

For DAB staining, epidermal layers of rice leaf sheaths were vacuum-infiltrated with 1 mg mL^–1 DAB (Sigma-Aldrich, St. Louis, MO, United States) solution for 5 min, followed by overnight destaining with ethanol:acetic acid:glycerol (3:1:1, v/v/v). ROS localization in the DAB-stained epidermal cells was observed under a microscope. The DAB-stained cells were categorized into two phenotypes: Type I, infected cells that display no or weak DAB staining; and Type II, infected cells that display strong DAB staining. DAB-stained 500 cells with different phenotypes were counted from each of infected sheaths. The counted cell numbers were then converted into the percentages of DAB-stained cells.

The chemiluminescence assay was used to measure ROS production in rice leaf sheaths as described previously (Singh et al., 2016; Dangol et al., 2019) with minor modifications. Epidermal layers of treated and M. oryzae-inoculated rice leaf sheaths were cut into small pieces (0.5 cm length) and incubated in 1 mL of sterilized Milli-Q water for 5 min to remove wound-induced ROS. Then, a piece of epidermal layer was added into a mixture of 30 μL luminol (Bio-Rad, Hercules, CA, United States), 1 μL horseradish peroxidase (Jackson Immunoresearch, West Grove, PA, United States), and 69 μL Milli-Q water in each well of a 96-well plate. Chemiluminescence (RLU, relative luminescent units) was detected from the ROS signals after 5 min incubation using a GloMax^® 96 Microplate Luminometer (Promega, Madison, WI, United States).

The malondialdehyde (MDA) assay was performed to determine lipid peroxidation in rice leaf sheath tissues as described previously (Zhang et al., 2009; Dangol et al., 2019). Lipid peroxidation is the degradation of lipids due to oxidative damage in plant cells. Briefly, the ground tissue powder of rice leaf sheath was mixed with the reaction solution [0.5% (w/v) thiobarbituric acid, 20% (v/v) trichloroacetic acid (TCA), and 0.25 mL 175 mM NaCl in 2 mL of 50 mM Tris-Cl, pH 8.0]. The mixed reaction was then incubated in boiling water for 5 min, cooled on ice for 5 min, and centrifuged at 14,000 × g for 5 min. The MDA concentration (C) in the resulting supernatant was determined by measuring supernatant absorbances (OD, optical density) at 450, 532, and 600 nm, and then calculating MDA concentration according to the following equation: C = 6.45 × (OD₅₃₂–OD₆₀₀) – (0.56 × OD₄₅₀).

Prussian blue staining was performed to detect ferric ion (Fe³⁺) in rice leaf sheaths as described previously (Liu et al., 2007; Dangol et al., 2019). Briefly, epidermal layers of rice leaf sheaths were excised and incubated in equal volumes (1:1, v/v) of 7% potassium ferrocyanide and 2% hydrochloric acid (HCl) for 15 h at room temperature. Prussian blue (ferric ferrocyanides, which combine with Fe³⁺ inside leaf sheath epidermal cells) was observed as a bright blue color under a fluorescence microscope. Prussian blue-stained cells were categorized into two phenotypes: Type I, cells that contain IH but are weakly or not Prussian blue-stained; and Type II, strongly Prussian blue-stained cells with only a few poor hyphae. Prussian blue-stained 500 cells with different phenotypes were counted from each of infected sheaths. The counted cell numbers were then converted into the percentages of Prussian blue-stained cells.

The full length cDNA of OsMEK2, OsMPK1, and OsWRKY90 were amplified from the rice cDNA library with the gene-specific primers containing attB1 and attB2 sites, as described in Supplementary Table 1. The amplified PCR products were used as a template for the second PCR using attB1 and attB2 primers. The second PCR products were sub-cloned into the pDONR^TM201 entry vector using BP clonase (Invitrogen) to create entry clones. The entry clones were recombined into the Gateway binary vector pGWB552 tagged with G3 green fluorescent protein (G3GFP) using LR clonase (Invitrogen) (Nakagawa et al., 2007; Dangol et al., 2017).

The binary plasmids containing OsMEK2, OsMPK1, and OsWRKY90 were transformed into A. tumefaciens GV3101. Recombinant agrobacteria were prepared for infiltration using a protocol as described previously with slight modification (Sainsbury and Lomonossoff, 2008). Briefly, single colonies of recombinant agrobacteria were incubated into the liquid LB media (10 g/L tryptone, 5 g/L yeast extract; 10 g/L NaCl, pH 7) containing spectinomycin (100 μg/L) for overnight at 28°C with continuous shaking. Harvested recombinant agrobacteria were resuspended to an OD₆₀₀ = 0.2 in MMA (10 mM MES pH 5.6, 10 mM MgCl₂, 150 μM acetosyringone). The agrobacterial suspension was incubated for 2 h at room temperature, and infiltrated into the abaxial leaves of 6-week-old Nicotiana benthamiana plants using a blunt tipped plastic syringe. Subcellular localization of 00:GFP, OsMEK2:GFP, OsMPK1:GFP, and OsWRKY90:GFP in N. benthamiana epidermal cells 36 h after agroinfiltration were microscopically observed following 4′,6-diamidino-2-phenylindole (DAPI, 5 μg/ml) staining for 10 min. Nuclear localization of the proteins was visualized by counterstaining the nuclei of the cells with DAPI.

Images were captured using a fluorescence microscope (Zeiss equipped with Axioplan 2; Campbell, CA, United States) with 40× oil-immersion objective lens. CM-H₂DCFDA-specific fluorescence was visualized under the fluorescence microscope using a combination of excitation (450–490 nm) and emission (515–565 nm) green fluorescence (GF) filters. Subcellular images were also taken using a fluorescence microscope (Olympus, Japan) using bright field, GF (Ex/Em: 488/498–548 nm), and DAPI (Ex/Em: 405/421–523 nm) filters.

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: OsMEK1 (Os01g32660), OsMEK2 (Os06g05520), OsMEK3 (Os03g12390), OsMEK4 (Os02g46760), OsMEK5 (Os06g09190), OsMEK6 (Os02g54600), OsMEK7 (Os06g09180), OsMEK8 (Os06g27890), OsMPK1 (Os06g06090), OsMPK6 (Os10g38950), OsWNK1 (Os07g38530), OsWRKY90 (Os09g30400), OsNADP-ME (Os01g52500), OsRbohB (Os01g25820), OsPR-1b (Os01g28450), OsPAL1 (Os04g43760), OsAPX1 (Os0.g17690), OsAPX2 (Os07g49400), OsUbiqutin (Os06g46770), 18S rRNA (XR_003238819.1), AtMKK1 (At4g26070), AtMKK2 (At4g29810), AtMEK3 (NP_198860), AtMEK4 (At1g51660), AtMEK5 (At3g21220), AtMKK6 (At5g56580), AtMKK9 (At1g73500), AtWNK9 (At3g04910), NtNPK2 (BAA06731), SlMKK1 (NP_001234744), SlMKK2 (NP_001234588), NbMEK2 (LOC107818847), and NtMEK2 (AF325168).

In our previous study, we isolated rice mitogen-activated protein kinase kinases (MAPKKs or MEKs) that interacted with OsMAPKs using yeast two-hybrid analysis (Singh et al., 2012). Amino acid sequences of the isolated rice MEKs were aligned with those of Arabidopsis AtMAPKKs, and subsequently categorized into Groups A–D (Ichimura et al., 2002) (Supplementary Figure 1). The plant MAPKKs contained 11 conserved subdomains (Supplementary Figure 2). All of the aligned MAPKKs contained the active site domain [D(I/L/V)KP] and the conserved motif (S/T-X₅-S/T, where X represents any amino acid residue) (Supplementary Figure 1A). The rice MAPKKs OsMEK1 and OsMEK2 belonged to Group A serine (S)/threonine (T) kinases. OsMEK1 amino acid sequence shared 54% and 53% homology with AtMKK1 and AtMKK2, respectively, whereas OsMEK2 had 62% sequence homology with both AtMKK1 and AtMKK2. A phylogenetic tree was generated to compare OsMEKs with Arabidopsis MAPKKs (Kumar et al., 2016) (Supplementary Figure 1B). OsMEK2 shares 65–66% homology with N. benthamiana NbMEK2 and tomato SlMKK1 (Supplementary Figure 2). OsMEK2 was phylogenetically close to NbMEK2, SlMKK1, AtMKK1 and AtMKK2 (Supplementary Figure 3). Based on the sequence alignment data of rice MAPKKs, OsMEK2 was selected to investigate whether rice MAPKKs are required for ferroptotic cell death signaling in this study.

OsMEK2 was knocked out in rice cultivar DJ by T-DNA insertion mutagenesis (Jeon et al., 2000). The OsMEK2 genomic DNA sequence contains nine exons and eight introns (Supplementary Figure 4). The T-DNA insertion mutant, ΔOsmek2, was identified in the intronic region between the sixth and seventh exons (Figure 1A). The genotypes of ΔOsmek2 (M5) progeny were analyzed with primer sets LP + RP and LP + LB to detect transgene and homo/hetero selection, respectively (Figure 1A). The ΔOsmek2 #2 andΔOsmek2 #4 mutants (M5) were identified as T-DNA insertion homozygous plants that lacked the specific PCR products.

OsMEK2 expression in ΔOsmek2 knock-out and 35S:OsMEK2 overexpression lines was examined by quantitative real-time RT-PCR and immunoblotting (Figures 1B,C). The qRT-PCR and immunoblot analyses indicated that OsMEK2 was not expressed in ΔOsmek2 #2 and #4 knock-out lines, but distinctly expressed in 35S:OsMEK2 #4 and #6 overexpression lines, compared to the wild-type rice DJ. These combined data indicate that the OsMEK2 gene is knocked out in the selected ΔOsmek2 #2 and #4 lines, but overexpressed in 35S:OsMEK2 #4 and #6 lines. Avirulent M. oryzae 007 infection caused susceptible response in ΔOsmek2 #2 and #4 lines, but resistant response in 35S:OsMEK2 #4 and #6 overexpression lines (Supplementary Figure 5). M. oryzae 007 grew well and produced invasive hyphae (IH) in the leaf sheath cells of ΔOsmek2 #2 and #4 knock-out plants, but induced hypersensitive cell death in wild-type (WT) rice DJ and 35S:OsMEK2 #4 and #6 overexpression plants (Supplementary Figures 5A,B). The ΔOsmek2 #2 and 35S:OsMEK2 #4 lines were selected to use in this study, because ΔOsmek2 #2 and #4 lines and 35S:OsMEK2 #4 and #6 lines exhibited the same susceptible and resistant responses to M. oryzae 007 infection, respectively.

Quantitative real-time RT-PCR analyses showed that avirulent M. oryzae 007 infection triggered the induction of OsMEK2 expression at early infection times (1–12 hpi). During M. oryzae 007 infection, OsMEK2 expression was up-regulated from 1 to 12 hpi and then back to the background level at 24 hpi (Figure 2). In contrast, OsMEK2 expression by virulent M. oryzae PO6-6 infection remained unchanged at 1, 6, and 12 hpi, but up-regulated at 24 hpi. Notably, infection with avirulent M. oryzae 007 significantly reduced OsMEK2 expression at late infection stages (72–96 hpi). These results suggest that early induction of OsMEK2 expression is involved in rice defense signaling in the incompatible rice–M. oryzae interaction (Figure 2).

We investigated whether OsMEK2 is required for cell death and resistant responses to M. oryzae 007 infection using ΔOsmek2 #2 and 35S:OsMEK2 #4 rice plants (Figure 3). M. oryzae 007 grew poorly and caused cell death responses in leaf sheath epidermal cells of rice DJ and 35S:OsMEK2 #4 overexpression plants at 48 hpi (Figures 3A,B). By contrast, the blast fungus grew well with plentiful invasive hyphae (IH) in the invaded ΔOsmek2 #2 leaf sheath cells. Avirulent M. oryzae 007 infection induced significantly more hypersensitive cell death in rice DJ and 35S:OsMEK2 #4 leaf sheaths than in ΔOsmek2 #2 leaf sheaths (Figure 3B). Whole-leaf disease phenotypes were observed at 5 days after inoculation with M. oryzae 007 (Figure 3C). Rice DJ and 35S:OsMEK2 #4 leaves displayed a typical resistant reaction with small necrotic, and brownish restricted lesions. By contrast, ΔOsmek2 #2 mutant leaves displayed a typical susceptible reaction with large grayish lesions (Figure 3C). These combined results indicate that OsMEK2 knock-out in rice DJ rendered resistance ineffective and induced susceptibility (disease) in response to avirulent M. oryzae infection. However, OsMEK2 overexpression in rice DJ enhanced the cell death and resistance responses to rice blast disease.

A previous study reported that rice OsMAP2K2 (OsMEK2) interacted with and phosphorylated OsMAPKs, such as OsMPK1 and OsMPK6 (Singh et al., 2012). We analyzed OsMPK1, OsMPK6, and OsWRKY90 expression in leaf sheaths of rice DJ, ΔOsmek2 #2 knock-out and 35S:OsMEK2 #4 overexpression plants during avirulent M. oryzae 007 infection (Figure 4). OsMEK2 knock-out in rice DJ plants distinctly downregulated OsMPK1 expression throughout the course of M. oryzae infection. However, OsMEK2 overexpression did not upregulate expression of OsMPK1 and OsMPK6 in rice DJ plants. By contrast, OsMPK6 downregulation in ΔOsmek2 #2 leaf sheath cells was observed at early infection stages 3–12 hpi. OsMPK6 (or OsMPK1) activation by OsMKK10-2 is required for the induction of OsWRKY45 expression and blast resistance in rice (Ueno et al., 2015). OsMPK1 is the pathogen-responsive MAPK that is involved in disease resistance (Singh et al., 2012; Ueno et al., 2015). Bimolecular fluorescence complementation (BiFC) analysis in rice leaf sheath indicates that OsMPK1 physically interacts with the OsWRKY80 transcription factor (Singh et al., 2012) and subsequently OsWRKY90 (Shen et al., 2012) as its downstream target. In plant disease resistance networks, WRKY transcription factors can associate with MAPK cascades and regulate downstream defense-related genes in the nucleus (Pandey and Somssich, 2009; Ishihama et al., 2011; Jalmi and Sinha, 2016). Avirulent M. oryzae 007 infection significantly upregulated OsWRKY90 expression in rice DJ and 35S:OsMEK2 #4 leaf sheaths, but did not affect OsWRKY90 expression in ΔOsmek2 #2 leaf sheaths at all tested time points after inoculation (Figure 4). This indicates that OsMEK2, the rice MAP2K, targets the OsWRKY90 transcription factor to function as a positive regulator of resistance to M. oryzae infection. Rice plant resistance to M. oryzae infection is markedly enhanced by overexpression of OsWRKY45, OsWRKY53, and OsWRKY89 (Chujo et al., 2007; Shimono et al., 2007; Wang et al., 2007).

We next investigated the expression patterns of some defense-related genes that are induced in response to M. oryzae 007 infection in rice, such as pathogenesis-related protein 1b (OsPR-1b), phenylalanine ammonia lyase1 (OsPAL1), ascorbate peroxidase1 (OsAPX1), and OsAPX2 (Nakashita et al., 2001; Agrawal et al., 2003; Xie et al., 2011). OsPR-1b expression was induced in rice DJ and 35S:OsMEK2 #4 at all tested times, whereas it was only induced in ΔOsmek2 #2 at 96 hpi (Supplementary Figure 6). OsPAL1 was distinctly induced in 35S:OsMEK2 #4 leaf sheaths during infection. OsPAL1 expression patterns did not significantly differ in rice DJ and ΔOsmek2 #2 leaf sheath cells at 12–96 hpi. OsAPX1 and OsAPX2 expression was gradually upregulated in ΔOsmek2 #2 leaf sheath cells at 12–72 hpi (Supplementary Figure 6). These results indicate that OsMEK2 expression positively regulates OsPR-1b and OsPAL1 expression in rice during M. oryzae infection.

We analyzed ROS and ferric ion (Fe³⁺) accumulation and lipid [malondialdehyde (MDA)] peroxidation in leaf sheath cells of rice DJ, 35S:OsMEK2 #4 and ΔOsmek2 #2 plants during avirulent M. oryzae 007 infection to determine whether OsMEK2 is involved in iron- and ROS-dependent ferroptotic cell death (Figure 5). CM-H₂DCFDA (green fluorescence) and DAB (dark brown) staining revealed that ROS (H₂O₂) strongly accumulated inside and around invasive hyphae (IH) in rice DJ and 35S:OsMEK2 #4 epidermal cells at 30–48 hpi (Figure 5A). By contrast, ROS did not accumulate around invasive hyphae (IH) in ΔOsmek2 #2 epidermal cells after avirulent M. oryzae 007 infection. The ROS-sensitive CM-H₂DCFDA dye is an indicator that can be used to monitor ROS localization in living plant cells (Kristiansen et al., 2009). CM-H₂DCFDA-specific ROS-localized fluorescence was clearly visible around invasive hyphae (IH) and cellular membranes in rice DJ and 35S:OsMEK2 #4 cells, whereas ROS-localized fluorescence was absent or weakly visible around invasive hyphae (IH) in ΔOsmek2 #2 cells at 30 hpi (Figure 5A). DAB is oxidized by H₂O₂ in the presence of peroxidase to generate a dark brown precipitate, which indicates the presence and distribution of H₂O₂ in plant cells (Fryer et al., 2002; Kristiansen et al., 2009). We classified DAB-stained cells into two phenotypes: Type I infected cells display no or weak DAB staining, and Type II infected cells display strong DAB staining (Figure 5B). Most of the infected cells displayed strong brown staining (Type II phenotype) in rice DJ and 35S:OsMEK2 #4 cells. By contrast, significantly fewer ΔOsmek2 #2 cells displayed DAB staining at 48 hpi. A chemiluminescent assay with a luminometer revealed that ROS levels were significantly lower in ΔOsmek2 #2 cells than in DJ and 35S:OsMEK2 #4 cells at 48 hpi (Figures 5A–C).

Ferric ion (Fe³⁺) accumulation and localization in rice cells was detected by Prussian blue (blue color) staining of rice leaf sheath cells at 48 hpi with avirulent M. oryzae 007 (Figure 5A). Rice DJ and 35S:OsMEK2 #4 epidermal cells displayed strong blue staining, whereas ΔOsmek2 #2 epidermal cells did not display blue stain. Next, we analyzed oxidative damage and lipid (MDA) peroxidation in rice leaf sheath cells at 48 hpi with M. oryzae 007 (Figure 5D) by performing the MDA assay as described previously (Zhang et al., 2009; Dangol et al., 2019). Lipid peroxidation levels were significantly lower in ΔOsmek2 #2 cells than in rice DJ. The MDA level in 35S:OsMEK2 #4 was similar to that in rice DJ. These combined results indicate that OsMEK2 has crucial roles in ROS and Fe³⁺ accumulation and lipid peroxidation during the ferroptotic cell death response in rice.

Erastin is a small molecule inducer that triggers ferroptotic cell death in mammals and plants (Dixon et al., 2012; Dangol et al., 2019). Treatment with 10 μM erastin triggered ROS (H₂O₂) and Fe³⁺ accumulation and cell death response in ΔOsmek2 #2 leaf sheaths during avirulent M. oryzae 007 infection (Figure 6). However, mock (water) or 10 μM erastin treatment did not trigger ROS (H₂O₂) and Fe³⁺ accumulation in healthy rice DJ leaf sheaths (Supplementary Figure 7). CM-H₂DCFDA and DAB staining detected H₂O₂ accumulation in ΔOsmek2 #2 leaf sheath cells at 30∼48 hpi with M. oryzae conidial suspension containing 10 μM erastin (Figures 6A,B). Erastin treatment during M. oryzae 007 infection induced H₂O₂ accumulation in ΔOsmek2 #2 cells at 48 hpi as detected with a luminometer (Figure 6D). Erastin induced Fe³⁺ accumulation and increased the number of Prussian blue-stained cells in ΔOsmek2 #2 leaf sheaths at 48 hpi (Figures 6A,C). Iron-dependent MDA peroxidation was upregulated at 48 hpi in ΔOsmek2 #2 leaf sheath cells by treating with erastin (Figure 6E). Erastin treatment significantly enhanced the cell death response in ΔOsmek2 #2 cells during M. oryzae infection (Figures 6A,F). These combined results indicate that erastin triggers iron- and lipid ROS-dependent, but OsMEK2-independent, ferroptotic cell death in rice during M. oryzae infection.

Reactive oxygen species (H₂O₂) and Fe³⁺ did not accumulate in healthy rice DJ leaf sheaths at 72 and 92 h after treatment with 10 μM erastin (Supplementary Figure 7). Erastin treatment strongly induced HR cell death, ROS and Fe³⁺ accumulation, and lipid peroxidation in ΔOsmek2 #2 leaf sheaths at 72 and 96 hpi with avirulent M. oryzae 007, similar to that observed in rice DJ (Figures 7, 8). By contrast, M. oryzae 007 infection induced disease-related cell death but not Fe³⁺ accumulation in erastin-untreated leaf sheaths of the susceptible ΔOsmek2 #2 cells at 72 and 96 hpi (Figures 7A,C,D). However, the chemiluminescent assay indicated that the high ROS levels observed in the ΔOsmek2 #2 cells were similar to those observed in rice DJ and erastin-treated ΔOsmek2 #2 leaf sheaths at 72 and 96 hpi (Figure 7B). ROS accumulation, MDA peroxidation, and cell death phenotypes were distinctly enhanced in the ΔOsmek2 #2 cells (Figures 7A,C, 8A–C); however, increased Fe³⁺ accumulation was not observed at 72 and 96 hpi (Figures 7A,D). Avirulent M. oryzae 007 infection did not increase the number of Prussian blue-stained cells in ΔOsmek2 #2 leaf sheaths at 72 and 96 hpi (Figure 7D). These combined results indicate that disease-related cell death is ROS-dependent but iron-independent in the compatible rice–M. oryzae interaction. Increased ROS production and lipid peroxidation in M. oryzae-infected tissues may induce susceptibility-related cell death that facilitates subsequent fungal invasion and infection. However, intracellular iron accumulation may not be required for disease-related cell death in compatible rice–M. oryzae interactions.

Rice MAP kinase (OsMPK1) is an interactor of OsMEK2 and actively involved in M. oryzae infection (Singh et al., 2012; Ueno et al., 2015). Genomic DNA sequence of OsMPK1 contains six exons and five introns (Supplementary Figure 8). Amino acid sequence alignments of OsMPK1 with other plant MPKs indicated that OsMPK1 shares 67.95–96.20% homology with MAPKs of rice, Arabidopsis, tomato, and maize (Supplementary Figure 9). In particular, OsMPK1 has high levels of identity with OsMPK6 (96.20%) and AtMPK6 (83.76%) (Supplementary Figure 9). OsMPK1 was also phylogenetically close to OsMPK6 and AtMPK6 (Supplementary Figure 10). We overexpressed OsMPK1 in the susceptible rice cultivar Nipponbarre (NB) under the control of CaMV 35S promoter. OsMPK1 was distinctly overexpressed in leaf sheath cells of 35S:OsMPK1-transformed plants (Supplementary Figure 11). 35S:OsMPK1 overexpression induced a hypersensitive cell death with poorly grown invasive hyphae (IH) in leaf epidermal cells at 48 hpi (Figures 9A,B). DAB and CM-H₂DCFDA staining showed accumulation of ROS around the IH in 35S:OsMPK1 leaf sheath cells at 36–48 hpi during virulent M. oryzae PO6-6 infection (Figures 9A,D). Chemiluminescence assay with a luminometer revealed that ROS levels distinctly increased in 35S:OsMPK1 overexpression cells at 48 hpi (Figure 9E). Prussian blue staining of Fe³⁺ showed strong accumulation of ferric ion in 35S:OsMPK1 cells at 48 hpi (Figures 9A,C). Lipid (MDA) peroxidation levels were significantly higher in 35S:OsMPK1 overexpression cells than in rice NB cells at 48 hpi (Figure 9F). By contrast, ferostatin-1(Fer-1) treatments distinctly inhibited iron- and ROS-dependent ferroptotic cell death during infection, which ultimately led to the restored normal hyphal growth in 35S:OsMPK1 overexpression cells (Figure 9). ROS and ferric ion accumulation and lipid peroxidation nearly disappeared in Fer-1-treated 35S:OsMPK1 leaf sheaths during infection (Figures 9A,B,D–F). The combined results indicate that OsMPK1 is involved in ROS and Fe³⁺ accumulation and lipid peroxidation leading to the ferroptotic cell death during M. oryzae infection.

We recently reported that rice NADP-malic enzyme (OsNADP-ME) and respiratory burst oxidase homolog (OsRboh, NADPH-oxidase) are involved in Fe³⁺ and ROS accumulation during cell death and defense responses in rice (Dangol et al., 2019). Interaction of N. benthamiana WRKY8 with MAPKs induce the downstream target genes NADP-ME and Rboh, resulting in the ROS burst (Yoshioka et al., 2003; Ishihama et al., 2011). RbohB activation via MAPK cascades is required for the pathogen-responsive ROS burst (Adachi and Yoshioka, 2015). Here, we analyzed the expression of OsNADP-ME2-3 (Singh et al., 2016) and OsRbohB (Wong et al., 2007) in wild-type rice DJ, ΔOsmek2 #2 knock-out and 35S:OsMEK2 #4 overexpression plants during avirulent M. oryzae 007 infection (Figure 10). OsNADP-ME2-3 expression patterns did not differ in rice DJ and ΔOsmek2 #2 plants, except for a reduction in ΔOsmek2 #2 plants at 12 hpi. However, OsNADP-ME2-3 was distinctly expressed in 35S:OsMEK2 #4 plants at 12 and 72 hpi. OsRbohB expression was significantly downregulated in ΔOsmek2 #2 plants, but distinctly upregulated at 96 hpi, compared to that in rice DJ (Figure 10). These combined results indicate that OsMEK2 expression positively regulates OsNADP-ME and OsRbohB expression during avirulent M. oryzae infection.

The subcellular localization study of MAP kinase signaling proteins is important for understanding their biological functions in plant cells. In this study, we investigated subcellular localization of green fluorescent protein (GFP)-tagged 35S:OsMEK2 (OsMEK2:GFP), OsMPK1:GFP, and OsWRKY90:GFP in N. benthamiana leaves using A. tumefaciens-mediated transient expression (Figure 11). The nuclei inside cells were counterstained with DAPI to help verify nuclear localization of GFP-tagged proteins. The control GFP construct (00:GFP) was ubiquitously detected in the cytoplasm of N. benthamiana cells. OsMEK2:GFP was localized mainly to the cytoplasm, but also to some nuclei in N. benthamiana cells. OsMPK1:GFP was localized to both the cytoplasm and nuclei. However, the OsWRKY90:GFP transcription factor was located inside the nuclei, but not in the cytoplasm. These results indicate that OsMEK2 interacts with OsMPK1 in the cytoplasm, and OsMPK1 moves into the nuclei to interact with the OsWRK90 transcription factor.