A cell death assay in barley and wheat protoplasts for identification and validation of matching pathogen AVR effector a
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(2019) 15:118 Saur et al. Plant Methods https://doi.org/10.1186/s13007-019-0502-0
Open Access
METHODOLOGY
A cell death assay in barley and wheat protoplasts for identification and validation of matching pathogen AVR effector and plant NLR immune receptors Isabel M. L. Saur1* , Saskia Bauer1, Xunli Lu1,3 and Paul Schulze‑Lefert1,2*
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. Keywords: NLR-type immune receptor, Pathogen avirulence effector, Cell death, Race-specific disease resistance, Barley, Wheat, Leaf protoplasts Background Monocotyledonous wheat (Triticum durum, Triticum aestivum) and barley (Hordeum vulgare) are important crops worldwide and diseases caused by infectious pathogens threaten their cultivation. The genomes of bacterial, fungal, and oomycete plant pathogens encode numerous virulence factors (so-called effectors) that either interfere with the plant immune system or manipulate the metabolism of their hosts, ultimately leading to disease development and proliferation of the pathogen [1]. Disease resistance to host-adapted pathogens is often *Correspondence: [email protected]; [email protected] 1 Department of Plant‑Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany Full list of author information is available at the end of the article
mediated through the recognition of pathogen effectors by plant-encoded nucleotide-binding and leucinerich repeat receptors (NLR) [2]. NLRs detect either the effector structure or effector-mediated modifications of additional host proteins (guards or decoys) [3, 4]. Effectors recognized by NLRs are termed avirulence (AVR) effectors. Usually, NLR-mediated AVR effector recognition is associated with a rapid host cell death at the site of attempted infection, called the hypersensitive response. Molecular isolation of NLRs and introgression of the corresponding genes into economically relevant crop varieties can contribute significantly to minimizing lo
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