1. Volume 12, Issue 2, Pages 248-262 (February 2019)
A Plant Immune Receptor Adopts a Two-Step Recognition Mechanism to Enhance Viral Effector Perception 
Jia Li, Haining Huang, Min Zhu, Shen Huang, Wenhua Zhang, Savithramma P. Dinesh-Kumar, Xiaorong Tao 
Molecular Plant 
Volume 12, Issue 2, Pages (February 2019)
DOI: /j.molp
Copyright © 2019 The Authors Terms and Conditions

2. Figure 1
Role of the Extended N-Terminal Solanaceae Domain of Sw-5b in the Recognition of the Tospovirus NSm Effector.
(A) Solanaceae Domain (SD) swapping analysis of Sw-5b and the Sw-5b paralog from Heinz1706 in N. benthamiana leaves (top and bottom left panels). YFP-tagged Sw-5b (i) induces hypersensitive response programmed cell death (HR-PCD) when co-expressed with NSm in N. benthamiana leaves. Sw-5bHeinz (ii), chimeric SDHeinz-CC-NB-ARC-LRRSw-5b (iii), and SDSw-5b-CC-NB-ARC-LRRHeinz (iv) failed to induce HR-PCD when co-expressed with NSm. The expression of each chimeric protein in infiltrated leaves was confirmed by immunoblot assay using YFP and NSm antibodies (bottom right panels). The sizes of proteins in kDa are shown on the left. Ponceau S staining was used to show the amount of protein loaded.
(B) In vitro GST pull-down analysis of Sw-5b SD interaction with NSm. GST-SD, GST, and NSm-6×HIS were expressed in E. coli. The crude extract of GST-SD or GST was mixed with the crude extract of NSm-6×HIS and pulled down with glutathione–Sepharose beads. GST-SD pulled down NSm-6×HIS. GST alone failed to pull down NSm-6×HIS. The sizes of proteins in kDa are shown on the right.
(C) Co-immunoprecipitation (coIP) analysis of the association between Sw-5b SD and NSm in planta. FLAG-tagged SD was co-expressed with NSm in N. benthamiana leaves and co-immunoprecipitation was performed. Leaf tissues expressing FLAG-SD or NSm alone were used as a negative control. IB, immunoblot with specific antibody; IP, immunoprecipitation with specific antibody. The sizes of proteins in kDa are shown on the right. Ponceau S staining was used to show the amount of protein loaded.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2019 The Authors Terms and Conditions

3. Figure 2 The SD1 Subdomain of Sw-5b Specifically Interacts with NSm.
(A) Sw-5b SD1 associates with NSm in planta. Sw-5b SD1 fused to the FLAG tag co-immunoprecipitated NSm when co-expressed in N. benthamiana leaves (lane 1). Leaves expressing FLAG-SD1 or NSm alone were used as a control.
(B) CoIP analysis of the association of Sw-5b SD2 with NSm in N. benthamiana in comparison with the association of SD1 with NSm using FLAG-specific and NSm-specific antibodies.
(C) CoIP analysis of the specificity of the association between Sw-5b SD1 and NSm in N. benthamiana. FLAG-SD1 was co-expressed with NSmWT or resistance-breaking NSmC118Y and NSmT120N mutants in N. benthamiana leaves and coIP assays were performed using FLAG-specific and NSm-specific antibodies.
(D) GST pull-down analysis of the specificity of the interaction between GST-SD1 and NSm in vitro. GST-SD1 expressed in E. coli was mixed with the crude extracts of E. coli expressing NSmWT-6×HIS, NSmC118Y-6×HIS, or NSmT120N-6×HIS and pulled down with glutathione–Sepharose beads followed by immunoblot analysis using GST-specific and 6×HIS-specific antibodies.
(E) Specificity of receptor activation by Sw-5b SD1 and NSm in N. benthamiana. YFP-tagged NB-ARC-LRRR927A, CC-NB-ARC-LRRR927A, and SD-CC-NB-ARC-LRRR927A were expressed alone or co-expressed with NSm in N. benthamiana. SD-CC-NB-ARC-LRRR927A co-expressed with NSmC118Y or NSmT120N was used to test the specificity of the interaction between SD and NSm in relieving CC-mediated inhibition. The expression of individual proteins was confirmed by performing an immunoblot assay using YFP- and NSm-specific antibodies. The sizes of proteins in kDa are shown on the left. Ponceau S staining was used to show the amount of protein loaded.
In (A) to (D), the sizes of proteins in kDa are shown on the right. In (A) to (C), IB represents immunoblot with specific antibody; IP, immunoprecipitation with specific antibody. Ponceau S staining is shown as a protein loading control.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2019 The Authors Terms and Conditions

4. Figure 3 NSm Recognition Sites in Sw-5b SD.
(A) Sequence alignment of the SD regions of Sw-5b and Sw-5b Heinz. Boxed regions indicate 16 different polymorphic sites/residues between Sw-5b and Sw-5b Heinz.
(B) Functional analysis of the 16 polymorphic sites in the Sw-5b SD. Each polymorphic site/residues in the SD of full-length Sw-5b was substituted with the corresponding site/residues of the Sw-5bHeinz paralog, and expressed alone or co-expressed with NSm in N. benthamiana plants.
(C) Sw-5b in which the polymorphic site 8 (M8) was substituted with the cognate site of Sw-5bHeinz failed to induce HR-PCD in the presence of NSm in N. benthamiana. Expression of YFP-tagged Sw-5b, Sw-5b mutants, and NSm was detected using YFP- and NSm-specific antibodies. The protein size is indicated on the left. Ponceau S staining was used to show the amount of protein loaded.
(D) FLAG-SD1WT or FLAG-SD1M8 was co-expressed with NSm in N. benthamiana leaves and coIP analysis was performed using FLAG-specific and NSm-specific antibodies. IB, immunoblot with specific antibody; IP, immunoprecipitation with specific antibody. The sizes of proteins in kDa are shown on the right. Ponceau S staining was used to show the amount of protein loaded.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2019 The Authors Terms and Conditions

5. Figure 4
Role of NB-ARC-LRR in NSm Recognition and Activation of Sw-5b.
(A) Swapping analysis of the CC domain and NB-ARC-LRR region of Sw-5b and the Sw-5b paralog from Heinz1706. Expression of YFP-tagged Sw-5b (i) or chimeric SDSw-5b-CCHeinz-NB-ARC-LRRSw−5b (iii) triggered strong HR-PCD in the presence of NSm in N. benthamiana leaves. Sw-5bHeinz (ii), chimeric SDHeinz-CCSw-5b-NB-ARC-LRRHeinz (iv), SDSw-5b-CCSw-5b-NB-ARC-LRRHeinz (v), and SDHeinz-CCHeinz-NB-ARC-LRRSw-5b (vi) failed to induce HR when co-expressed with NSm.
(B) GST pull-down analysis of the interaction between Sw-5b NB-ARC-LRR and NSm. GST-NB-ARC-LRR, GST, and NSm-6×HIS were expressed in E. coli. The crude extract of GST-NB-ARC-LRR or GST was mixed with the crude extract of NSm-6×HIS, and proteins were pulled down with GST beads. NSm-6×HIS was pulled down with GST-NB-ARC-LRR. GST alone failed to pull down NSm-6×HIS. The sizes of proteins in kDa are shown on the left.
(C) GST pull-down analysis of the specificity of the interaction between GST-NB-ARC-LRR and NSm in vitro. GST-NB-ARC-LRR, NSmWT-6×HIS, and NSm mutants were expressed in E. coli. The crude extract of GST-NB-ARC-LRR was mixed with the crude extract of NSmWT-6×HIS, NSmC118Y-6×HIS, or NSmT120N-6×HIS, then proteins were pulled down with GST, and immunoblot analysis was performed using GST-specific and 6×HIS-specific antibodies. The sizes of proteins in kDa are shown on the right.
(D) Quantification of the ADP and ATP occupancies of Sw-5b NB-ARC-LRRWT, NB-ARC-LRRK568R (P-loop mutant), and NB-ARC-LRRR927A (auto-active mutant). The ADP/ATP occupancies of NB-ARC-LRR and its mutants expressed and purified from E. coli were determined after glutathione affinity purification. GST was used as a negative control. The data obtained from at least three independent experiments were used to calculate the mean. ADP and ATP occupancies were calculated as a percentage of protein molecules bound with each nucleotide.
(E) Quantification of the ADP and ATP occupancies of Sw-5b NB-ARC-LRR in the presence of NSmWT, NSmC118Y, or NSmT120N. The crude extract of GST-NB-ARC-LRR prepared from E. coli was mixed with the crude extract of NSmWT, NSmC118Y, or NSmT120N for 2 h at 4°C, then glutathione affinity chromatography purification was performed. The ADP/ATP occupancies of purified NB-ARC-LRR in the presence of NSm or NSm mutants were determined. Extract from E. coli expressing the empty vector was added to NB-ARC-LRR as a negative control. The same results were obtained in at least three independent experiments.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2019 The Authors Terms and Conditions

6. Figure 5
Co-expression of SD-CC and NB-ARC-LRR Reconstitutes HR-PCD in the Presence of NSm.
(A) SD-CC and NB-ARC-LRR were co-expressed with NSm in N. benthamiana. The HR-PCD phenotype in an infiltrated leaf was photographed at 5 dpi.
(B) Sw-5b SD-CC fused to the FLAG tag (FLAG-SD-CC) was used to immunoprecipitate YFP-NB-ARC-LRRK568R in the presence or absence of NSm.
(C) The effect of NSmWT or the resistance-breaking mutants NSmC118Y and NSmT120N on the physical interaction between NB-ARC-LRR and SD-CC. Sw-5b SD-CC fused to the FLAG tag (FLAG-SD-CC) was used to immunoprecipitate YFP-NB-ARC-LRRK568R in the presence of NSmWT, NSmC118Y, or NSmT120N.
(D) Sw-5b CC fused to the FLAG tag (FLAG-CC) was used to immunoprecipitate YFP-NB-ARC-LRR in the presence or absence of NSm.
IB, immunoblot with specific antibody; IP, immunoprecipitation with specific antibody. The sizes of proteins in kDa are shown on the right. Ponceau S staining is shown as a protein loading control.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2019 The Authors Terms and Conditions

7. Figure 6
The Sw-5b SD-CC Significantly Enhances the Ability of the NB-ARC-LRR to Detect the NSm Effector and Facilitates its Activation.
(A) The same amount of Agrobacterium carrying NB-ARC-LRR (OD600 = 0.1) was infiltrated with different concentrations of Agrobacterium carrying NSm (OD600 = 0.1, 0.05, or 0.025) in the presence of the p2300 empty vector (EV), SDWT-CC, or the SDM8-CC mutant into leaves of 10-week-old N. benthamiana plants. The infiltrated area for each treatment is marked with a dotted circle. The infiltrated leaves were photographed at 5 dpi. Immunoblot detection of FLAG-NB-ARC-LRR, SD-CC, and NSm proteins using a FLAG-specific and an NSm-specific antibody, respectively, is shown in the lower panel. The sizes of proteins are indicated on the left. Ponceau S staining is shown as a protein loading control.
(B) Comparison of the NSm binding strength between SD1 and NB-ARC-LRRK568R using coIP analysis in N. benthamiana. FLAG-SD1 or FLAG-NB-ARC-LRRK568R was co-expressed with NSm in N. benthamiana leaves and coIP assays were performed using FLAG-specific and NSm-specific antibodies. IB, immunoblot with specific antibody; IP, immunoprecipitation with specific antibody. The sizes of proteins in kDa are shown on the right. Ponceau S staining is shown as a protein loading control.
(C) FLAG-NB-ARC-LRR was co-expressed with NSm in the presence of HA-SDWT-CC or HA-SDM8-CC in N. benthamiana leaves and coIP analysis was performed using FLAG-, HA-, and NSm-specific antibodies. Star indicates a non-specific band. IB, immunoblot with specific antibody; IP, immunoprecipitation with specific antibody. The sizes of proteins are indicated on the right. Ponceau S staining is shown as a protein loading control.
(D) The two-step recognition model for the enhancement of Sw-5b NLR sensitivity in detecting a viral pathogen effector. In this two-step recognition model, both the extended SD and the NB-ARC-LRR region of Sw-5b play critical roles in direct ligand recognition. The Sw-5b SD acts as an extra sensor, whereas the Sw-5b NB-ARC-LRR functions as both a sensor and an activator. (i) The Sw-5b NB-ARC-LRR region detects NSm and becomes activated, leading to defense when NSm levels are high. (ii) The Sw-5b NB-ARC-LRR region is unable to induce defense when NSm levels are low. (iii) In the presence of a low amount of NSm effector, the Sw-5b SD directly interacts with NSm and facilitates NSm detection and transformation of NB-ARC-LRR into an active state, leading to a robust defense response. Sw-5b NLR uses this two-step recognition mechanism to significantly enhance its perception of a viral pathogen effector.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2019 The Authors Terms and Conditions