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Pathogen pressure on hosts can lead to the evolution of genes regulating the innate immune response. By characterizing naturally occurring polymorphisms in immune receptors, we can better understand the molecular determinants of pathogen recognition. ZAR1 is an ancient Arabidopsis thaliana NLR (Nucleotide-binding [NB] Leucine-rich-repeat [LRR] Receptor) that recognizes multiple secreted effector proteins from the pathogenic bacteria Pseudomonas syringae and Xanthomonas campestris through its interaction with receptor-like cytoplasmic kinases (RLCKs). ZAR1 was first identified for its role in recognizing P. syringae effector HopZ1a, through its interaction with the RLCK ZED1. To identify additional determinants of HopZ1a recognition, we performed a computational screen for ecotypes from the 1001 Genomes project that were likely to lack HopZ1a recognition, and tested ~300 ecotypes. We identified ecotypes containing polymorphisms in ZAR1 and ZED1. Using our previously established Nicotiana benthamiana transient assay and Arabidopsis ecotypes, we tested for the effect of naturally occurring polymorphisms on ZAR1 interactions and the immune response. We identified key residues in the NB or LRR domain of ZAR1 that impact the interaction with ZED1. We demonstrate that natural diversity combined with functional assays can help define the molecular determinants and interactions necessary to regulate immune induction in response to pathogens., (© 2020 John Wiley & Sons Ltd.)

The intracellular immune receptor Rx1 of potato ( Solanum tuberosum ), which confers effector-triggered immunity to Potato virus X , consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality. Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling., (© 2018 American Society of Plant Biologists. All rights reserved.)

Plant disease immunity heavily depends on the recognition of plant pathogens and the subsequent activation of downstream immune pathways. Nod-like receptors are often crucial in this process. Tsw, a Nod-like resistance gene from Capsicum chinense conferring resistance against Tomato spotted wilt virus (TSWV), belongs to the small group of Nod-like receptors with unusually large LRR domains. While typical protein domain dimensions rarely exceed 500 amino acids due to stability constraints, the LRR of these unusual NLRs range from 1,000 to 3,400 amino acids and contain over 30 LRR repeats. The presence of such a multitude of repeats in one protein is also difficult to explain considering protein functionality. Interactions between the LRR and the other NLR domains (CC, TIR, NBS) take place within the first 10 LRR repeats, leaving the function of largest part of the LRR structure unexplained. Herein we discuss the structural modeling limits and various aspects of the structurefunction relation conundrums of large LRRs focusing on Tsw, and raise questions regarding its recognition of its effector NSs and the possible inhibition on other domains as seen in other NLRs. [ABSTRACT FROM AUTHOR]