Bypassing Both Surface Attachment and Surface Recognition Requirements for Appressorium Formation by Overactive Ras Signaling in Magnaporthe oryzae

Magnaporthe oryzae forms a highly specialized infection structure called an appressorium for plant penetration. In M. oryzae and many other plant-pathogenic fungi, surface attachment and surface recognition are two essential requirements for appressorium formation. Development of appressoria in the air has not been reported. In this study, we found that expression of a dominant active MoRAS2G18V allele in M. oryzae resulted in the formation of morphologically abnormal appressoria on nonconducive surfaces, in liquid suspensions, and on aerial hyphae without attachment to hard surfaces. Both the Pmk1 mitogen-activated protein kinase cascade and cAMP signaling pathways that regulate surface recognition and appressorium morphogenesis in M. oryzae were overactivated in the MoRAS2G18V transformant. In mutants deleted of PMK1 or CPKA, expression of MoRAS2G18V had no significant effects on appressorium morphogenesis. Furthermore, expression of dominant MoRAS2 in Colletotrichum graminicola and C. gloeosporioides also caused the formation of appressorium-like structures in aerial hyphae. Overall, our data indicate that MoRas2 functions upstream from both the cAMP-PKA and Pmk1 pathways and overactive Ras signaling leads to improper activation of these two pathways and appressorium formation without surface attachment in appressorium-forming pathogens.

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Magnaporthe oryzae Auxiliary Activity Protein MoAa91 Functions as Chitin-Binding Protein To Induce Appressorium Formation on Artificial Inductive Surfaces and Suppress Plant Immunity

mBio ◽

10.1128/mbio.03304-19 ◽

2020 ◽

Vol 11 (2) ◽

Author(s):

Ying Li ◽

Xinyu Liu ◽

Muxing Liu ◽

Yang Wang ◽

Yibin Zou ◽

...

Keyword(s):

Magnaporthe Oryzae ◽

Binding Protein ◽

Plant Immunity ◽

G Protein Signaling ◽

Protein Signaling ◽

Appressorium Formation ◽

Protein Precursor ◽

Content Type ◽

Surface Recognition ◽

Appressorium Development

ABSTRACT The appressoria that are generated by the rice blast fungus Magnaporthe oryzae in response to surface cues are important for successful colonization. Previous work showed that regulators of G-protein signaling (RGS) and RGS-like proteins play critical roles in appressorium formation. However, the mechanisms by which these proteins orchestrate surface recognition for appressorium induction remain unclear. Here, we performed comparative transcriptomic studies of ΔMorgs mutant and wild-type strains and found that M. oryzae Aa91 (MoAa91), a homolog of the auxiliary activity family 9 protein (Aa9), was required for surface recognition of M. oryzae. We found that MoAA91 was regulated by the MoMsn2 transcription factor and that its disruption resulted in defects in both appressorium formation on the artificial inductive surface and full virulence of the pathogen. We further showed that MoAa91 was secreted into the apoplast space and was capable of competing with the immune receptor chitin elicitor-binding protein precursor (CEBiP) for chitin binding, thereby suppressing chitin-induced plant immune responses. In summary, we have found that MoAa91 is a novel signaling molecule regulated by RGS and RGS-like proteins and that MoAa91 not only governs appressorium development and virulence but also functions as an effector to suppress host immunity. IMPORTANCE The rice blast fungus Magnaporthe oryzae generates infection structure appressoria in response to surface cues largely due to functions of signaling molecules, including G-proteins, regulators of G-protein signaling (RGS), mitogen-activated protein (MAP) kinase pathways, cAMP signaling, and TOR signaling pathways. M. oryzae encodes eight RGS and RGS-like proteins (MoRgs1 to MoRgs8), and MoRgs1, MoRgs3, MoRgs4, and MoRgs7 were found to be particularly important in appressorium development. To explore the mechanisms by which these proteins regulate appressorium development, we have performed a comparative in planta transcriptomic study and identified an auxiliary activity family 9 protein (Aa9) homolog that we named MoAa91. We showed that MoAa91 was secreted from appressoria and that the recombinant MoAa91 could compete with a chitin elicitor-binding protein precursor (CEBiP) for chitin binding, thereby suppressing chitin-induced plant immunity. By identifying MoAa91 as a novel signaling molecule functioning in appressorium development and an effector in suppressing host immunity, our studies revealed a novel mechanism by which RGS and RGS-like proteins regulate pathogen-host interactions.

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Pleiotropic Function of the Putative Zinc-Finger Protein MoMsn2 in Magnaporthe oryzae

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-09-13-0271-r ◽

2014 ◽

Vol 27 (5) ◽

pp. 446-460 ◽

Cited By ~ 28

Author(s):

Haifeng Zhang ◽

Qian Zhao ◽

Xianxian Guo ◽

Min Guo ◽

Zhongqiang Qi ◽

...

Keyword(s):

Cell Wall ◽

Magnaporthe Oryzae ◽

Stress Responses ◽

Zinc Finger Protein ◽

Affinity Purification ◽

Hyphal Growth ◽

Mitogen Activated Protein Kinase ◽

Phenotypic Characterization ◽

Cell Wall Biosynthesis ◽

Appressorium Formation

The mitogen-activated protein kinase MoOsm1–mediated osmoregulation pathway plays crucial roles in stress responses, asexual and sexual development, and pathogenicity in Magnaporthe oryzae. Utilizing an affinity purification approach, we identified the putative transcriptional activator MoMsn2 as a protein that interacts with MoOsm1 in vivo. Disruption of the MoMSN2 gene resulted in defects in aerial hyphal growth, conidial production, and infection of host plants. Quantitative reverse transcription-polymerase chain reaction analysis showed that the expression of several genes involved in conidiophore formation was reduced in ΔMomsn2, suggesting that MoMsn2 might function as a transcriptional regulator of these genes. Subsequently, MoCos1 was identified as one of the MoMsn2 targets through yeast one-hybrid analysis in which MoMsn2 binds to the AGGGG and CCCCT motif of the MoCOS1 promoter region. Phenotypic characterization showed that MoMsn2 was required for appressorium formation and penetration and pathogenicity. Although the ΔMomsn2 mutant was tolerant to the cell-wall stressor Calcofluor white, it was sensitive to common osmotic stressors. Further analysis suggests that MoMsn2 is involved in the regulation of the cell-wall biosynthesis pathway. Finally, transcriptome data revealed that MoMsn2 modulates numerous genes participating in conidiation, infection, cell-wall integrity, and stress response. Collectively, our results led to a model in which MoMsn2 mediates a series of downstream genes that control aerial hyphal growth, conidiogenesis, appressorium formation, cell-wall biosynthesis, and infection and that also offer potential targets for the development of new disease management strategies.

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The CfMK1 Gene Regulates Reproduction, Appressorium Formation, and Pathogenesis in a Pear Anthracnose-Causing Fungus

Journal of Fungi ◽

10.3390/jof8010077 ◽

2022 ◽

Vol 8 (1) ◽

pp. 77

Author(s):

Chaohui Li ◽

Weibo Sun ◽

Shulin Cao ◽

Rongxian Hou ◽

Xiaogang Li ◽

...

Keyword(s):

Cell Wall ◽

Germination Rate ◽

Hyphal Growth ◽

Pathogenic Fungi ◽

Mitogen Activated Protein Kinase ◽

Economic Losses ◽

Deletion Mutants ◽

Appressorium Formation ◽

Mapk Cascades ◽

Mitogen Activated Protein

Colletotrichum fructicola, the causal agent of pear anthracnose, causes significant annual economic losses. Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction pathways that play a crucial role in mediating cellular responses to environmental and host signals in plant pathogenic fungi. In this study, we identified an ortholog of the FUS3/KSS1-related MAPK gene, CfMK1, and characterized its function in C. fructicola. The Cfmk1 deletion mutants exhibited poorly developed aerial hyphae, autolysis, no conidial mass or perithecia on solid plates. However, the conidiation of the Cfmk1 mutant in PDB liquid medium was normal compared with that of the wild type (WT). Conidia of the Cfmk1 mutant exhibited a reduced germination rate on glass slides or plant surfaces. The Cfmk1 deletion mutants were unable to form appressoria and lost the capacity to penetrate plant epidermal cells. The ability of the Cfmk1 mutants to infect pear leaves and fruit was severely reduced. Moreover, RNA sequencing (RNA-seq) analysis of the WT and Cfmk1 mutant was performed, and the results revealed 1886 upregulated and 1554 downregulated differentially expressed genes (DEGs) in the mutant. The DEGs were significantly enriched in cell wall and pathogenesis terms, which was consistent with the defects of the Cfmk1 mutant in cell wall integrity and plant infection. Overall, our data demonstrate that CfMK1 plays critical roles in the regulation of aerial hyphal growth, asexual and sexual reproduction, autolysis, appressorium formation, and pathogenicity.

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Activation of Mst11 and Feedback Inhibition of Germ Tube Growth in Magnaporthe oryzae

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-12-14-0391-r ◽

2015 ◽

Vol 28 (8) ◽

pp. 881-891 ◽

Cited By ~ 10

Author(s):

Linlu Qi ◽

Yangseon Kim ◽

Cong Jiang ◽

Yang Li ◽

Youliang Peng ◽

...

Keyword(s):

Magnaporthe Oryzae ◽

Feedback Inhibition ◽

Mapk Pathway ◽

Regulatory Region ◽

Kinase Domain ◽

Terminal Region ◽

Site Directed Mutagenesis ◽

Mitogen Activated Protein Kinase ◽

Appressorium Formation ◽

Germ Tubes

Appressorium formation and invasive growth are two important steps in the infection cycle of Magnaporthe oryzae that are regulated by the Mst11-Mst7-Pmk1 mitogen-activated protein kinase (MAPK) pathway. However, the molecular mechanism involved in the activation of Mst11 MAPK kinase kinase is not clear in the rice blast fungus. In this study, we functionally characterized the regulatory region of Mst11 and its self-inhibitory binding. Deletion of the middle region of Mst11, which contains the Ras-association (RA) domain and two conserved phosphorylation sites (S453 and S458), blocked Pmk1 activation and appressorium formation. However, the MST11ΔRA transformant MRD-2 still formed appressoria, although it was reduced in virulence. Interestingly, over 50% of its germ tubes branched and formed two appressoria by 48 h, which was suppressed by treatments with exogenous cAMP. The G18V dominant active mutation enhanced the interaction of Ras2 with Mst11, suggesting that Mst11 has stronger interactions with the activated Ras2. Furthermore, deletion and site-directed mutagenesis analyses indicated that phosphorylation at S453 and S458 of Mst11 is important for appressorium formation and required for the activation of Pmk1. We also showed that the N-terminal region of Mst11 directly interacted with its kinase domain, and the S789G mutation reduced their interactions. Expression of the MST11S789G allele rescued the defect of the mst11 mutant in plant infection and resulted in the formation of appressoria on hydrophilic surfaces, suggesting the gain-of-function effect of the S789G mutation. Overall, our results indicate that the interaction of Mst11 with activated Ras2 and phosphorylation of S453 and S458 play regulatory roles in Mst11 activation and infection-related morphogenesis, possibly by relieving its self-inhibitory interaction between its N-terminal region and the C-terminal kinase domain. In addition, binding of Mst11 to Ras2 may be involved in the feedback inhibition of cAMP signaling and further differentiation of germ tubes after appressorium formation.

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Identification of the functional components of the Ras signaling pathway regulating pituitary cell-specific gene expression.

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1553 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1553-1565 ◽

Cited By ~ 54

Author(s):

K E Conrad ◽

J M Oberwetter ◽

R Vaillancourt ◽

G L Johnson ◽

A Gutierrez-Hartmann

Keyword(s):

Gene Expression ◽

Pituitary Cell ◽

Mitogen Activated Protein Kinase ◽

Specific Gene ◽

Ras Signaling ◽

Raf Kinase ◽

Prolactin Gene ◽

Ras Signaling Pathway ◽

Mitogen Activated Protein ◽

Prolactin Promoter

Ras, a small GTP-binding protein, is required for functional receptor tyrosine kinase signaling. Ultimately, Ras alters the activity of specific nuclear transcription factors and regulates novel patterns of gene expression. Using a rat prolactin promoter construct in transient transfection experiments, we show that both oncogenic Ras and activated forms of Raf-1 kinase selectively stimulated the cellular rat prolactin promoter in GH4 rat pituitary cells. We also show that the Ras signal is completely blocked by an expression vector encoding a dominant-negative Raf kinase. Additionally, using a molecular genetic approach, we determined that inhibitory forms of p42 mitogen-activated protein kinase and an Ets-2 transcription factor interfere with both the Ras and the Raf activation of the rat prolactin promoter. These findings define a functional requirement for these signaling constituents in the activation of the prolactin gene, a cell-specific gene which marks the lactotroph pituitary cell type. Further, this analysis allowed us to order the components in the Ras signaling pathway as it impinges on regulation of prolactin gene transcription as Ras-->Raf kinase-->mitogen-activated protein kinase-->Ets. In contrast, we show that intact c-Jun expression inhibited the Ras-induced activation of the prolactin promoter, defining it as a negative regulator of this pathway, whereas c-Jun was able to enhance the Ras activation of an AP-1-driven promoter in GH4 cells. These data show that c-Jun is not the nuclear mediator of the Ras signal for the highly specialized, pituitary cell-specific prolactin cellular promoter. Thus, we have defined a model system which provides an ideal paradigm for studying Ras/Raf signaling pathways and their effects on neuroendocrine cell-specific gene regulation.

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Signaling through mitogen-activated protein kinase and Rac/Rho does not duplicate the effects of activated Ras on skeletal myogenesis.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.3547 ◽

1997 ◽

Vol 17 (7) ◽

pp. 3547-3555 ◽

Cited By ~ 59

Author(s):

M B Ramocki ◽

S E Johnson ◽

M A White ◽

C L Ashendel ◽

S F Konieczny ◽

...

Keyword(s):

Map Kinase ◽

Signaling Pathways ◽

Transcriptional Activation ◽

Intracellular Signaling ◽

Mitogen Activated Protein Kinase ◽

Ras Signaling ◽

Negative Effects ◽

Intracellular Signaling Pathway ◽

Helix Loop Helix ◽

Mitogen Activated Protein

The ability of basic helix-loop-helix muscle regulatory factors (MRFs), such as MyoD, to convert nonmuscle cells to a myogenic lineage is regulated by numerous growth factor and oncoprotein signaling pathways. Previous studies have shown that H-Ras 12V inhibits differentiation to a skeletal muscle lineage by disrupting MRF function via a mechanism that is independent of the dimerization, DNA binding, and inherent transcriptional activation properties of the proteins. To investigate the intracellular signaling pathway(s) that mediates the inhibition of MRF-induced myogenesis by oncogenic Ras, we tested two transformation-defective H-Ras 12V effector domain variants for their ability to alter terminal differentiation. H-Ras 12V,35S retains the ability to activate the Raf/MEK/mitogen-activated protein (MAP) kinase cascade, whereas H-Ras 12V,40C is unable to interact directly with Raf-1 yet still influences other signaling intermediates, including Rac and Rho. Expression of each H-Ras 12V variant in C3H10T1/2 cells abrogates MyoD-induced activation of the complete myogenic program, suggesting that MAP kinase-dependent and -independent Ras signaling pathways individually block myogenesis in this model system. However, additional studies with constitutively activated Rac1 and RhoA proteins revealed no negative effects on MyoD-induced myogenesis. Similarly, treatment of Ras-inhibited myoblasts with the MEK1 inhibitor PD98059 revealed that elevated MAP kinase activity is not a significant contributor to the H-Ras 12V effect. These data suggest that an additional Ras pathway, distinct from the well-characterized MAP kinase and Rac/Rho pathways known to be important for the transforming function of activated Ras, is primarily responsible for the inhibition of myogenesis by H-Ras 12V.

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Early interactions, adhesion, and establishment of the infection court by Erysiphe graminis

Canadian Journal of Botany ◽

10.1139/b95-302 ◽

1995 ◽

Vol 73 (S1) ◽

pp. 609-615 ◽

Cited By ~ 29

Author(s):

Ralph L. Nicholson ◽

Hitoshi Kunoh

Keyword(s):

Hydrophobic Surface ◽

Surface Hydrophobicity ◽

Infection Process ◽

Erysiphe Graminis ◽

Hydrophilic Surface ◽

Colletotrichum Graminicola ◽

Appressorium Formation ◽

Barley Leaf ◽

Powdery Mildew Pathogen ◽

Suitable Substratum

The establishment of a fungal pathogen on the surface of its host is essential to the success of the infection process. For many fungi, establishment on the host is an active process that may depend on recognition of the host surface through chemical or topographic signals. Events that allow for establishment may be considered to represent the "preparation of the infection court" by the pathogen. This sometimes involves the adhesion of the pathogen to the host and possibly the alteration of the host's surface topography or chemistry. Adhesion is often presumed to be a single, chemically mediated event associated with germ tube or appressorium formation. However, adhesion of ungerminated propagules may also occur, and evidence suggests that it is mediated by the release of adhesive materials directly from the propagule upon contact with a suitable substratum. Fungi may require either a hydrophobic or a hydrophilic surface to initiate the infection process. The barley powdery mildew pathogen, Erysiphe graminis, requires a hydrophilic surface for appressorium formation, yet the barley leaf is extremely hydrophobic. The problem is resolved by the release of an exudate from conidia that makes the hydrophobic leaf surface hydrophilic. In contrast, Colletotrichum graminicola requires a hydrophobic surface for the initiation of its infection process. Ungerminated conidia of this fungus release materials that allow for the rapid adhesion of conidia, which ensures that germination and appressorium formation occur, initiating the infection process. For both fungi, these events happen well in advance of germination and establish the pathogen at the site of the infection court. Key words: adhesion, cuticle, cutinase, surface hydrophobicity, infection process.

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Protein Kinase A Operates a Molecular Switch That Governs Yeast Pseudohyphal Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.22.12.3981-3993.2002 ◽

2002 ◽

Vol 22 (12) ◽

pp. 3981-3993 ◽

Cited By ~ 137

Author(s):

Xuewen Pan ◽

Joseph Heitman

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Pathogenic Fungi ◽

Mitogen Activated Protein Kinase ◽

Signaling Cascades ◽

Mitogen Activated Protein ◽

Eukaryotic Organisms ◽

Pseudohyphal Differentiation ◽

Developmental Switch ◽

Kinase A

ABSTRACT The yeast Saccharomyces cerevisiae undergoes a dimorphic filamentous transition in response to nutrient cues that is affected by both mitogen-activated protein kinase and cyclic AMP-protein kinase A signaling cascades. Here two transcriptional regulators, Flo8 and Sfl1, are shown to be the direct molecular targets of protein kinase A. Flo8 and Sfl1 antagonistically control expression of the cell adhesin Flo11 via a common promoter element. Phosphorylation by the protein kinase A catalytic subunit Tpk2 promotes Flo8 binding and activation of the Flo11 promoter and relieves repression by prohibiting dimerization and DNA binding by Sfl1. Our studies illustrate in molecular detail how protein kinase A combinatorially effects a key developmental switch. Similar mechanisms may operate in pathogenic fungi and more complex multicellular eukaryotic organisms.

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A Feed-Forward Subnetwork Emerging from Integrated TOR- and cAMP/PKA-Signaling Architecture Reinforces Magnaporthe oryzae Appressorium Morphogenesis

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-10-18-0287-r ◽

2019 ◽

Vol 32 (5) ◽

pp. 593-607 ◽

Cited By ~ 3

Author(s):

Guangchao Sun ◽

Xiaobo Qi ◽

Richard A. Wilson

Keyword(s):

Cell Cycle ◽

Adenylate Cyclase ◽

Magnaporthe Oryzae ◽

Environmental Conditions ◽

Germ Tube ◽

Nutrient Status ◽

Specific Cell ◽

Appressorium Formation ◽

G2 Arrest ◽

Feed Forward

Appressoria are important mediators of plant–microbe interactions. In the devastating rice blast pathogen Magnaporthe oryzae, appressorial morphogenesis from germ tube tips requires activated cAMP/PKA signaling and inactivated TOR signaling (TORoff). TORoff temporarily arrests G2 at a metabolic checkpoint during the single round of mitosis that occurs following germination. G2 arrest induces autophagy and appressorium formation concomitantly, allowing reprogression of the cell cycle to G1/G0 quiescence and a single appressorial nucleus. Inappropriate TOR activation abrogates G2 arrest and inhibits cAMP/PKA signaling downstream of cPKA. This results in multiple rounds of germ tube mitosis and the loss of autophagy and appressoria formation. How cAMP/PKA signaling connects to cell cycle progression and autophagy is not known. To address this, we interrogated TOR and cAMP/PKA pathways using signaling mutants, different surface properties, and specific cell cycle inhibitors and discovered a feed-forward subnetwork arising from TOR- and cAMP/PKA-signaling integration. This adenylate cyclase-cAMP-TOR-adenylate cyclase subnetwork reinforces cAMP/PKA-dependent appressorium formation under favorable environmental conditions. Under unfavorable conditions, the subnetwork collapses, resulting in reversible cell cycle-mediated germ tube growth regardless of external nutrient status. Collectively, this work provides new molecular insights on germ tube morphogenetic decision-making in response to static and dynamic environmental conditions.

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MAP Kinase OsMEK2 and OsMPK1 Signaling for Ferroptotic Cell Death in Rice-Magnaporthe oryzae Interactions

10.1101/2020.06.26.174292 ◽

2020 ◽

Author(s):

Sarmina Dangol ◽

Raksha Singh ◽

Khoa Nam Nguyen ◽

Yafei Chen ◽

Juan Wang ◽

...

Keyword(s):

Cell Death ◽

Map Kinase ◽

Signaling Pathways ◽

Magnaporthe Oryzae ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Microbial Pathogens ◽

Blast Disease ◽

Related Cell ◽

Mitogen Activated Protein

ABSTRACTMitogen-activated protein kinase (MAPK) signaling is required for plant cell death responses to invading microbial pathogens. Ferric ions and reactive oxygen species (ROS) accumulate in rice (Oryza sativa) tissues undergoing cell death during Magnaporthe oryzae infection. Here, we report that rice MAP kinase (OsMEK2 and OsMPK1) signaling cascades are involved in iron- and ROS-dependent ferroptotic cell death responses of rice to M. oryzae infection. OsMEK2 interacted with OsMPK1 in the cytoplasm, and OsMPK1 moved from the cytoplasm into the nucleus to bind to the OsWRKY90 transcription factor. OsMEK2 expression may trigger OsMPK1-OsWRKY90 signaling pathways in the nucleus. Avirulent M. oryzae infection in ΔOsmek2 mutant rice did not trigger iron and ROS accumulation and lipid peroxidation, and also downregulated OsMPK1, OsWRKY90, OsRbohB, and OsPR-1b expression. However, OsMEK2 overexpression induced ROS-and iron-dependent cell death in rice during M. oryzae infection. The downstream MAP kinase (OsMPK1) overexpression induced ROS- and iron-dependent ferroptotic cell death in the compatible rice-M. oryzae interaction. These data suggest that the OsMEK2-OsMPK1-OsWRKY90 signaling cascade is involved in the ferroptotic cell death in rice. The small-molecule inducer erastin triggered iron- and lipid ROS-dependent, but OsMEK2-independent, ferroptotic cell death in ΔOsmek2 mutant plants during M. oryzae infection. Disease-related cell death was lipid ROS-dependent and iron-independent in the ΔOsmek2 mutant plants. These combined results suggest that OsMEK2 and OsMPK1 expression positively regulates iron- and ROS-dependent ferroptotic cell death via OsMEK2-OsMPK1-OsWRKY90 signaling pathways, and blast disease (susceptibility)-related cell death was ROS-dependent but iron-independent in rice-M. oryzae interactions.

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