scholarly journals A cell death assay in barley and wheat protoplasts for identification and validation of matching pathogen AVR effector and plant NLR immune receptors

Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Isabel M. L. Saur ◽  
Saskia Bauer ◽  
Xunli Lu ◽  
Paul Schulze-Lefert
Keyword(s):  
Cell Death ◽  
Plant Disease Resistance ◽  
Fungal Pathogen ◽  
Fungal Pathogens ◽  
Plant Disease ◽  
Mesophyll Protoplast ◽  
Leucine Rich Repeat ◽  
Cell Death Assay ◽  
Host Cell Death ◽  
A Cell

Abstract Background Plant disease resistance to host-adapted pathogens is often mediated by host nucleotide-binding and leucine-rich repeat (NLR) receptors that detect matching pathogen avirulence effectors (AVR) inside plant cells. AVR-triggered NLR activation is typically associated with a rapid host cell death at sites of attempted infection and this response constitutes a widely used surrogate for NLR activation. However, it is challenging to assess this cell death in cereal hosts. Results Here we quantify cell death upon NLR-mediated recognition of fungal pathogen AVRs in mesophyll leaf protoplasts of barley and wheat. We provide measurements for the recognition of the fungal AVRs AvrSr50 and AVRa1 by their respective cereal NLRs Sr50 and Mla1 upon overexpression of the AVR and NLR pairs in mesophyll protoplast of both, wheat and barley. Conclusions Our data demonstrate that the here described approach can be effectively used to detect and quantify death of wheat and barley cells induced by overexpression of NLR and AVR effectors or AVR effector candidate genes from diverse fungal pathogens within 24 h.

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

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 703 ◽  
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.

scholarly journals Plant mixed lineage kinase domain-like proteins limit biotrophic pathogen growth

2019 ◽  
Author(s):  
Lisa Mahdi ◽  
Menghang Huang ◽  
Xiaoxiao Zhang ◽  
Ryohei Thomas Nakano ◽  
Leïla Brulé Kopp ◽  
...  
Keyword(s):  
Cell Death ◽  
Host Cell ◽  
Kinase Domain ◽  
Protein Family ◽  
Mixed Lineage Kinase ◽  
Helical Bundles ◽  
Cryo Electron Microscopy ◽  
Host Cell Death ◽  
A Cell ◽  
Immunity Mechanism

AbstractMixed lineage kinase domain-like (MLKL) protein mediates necroptotic cell death in vertebrates. We report here the discovery of a conserved protein family across seed plants that is structurally homologous to vertebrate MLKL. TheArabidopsis thalianagenome encodes three MLKLs with overlapping functions in limiting growth of obligate biotrophic fungal and oomycete pathogens. Although displaying a cell death activity mediated by N-terminal helical bundles, termed HeLo domain,AtMLKL-dependent immunity can be separated from host cell death. Cryo-electron microscopy structures ofAtMLKLs reveal a tetrameric configuration, in which the pseudokinase domain and brace region bury the HeLo-domains, indicative of an auto-repressed complex. We also show the association of twoAtMLKLs with microtubules. These findings, coupled with resistance-enhancing activity and altered microtubule association of a phosphomimetic mutation in the pseudokinase domain ofAtMLKL1, point to a cell death-independent immunity mechanism.One Sentence SummaryPlants have a protein family that is structurally homologous to vertebrate mixed lineage kinase domain-like protein, which induces necroptotic cell death, but these plant proteins can confer immunity without host cell death.

scholarly journals Characterization of extracellular vesicles produced by Aspergillus fumigatus protoplasts

2020 ◽  
Author(s):  
Juliana Rizzo ◽  
Thibault Chaze ◽  
Kildare Miranda ◽  
Robert W. Roberson ◽  
Olivier Gorgette ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Extracellular Vesicles ◽  
Fungal Pathogen ◽  
Fungal Pathogens ◽  
Sugar Metabolism ◽  
Biologically Active ◽  
Thick Cell Wall ◽  
A Cell

AbstractExtracellular vesicles (EVs) are outer membranous compartments produced by yeast and mycelial forms of several fungal species. One of the difficulties to perceive the role of EVs during the fungal life is the fact that an active secretion of these EVs has not been clearly demonstrated in situ due to the presence of a thick cell wall. One alternative to have a better access to these vesicles is to use protoplasts. This approach has been investigated here with Aspergillus fumigatus, one of the most common opportunistic fungal pathogens worldwide. Analysis of regenerating protoplasts by scanning electron microscopy and fluorescence microscopy indicated the occurrence of outer membrane projections in association with surface components and the release of particles with properties resembling those of fungal EVs. EVs in culture supernatants were characterized by transmission electron microscopy and nanoparticle tracking analysis. Proteomic and glycome analysis of EVs revealed the presence of a complex array of enzymes related to lipid / sugar metabolism, pathogenic processes, and cell wall biosynthesis. Our data indicate that i) EV production is a common feature of different morphological stages of this major fungal pathogen, and ii) protoplastic EVs are a promising tool to undertake studies of vesicle functions in fungal cells.IMPORTANCEFungal cells use extracellular vesicles (EVs) to export biologically active molecules to the outer space. Since fungal cells are encaged in a thick cell wall, it is reasonable to expect that this structure might impact the vesicle-mediated molecular export. In this study, we used protoplasts of Aspergillus fumigatus, a major fungal pathogen, as a model to evaluate EV production in the absence of a cell wall. Our results demonstrated that wall-less A. fumigatus exports plasma membrane-derived EVs containing a complex combination of proteins and glycans. Our study is the first to characterize fungal EVs in the absence of a cell wall. Our results suggest that protoplasts are a promising model for functional studies of fungal vesicles.

scholarly journals Plant Disease Resistance Inducing Activity of 7-Oxo- and 7-Hydroxysterols

1995 ◽  
Vol 50 (3-4) ◽  
pp. 257-262 ◽  
Author(s):  
H. Schabdach ◽  
S. Johne ◽  
U. Steiner ◽  
K. Seifert
Keyword(s):  
Plant Disease Resistance ◽  
Fungal Pathogens ◽  
Plant Disease ◽  
Puccinia Striiformis ◽  
Cochliobolus Sativus ◽  
Puccinia Hordei ◽  
Challenge Inoculation ◽  
Enhanced Resistance ◽  
Induce Resistance ◽  
Puccinia Striiformis West

The 7-oxosterols 1-2 and the 7-hydroxysterols 3-6 induce resistance toward the fungal pathogens Puccinia striiformis West, and Puccinia hordei Otth in barley and wheat. Primary leaves of the plants were sprayed with solutions of the compounds ( 10-4 mol/l in 1% aqu. ethanol) followed, 2 days later, by challenge inoculation with the fungal pathogens. The results indicate that 7α- and 7β-hydroxylated epimers of β-sitosterol and cholesterol show the highest value of induced resistance (39-49% reduction of infection sites). No enhanced resistance toward the fungi Erysiphe graminis DC f.sp. tricitic and hordei and Cochliobolus sativus Ito & Kuribayashi was observed.

O05. S-nitrosothiols regulate hypersensitive cell death during the plant disease resistance response

Nitric Oxide ◽  
2006 ◽  
Vol 14 (4) ◽  
pp. 2
Author(s):  
Gary John Loake ◽  
Byung Wook Yun ◽  
Angela Feechan ◽  
Jacqueline Pallas ◽  
Eunjung Kwon
Keyword(s):  
Cell Death ◽  
Disease Resistance ◽  
Plant Disease Resistance ◽  
Plant Disease ◽  
Resistance Response ◽  
Hypersensitive Cell Death

Staying alive – is cell death dispensable for plant disease resistance during the hypersensitive response?

2016 ◽  
Vol 93 ◽  
pp. 75-84 ◽  
Author(s):  
András Künstler ◽  
Renáta Bacsó ◽  
Gábor Gullner ◽  
Yaser Mohamed Hafez ◽  
Lóránt Király
Keyword(s):  
Cell Death ◽  
Disease Resistance ◽  
Hypersensitive Response ◽  
Plant Disease Resistance ◽  
Plant Disease

scholarly journals Suppressors of the Arabidopsis lsd5 Cell Death Mutation Identify Genes Involved in Regulating Disease Resistance Responses

Genetics ◽  
1999 ◽  
Vol 151 (1) ◽  
pp. 305-319
Author(s):  
Jean-Benoit Morel ◽  
Jeffery L Dangl
Keyword(s):  
Cell Death ◽  
Disease Resistance ◽  
Pseudomonas Syringae ◽  
Plant Disease Resistance ◽  
Plant Disease ◽  
Disease Susceptibility ◽  
Bacterial Pathogen ◽  
Hypersensitive Reaction ◽  
Cell Death Phenotype ◽  
New Mutations

Abstract Cell death is associated with the development of the plant disease resistance hypersensitive reaction (HR). Arabidopsis lsd mutants that spontaneously exhibit cell death reminiscent of the HR were identified previously. To study further the regulatory context in which cell death acts during disease resistance, one of these mutants, lsd5, was used to isolate new mutations that suppress its cell death phenotype. Using a simple lethal screen, nine lsd5 cell death suppressors, designated phx (for the mythological bird Phoenix that rises from its ashes), were isolated. These mutants were characterized with respect to their response to a bacterial pathogen and oomycete parasite. The strongest suppressors—phx2, 3, 6, and 11-1—showed complex, differential patterns of disease resistance modifications. These suppressors attenuated disease resistance to avirulent isolates of the biotrophic Peronospora parasitica pathogen, but only phx2 and phx3 altered disease resistance to avirulent strains of Pseudomonas syringae pv tomato. Therefore, some of these phx mutants define common regulators of cell death and disease resistance. In addition, phx2 and phx3 exhibited enhanced disease susceptibility to different virulent pathogens, confirming probable links between the disease resistance and susceptibility pathways.

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