Plant NLR immune receptor Tm-22 activation requires NB-ARC domain-mediated self-association of CC domain

Abstract

The nucleotide-binding, leucine-rich repeat-containing (NLR) class of immune receptors of plants and animals recognize pathogen-encoded proteins and trigger host defenses. Although animal NLRs form oligomers upon pathogen recognition to activate downstream signaling, the mechanisms of plant NLR activation remain largely elusive. Tm-22 is a plasma membrane (PM)-localized coiled coil (CC)-type NLR and confers resistance to Tobacco mosaic virus (TMV) by recognizing its viral movement protein (MP). In this study, we found that Tm-22 self-associates upon recognition of MP. The CC domain of Tm-22 is the signaling domain and its function requires PM localization and self-association. The nucleotide-binding (NB-ARC) domain is important for Tm-22 self-interaction and regulates activation of the CC domain through its nucleotide-binding and self-association. (d)ATP binding may alter the NB-ARC conformation to release its suppression of Tm-22 CC domain-mediated cell death. Our findings provide the first example of signaling domain for PM-localized NLR and insight into PM-localized NLR activation.

Fig 1. Tm-2² self-associates upon activation.

(A) Self-association of Tm-2² was induced by TMV MP effector. (B) Tm-2² (D481V) auto-active mutant self-associated in the absence of TMV MP. (C) Functional P-loop motif was required for TMV MP-induced Tm-2² self-association. Indicated protein samples were subjected to anti-Myc immunoprecipitation (IP), followed by immunoblotting (IB) using the indicated antibodies. Protein molecular weight (kDa) marker is indicated on the right.

Fig 2. Domain interactions of Tm-2².

Co-IP experiments were performed to detect the self-association of CC (A), NB-ARC (B), or LRR domain (C). CC interacted with NB-ARC (D), LRR interacted with CC and NB-ARC (E). All the constructs were transiently expressed in N. benthamiana for 48 hours, and leaves were collected for co-IP by using anti-GFP beads. Co-IP samples were detected by IB with anti-Myc or anti-GFP antibody.

Fig 3. Tm-2² CC domain triggers cell death upon PM localization.

(A) The confocal images of CC-YFP-mRop and CC-YFP-Rop. Bar = 50 μm. (B) Only CC-YFP-Rop induced cell death in N. benthamiana leaves. Solid line circle indicates cell death; dashed line circle indicates no cell death at the infiltrated region. (C) Cell death phenotype of Tm-2² or its domains fused to Myc-Rop or Myc-mRop. Only CC domain fusion with Myc-Rop induced cell death. The red box indicates cell death triggered by the fusion protein. The cell death phenotypes were photographed at 3 days post infiltration (dpi).

Fig 4. Enhanced self-association promotes Tm-2² CC-mediated cell death.

(A) Cell death mediated by CC fused with YFP-Rop or monomeric YFP-Rop (mYFP-Rop). The leaves were stained by trypan blue to facilitate observation of cell death. (B) The confocal images of CC-mYFP-Rop. Bar = 50 μm. (C) The protein levels of different fusion proteins. Rubisco L (RbcL) was stained by Ponceau S as loading control. (D) Cell death mediated by CC fused with dimerization peptide (DP) or mutant DP. CC fused to DP exhibited stronger cell death than CC fused to mDP. (E) DP enhanced the self-association of CC fusion proteins. Myc-Rop or YFP-Rop tagged CC fusion proteins were transiently expressed in pair. Proteins extracted from leaves were subjected to IP with anti-GFP beads, followed by IB using anti-Myc or anti-GFP antibody.

Fig 5. Tm-2² NB-ARC domain regulates CC-mediated cell death.

(A) Cell death mediated by CC with different NB-ARC extensions. Different YFP-Rop tagged CC extension constructs are shown schematically on the left side. All constructs were agro-inoculated into N. benthamiana leaves, and the cell death phenotypes were photographed at 3 dpi. “S” indicates strong cell death; “W” indicates weak partial cell death; “-” indicates no cell death observed. (B) Ion leakage caused by different constructs was measured at different time after agro-inoculation. The error bars indicate the standard deviation from 3 replicates. The experiment was performed at least three times with similar results.

Fig 6. Nucleotide binding state switches CC-NB-ARC activation.

(A) Self-association ability of CC, CC-NB-ARC or CC-NB-ARC (D481V). All domains were fused with Myc-Rop or YFP-Rop. CN stands for CC-NB-ARC. After co-expression, protein extracts were subjected to IP with anti-GFP beads, followed by IB with anti-GFP or anti-myc antibody. (B) and (C), K191R mutation repressed cell death signaling and impaired self-association of CC-NB-ARC (D481V)-Rop. Indicated protein samples were subjected to IP with anti-GFP beads, followed by IB with anti-Myc or anti-GFP antibody.

Fig 7. Tm-2² requires NB-ARC self-association for its function.

(A) Structure modeling of Tm-2² NB-ARC domain. Green, the predicted structure of Tm-2² NB-ARC domain; purple, the structure of ZAR1 (PDB ID: 6J5T); Orange, helix-loop-helix located at the center of ZAR1 inter-subunit-interface. Right orange ring shows the indicated helix-loop-helix motifs among the ZAR1 pentamer center [14]. (B) Potential oligomer surface mutations L233A, L242A and L246A weakened the self-association of NB-ARC (D481V). Numbers below the panel indicate the relative intensity analyzed by ImageJ. Indicated protein samples were subjected to IP with anti-Myc beads, followed by IB with anti-Myc or anti-HA antibody. (C) Ion leakage measurement showed L233A, L242A and L246A weakend full-length Tm-2² mediated cell death in the presence of TMV MP. The error bar indicates the standard deviation from 3 technical repetitions. The experiment was performed at least three times with similar results. (D) Trypan blue staining of infiltrated leaves collected at 30 hpi showed that for full-length proteins, L233A, L242A and L246A mutations weakened the cell death triggered by Tm-2²/MP. The expression of Tm-2² and its mutants were detected by anti-Myc antibody. RbcL was stained by Ponceau S as loading control. (E) L233A, L242A and L246A mutations weakened TMV MP induced full-length Tm-2² self-association. Indicated protein samples were subjected to IP with anti-Myc beads, followed by IB with anti-Myc, anti-HA or anti-GFP antibody.

Fig 8. A model for Tm-2² activation.

(A) Tm-2² localizes at the PM with multiple domain interactions. (B) LRR recognizes its effector MP through direct or indirect interaction. (C) Tm-2² binds (d)ATP instead of ADP accompanied with conformational change. (D) At least two Tm-2² molecules self-associate, thus leading to the self-association of CC domains for further defense signaling.