Protoplast Cell Death Assay to Study Magnaporthe oryzae AVR Gene Function in Rice

ABSTRACTRice blast disease caused by Magnaporthe oryzae is a devastating disease of cultivated rice worldwide. Infections by this fungus lead to a significant reduction in rice yields and threats to food security. To gain better insight into growth and cell death in M. oryzae during infection, we characterized two predicted M. oryzae metacaspase proteins, MoMca1 and MoMca2. These proteins appear to be functionally redundant and are able to complement the yeast Yca1 homologue. Biochemical analysis revealed that M. oryzae metacaspases exhibited Ca2+ dependent caspase activity in vitro. Deletion of both MoMca1 and MoMca2 in M. oryzae resulted in reduced sporulation, delay in conidial germination and attenuation of disease severity. In addition, the double ΔMomca1mca2 mutant strain showed increased radial growth in the presence of oxidative stress. Interestingly, the ΔMomca1mca2 strain showed an increase accumulation of insoluble aggregates compared to the wild-type strain during vegetative growth. Our findings suggest that MoMca1 and MoMca2 promote the clearance of insoluble aggregates in M. oryzae, demonstrating the important role these metacaspases have in fungal protein homeostasis. Furthermore, these metacaspase proteins may play additional roles, like in regulating stress responses, that would help maintain the fitness of fungal cells required for host infection.IMPORTANCEMagnaporthe oryzae causes rice blast disease that threatens global food security by resulting in the severe loss of rice production every year. A tightly regulated life cycle allows M. oryzae to disarm the host plant immune system during its biotrophic stage before triggering plant cell death in its necrotrophic stage. The ways M. oryzae navigates its complex life cycle remains unclear. This work characterizes two metacaspase proteins with peptidase activity in M. oryzae that are shown to be involved in the regulation of fungal growth and development prior to infection by potentially helping maintain fungal fitness. This study provides new insight into the role of metacaspase proteins in filamentous fungi by illustrating the delays in M. oryzae morphogenesis in the absence of these proteins. Understanding the mechanisms by which M. oryzae morphology and development promote its devastating pathogenicity may lead to the emergence of proper methods for disease control.

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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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Live-cell fluorescence imaging to investigate the dynamics of plant cell death during infection by the rice blast fungus Magnaporthe oryzae

BMC Plant Biology ◽

10.1186/s12870-016-0756-x ◽

2016 ◽

Vol 16 (1) ◽

Cited By ~ 46

Author(s):

Kiersun Jones ◽

Dong Won Kim ◽

Jean S. Park ◽

Chang Hyun Khang

Keyword(s):

Cell Death ◽

Fluorescence Imaging ◽

Plant Cell ◽

Magnaporthe Oryzae ◽

Rice Blast ◽

Rice Blast Fungus ◽

Live Cell ◽

Blast Fungus

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A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids

Cells ◽

10.3390/cells9030703 ◽

2020 ◽

Vol 9 (3) ◽

pp. 703 ◽

Cited By ~ 5

Author(s):

Robin Demuynck ◽

Iuliia Efimova ◽

Abraham Lin ◽

Heidi Declercq ◽

Dmitri V. Krysko

Keyword(s):

Cell Death ◽

Three Dimensional ◽

Drug Efficacy ◽

Cost Effective ◽

3D Cultures ◽

Cell Death Assay ◽

Tumour Spheroids ◽

A Cell ◽

Triton X

The failure of drug efficacy in clinical trials remains a big issue in cancer research. This is largely due to the limitations of two-dimensional (2D) cell cultures, the most used tool in drug screening. Nowadays, three-dimensional (3D) cultures, including spheroids, are acknowledged to be a better model of the in vivo environment, but detailed cell death assays for 3D cultures (including those for ferroptosis) are scarce. In this work, we show that a new cell death analysis method, named 3D Cell Death Assay (3DELTA), can efficiently determine different cell death types including ferroptosis and quantitatively assess cell death in tumour spheroids. Our method uses Sytox dyes as a cell death marker and Triton X-100, which efficiently permeabilizes all cells in spheroids, was used to establish 100% cell death. After optimization of Sytox concentration, Triton X-100 concentration and timing, we showed that the 3DELTA method was able to detect signals from all cells without the need to disaggregate spheroids. Moreover, in this work we demonstrated that 2D experiments cannot be extrapolated to 3D cultures as 3D cultures are less sensitive to cell death induction. In conclusion, 3DELTA is a more cost-effective way to identify and measure cell death type in 3D cultures, including spheroids.

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A High-Throughput, Multiplex Cell Death Assay Using an RNAi Screening Approach

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot080267 ◽

2014 ◽

Vol 2014 (6) ◽

pp. pdb.prot080267-pdb.prot080267 ◽

Cited By ~ 1

Author(s):

K. J. Falkenberg ◽

D. N. Saunders ◽

K. J. Simpson

Keyword(s):

Cell Death ◽

High Throughput ◽

Rnai Screening ◽

Cell Death Assay ◽

Screening Approach

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Ethylene Biosynthesis and Signaling Is Required for Rice Immune Response and Basal Resistance Against Magnaporthe oryzae Infection

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-06-16-0121-r ◽

2016 ◽

Vol 29 (11) ◽

pp. 831-843 ◽

Cited By ~ 19

Author(s):

Emily E. Helliwell ◽

Qin Wang ◽

Yinong Yang

Keyword(s):

Cell Death ◽

Magnaporthe Oryzae ◽

Ethylene Production ◽

Ethylene Biosynthesis ◽

Blast Disease ◽

Intermediate Phenotype ◽

Slight Reduction ◽

Callose Deposition ◽

Fungal Cell ◽

Basal Resistance

Recent studies have suggested that ethylene enhances host resistance to fungal pathogen Magnaporthe oryzae, the causal agent of rice blast disease. Among the six 1-aminocyclopropane-1-carboxylic acid synthase genes in rice, OsACS1 and OsACS2 are induced within 24 h of inoculation by M. oryzae. This induction occurs simultaneously with an increase in ethylene production that is noticeable 12 h postinoculation. The purpose of this study was to examine the dynamics of ethylene production and signaling in wild type and RNA interference–mediated suppression lines deficient in ethylene production (acs2) or signaling (eil1) after challenge with M. oryzae as well as fungal cell-wall elicitors. Ethylene-insensitive mutant lines show an attenuated basal defense response including lower basal expression of the genes encoding a chitin-binding receptor, pathogenesis-related (PR) proteins, and the enzymes involved in the synthesis of diterprenoid phytoalexins, a reduction on early hypersensitive response (HR)-like cell death, and reduced incidence of callose deposition. Ethylene-deficient mutants showed an intermediate phenotype, with a significant reduction in expression of defense-related genes and callose deposition, but only a slight reduction in HR-like cell death. As a result, all ethylene-insensitive mutants show increased susceptibility to M. oryzae, whereas the ethylene-deficient lines show a slight but less significant increase in disease severity. These results show that ethylene signaling and, to some extent, ethylene production are required for rice basal resistance against the blast fungus Magnaporthe oryzae.

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Reaction of wheat plants and alternative hosts to Magnaporthe oryzae

Arquivos do Instituto Biológico ◽

10.1590/1808-1657000952017 ◽

2018 ◽

Vol 85 (0) ◽

Author(s):

Lucas Gustavo Yock Durante ◽

Lilian Maria Arruda Bacchi ◽

Jessica Evangelista de Souza ◽

Felipe André Sganseria Graichen

Keyword(s):

Cell Death ◽

Magnaporthe Oryzae ◽

Plant Pathogen ◽

Host Plants ◽

Blast Disease ◽

Plant Responses ◽

Defensive Response ◽

Species Pair ◽

Digitaria Sanguinalis ◽

Plant Pathogen Interactions

ABSTRACT: Blast disease, caused by the fungus Magnaporthe oryzae, has a major impact on wheat farming. The study of plant responses to pathogens has improved the management of this disease. Moreover, it is important to identify potential host plants in the crops’ vicinity and to understand reactions caused by plant-pathogen interactions. The objective of this study was to assess the histopathology of wheat plants, Digitaria insularis and Digitaria sanguinalis inoculated with M. oryzae isolates obtained either rice or wheat plants. Thirty-three days after sowing, greenhouse-grown plants of all three species were inoculated with each M. oryzae isolate. The observed effects (48 hours after inoculation) differed depending on the particular interaction between each pathogen isolate-plant species pair. For instance, wheat and D. sanguinalis had the weakest defensive response against spore germination, production of melanized appressoria, and appressorial penetration, with average values above 87, 90, and 43%, respectively, for these events in these plants. Furthermore, germination and appressoria melanization were more aggressive in the rice isolate than in the wheat isolate. Additionally, evidence for a defensive response (such as cell death) was observed in wheat plants inoculated with rice isolates. However, such a response was absent in plants inoculated using wheat isolates, presumably because pathogen recognition failed.

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