Distinct modes of derepression of an <i>Arabidopsis</i> immune receptor complex by two different bacterial effectors


Supporting Information SI Materials and Methods

Plant Materials and Growth Conditions Nicotiana tabacum (cultivar 'Petite Gerard') and Nicotiana benthamiana (Nb) were sown on soil, and grown at 24°C, 55% relative humidity with 16/8 h light/dark photoperiod. Leaves of 4-5-week-old plants were used for Agrobacterium tumefaciens-mediated transient transformation for HR, immunoblots or co-IP assays. 
). ). Prior to amplification, the DNA templates were 'domesticated' to remove internal BsaI sites without altering protein sequences. Using the Golden Gate assembly(1), these domain modules were assembled into full-length R genes with a C-terminal HF tag module (pICSL50001) and expressed under a 35S (CaMV) promoter in binary vectors, pICSL86922 or pICSL86977. A unique set of compatible 4 bp overhangs at the module junctions and in the vector are designed to ensure the assembly in a desired order. The domain breakpoint amino acids were chosen so that they match between RRS1 & RRS1B and RPS4 & RPS4B, so do the overhangs, enabling chimeric R gene assembly. ). Similar cloning techniques were used to generate constructs expressing truncated R proteins or selected domains. AvrRps4 (P. syringae pv. Pisi) and PopP2 (R. solanacearum) were PCR-amplified from published plasmids (2) and assembled with a C-terminal mCherry tag into binary vectors pICH86988 and pICSL86977 respectively (35S promoter). Mutations in genes were generated using Goldern Gate cloning, with primers carrying a BsaI site and the desired mutations, or otherwise previously reported. 

For co-IP and cell biology experiments, various Golden Gate compatible epitope tag modules were used. C-terminal HF, GFP, mCherry tags for co-IP; N-and/or C-terminal cCFP, nVenus, nCerulean tags for multi-color BiFC(3); N-and C-terminal eCFP and YFP tags for FRET. Binary vector pICSL86900 was used to accommodate an N-and a C-terminal tag simultaneously. Interdomain tag modules were cloned as if they were domain modules, carrying appropriate overhangs matching the domain junctions. For FRET studies, we first built level 1 constructs carrying eCFP:D456-R:YFP (pICH47732) and AvrRps4:mCherry (pICH47751), then assembled them together on the same T-DNA in a level 2 binary vector (pICSL4723) using Golden Gate assembly, and similarly with the controls 35S:mCherry, EEAA:mCh and KRVY:mCh. 

Agrobacterium-Mediated Tobacco Transient Assays (HR) Agrobacterium.

tumefaciens strains carrying denoted binary constructs were streaked on selective Lmedium agar plates and grown for 24-48 h. Cultures were grown in liquid L-medium for 24 h in a shaking incubator (200 rpm, 28°C). Cells were harvested by centrifugation and re-suspended in infiltration medium (10mM MgCl 2 , 10mM MES, pH 5.6). For coexpression, each bacterial suspension carrying individual constructs was adjusted to OD 600 =0.5 (2.5x10 8 cfu/mL) in the final mix for infiltration. Leaf sections of 5-weekold N. tabacum (tobacco) leaves were infiltrated with 1 mL needle-less syringe. HR phenotypes were photographed at 3-4 dpi. 

Immunoblot and Co-immunoprecipitation (co-IP) Assays

Protein samples were prepared from Nb leaves 2 days after Agrobacterium-mediated transformation. Plant materials were flash-frozen and ground in liquid nitrogen. Total proteins were extracted in GTEN buffer (10% glycerol, 150 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl) supplemented with 10 mM DTT, 0.2% (vol/vol) Nodinet-40, Anti-protease tablet (Complete EDTA-free Roche) and 2% PVPP (Polyvinylpolypyrrolidone). 
Homogenisation was achieved by mixing on a rotator for 20 min at 4°C. The lysates were then centrifuged for 20 min at 5000 g at 4°C, and the supernatant was filtered through 0.45 mm filters. The filtered protein extract was mixed with 3xSDS loading buffer (30% glycerol, 3.3% SDS, 94 mM Tris-HCl pH 6.8, 0.05% (vol/vol) bromophenol blue) supplemented with 10 mM DTT for SDS-PAGE (SDS-polyacrylamide gel electrophoresis) and immunoblot analyses as input samples or for Westerns. 
Immunoprecipitations were conducted on 1.5 ml of filtered extract incubated with 30 μl agarose beads (Anti-FLAG M2, Sigma; Anti-GFP, Chromotek) for 2 h at 4°C under gentle agitation. Antibodies-coupled agarose beads were then collected via centrifugation at 7000 rpm for 30 seconds. Beads were washed three times in washing buffer (GTEN buffer supplemented with 10 mM DTT, 0.2% Nodinet-40 and Antiprotease tablet), and re-suspended in SDS-loading buffer with 10 mM DTT. The immunoprecipitated samples were released from the beads and denatured by boiling at 95°C for 10 min. Proteins were separated by SDS-PAGE and analysed by immunoblotting. Tris-Glycine polyacrylamide (PAA) gels were prepared with 5% polyacrylamide for the stacking gel, and 10 or 12% polyacrylamide for resolving gels in this study. The pre-stained protein ladder (PageRuler, ThermoFisher) was used as molecular weight marker. Proteins were transferred to Immobilon-P PVDF membranes (Merck Millipore), using a semi-dry transfer apparatus supplied by Trans-Blot Turbo (Bio-Rad). Membranes were blocked for 1 h at room temperature or overnight at 4 °C in TBST (Tris-Buffered Saline with 0.1% Tween) containing 5% (w/v) non-fat dry milk. 
Membrane incubation with Horseradish Peroxidate (HRP) conjugated antibodies (Anti-FLAG M2, 1:10000 dilution, Sigma; Anti-GFP, 1:10000 dilution, Santa Cruz Biotechnology) was carried out in TBST supplemented with 5% milk by gentle agitation at room temperature for 1 h. The membrane was then rinsed 3 times in TBST (10 min), and once in TBS (Tris-Buffered Saline). Chemiluminescence detection for proteins of interest was carried out firstly by incubating the membrane with developing reagents (SuperSignal West Pico & West Femto), and then exposing it to X-ray film (Fuji) or by imaging using ImageQuant LAS 4000 (Life Sciences). For microscopic analyses, leaf sections were sampled 2 dpi with water as imaging medium. Live-cell imaging was conducted by a Leica DM6000B/TCS SP5 laserscanning confocal microscope (Leica microsystems, Bucks, UK), using a 63x water immersion objective with NA=1.2 (Leica PL-APO CS2). Fluorescence emissions were collected between 465-485 nm for CFP, 680-700 nm, 530-550 nm for YFP, and 580-620 nm for mCherry. Images acquired at laser power of 15-20% transmittance for 458 nm (HYD1), 10-15% for 514 nm (HYD1), 3-8% for 561 nm (HYD3);1024 x 1024 8-bit pixel format; scan speed 400 Hz; zoom factor 3-4. Same setting was used for each experiment. Image analyses and signal intensity quantifications were performed with FIJI 2.0.0 (http://fiji.sc/Fiji). 

Bimolecular Fluorescence Complementation (BiFC) Assays


Fluorescence Resonance Energy Transfer (FRET) Assay For transient expression,

A. tumefacien strains carrying indicated FRET constructs were used for infiltration of Nb leaf sections at OD 600 =0.5. For FRET analyses, leaf disks were sampled 2 dpi and imaged with a 63x water immersion objective with NA=1.2 (Leica PL-APO CS2) by a Leica TCS SP5 confocal microscope. For each cell, we confirmed fluorescence of the donor (CFP), acceptor (YFP) and the effector (mCherry) prior to FRET analyses. Leica FRET-AB wizard software (LAS AF 2.7.3.9723) was set up for acquisition of pre-bleach and post-bleach images, acceptor photobleaching and for FRET efficiency quantification. Settings were optimised according to (4). Briefly, in the event of FRET, the donor encounters a quenching of fluorescence due to its energy transfer to the acceptor. After bleaching the acceptor using 514 nm laser lines at 100% transmittance and 20 iterations, the donor fluorescence will be unquenched. Pre-and post-bleach scans were at laser power 8% transmittance for 458 nm (HYD1), 20% for 514 nm (PMT trans), 3% for 561 nm (HYD3); 512x512 8-bit pixel format; scan speed 400 Hz, pinhole of 111.4 um; zoom factor of 3.5-4. FRET efficiencies were quantified as FRET eff = (D post -D pre )/D post . D pre and D post are the fluorescence intensity of the donor before and after photobleaching respectively. For each nucleus, three randomly selected regions of interest (ellipse d=5 um) in the FRET efficiency heat map (FRET-AB wizard software) were averaged to represent the FRET efficiency value. 

Protein Engineering with E9 and Im9

The coding regions of RRS1TIR-NB-LRR, Im9 and D456 were PCR-amplified using primers indicted in Table S1 and cloned into pICSL010005 by Golden Gate assembly, resulting in level 0 module of RRS1-R(Im9). 
The fragments encoding enzymatically inactive E9(H103A) gene with an N-terminal 3xFLAG epitope tag and a TEV cleavage site were amplified by PCR using primers containing these sequences and then cloned into pICSL010003, resulting in level 0 module of E9. Mutants of E9 and Im9 were generated by PCR extension with primers containing the desired mutations and flanking BbsI site using Golden Gate cloning and mutated variants were verified by sequencing. TEV was amplified by PCR from the pET-30a-TEV plasmid template and then sub-cloned into binary vector pICSL86977 together with the 3×HA tag by Golden Gate cloning to generate TEV protease expression construct. For TEV protease-mediated cleavage, TEV was transiently coexpressed with RRS1-R(Im9-E9) by agroinfiltration. S2. (E) Percentage representations of cell death scores of (D) at 4 dpi. Stacked bars are colorcoded to show the proportions (%) of each cell death scale (0-5) out of the total infiltrated panels scored. Total panels scored are 9-16. 

Quantifiaction and Statistical Analysis

-R-TIR pCR8- TOPO R1-TIR-F CACCGGTCTCTAATGACCAATTGTGAAAAGGATG RRS1-R-TIR R1-TIR-R TACGGTCTCATCCAATTCGTCCAACATAAAAG RRS1-R-NB-ARC R1-NB-F CACCGGTCTCTTGGAATCTATTCGAAGCTGCT RRS1-R-NB-ARC R1-NB-R TACGGTCTCAGGTTCCACCCCAAAGTTTCTG RRS1-R-LRR R1-L-F CACCGGTCTCTAACCAAGGTAAGCAATCTCT RRS1-R-LRR R1-L-R TACGGTCTCAGTAGCTTCTCCGAGTCAGAAC RRS1-R-DOM4 R1-D4-F CACCGGTCTCTCTACCTATGCATTACAAGTTC RRS1-R-DOM4 R1-D4-R TCCGGTCTCACTTTTTTGGTACGTCAGACA RRS1-R-DOM5 R1-W-F CGCCGGTCTCTAAAGTAAGTTCTCTTTTTCTATC RRS1-R-DOM5 R1-W-R TCCGGTCTCAATGGTTATGCTCAGATAGGTA RRS1-R-DOM6 R1R-D6-F CAAGGTCTCTCCATCCACGGCCCACTAAA RRS1-R-DOM6 R1R-D6-R TAGGTCTCACGAACCATAATCGAAGAATGTTGACCAAGG RRS1B-TIR R1B-TIR-F CACCGGTCTCTAATGATCGAGAGTGAGCAAATC RRS1B-TIR R1B-TIR-R TCCGGTCTCATCCAATTCGTTCCATATAA RRS1B-NB-ARC R1B-NB-F CACCGGTCTCTTGGAATCTACTCGAAGCTG RRS1B-NB-ARC R1B-NB-R TACGGTCTCAGGTTCCACTCCAAAGTTTCT RRS1B-LRR R1B-L-F CACCGGTCTCTAACCAAGGTAAGCAATCTCA RRS1B-LRR R1B-L-R TACGGTCTCAGTAGCTTCTTGAAGTCCAAAC RRS1B-DOM4 R1B-D4-F CACCGGTCTCTCTACCAGTGCATTACACATT RRS1B-DOM4 R1B-D4-R TACGGTCTCACTTTCGGCGACTCTTTGAAC RRS1B-DOM5 R1B-D5-F CGCCGGTCTCTAAAGTATGTTGCTTAGTTT RRS1B-DOM5 R1B-D5-R TACGGTCTCAATGGTTATGCTCAGAGATGT RRS1B-DOM6 R1B-D6-F CAAGGTCTCTCCATCCATTCCCCACTCTAC RRS1B-DOM6 R1B-D6-R TAGGTCTCACGAACCTCTATTCAGAATTTTACTAG RPS4-TIR R4-TIR-F CACCGGTCTCTAATGGAGACATCATCTATTTCC RPS4-TIR R4-TIR-R TATGGTCTCATCCCTCCGGTGGTATTCC RPS4-NB-ARC R4-N-F CAATGGTCTCTGGGAAGTCACAACGCCGT RPS4-NB-ARC R4-N-R TGCCGGTCTCAGTGTGTCCTATTACAGAAAA RPS4-LRR R4-L-F CAACGGTCTCTACACCATGCTTAAGGTGGGT RPS4-LRR R4-L-R TCCGGTCTCATGGAACTGATGTAAGACTCGT RPS4-CTD R4-C-F CACCGGTCTCTTCCAGAGTTTCCACCAAAT RPS4-CTD R4-C-R TATGGTCTCACGAACCGAAATTCTTAACCGTGTG RPS4-TIR R4-TIR-F CAAAGGTCTCTAATGGCGGCGTCGTCGTCCT RPS4-TIR R4-TIR-R TACGGTCTCATCCCTCCAATGAAACTTTCTTCA RPS4-NB-ARC R4-N-F CAAGGTCTCTGGGAAGCCAAAAGGTGGTGTC RPS4-NB-ARC R4-N-R TACCGGTCTCAGTGTATCCTGAGCAGTAAAA RPS4-LRR R4-L-F CACCGGTCTCTACACCAGTCTTAAAATGGGT RPS4-LRR R4-L-R TACCGGTCTCATGGAACATATGTAAGCTTCG RPS4-CTD R4-C-F CAAAGGTCTCTTCCAGAGTTGCCACCAACTC RPS4-CTD R4-C-R TACCGGTCTCACGAACCACTAGTGACTTTATCTTCTA WRKY41 W41-F CACCGGTCTCAGGGAGATATCTTCAGCTGGAG WRKY41 W41-R CACCGGTCTCACGAACCAATCGAATTGTGGAAAAAAG Cloning for expressing individual domains RRS1-R-NB-ARC pICSL 86977 R1-NB-F CACGGTCTCTAATGATCTATTCGAAGCTGCTG RRS1-R-NB-ARC R1-NB-R TAGGTCTCACGAACCGGTTCCACCCCAAAGTTT RRS1-R-LRR R1-L-F CACGGTCTCTAATGAAGAACCTGGAGATGTTGAGG RRS1-R-LRR R1-L-R TAGGTCTCACGAACCAAGCTTCTCCGAGTCAGAAC RRS1-R-DOM4 R1-D4-F CACCGGTCTCTAATGCCTATGCATTACAAGTTCAAC RRS1-R-DOM4 R1-D4-R TAGGTCTCACGAACCCTTTTTTGGTACGTCAGACA RRS1-R-DOM5 R1-D5-F CGCCGGTCTCTAAAGTAAGTTCTCTTTTTCTATC RRS1-R-DOM5 R1-D5-R TACGGTCTCACGAACCATGGTTATGCTCAGATAGG RRS1-R-DOM6 R1R-D6-F CAGGTCTCTAATGCCACGGCCCACTAAACGC RRS1-R-DOM6 R1R-D6-R TAGGTCTCACGAACCATAATCGAAGAATGTTGACCAAGG RRS1-S-DOM6 R1S-D6-R TAGGTCTCACGAACCGCAGATGGAGGAGGAAGTGGAAC RRS1B-DOM4 R1B-D4-F CACCGGTCTCTAATGGTGCATTACACATTTAGTAATTG RRS1B-DOM4 R1B-D4-R TAGGTCTCACGAACCCTTTCGGCGACTCTTTGAA RRS1B-DOM5 R1B-D5-F CACGGTCTCTAATGAACAACAAGGAAAAGAGAGT RRS1B-DOM5 R1B-D5-R TAGGTCTCACGAACCATGGTTATGCTCAGAGATGT RRS1B-DOM6 R1B-D6-F CAAGGTCTCTAATGCCATTCCCCACTCTACGCA RRS1B-DOM6 R1B-D6-R TAGGTCTCACGAACCTCTATTCAGAATTTTACTAG RPS4-CTD R4-C-F CACCGGTCTCTTCCAGAGTTTCCACCAAAT RPS4-CTD R4-C-R TATGGTCTCACGAACCGAAATTCTTAACCGTGTG RPS4B-CTD R4B-C-F CAAAGGTCTCTTCCAGAGTTGCCACCAACTC RPS4B-CTD R4B-C-R TACCGGTCTCACGAACCACTAGTGACTTTATCTTCTA Cloning effectors AvrRps4 pICH 86988 A4-F GAGGGTCTCAAATGACTCGAATTTCAACCAGTTCAG AvrRps4 A4-R GAGGGTCTCACGAACCTTGGTTGATTCTGCGGTCTT PopP2 pICSL 86977 P2-F TAGGTCTCTAATGAAGGTCAGTAGCGCAAAC PopP2 P2-R CAGGTCTCACGAACCGTTGGTATCCAATAGGG Cloning for E9/Im9 protein engineering RRS1-TIR-NB-LRR pICSL 01005 D123-F AATGAAGACATAATGACCAATTGTGAAAAGGAT RRS1-TIR-NB-LRR D123-R AATGAAGACATCAAGCTTCTCCGAGTCAGAACCATG Im9 Im9-F AATGAAGACATAGATCCGCCCTGTTTAAATCCTGACTTA Im9 Im9-R AATGAAGACATCGACCCGCCCTGTTTAAATCCTGACTTA RRS1-R-D456 D456-F AATGAAGACATATCTCCTATGCATTACAAGTTCAA RRS1-R-D456 D456-R AATGAAGACATCGAACCATAATCGAAGAATGTTGACC E9 pICSL 01003 E9-F AATGAAGACATGGGTGGTTCTATGGAGAGTAAACGGAATAAG E9 E9-R AATGAAGACATAAGCTTACTTACCTCGGTGAATATC 3xFLAG_TEV cleavage cite 3F_TEV-F AATGAAGACATTTCGGATTATAAGGACCATGACGGAGACTATA AG 3xFLAG_TEV cleavage cite 3F_TEV-R AATGAAGACATACCCTGAAAATACAAATTCTCCTTATCGTCATC 



  For transient expression, A. tumefacien strains carrying indicated BiFC constructs were used for infiltration of Nb leaf sections at OD 600 =0.5. D456-R or full-length RRS1-R was tagged with the C-terminal part of CFP (cCFP) as a bait, and the N-terminal part of either Venus (nVenus) or Cerulean (nCerulean) as a prey respectively. Interaction of the cCFP with nVenus generates yellow fluorescence (YFP), and interaction of cCFP with nCerulean generates blue fluorescence (CFP)(3). Effector proteins (or their mutant controls) tagged with mCherry were co-expressed with the R proteins in the BiFC assays. All control experiments (effector mutants or mCherry) were conducted on different sections of the same leaf, with each experiment replicated independently for at least 3 times. 




  Fig. S5, 6 ,7) and statistical analysis (Fig. 5, 6, Fig. S5 , 6). For stacked bars representing HR scoring proportions, number of total leaf panels scored are indicated in figure legends. Each sample combines data from at least three independent biological replicates. For microscopy analyses, each data point shown in the boxwhisker plots represent a signal cell analysis. Number of biological replicates for each experiment are shown in the figures. Due to unbalanced design in some experiments (unequal number of observations for each treatment), linear mixed-effects model (R package lme) were used to compare the means. For multiple pairwise comparisons, general linear hypotheses (glht, R multicomp package) with Tukey comparisons of means were used. Significance was indicated in the figures: means with the same letter are not significantly different (P<0.001). P-values for all pairwise comparisons for Fig. 5, 6 are summarised in TableS2. 




  Fig. S1. RRS1 WRKY domain (DOM5) is required for autoinhibition and domain 4 (DOM4) for activation of the RRS1/RPS4 complex. (A) Schematic diagrams of domain structures for RPS4, RRS1-R, RRS1-S, RPS4B and RRS1B. Amino acid (aa) numbers indicate the boundaries for domain swaps, deletions and domain expression in this study. See also Table S1. (B) Sequence alignment of the WRKY domain and C-terminal amino acids of RRS1-R and RRS1B and WRKY41. D56 of RRS1-R and RRS1B is highlighted in blue (DOM5= WRKY) and yellow (DOM6). Sequence of WRKY41 displayed was used to replace D56 of RRS1-R in RRS1-R (AAAAW41). (C) Replacement of the DOM5 and/or DOM6 of RRS1B causes RPS4B-dependent autoactivity. Each tobacco leaf section was infiltrated to express a chimeric RRS1B with or without RPS4B. HRs were assessed at 4 dpi. Photos shown are representative of 3 consistent replicates. (D) Immunoblots show protein accumulations of truncated RRS1-R and RRS1-R chimeras in the absence of RPS4 after transient expression in Nb leaves at 2 dpi. All proteins are tagged with HF. Ponceau S staining of the large subunit of RuBisCo provides the loading control. (E) Co-IP assays to evaluate the association of D56-R:GFP with HF-tagged TIR, NBARC, LRR and DOM4 after transient co-expression in Nb leaves. All domains are from RRS1-R. GUS:HF was used as a control. Immunoblots show protein accumulation in total extracts (input) and after IP with anti-FLAG beads (IP-FLAG). Asterisks mark protein bands that indicate associations. (F) Co-expression of RRS1-R fragments in trans cannot reconstitute the activity of intact proteins. Each leaf section was co-infiltrated to express in trans two RRS1-R fragments together with RPS4, assessing their activity and AvrRps4 responsiveness. HRs were assessed at 4 dpi. Photos shown are representative of 3 consistent replicates. 


Fig. S5 .

 S5 Fig. S4. Interactions between RPS4 CTD and RRS1 DOM4 are not influenced by mutations or domain swaps that compromise RPS4/RRS1 function. (A) Co-IP assays to assess associations between CTD:HF from RPS4 (A) or RPS4B (B) and DOM4:GFP from RRS1 (A) or RRS1B after transient co-expression in Nb leaves. GUS:HF was used as a control. Both CTDs co-IP with both DOM4s, and domains from matching pairs do not show stronger association affinity compared to from non-matching pairs. (B) Co-IP assays to assess associations of CTD:HF and DOM4:GFP where one or both carry a mutation (in red). All CTD and DOM4 mutants co-IP with similar affinity compared to their wild type (WT) counterparts. Immunoblots show protein accumulations in total extracts (input) and after IP-GFP. Asterisks mark bands that indicate associations. (C) Co-IP assays reveal effector interference of RPS4 CTD:GFP and RRS1-R D456-R:HF associations. Effectors are tagged with mCh. Controls include AvrRps4 mutant EEAA, PopP2 mutant C321A, and mCherry. Immunoblots show protein accumulation in input and after IP-FLAG or IP-GFP. Asterisks mark bands that indicate association. 


Fig. S6 .

 S6 Fig. S6. BiFC and FRET analyses reveal more 'open' conformations of D456(ABA) and D456(ABB) compared to D456-R(AAA). (A-B) BiFC signals of cCFP:D456(ABA):nCer, cCFP:D456(ABB):nCer and cCFP:D456(BBB):nCer are significantly lower than cCFP:D456-R(AAA):nCer after transient expression in Nb leaves at 2 dpi. nCer; nCerulean. CFP signal indicates cCFP/nCer interactions upon close proximity of the tagged domains. BF, bright field. Representative images are shown (A). Box plots show quantifications of CFP signals (D). Signal intensity was quantified as average grey value of each nuclei and then normalized to the mean of the control sample (AAA) within each biological replicate. Data points, color-coded for different biological replicates, represent Log 10 of the normalized values. Statistical analysis used linear mixed-effects model (lme), followed by tests for general linear hypotheses (glht) with Tukey multiple comparisons. Means with the same letter are not significantly different (P<0.001). (C) FRET efficiencies of eCFP:D456(ABA):YFP and eCFP:D456(ABB):YFP are significantly lower than eCFP:D456(AAA):YFP after transient expression in Nb leaves at 2dpi. Data points, pooling several biological replicates (color-coded), each represent a single cell FRET efficiency (%) quantified by FRET-AB. Statistical analysis used linear mixed-effects model (lme), followed by tests for general linear hypotheses (glht) with Tukey comparisons. Means with the same letter are not significantly different (P<0.001). 




  Forward primer) R (Reverse primer). b. Name of primers with underlined domains indicate purpose of cloning for individual domain expression, which differs from cloning for domain modules for full length R gene assembly. 


Table S1 . Primers used for plasmid construction in this study

 S1 . F (Forward primer) R (Reverse primer). Name of primers with underlined domains indicate purpose of cloning for individual domain expression, which differs from cloning for domain modules for full length R gene assembly. 



Table S2 . P-values of pairwise comparisons tested by general linear hypothesis.

 S2 
	Significance codes: *** P< 0.001, ** P< 0.01, * P< 0.05.

Table S1 . Primers used for plasmid construction in this study. Related to Figure 1-7

 S1 
	Purpose	Gene/ Fragments Vector	Name	Sequence (5'-3')
		RRS1		
	Cloning			
	domain			
	modules for			
	full length R			
	gene			
	assembly			

Table S2 . P-values of pairwise comparisons tested by general linear hypothesis. Related to Figure 5 and 6.

 S2 
	Figure	Pairwise comparisons	P-value	Significance code
		EEAA -AvrRps4	< 0.001	***
		KRVY -AvrRps4	0.00115	**
	5B	mCh -AvrRps4 KRVY -EEAA	< 0.001 0.26617	***
		mCh -EEAA	< 0.001	***
		mCh -KRVY	< 0.001	***
		EEAA -AvrRps4	0.698	
		KRVY -AvrRps4	0.976	
	5C	mCh -AvrRps4 KRVY -EEAA	< 0.001 0.939	***
		mCh-EEAA	0.019	*
		mCh -KRVY	< 0.001	***
		EEAA -AvrRps4	< 0.001	***
		KRVY -AvrRps4	< 0.001	***
	5F	mCh -AvrRps4 KRVY -EEAA	< 0.001 0.9270	***
		mCh-EEAA	0.0289	*
		mCh -KRVY	0.1270	
		EEAA -AvrRps4	0.873	
		KRVY -AvrRps4	0.199	
	5G	mCh -AvrRps4 KRVY -EEAA	< 0.001 0.651	***
		mCh-EEAA	< 0.001	***
		mCh -KRVY	< 0.001	***
		PopP2 -mCh	0.4071	
	5I D456-R	C321A -mCh	0.0023	**
		C321A -PopP2	0.0494	*
		PopP2 -mCh	0.31024	
	5I D456-S	C321A -mCh	0.00735	**
		C321A -PopP2	0.16788	
		PopP2 -mCh	< 1x10 -4	***
	5J D456-R	C321A -mCh	< 1x10 -4	***
		C321A -PopP2	0.523	
		PopP2 -mCh	< 1x10 -4	***
	5J D456-S	C321A -mCh	< 1x10 -4	***
		C321A -PopP2	0.555	
		EEAA -AvrRps4	<0.001	***
		KRVY -AvrRps4	0.193	
	6C	mCh -AvrRps4 KRVY -EEAA	<0.001 0.101	***
		mCh-EEAA	0.751	
		mCh -KRVY	<0.001	***
	a. Significance codes: *** P< 0.001, ** P< 0.01, * P< 0.05.
Yan Ma, Hailong Guo, Lanxi Hu, Paula Pons Martinez, Panagiotis N Moschou, Volkan Cevik, Pingtao Ding, Zane Duxbury, Panagiotis F Sarris, Jonathan D G Jones
Distinct modes of derepression of an <i>Arabidopsis</i> immune receptor complex by two different bacterial effectors