Structural and biochemical studies of an NB-ARC domain from a plant NLR immune receptor

Abstract

Plant NLRs are modular immune receptors that trigger rapid cell death in response to attempted infection by pathogens. A highly conserved n ucleotide- b inding domain shared with A PAF-1, various R -proteins and C ED-4 (NB-ARC domain) is proposed to act as a molecular switch, cycling between ADP (repressed) and ATP (active) bound forms. Studies of plant NLR NB-ARC domains have revealed functional similarities to mammalian homologues, and provided insight into potential mechanisms of regulation. However, further advances have been limited by difficulties in obtaining sufficient yields of protein suitable for structural and biochemical techniques. From protein expression screens in Escherichia coli and Sf9 insect cells, we defined suitable conditions to produce the NB-ARC domain from the tomato NLR NRC1. Biophysical analyses of this domain showed it is a folded, soluble protein. Structural studies revealed the NRC1 NB-ARC domain had co-purified with ADP, and confirmed predicted structural similarities between plant NLR NB-ARC domains and their mammalian homologues.

Molecular evidence of the avocado defense response to Fusarium kuroshium infection: a deep transcriptome analysis using RNA-Seq

“…5A). Regarding the NBS domain, although the only known 3D structure other than ZAR1 is from the tomato protein NRC1 (Steele, Hughes & Banfield, 2019), we demonstrate that at least the differentially expressed avocado UniGenes that encode CC-type NLR proteins can be modeled in both their active and inactive forms (Fig. 6) when the resolved 3D structures for ZAR1 (PDB: 6j5t.1.…”

Molecular actions of NLR immune receptors in plants and animals

“…LRR-mediated inhibition, however, is not essential for autoinhibition of some NLRs. This is evidenced by the crystal structure of the ADP-bound NRC1 NOD which is nearly identical with that of the inactive ZAR1 NOD (Figure 2A and B) (Steele et al, 2019). Insights into the autoinhibition mechanism of animal NLRs were provided by the crystal structures of mouse NLRC4 (mNLRC4) and rabbit NOD2 (OcNOD2), and the cryo-EM structure of human NLRP3 (hNLRP3)-NEK7 complex (Hu et al, 2013;Maekawa et al, 2016;Sharif et al, 2019).…”

“…Compared to the dense contacts made by ADP with NBD and HD1, only a single hydrogen bond is formed between ADP and His488 of WHD ( Figure 2B). The histidine residue is conserved among plant NLRs and from the "MHD" motif (Tameling et al, 2006;Williams et al, 2011), supporting an important role of this motif in keeping plant NLRs inactive (Steele et al, 2019;Wang et al, 2019b). ZAR1…”

“…The cryo-EM structure of the inactive plant NLR-containing complex ZAR1-RKS1 and the crystal structure of the inactive NOD module of NRC1 (NRC1 NOD ) provided insight into the autoinhibition mechanism of plant NLRs (Steele et al, 2019;Wang et al, 2019b). The CNL ZAR1 in Arabidopsis forms a constitutive complex with the pseudokinase RKS1 to mediate immunity induced by the Xanthomonas campestris pathovar campestris effector AvrAC (Feng et al, 2012;Wang et al, 2015).…”

Sugarcane mosaic virus mediated changes in cytosine methylation pattern and differentially transcribed fragments in resistance-contrasting sugarcane genotypes

“…The GO terms “protein phosphorylation” and “cell surface receptor signaling pathway” assigned to the transcripts aligned with DMFs 5000_06 and 5000_09 suggested that the changes in cytosine methylation may play roles in plant immunity triggered by pathogen-associated molecular patterns (PAMPs)—the PAMP-triggered immunity (PTI)—since the detection of PAMPs involves surface-localized pattern recognition receptors (PRRs), whereas concurrent signal transmission occurs via phosphorylation cascades [ 48 ]. Moreover, the transcript aligned with DMF 5000_18 was assigned to the pfam motif NB-ARC, which is posited as a regulatory domain of nucleotide-binding leucine-rich repeat proteins [ 49 ], likely corresponding to antiviral ETI mechanisms [ 50 ]. Many proteins from the nucleotide-binding leucine-rich repeat class conferring resistance against viruses have been identified [ 50 ], whereas, only recently, the activation of plant PTI against viruses has been reported [ 51 ].…”