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Random mutagenesis of the nucleotide‐binding domain of NRC 1 ( NB ‐ LRR Required for Hypersensitive Response‐Associated Cell Death‐1), a downstream signalling nucleotide‐binding, leucine‐rich repeat ( NB ‐ LRR ) protein, identifies gain‐of‐function mutations in the nucleotide‐binding pocket
Author(s) -
Sueldo Daniela J.,
Shimels Mahdere,
Spiridon Laurentiu N.,
Caldararu Octav,
Petrescu AndreiJose,
Joosten Matthieu H. A. J.,
Tameling Wladimir I. L.
Publication year - 2015
Publication title -
new phytologist
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.742
H-Index - 244
eISSN - 1469-8137
pISSN - 0028-646X
DOI - 10.1111/nph.13459
Subject(s) - leucine rich repeat , hypersensitive response , biology , nicotiana benthamiana , mutagenesis , elicitor , leucine zipper , cyclic nucleotide binding domain , nucleotide , genetics , mutant , effector , programmed cell death , biochemistry , microbiology and biotechnology , peptide sequence , gene , apoptosis
Summary Plant nucleotide‐binding, leucine‐rich repeat ( NB ‐ LRR ) proteins confer immunity to pathogens possessing the corresponding avirulence proteins. Activation of NB ‐ LRR proteins is often associated with induction of the hypersensitive response ( HR ), a form of programmed cell death. NRC 1 ( NB ‐ LRR Required for HR ‐Associated Cell Death‐1) is a tomato ( Solanum lycopersicum ) NB ‐ LRR protein that participates in the signalling cascade leading to resistance to the pathogens Cladosporium fulvum and Verticillium dahliae . To identify mutations in NRC 1 that cause increased signalling activity, we generated a random library of NRC 1 variants mutated in their nucleotide‐binding domain and screened them for the ability to induce an elicitor‐independent HR in Nicotiana tabacum . Screening of 1920 clones retrieved 11 gain‐of‐function mutants, with 10 of them caused by a single amino acid substitution. All substitutions are located in or very close to highly conserved motifs within the nucleotide‐binding domain, suggesting modulation of the signalling activity of NRC 1. Three‐dimensional modelling of the nucleotide‐binding domain of NRC 1 revealed that the targeted residues are centred around the bound nucleotide. Our mutational approach has generated a wide set of novel gain‐of‐function mutations in NRC 1 and provides insight into how the activity of this NB ‐ LRR is regulated.