structure-function analysis of zar1 immune …structure-function analysis of zar1 immune receptor...

Structure-function analysis of ZAR1 immune receptor reveals key molecular interactions for 1

activity 2

3

Running title: ZAR1 molecular interactions 4

5

6

Maël Baudin1, Karl J. Schreiber1†, Eliza C. Martin2†, Andrei J. Petrescu2 and Jennifer D. 7

Lewis1,3* 8

9

1Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, USA 10

2Department of Bioinformatics and Structural Biochemistry, Institute of Biochemistry of the 11

Romanian Academy, Bucharest, Romania 12

3Plant Gene Expression Center, United States Department of Agriculture, Albany, USA 13

14

†These authors contributed equally 15

*Corresponding author: [email protected] 16

17

18

One sentence-summary 19

Structure-informed analyses reveal multiple finely-tuned intramolecular interactions that 20

regulate the activity of the immune receptor ZAR1. 21

22

Author contributions 23

MB, ECM, AJP, KJS and JDL designed research. MB performed in planta experiments. 24

KJS performed yeast experiments. ECM and AJP contributed new analytic/computational tools. 25

MB, ECM, AJP, KJS and JDL analyzed data. MB, AJP, KJS and JDL wrote the paper. 26

27

Funding 28

Research on plant immunity in the Lewis laboratory was supported by the USDA ARS 29

2030-21000-046-00D and 2030-21000-050-00D (JDL), and the NSF Directorate for Biological 30

certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was notthis version posted March 29, 2019. . https://doi.org/10.1101/592824doi: bioRxiv preprint

https://doi.org/10.1101/592824

Sciences IOS-1557661 (JDL). ECM and AJP acknowledge financial support from UEFISCDI 31

grant PN-III-ID-PCE-2016-0650 and the Romanian Academy programs 1 & 2 of IBAR. 32

33


https://doi.org/10.1101/592824

ABSTRACT 34

NLR (Nucleotide-binding [NB] Leucine-rich repeat [LRR] Receptor) proteins are critical 35

for inducing immune responses in response to pathogen proteins, and must be tightly regulated to 36

prevent spurious activation in the absence of a pathogen. The ZAR1 NLR recognizes diverse 37

effector proteins from Pseudomonas syringae, including HopZ1a, and Xanthomonas species. 38

Receptor-like cytoplasmic kinases (RLCKs) such as ZED1, interact with ZAR1 and provide 39

specificity for different effector proteins, such as HopZ1a. We previously developed a transient 40

expression system in Nicotiana benthamiana, that allowed us to demonstrate ZAR1 function is 41

conserved from the Brassicaceae to the Solanaceae. Here, we combined structural modeling of 42

ZAR1, with molecular and functional assays in our transient system, to show that multiple 43

intramolecular and intermolecular interactions regulate ZAR1 activity. We identified new 44

determinants required for the formation of the ZARCC dimer and its activity. Lastly, we 45

characterized new intramolecular interactions between ZAR1 subdomains that participate in 46

keeping ZAR1 immune complexes inactive. This work identifies molecular constraints on 47

immune receptor function and activation. 48

49


https://doi.org/10.1101/592824

INTRODUCTION 50

The plant immune system activates rapid and highly effective defense mechanisms upon 51

detection of pathogens in the vicinity of host cells (Jones and Dangl, 2006; Wu et al., 2018). 52

Plants detect highly conserved pathogen-associated molecular patterns (PAMPs) using cell 53

surface-localized pattern recognition receptors (PRRs), in a process called Pattern Triggered 54

Immunity (PTI) (Segonzac and Zipfel, 2011; Monaghan and Zipfel, 2012). Many 55

phytopathogenic bacteria encode a molecular syringe called the type III secretion system, which 56

injects effector proteins into the plant cell (Galán and Wolf-Watz, 2006). Effector proteins 57

primarily function to suppress PTI and promote bacterial virulence (Mudgett, 2005; Grant et al., 58

2006; Block et al., 2008; Lewis et al., 2009; Deslandes and Rivas, 2012; Xin and He, 2013; 59

Macho, 2016). Plants sense pathogen activities through the cytoplasmic detection of effector 60

proteins by a family of proteins that contain a nucleotide-binding-Apaf1-R protein-CED4 61

(NBARC) domain and a leucine-rich repeat domain (LRR) and are termed NLR proteins 62

(Schreiber et al., 2016a; Khan et al., 2017; Urbach and Ausubel, 2017; Jones et al., 2016). 63

Effector-triggered immunity (ETI) is particularly strong and often results in a form of localized 64

programmed cell death called the hypersensitive response (HR) (Heath, 2000). 65

Plants encode numerous NLR genes, which use different well-characterized strategies to 66

protect plants against pathogens (Kourelis and van der Hoorn, 2018) (Cesari, 2018). The most 67

straightforward way of detecting the effector is the physical binding of the NLR protein with the 68

microbial effector following a ligand-receptor model (Cesari, 2018). However, in most cases, the 69

recognition is indirect and involves another host protein called the “guardee” that is monitored 70

by the NLR protein. The “guardee” can be a virulence target that is modified by the effector to 71

promote disease in the absence of the NLR protein (the guard model) (Khan et al., 2016; Dangl 72

and Jones, 2001; Van Der Biezen and Jones, 1998; Schreiber et al., 2016a). In some cases, the 73

plant evolves a protein that mimics a virulence target (a “decoy”) to trap the effector; here the 74

decoy is not targeted by the pathogen to promote host susceptibility in the absence of the NLR 75

protein (decoy model) (van der Hoorn and Kamoun, 2008; Cesari, 2018; Schreiber et al., 2016a). 76

In addition, some NLR proteins encode an additional domain that mimics the effector target 77

(integrated domain/decoy model) (Kroj et al., 2016; Baggs et al., 2017). 78

In addition to the NBARC and LRR domains, NLRs typically contain an additional N-79

terminal domain, a Toll/interleukin1 receptor (TIR) or coiled coil (CC) domain, which roughly 80


https://doi.org/10.1101/592824

define two main groups of NLRs (McHale et al., 2006; Meyers et al., 2003). These N-terminal 81

domains are often associated with triggering downstream signaling as the overexpression of 82

several CC and TIR domains alone induces a HR (Frost et al., 2004; Swiderski et al., 2009; 83

Williams et al., 2014; Wróblewski et al., 2018). HR activation by CC or TIR domains has been 84

correlated, in some cases, with homodimerization, which has also been observed in mammalian 85

NLRs (Maekawa et al., 2011; Bernoux et al., 2011; Bentham et al., 2017; Zhang et al., 2017; 86

Jones et al., 2016). The NBARC domain acts as a molecular switch, binding ADP in the inactive 87

state and ATP in the active state of the protein (Wendler et al., 2012; Sukarta et al., 2016). 88

Mutations that affect the nucleotide-bound state of the NBARC domain often result in auto-89

activity or loss of function, which demonstrates the crucial role of this domain (Tameling et al., 90

2006; Tameling et al., 2002; Bendahmane et al., 2002; DeYoung and Innes, 2006). The C-91

terminal LRR domain interacts with the effector in the case of direct recognition (Schreiber et 92

al., 2016b; Ravensdale et al., 2012), or with the “guardee” in cases of indirect recognition 93

(Baudin et al., 2017; Qi et al., 2012; Collier and Moffett, 2009). Since NLR-activated 94

downstream signaling often results in cell death and a high fitness cost for the plant, NLRs need 95

to be tightly regulated in the absence of pathogens. NLRs are maintained in an “off” state by 96

intra-molecular interactions (Takken and Goverse, 2012), including between the ARC2 97

subdomain of the NBARC and the N-terminal part of the LRR (Slootweg et al., 2013; Sukarta et 98

al., 2016; Rairdan et al., 2008; Ade et al., 2007; Leister et al., 2005), and between the CC or TIR 99

domains and the NBARC domain (Wang et al., 2015a; Qi et al., 2012; Moffett et al., 2002). 100

Structure-function analyses of several receptors led to the development of models for NLR 101

activation based on the equilibrium between the ADP and ATP bound states (Bernoux et al., 102

2016; Takken and Goverse, 2012; Sukarta et al., 2016; Zhang et al., 2017). Structural modeling 103

based on the crystal structures of CC, NBS and LRR domain homologues helps provide insights 104

into the conformational changes that occur during NLR activation (Takken and Goverse, 2012; 105

Slootweg et al., 2013). 106

HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) is a canonical CC-type NLR that was 107

first identified in Arabidopsis thaliana, and is involved in the immune responses against HopZ1a 108

and HopF2a, effectors from the phytopathogenic bacteria Pseudomonas syringae (Lewis et al., 109

2010; Seto et al., 2017) and AvrAC, an effector from the phytopathogenic bacteria Xanthomonas 110

campestris (Wang et al., 2015b). Recognition of diverse effectors is possible due to the 111


https://doi.org/10.1101/592824

association of ZAR1 with Receptor-Like Cytoplasmic Kinases (RLCK) that are encoded in a 112

genomic cluster in Arabidopsis and that serve as adaptors/baits for the effectors (Lewis et al., 113

2013; Wang et al., 2015b; Seto et al., 2017). HopZ1a triggers a strong HR, that depends on the 114

RLCK ZED1 (HOPZ-EFFECTOR-TRIGGERED IMMUNITY DEFICIENT 1) and ZAR1 115

(Lewis et al., 2008, 2010, 2013; Baudin et al., 2017). HopZ1a acetylates ZED1 which is 116

hypothesized to trigger ZAR1 activation, likely through conformational changes (Lewis et al., 117

2013; Baudin et al., 2017). The recognition of AvrAC and HopF2a is more complex and involves 118

other proteins that mediate a connection between the effector and the ZAR1-RLCK complex 119

(Wang et al., 2015b; Seto et al., 2017). In the case of AvrAC, the effector interacts with and 120

uridylylates the PBL2 (PBS1-LIKE 2) kinase which then interacts with the RLCK RKS1 121

(RESISTANCE RELATED KINASE 1) and ZAR1 (Wang et al., 2015b). For HopF2a, the ZRK3 122

(ZED1-RELATED KINASE 3) RLCK does not interact directly with the effector, which 123

suggests that another RLCK like PBL2 might be involved (Seto et al., 2017). N. benthamiana 124

ZAR1 (NbZAR1) was recently identified together with the RLCK JIM2 (XOPJ4 IMMUNITY 2) 125

as being required for the recognition of the Xanthomonas perforans effector XopJ4, which is a 126

member of the HopZ/YopJ superfamily of effector proteins (Lewis et al., 2009; Schultink et al., 127

2019). Spurious activation of ZAR1 is, in part, controlled by intermolecular interactions with 128

ZED1/RKS1/ZRK3/JIM2 and intramolecular interactions within ZAR1 (Baudin et al., 2017). 129

The downstream signaling activated by ZAR1 is still uncharacterized, and acts independently of 130

most pathways identified so far (Lewis et al., 2010; Macho et al., 2010). 131

In this paper, we used structural modeling together with molecular assays and functional 132

studies to decipher the interactions and conformational changes that regulate ZAR1 activity. We 133

previously established a N. benthamiana transient expression system that allowed us to evaluate 134

the contributions of ZAR1 and ZED1 to HopZ1a recognition and demonstrated that ZAR1-135

mediated recognition is conserved from the Brassicaceae to the Solanaceae (Baudin et al., 2017). 136

We took advantage of this system to evaluate the impact of domain truncations and loss-of-137

function mutations on ZAR1 intramolecular interactions and activity. Finally, our interdomain 138

contact data provide new insights into our understanding of ZAR1 architecture. This work will 139

help identify the constraints on immune receptor function and activation. 140

141

RESULTS 142


https://doi.org/10.1101/592824

143

Model-based identification of ZAR1CC structural determinants for homodimerization 144

We previously showed that the overexpression of the CC domain of ZAR1 (ZAR1CC 145

amino acids 1-144) induces a HR in N. benthamiana when ZAR1CC was cloned as an in-frame 146

fusion to YFP (Baudin et al., 2017). We further demonstrated that ZAR1CC and a shorter version 147

of ZAR1CC (ZAR1CC2, amino acids 1-121) homodimerize in planta (ZAR1CC) and in yeast 148

(ZAR1CC or ZAR1CC2) (Baudin et al., 2017). The CC domains of several NLRs, including 149

MLA10 (MILDEW LOCUS A 10) from Hordeum vulgare (barley) and Rp1 (RESISTANCE to 150

PUCCINIA 1) from Zea mays (maize), also homodimerize and are autoactive when expressed 151

alone or as a fusion with YFP (Maekawa et al., 2011; Wang et al., 2015a; Bai et al., 2012). 152

To better understand the structural dynamics that lead to ZAR1CC dimerization and 153

autoactivation, we examined the effects of mutations introduced into critical parts of the CC 154

structure identified by molecular modeling. Based on Rx [RESISTANCE to PVX, Solanum 155

tuberosum (potato)] and Sr33 [STEM RUST 33, Triticum aestivum (wheat)] templates, ZAR1CC 156

can be modeled in the monomeric state as a four short helices bundle structure interconnected by 157

three turns (1CC4α: α1a-t1-α1b-t2-α2-t3-α3) (Figure 1A, S1A, S1B). Based on the MLA10 158

template, the dimeric state can be modeled as two intertwined long helices, where the second 159

helix is broken by a short coil break, separated by only one central turn (2CC2α: α1-t2-α2-c-α3) 160

(Figure 1A, S1B). The two peripheral short helices of 1CC4α (α1a and α3) perfectly 161

superimpose the beginning of α1 and α3 in the second monomer of the dimer (Casey et al., 2016) 162

(Figure 1A). This suggests that the regions corresponding to t1 and t3 in the 1CC4α structure 163

might play a role in dimerization. In addition, the first helix in the 1CC4α monomeric state is the 164

only helix region of the CC domain that is not constrained at both ends by the rest of ZAR1, and 165

is free to move at its N-terminal end. Therefore, its hydrophobic zipping to the rest of the CC 166

domain, and the propensity of α1a and α1b to form a turn or a helix might be critical in 167

dimerization or protein-protein interactions. 168

To better understand the role of different motifs in the CC domain, we generated five 169

versions of ZAR1CC (hereafter ZAR1CCH1, ZAR1CCH2a, ZAR1CCH2b, ZAR1CC2, ZAR1CCED) 170

(Figure 1B, S1C). ZAR1CCH1 has mutations in the lateral loops, in which the amino acids that are 171

predicted to form salt bridges have been mutated. To reduce the probability of transient 172

interactions between the two monomers at the α1a-α1b/α2-α3 site, we introduced glutamine 173


https://doi.org/10.1101/592824

mutations which eliminated the favorable charge-charge interaction network between two 174

monomers in that region. For ZAR1CCH2a and ZAR1CCH2b, hydrophobic amino acids of the first 175

helix (α1a) were replaced to investigate whether increased mobility at α1a-α1b region affects the 176

mechanism of dimerization. For ZAR1CCH2a, the first loop (t1) was modified to increase the local 177

helix propensity and stabilize the interaction. As in the MLA10 dimeric structure, this region 178

corresponds to an uninterrupted α1a-α1b helix. For ZAR1CCH2b, the loop region between t1a and 179

t1b was modified to be less prone in adopting a helical fold and therefore should be destabilizing. 180

ZAR1CCED targets the conserved EDVID motif (amino acids 73-77), which is found in many CC-181

type NLRs (Bai et al., 2012; Rairdan et al., 2008; Baudin et al., 2017; El Kasmi et al., 2017) 182

(Figure S1A, S1C). Finally, we generated a shorter version of the CC domain, ZARCC2 that can 183

still dimerize in yeast (Baudin et al., 2017). 184

To independently test the dimerization capacity of each construct, we conducted yeast 185

two-hybrid assays (Y2H). We cloned the five versions of ZAR1CC as a fusion to the activation 186

domain or DNA-binding domain in the LexA yeast two-hybrid system. For each combination, 187

the same optical density of yeast was spotted on the plates to quantitate the interaction strength. 188

The ZAR1CCH1, ZAR1CCH2a, and ZAR1CCED mutations showed similar levels of yeast growth that 189

were weaker than that of wild type ZAR1CC (Figure 1C). The ZAR1CCH2b mutations had the most 190

substantial effect on yeast growth compared to the other constructs (Figure 1C). Mutation of the 191

EDVID motif only slightly reduced the interaction strength, indicating that this motif is not 192

absolutely required for dimerization (Figure 1C). ZAR1CC did not interact with an unrelated 193

NLR, At5g48620, indicating that homodimerization is specific (Figure 1C). The lack of 194

interactions was not due to a lack of expression (Figure S2). 195

Our data indicate that CC dimerization is impacted by mutations which affect charge-196

charge interactions around t1 and t3 in ZAR1CCH1, or by disrupting the hydrophobic zipping in 197

α1a in ZAR1CCH2a and ZAR1CCH2b. The mutations in ZAR1CCH2b that increase local flexibility of 198

the α1a-α1b region have a stronger effect on shifting the equilibrium towards the monomeric 199

state, compared to the mutations in ZAR1CCH2a that increase the helical propensity within the 200

same region. 201

202

Impact of ZAR1CC architecture on its activity 203


https://doi.org/10.1101/592824

The ability of ZAR1CC to induce autoactivity is dependent on the presence of a YFP 204

epitope, suggesting that the weak dimerization capacity of YFP stabilizes ZAR1CC 205

oligomerization (Baudin et al., 2017). However, no clear correlation between ZAR1CC 206

dimerization and autoactivity has been demonstrated so far. We took advantage of the mutations 207

we generated in the ZAR1CC domain (Figure 1B) to test their capacity for autoactivity as a 208

ZAR1CC-YFP fusion. When over-expressed in N. benthamiana leaves, the YFP fusions of 209

ZAR1CCH1, ZAR1CCH2a, ZAR1CCH2b, ZAR1CC2 and ZAR1CCED did not lead to autoactivity, while 210

wild type ZAR1CC-YFP still induced autoactivity (Figure 2A). The absence of a HR-like 211

phenotype was not due to the absence of expression as all constructs are equally expressed 212

(Figure 2B). Consistent with reduced dimerization in ZAR1CCH1or ZAR1CCH2a, or even less 213

dimerization in ZAR1CCH2b, mutations in all of these constructs disrupted autoactivity of the CC 214

domain. Interestingly, both ZAR1CC2-YFP and ZAR1CCED-YFP were also unable to induce 215

autoactivity (Figure 2A), even though ZAR1CC2 could dimerize like wild type and ZAR1CCED 216

dimerized poorly (Figure 1C). These data indicate that the C-terminal portion of the CC domain 217

and the EDVID motif are required to activate downstream signaling, and that dimerization is not 218

sufficient to activate downstream signaling. 219

To analyze the effect of these ZAR1CC mutations on ZAR1 function, we introduced the 220

ZAR1CC mutations into full-length ZAR1 (hereafter ZAR1H1, ZAR1H2a, ZAR1H2b, and ZAR1ED) 221

and tested them for complementation of NbZAR1 silencing in N. benthamiana as previously 222

described (Baudin et al., 2017). HopZ1a and ZED1 were co-expressed with the ZAR1 mutants, 223

ZAR1 as a positive control, or empty vector as a negative control, in N. benthamiana leaves 224

silenced for NbZAR1 by virus-induced gene silencing (VIGS). To quantify the HR intensity, and 225

thus the complementation capacity of each construct, we measured ion leakage into the media 226

over time. Wild type ZAR1 showed high levels of conductivity (Figure 2C), as previously 227

observed (Baudin et al., 2017). ZAR1H1 showed moderate conductivity that was weaker than and 228

statistically different from wild type ZAR1 (Figure 2C). The ZAR1H2a, ZAR1H2b and ZAR1ED 229

constructs resulted in conductivity measurements that were similar to empty vector (Figure 2C). 230

Therefore, ZAR1H1 was able to partially complement NbZAR1-VIGS while ZAR1H2a, ZAR1H2b 231

and ZAR1ED did not display any complementation of NbZAR1-VIGS (Figure 2C). The 232

differences observed in the conductivity assay were not due to differences in expression level as 233

all the ZAR1 constructs accumulated to similar levels (Figure 2D). 234


https://doi.org/10.1101/592824

To investigate if ZAR1CC-YFP-induced autoactivity requires the presence of the 235

endogenous NbZAR1, we expressed ZAR1CC-YFP in silenced NbZAR1-VIGS or control GUS-236

VIGS plants. We observed a strong HR in both GUS-VIGS and NbZAR1-VIGS plants indicating 237

that endogenous NbZAR1 is not needed for ZAR1CC-YFP autoactivity (Figure S3). VIGS 238

silencing was efficient because co-expressed HopZ1a and ZED1 were not able to induce the HR 239

in NbZAR1-VIGS plants while they showed a strong HR in GUS-VIGS plants (Figure S3). 240

Taken together, these data indicate that the first helix (α1a) and the EDVID motif of 241

ZAR1CC are particularly important as mutations in these regions lead to a non-functional ZAR1 242

protein. The mutations in the ZAR1H1 construct have a less significant impact, as ZAR1CCH1 was 243

still able to dimerize and ZAR1H1 was still able to partially complement NbZAR1-VIGS. In 244

addition, ZAR1CC-YFP autoactivity is independent of NbZAR1. 245

246

Autoactivity of ZAR1CC-YFP is inhibited by the ZAR1NBARC domain 247

We previously showed that ZAR1CCNBARC1-YFP did not elicit a HR-like phenotype, 248

indicating that the NBARC1 domain (amino acids 145-391) suppresses autoactivity of the CC 249

domain (Baudin et al., 2017). We hypothesized that the inhibitory effect of ZAR1NBARC on 250

ZAR1CC autoactivity was due to an intramolecular interaction between these two domains. To 251

test this hypothesis, we performed Y2H assays between ZAR1CC and different ZAR1NBARC 252

truncations. We constructed ZAR1NBARC variants that contained the NB subdomain alone 253

(ZAR1NB, amino acids 145-315), the NB subdomain and the first ARC subdomain (ZAR1NBARC1, 254

amino acids 145-391) or the NB subdomain and both ARC subdomains (ZAR1NBARC1+2, amino 255

acids 145-504), as in-frame fusions to the activation domain. Each was tested for an interaction 256

with ZAR1CC cloned as an in-frame fusion to the DNA-binding domain in yeast. We observed 257

significant yeast growth for all three truncations of the NBARC domain when co-expressed with 258

ZAR1CC but not with the negative control At5g48620CC (Figure 3A). The full-length NBARC 259

domain (ZAR1NBARC1+2) showed the strongest interaction with ZAR1CC (Figure 3A). All 260

constructs were expressed at similar levels in yeast (Figure S2). 261

To confirm these interactions in planta, we carried out co-immunoprecipitation 262

experiments (CoIP). We cloned NBARC variants as in-frame fusions with a 3xFlag epitope and 263

ZAR1CC as an in-frame with a 5xMyc epitope. We co-expressed ZAR1CC-5xMyc with each 264

NBARC variant or empty vector (EV) in N. benthamiana leaves, followed by 265


https://doi.org/10.1101/592824

immunoprecipitation of protein complexes using anti-Flag magnetic beads. As seen in yeast 266

(Figure 3A), ZAR1CC interacted with all three variants and showed the strongest interaction with 267

ZAR1NBARC1+2 (Figure 3B). Lastly, we confirmed these interactions using bimolecular 268

fluorescence complementation (BiFC) in N. benthamiana. We cloned NBARC variants as in-269

frame fusions with the N-terminal part of YFP (n-YFP) and ZAR1CC as an in–frame fusion with 270

the C-terminal part of YFP (cYFP), and co-expressed these constructs in N. benthamiana leaves. 271

We observed strong fluorescence when ZAR1CC-cYFP was co-expressed with all three variants 272

of the NBARC domain which further validates the Y2H and CoIP experiments and confirms that 273

the CC and NBARC domains of ZAR1 interact (Figure S4A). 274

To explore structural determinants in the CC domain that might be required for the 275

interaction with ZAR1NBARC, we then tested for an interaction between our ZAR1CC mutants 276

(Figure 1B) and ZAR1NBARC1+2 in Y2H assays. While wild type ZAR1CC was able to interact 277

with ZAR1NBARC1+2 as we showed previously, ZAR1CCH1, ZAR1CCH2a and ZAR1CCH2b were 278

strongly impaired in their ability to interact with ZAR1NBARC1+2 (Figure 3C). Interestingly, 279

ZAR1CCED was completely unable to interact with ZAR1NBARC1+2, suggesting that this motif 280

plays a particularly important role in the intramolecular interaction. 281

To further delineate what part of the NBARC domain was necessary for suppression of 282

ZAR1CC autoactivity, we cloned C-terminal truncations of ZAR1 as in-frame fusions with YFP 283

and expressed them under a dexamethasone-inducible promoter in N. benthamiana. We 284

compared ZAR1CC with ZAR1CCNBARC variants that contained the CC and NB domains 285

(ZAR1CCNB, amino acids 1-315), the CC, NB and first ARC subdomain (ZAR1CCNBARC1, amino 286

acids 1-391) or the CC, NB and both ARC domains (ZAR1CCNBARC1+2, amino acids 1-504) 287

(Figure 3C). ZAR1CC-YFP induced a strong HR-like phenotype, while ZAR1CCNB-YFP, 288

ZAR1CCNBARC1-YFP and ZAR1CCNBARC1+2-YFP did not display a HR-like phenotype (Figure 289

3D). These data indicate that the NB subdomain is sufficient to suppress ZAR1CC autoactivity. 290

The absence of autoactivity was not due to the absence of protein expression as all the constructs 291

showed similar expression levels (Figure S4B). 292

Taken together, our data show that the ZAR1CC and ZAR1NBARC1+2 interaction requires 293

the EDVID motif in ZAR1CC, and that proper folding of the ZAR1CC domain contributes to 294

interactions with ZAR1NBARC1+2. As well, the NB domain alone was sufficient to suppress the 295

autoactivity of ZAR1CC-YFP. 296


https://doi.org/10.1101/592824

297

Effect of loss- and gain-of-function mutations on the ZAR1NBARC domain 298

We and others previously identified loss-of-function mutations in zar1 that resulted in a 299

lack of HopZ1a or AvrAC recognition (Lewis et al., 2013; Wang et al., 2015b; Baudin et al., 300

2017). Among these loss-of function mutations, K195N, V202M and S291N are located in the 301

NB subdomain, P359L is in the ARC1 subdomain, and L465F is in the ARC2 subdomain (Figure 302

4A). To determine if these mutations in the NBARC domain affected the interaction with 303

ZAR1CC, we constructed ZAR1NBARC1+2 variants carrying each EMS mutation with an in-frame 304

3xFlag tag, and co-expressed them with ZAR1CC-5xMyc in CoIP assays. We found that ZAR1CC 305

coimmunoprecipitated with all of them, indicating that these substitutions did not affect the 306

interaction between the CC and NBARC domain of ZAR1 (Figure 4B). Since the CC domain 307

could interact with the NBARC domain carrying each mutation, we tested whether the mutated 308

NBARC domain could still suppress autoactivity of the CC domain. To maximize any effect of 309

the mutation, we used the minimally interacting NBARC domain that would still include each 310

EMS mutation. The three NB mutations (K195N, V202M and S291N) were cloned into 311

ZAR1CCNB with an in-frame C-terminal YFP tag. The ARC1 mutation (P359L) was cloned into 312

ZAR1CCNBARC1 with an in-frame C-terminal YFP tag. The ARC2 mutation (L465F) was cloned 313

into ZAR1CCNBARC1+2 with an in-frame C-terminal YFP tag. None of the constructs showed 314

autoactive phenotypes as displayed by the positive control ZAR1CC-YFP when overexpressed in 315

N. benthamiana leaves (Figure 4C). The absence of phenotype was not due to the absence of 316

expression as all of the constructs expressed well in N. benthamiana (Figure S4C). Therefore, the 317

five loss-of function mutations in ZAR1NBARC1+2 do not play a role in ZAR1CC intramolecular 318

interactions or in NBARC suppression of ZAR1CC-YFP autoactivity. 319

In an effort to identify potential autoactive mutants of ZAR1, we mined the literature for 320

mutations in the NBARC domain that conferred autoactivity in other NLRs. The following sites 321

were identified as strong candidates for autoactivity: G194/K195 in the P-loop from a large-scale 322

screen of autoactive NLRs (Lolle et al., 2017), D283 in the Walker B/kinase2 motif from the 323

tomato NLR I-2 (DeYoung and Innes, 2006; Tameling et al., 2006), and the aspartate in the 324

MHD motif in multiple NLRs (DeYoung and Innes, 2006; Bendahmane et al., 2002; De La 325

Fuente Van Bentem et al., 2005; Dodds et al., 2006; Howles et al., 2005; Roberts et al., 2013; 326

Williams et al., 2011). We mutated the corresponding residues in ZAR1: G194A/K195A in the 327


https://doi.org/10.1101/592824

P-loop, D268E in the Walker B/kinase2 motif, D489V in the MHD motif, and tested whether 328

they induced a HR in N. benthamiana. Wild type AtZAR1 did not induce a HR-like phenotype, 329

as previously observed (Figure S5A, S5B) (Baudin et al., 2017). AtZAR1G194E/K195A, 330

AtZAR1D268E, and AtZAR1D489V also did not cause a HR-like phenotype, and all of the proteins 331

were expressed at similar levels (Figure S5B, S5C). 332

333

Interaction between the NBARC and LRR domains 334

The NBARC-LRR interaction is also important for maintaining the NLR in the “off” 335

state, as seen for Rx (Slootweg et al., 2013; Bendahmane et al., 2002), RPS5 (RESISTANCE TO 336

PSEUDOMONAS SYRINGAE 5) (Ade et al., 2007; Qi et al., 2012) and RPP1 (RESISTANCE 337

TO PERONOSPORA PARASITICA 1) (Schreiber et al., 2016b). We therefore tested if ZAR1 338

was similarly regulated by a NBARC-LRR interaction. We cloned ZAR1LRR (amino acids 505-339

852) as an in–frame fusion with a 5xMyc tag and co-expressed it with different truncations of the 340

NBARC domain as previously described. We observed the strongest interaction between 341

ZAR1LRR and ZAR1NB, a weaker interaction between ZAR1LRR and ZAR1NBARC1, and the 342

weakest interaction between ZAR1LRR and ZAR1NBARC1+2 (Figure 5A). To confirm these 343

interactions, we carried out BiFC assays in N. benthamiana. We observed a fluorescence signal 344

when ZAR1LRR was co-expressed with ZAR1NB, ZAR1NBARC1 and ZAR1NBARC1+2 (Figure S6). 345

To investigate the role of NBARC mutations on the intramolecular interaction with the 346

LRR domain, we carried out CoIP with the ZAR1NBARC1+2 constructs described in Figure 4A. All 347

of the mutants in the NBARC domain were able to coimmunoprecipitate with ZAR1LRR at a 348

similar level to wild type ZAR1NBARC1+2 (Figure 5C). Interestingly, the NBARC mutants were 349

not affected in their interaction with either the CC or the LRR domains (Figure 4B, 5C), 350

suggesting that these mutations affect distinct roles in ZAR1 function. 351

We then turned to mutations in the LRR domain, to evaluate their impact on the NBARC-352

LRR interaction. Previous genetic screens identified three loss-of-function substitutions in the 353

LRR domain of ZAR1 (Lewis et al., 2013; Wang et al., 2015b; Baudin et al., 2017) (Figure 5B). 354

We therefore cloned these mutations in ZAR1LRR-5xMyc and tested them for their ability to 355

interact with ZAR1NBARC1+2-3xFlag. As we observed for the NBARC mutations (Figure 4B), 356

none of the LRR mutations affected the interaction between ZAR1NBARC1+2 and ZAR1LRR (Figure 357


https://doi.org/10.1101/592824

5B). ZAR1LRR-P816Q co-immunoprecipitated more weakly than the other LRR mutants however it 358

was also most weakly expressed. 359

Taken together, these data show that the NBARC domain interacts with both the CC and 360

LRR domains, and that the loss-of function mutations identified in the NBARC and LRR 361

domains do not affect these intramolecular interactions. 362

363

Remote homology modeling of ZAR1NBARC and ZAR1LRR 364

To better understand potential effects of these mutations on ZAR1, we developed remote 365

homology models of the ZAR1 NBARC domain from crystal structures of closed ADP-bound 366

and open ATP-bound conformations of proteins in the NBARC domain superfamily. Structures 367

included: closed and open Apaf1 from human, mouse and Drosophila (hApaf1, mApaf1 and 368

dApaf1), open CED4 from C. elegans, closed and open NLRC4 from mouse, and closed NOD2 369

from rabbit (Riedl et al., 2005; Reubold et al., 2011; Pang et al., 2015; Cheng et al., 2016; Liu et 370

al., 2010; Maekawa et al., 2011; Hu et al., 2013; Cheng et al., 2017; Zhang et al., 2015). Despite 371

relatively low sequence homology (11-20% identity) among hApaf1 (5JUY - (Cheng et al., 372

2016)) , dApaf1 (3J9L - (Pang et al., 2015)), C. elegans CED4 (3LQQ - (Qi et al., 2010)) and 373

mouse NLRC4 (3JBL - (Zhang et al., 2015)), their overall 3D structure superposition was quite 374

good. The RMSD values were ~3Å RMSD for the hApaf1 core vs. dApaf1 and CED4, and 375

~14.5Å RMSD for the hApaf1 core vs. NLRC4. When the individual NB, ARC1 or ARC2 376

subdomains were compared, the RMSD values were better than the aforementioned values in all 377

cases. We used remote homology modeling to model the NBARC domain of ZAR1 based on the 378

Apaf1 structure, as they showed the highest percentage sequence homology (~20% identity) 379

among all experimental NBARC structures (Figure S7, 6A). The internal structure of each 380

subdomain was relatively similar in the ADP-bound and ATP-bound states, except for a 381

rearrangement of the ARC2 subdomain (Figure 6A). Most loss-of-function mutations were 382

located in or close to key motifs in the NBARC domain: K195 in the P-loop, S202 between the 383

P-loop and RNBS-A, S291 in RNBS-B, and P359 in the GLPL motif (Figure 6A, S7). 384

We then turned to the LRR domain of ZAR1 and used a joint fragment remote homology 385

modeling approach, as this was shown to be effective in assessing the 3D structural properties of 386

other LRR domains such as those in Lr10, Rx and Pm3 (Sela et al., 2012; Slootweg et al., 2013; 387

Sela et al., 2014). Using our structural database of LRRs, the closest local ZAR1 templates were: 388


https://doi.org/10.1101/592824

(a) the LRR region in polygalacturonase-inhibiting protein from Phaseolus vulgaris, (b) the LRR 389

protein from Leptospira interrogans, and (c) LRR repeats from a protein of Agrobacterium 390

tumefaciens (Vorobiev et al., 2007; Di Matteo et al., 2003; Miras et al., 2015). We generated a 391

composite model of ZAR1LRR structure by joining these local fragments into the overall LRR 392

frame based on the constraints imposed by the parallel beta strands of the ventral side of the LRR 393

motif and a maximal structural fit over the rest of the repeat (Figure 6B, S8). The overlapping 394

frame of the three templates runs from amino acids 510 to 823 and does not cover the last 28 395

residues. This analysis identified 13 LRR motifs from amino acids 510 to 823. The C-terminal 396

region (amino acids 824-852) was modeled with low confidence based exclusively on its 397

secondary structure propensity. Given that amino acids 843-846 are predicted in an extended 398

configuration and are located at a distance in sequence that is compatible with one LRR repeat, 399

the 824-852 amino acid extension was modeled here as an extra LRR repeat (Figure 6B, S8). 400

401

DISCUSSION 402

To prevent cell death in the absence of pathogens, immune receptors must remain in an 403

inactive state, while rapidly responding and transitioning to an active state upon pathogen 404

recognition. Here, we explored the molecular determinants of ZAR1 interactions using structural 405

modeling and functional assays in a transient assay system that we previously developed (Baudin 406

et al., 2017). Structural modeling of ZAR1 allowed us to generate hypotheses for intramolecular 407

interactions. We demonstrated experimentally that multiple intramolecular interactions 408

contribute to ZAR1 function. 409

We carried out targeted mutagenesis of the ZAR1CC domain to determine its possible 410

structure as a monomer and how the monomer transitioned to the dimeric form. Targeted 411

mutagenesis of ZAR1CC impacted the dimerization of this sub-domain (Figures 1, S1). In 412

particular, the ZAR1CCH2a and ZAR1CCH2b constructs disrupted hydrophobic zipping in α1a and 413

shifted the monomer-dimer equilibrium in favor of the monomeric state. This is consistent with 414

coimmunoprecipitation assays in Rx where mutations in the hydrophobic positions of the α1a 415

region (V6E/L9E/I13E) disrupted the interaction between the 2 halves of Rx (segment α1a-α1b 416

and segment α2-α3) (Slootweg et al., 2018). However, yeast two-hybrid assays with MLA10 417

showed that single mutations (I33E, L36E, or M43E) in the first helix were enough to impair 418

dimerization (Maekawa et al., 2011). For RPM1 (RESISTANCE TO PSEUDOMONAS 419


https://doi.org/10.1101/592824

SYRINGAE PV. MACULICOLA 1), mutation of M34 or M41 weakened dimerization of the 420

CC domain in coimmunoprecipitation assays, and mutation of 3 hydrophobic residues 421

(I31E/M34E/M41E) was necessary to prevent CC dimerization in BiFC and 422

coimmunoprecipitation assays (El Kasmi et al., 2017). It would be interesting to test the role of 423

these residues in dimerization as ZAR1 contains hydrophobic residues at similar positions 424

(Figure S1A). 425

All of the CC mutants as YFP fusions lost the ability to autoactivate immunity (Figure 426

2A, 2B). Since these mutations targeted different regions of the CC, these data indicate that the 427

CC-YFP autoactivation phenotype is very sensitive to small disruptions and the overall folding 428

of the domain. We therefore also tested the CC mutants in the context of full-length ZAR1, since 429

this assay can identify weak effects on ZAR1 function. We found that ZAR1H1 retained some 430

complementation of NbZAR1-VIGS, while the other mutants (ZAR1H2a, ZAR1H2b and ZAR1ED) 431

could not complement NbZAR1-VIGS. These data suggest that the α1a-α1b and α2-α3 loops 432

(mutated in ZAR1CCH1 and ZAR1H1) only make a partial contribution to the overall folding of 433

ZAR1CC. In addition, the α1a helix (mutated in ZAR1CCH2a, ZAR1H2a, ZAR1CCH2b and ZAR1H2b) 434

is important for ZAR1 function. Interestingly, the EDVID motif in ZAR1CC (mutated in 435

ZAR1CCED and ZAR1ED) partially contributed to homodimerization and was essential for the 436

interaction with ZAR1NBARC1, as well as the activation of downstream signaling (Figures 1, 2, 437

S1). The EDVID motif was first identified in Rx, an NLR that recognizes the coat protein of 438

Potato Virus X (Bendahmane et al., 1999; Rairdan et al., 2008). In Rx, the EDVID motif 439

contributes to intramolecular interactions between the CC and NBARC domains (Rairdan et al., 440

2008). In MLA10, the EDVID motif is surface-exposed and positioned at the dimer interface 441

(Maekawa et al., 2011), suggesting that it contributes to CC homodimerization. RPM1, an NLR 442

involved in the recognition of P. syringae effector AvrRpm1, also contains an EDVID motif that 443

may contribute to the self-association of CC domains (El Kasmi et al., 2017). In agreement with 444

previous studies, the EDVID motif in ZAR1 is predicted to be surface-exposed (Figure S1), and 445

is involved in CC dimerization (Figure 1C) and in the CC-NBARC interaction (Figure 3C). An 446

outstanding question in the field is how the CC domain of various NLRs can activate immunity. 447

TIR domains in NLRs have commonly been thought of as scaffolding proteins (Vajjhala et al., 448

2017). However, TIR domains in bacteria and Archaea were recently shown to act as NADase 449

enzymes (Essuman et al., 2018). CC domains have no homology to proteins with known 450


https://doi.org/10.1101/592824

enzymatic activity. Thus it remains unclear whether they act directly or indirectly as a signal for 451

immunity. Our data further support that the CC domain acts a signal for immunity because 452

ZAR1CC-YFP could still trigger a HR in NbZAR1-silenced plants (Figure S3). 453

We demonstrated that the CC and NBARC domains of ZAR1 interact (Figure 3A), and 454

that the NB domain is sufficient for the interaction and for suppression of ZAR1CC-YFP 455

autoactivity (Figure 3D). Interactions between the CC and NBARC domain have also been 456

observed for RPM1 (El Kasmi et al., 2017) which recognizes the P. syringae effector protein 457

AvrRpm1, RPS5 (Ade et al., 2007) which recognizes the P. syringae effector protein AvrPphB, 458

and the autoactive Rp1 NLR in maize (Wang et al., 2015a). Interestingly, the ZAR1CCH2a and 459

ZAR1CCH2b constructs, that carried nine mutations in the first helix, were still able to interact 460

very weakly with ZARNBARC1+2. In contrast, only 2 mutations in the first helix (Y3A/M10A) of 461

Rx were enough to completely disrupt the interaction between RxCC and RxNB-ARC1+2-LRR, and the 462

V6E/L9E/I13E mutations severely reduced the interaction (Slootweg et al., 2018). Unlike ZAR1, 463

the CC-NBARC interaction in Rx appears to be sufficiently rigid that expression of either half of 464

the CC domain of Rx1 (i.e. α1a-α1b or α2-α3) with Rx1NB-ARC1+2-LRR led to the reconstitution of 465

a functional Rx1 (Slootweg et al., 2018). 466

We sought to take advantage of loss-of-function mutations in ZAR1NBARC1+2 to identify 467

whether they contributed to the CC-NBARC intramolecular interaction. However, these five 468

mutations did not have any effect (Figure 4B). Consistent with this, the introduction of these 469

mutations in a ZAR1CCNBARC-YFP variant did not restore the autoactivity observed in ZAR1CC-470

YFP. In Rp1 (Wang et al., 2015a), mutations that led to the loss of the CC-NBARC interaction in 471

ZAR1 resulted in autoimmunity. Our loss-of-function mutations are found in or close to motifs 472

that are believed to be particularly important for nucleotide binding and ATP hydrolysis (Collier 473

and Moffett, 2009), including the P-loop (K195 and V202), the RNBS-B/kinase3 motif (S291) 474

and the GLPL motif (P359) (Figure 6A, S7). Given the contribution of these residues to 475

resistance, we speculate that they are critical for NBARC function in the activation of ZAR1 by 476

effectors. Since our predicted autoactive mutations did not confer autoactivity (Figure S7), we 477

were unable to test whether ZAR1 intramolecular or intermolecular interactions would be 478

affected in the active state of the NLR. 479

We further demonstrated that the NBARC and LRR domains of ZAR1 interact in planta. 480

The interaction was stronger with ZAR1NB alone but could still be observed with the full-length 481


https://doi.org/10.1101/592824

NBARC domain (Figure 5A). Interactions between the NBARC and LRR domains have also 482

been observed for RPS5 and RPP1 (Ade et al., 2007; Schreiber et al., 2016b), and for RPS5 this 483

interaction maintains the inactive state (Qi et al., 2012). Interestingly, for Rx1, an acidic loop in 484

the ARC2 domain is particularly important for interaction with a basic patch in the N-terminal 485

region of the LRR domain (Slootweg et al., 2013). However, ZAR1 behaves more like RPP1 486

(Schreiber et al., 2016b) than Rx1, because the full NBARC domain containing the ARC2 487

subdomain exhibits a weaker interaction with the LRR domain. As seen with the CC-NBARC 488

domain interactions (Figure 4B), mutations in the NBARC domain did not affect the interaction 489

with LRR domain (Figure 5C). 490

Structural analysis of the LRR domain of ZAR1 identified 13 LRRs, followed by an 491

extended region of the LRR domain, that could form another LRR segment or two helices 492

(Figure 6B, S8). We found that loss-of-function mutations in the LRR domain had no effect on 493

the interaction with the NBARC domain (Figure 5D). We previously showed that G645 and 494

P816, but not S831, are necessary for the interaction between ZAR1LRR and ZED1 (Baudin et al., 495

2017). The S831 mutation is located in the LRR extension (Figure 6B, S8). When we deleted this 496

region (in ZAR1∆1 containing aa1-824), ZAR1∆1 was no longer able to complement NbZAR1-497

VIGS (Baudin et al., 2017). While zar1S831F lacks an immune response to HopZ1a, ZAR1LRR 498

S831F can still interact with ZED1 (Baudin et al., 2017), and with ZAR1NBARC1+2 (Figure 5D). 499

These data suggest that the LRR extension plays a distinct but unknown role in ZAR1 function 500

and immunity. 501

Despite efforts by many groups to understand the dynamics and regulation of plant NLRs 502

(Bonardi and Dangl, 2012), there is still a broad diversity of protein-protein interactions and 503

higher-order macromolecular complexes that are critical for immunity. Oligomerization of NLRs 504

is common for mammalian NLRs (Lechtenberg et al., 2014), and has only been rarely 505

demonstrated for plant NLRs (Gutierrez et al., 2010). We propose the following model for ZAR1 506

interactions and activation (Figure 7). Inactive ZAR1 complexes are kept in an “off” state by 507

intramolecular interactions, and intermolecular interactions with ZED1. We speculate that ZAR1 508

exists in an equilibrium between ZAR1 alone and ZAR1-ZED1 or ZAR1-ZRK complexes. This 509

would provide sufficient plasticity for ZAR1 to exist as pre-activation complexes with different 510

ZRKs, allowing ZAR1 to “switch” between different RLCKs, and provide specificity for 511

different effector proteins. According to our model and experimental data, the inactive ZAR1 is 512


https://doi.org/10.1101/592824

tightly folded with both the LRR and CC domain interacting with the central NBARC domain 513

(Figure 7). We previously showed that both the CC and LRR domain of ZAR1 interact with 514

ZED1 in the absence of pathogens (Baudin et al., 2017). However, our data do not show whether 515

both domains interact with ZED1 at the same time, and how ZED1 impacts ZAR1 intramolecular 516

interactions. We propose that modification of the guardee by the effector induces a series of 517

conformational rearrangements, which allows binding of ATP by the NBARC domain and 518

dimerization of ZAR1CC. However, dimerization of ZAR1CC is not sufficient for autoactivity, 519

indicating that dimerization precedes autoactivity and the CC domain is more likely to act as a 520

signal. Although the lack of autoactive mutations for ZAR1 does not allow us to specifically 521

analyze the intramolecular interactions present in active complexes, we demonstrated that 522

interactions between the domains must have a certain versatility to allow for conformational 523

changes upon activation. Our data show that ZAR1 is regulated by subtle conformational 524

changes that are finely controlled, and that ZAR1 displays specificity in its intramolecular 525

interactions despite broad structural similarity with other NLRs. The ZAR1-ZED1 system 526

demonstrates the complex and intricate ways in which immune receptors are dynamically 527

regulated in vivo. 528

529

MATERIALS AND METHODS 530

531

Computational analysis and 3D modeling 532

The CC, NBARC and LRR domains of ZAR1 were modelled individually by remote 533

homology modeling workflows similar to those used to model equivalent domains in Lr10 534

(LEAF RUST 10 from Triticum dicoccoides [emmer wheat]), NRC1 (NB-LRR REQUIRED 535

FOR HYPERSENSITIVE RESPONSE-ASSOCIATED CELL DEATH 1 from Solanum 536

lycopersicum [tomato]), Rx and Pm3 (POWDERY MILDEW 3 from Triticum aestivum) (Sela et 537

al., 2012; Slootweg et al., 2013; Sela et al., 2014; Sueldo et al., 2015). In brief, profiles were 538

raised for sequence variability and sequence propensities for secondary structure, intrinsic 539

disorder, accessibility, linkers, coil-coil and turn local structure. For each profile, several 540

methods were used and the consensus was built to increase the prediction reliability as 541

previously described (Sela et al., 2012; Slootweg et al., 2013; Sela et al., 2014; Sueldo et al., 542

2015). These structural propensities were further used to refine sequence alignments with the 543


https://doi.org/10.1101/592824

closest domain templates in each case. Alignments were generated by using appropriate 544

BLOSUM matrices with either the EMBOSS-stretcher sequence-only pair-wise alignment 545

method (Myers and Miller, 1988) for single template models or PRALINE multiple sequence 546

alignment method enhanced by incorporating PSSM profiles and secondary structure predictions 547

(Bawono and Heringa, 2014) when dealing with multiple potential structural templates. 548

ZAR1CC was modeled in both the monomeric 4-bundle helix configuration (1CC4α) and 549

the 2-helix dimer conformation (2CC2α). The 1CC4α model was built starting from the Sr33 550

NMR structure (PDB: 2NCG, (Casey et al., 2016)) and the Rx crystal structure (PDB: 4M70, 551

(Hao et al., 2013)) with which ZAR1 shares 17% and 15% sequence identity respectively. The 552

2CC2α model was built from the dimer configuration of MLA10 structures (PBD: 3QFL, 553

(Maekawa et al., 2011)) with which ZAR1-CC sequence shares 19% identity. 554

ZAR1NBARC was modeled in both closed and opened conformations starting from human 555

Apaf1 structures in the closed ADP-bound state (PDB: 1Z6T, (Riedl et al., 2005)) and open 556

ATP-bound state (PDB: 5JUY, (Cheng et al., 2016)) respectively. We used Apaf1, as ZAR1 557

shares 20% sequence identity with Apaf1, which was the best score among all experimental 558

NBARC structures. 559

ZAR1LRR was modeled using the Optimized Joined Fragment Remote Homology 560

modeling (OJFRHM) method previously used to model the LRR domains of Lr10, Rx and Pm3 561

(Sela et al., 2012; Slootweg et al., 2013; Sela et al., 2014). From our structural LRR database, the 562

closest local ZAR1 templates were PBD: 2RA8, 1OGQ and 4U06 (Vorobiev et al., 2007; Di 563

Matteo et al., 2003; Miras et al., 2015). 564

All homology models were built with Discovery Studio software from Accelrys-Dassault 565

Systemes and Modeller v9.17 (Webb and Sali, 2014). Within the sequence conserved regions 566

(SCR), the model was raised by coordinate transfer and side chain rebuilding, while the sequence 567

variable regions (SVRs) were randomly generated and filtered by steric constraints, followed by 568

successive rounds of energy minimization (EM) and simulated annealing (SA). The global model 569

generated as above was further subjected to overall structural optimization by repeated rounds of 570

EM, SA and Molecular Dynamics in explicit solvent performed with CHARMM36 force field in 571

NAMD (Phillips et al., 2005), followed iteratively by model quality check until convergence. 572

Model quality was assessed with MolProbity (Davis et al., 2007) and PROCHECK V.3.5 573

(Laskowski et al., 1993) for crystallographic standards compliance. Trajectory analysis was 574


https://doi.org/10.1101/592824

performed using VMD (Humphrey et al., 1996) and all figures of predictive models’ structures 575

were created using PyMOL Molecular Graphics System v1.3. 576

577

Plasmid construction 578

Phusion polymerase (New England Biolabs) was used for all cloning, and all constructs 579

were confirmed by sequencing. Sequence analysis was performed with CLC Main Workbench. 580

For the VIGS constructs, we used a clone carrying a genomic fragment of NbZAR1, as described 581

in Baudin et al., 2017. The single mutations were generated using the Q5 Site-directed 582

mutagenesis kit (New England Biolabs). For ZAR1CCH1, ZAR1CCH2a ZAR1CCH2b and ZAR1CCED, 583

the gene fragments were synthesized as gBlocks by Integrated DNA Technologies, and then used 584

as templates for PCR. For the complementation assay, the ZAR1CC mutations were introduced in 585

the full-length gene using the internal restriction site EcoRI. ZAR1 variants were amplified by 586

PCR to add a 5’XhoI site and cloned into the pMac15 vector to maintain the frame for the vector-587

encoded C-terminal 5xMyc tag. The pMac15 vector was modified from pBD to contain a 5xMyc 588

tag between the StuI and SpeI sites, as described in Baudin et al., 2017. The ZED1 3xFlag 589

construct was cloned by a crossover PCR approach to add the 3xFlag tag in frame at the 3’end. 590

The PCR products were then cloned into the pMac14 vector, which was modified from pBD 591

(Aoyama and Chua, 1997; Lewis et al., 2008). The 3xFlag tagged NBARC variants were 592

amplified by PCR to add a 5’XhoI site and cloned into the pMac16 vector to maintain the frame 593

for the vector-encoded C-terminal 3xFlag tag. The pMac16 vector was modified from pBD to 594

contain a 3xFlag tag between the StuI and SpeI sites. For the split YFP and YFP fusions, the 595

genes were amplified by PCR to contain a 5’XhoI restriction site. The 3’ primers were designed 596

to maintain the reading frame of the C-terminal fusion. pBD-YFP, pBD-nYFP, and pBD-cYFP 597

were modified from pTA7002 (Aoyama and Chua, 1997) to add an HA tag and full-length YFP, 598

nYFP (residues 1-155), or cYFP (residues 156 to the stop codon) between the StuI and SpeI sites 599

(Lewis et al., 2014). 600

For protein interaction analyses in yeast, all constructs were prepared in the DupLEX-A 601

system (OriGene Technologies, Inc., Rockford, MD) using the vectors pEG202 and pJG4-5 to 602

express bait and prey proteins, respectively. All sequences were cloned into the EcoRI and XhoI 603

sites of these vectors. to maintain the frame of the vector-encoded fusions. The pJG4-5 vector 604


https://doi.org/10.1101/592824

contains a nuclear localization signal, B42 activation domain and HA tag at the 5’ end. The 605

pEG202 vector contains the LexA DNA binding domain at the 5’ end. 606

607

Yeast two-hybrid experiments 608

All constructs were transformed into yeast using Frozen-EZ Yeast Transformation II 609

reagents (Zymo Research Corp., Irvine, CA) to introduce pEG202 constructs into yeast strain 610

RFY206 (mating type A), while pJG4-5 constructs were introduced into strain EGY48 (mating 611

type α). Transformants were recovered on Synthetic Defined (SD)+Glucose media lacking 612

histidine and uracil to select for colonies containing pEG202 constructs, or lacking tryptophan to 613

select for pJG4-5 transformants. Yeast matings were performed in YPDA media at 30°C 614

overnight, followed by two rounds of diploid selection on SD+Glucose-HisUraTrp plates at 30°C 615

overnight. To assay for protein-protein interactions, diploid yeast were first resuspended in 150 616

μL of sterile distilled water and adjusted to an OD600 of 2, followed by three serial 1:5 dilutions 617

in water. Five microliters of each dilution were spotted on SD+Glucose-HisUraTrpLeu and 618

SD+Galactose+Raffinose-HisUraTrpLeu plates, with yeast growth indicating the activation of a 619

LEU2 reporter. Images of yeast plates were generally captured after three days of growth at 620

30°C. 621

622

Coimmunoprecipitation 623

In planta coimmunopurification was performed using 5 cm2 of N. benthamiana leaves 624

transiently expressing our genes of interest. Tissue was homogenized in liquid nitrogen, and 625

protein complexes were extracted using 1 mL of IP1 buffer (50 mM HEPES, 50 mM NaCl, 10 626

mM EDTA, 0.2% TritonX-100, and 0.1 mg/mL Dextran [Sigma-Aldrich D1037], pH 7.5). The 627

clarified extract was then mixed with 10 µL of anti-Flag magnetic beads [Sigma-Aldrich M8823] 628

for 3 h at 4°C. The coimmunoprecipitated proteins were washed four times with IP2 buffer (50 629

mM HEPES, 150 mM NaCl, 10 mM EDTA, and 0.1% Triton X-100, pH 7.5) on a magnetic 630

stand. IP1 and IP2 buffers were supplemented with proteinase inhibitor cocktail (1 mM PMSF, 1 631

mg/mL leupeptin [Sigma-Aldrich L2023], 1 mg/mL aprotinin [Sigma-Aldrich A6191], 1 mg/mL 632

antipain [Sigma-Aldrich A1153], 1 mg/mL chymostatin [Sigma-Aldrich C7268], and 1 mg/mL 633

pepstatin [Sigma-Aldrich P5315]). The immunopurified fraction was eluted incubating the beads 634

with 30 µL of 150 µg/mL 3xFlag peptide [Sigma-Aldrich F4799] for 30 minutes at room 635


https://doi.org/10.1101/592824

temperature. Finally, the input and immunopurified fractions were separated on 10% acrylamide 636

gels by SDS-PAGE and detected by immunoblot using α-c-Myc antibody (Abcam ab32072), or 637

α-Flag antibody (Sigma-Aldrich F1804) followed by the appropriate secondary antibody coupled 638

with horseradish peroxidase. 639

640

Plant material and transient expression 641

We followed protocols previously described in Baudin et al., 2017 with some slight 642

modifications as follows. Nicotiana benthamiana plants were grown in a growth chamber at 643

22°C with 9 h light (~130 µEm-2s-1) and 15 h dark cycles. The seeds were sterilized in 10% 644

bleach for 10 min and then washed ten times with sterile nanopure water. After germination on 645

soil, seedlings were transplanted onto individual pots and used 3 to 5 weeks after transplanting. 646

For the Agrobacterium-mediated transient expression, Agrobacterium tumefaciens GV2260 647

cultures were grown overnight at 28°C in LB broth with kanamycin and rifampicin. The next 648

day, the bacteria were resuspended in 1 mL of 10 mM MES, pH 5.6 and 200 µM of 649

acetosyringone and incubated in the dark for ~4 h. The cultures were then adjusted to an optical 650

density (λ=600 nm) of 1.0. The cultures containing each plasmid were mixed in equal volumes to 651

a final optical density of 0.25 per construct. For BiFC experiments, A. tumefaciens carrying the 652

gene silencing suppressor P19 was co-infiltrated with the constructs at an optical density of 0.25 653

(Voinnet et al., 2003). The underside of the leaves of 5- to 7-week-old N. benthamiana plants 654

were infiltrated by hand with a needleless syringe. For dexamethasone-inducible constructs, the 655

plants were sprayed with 20 µM dexamethasone (Sigma-Aldrich) 12 to 24 h after inoculation. 656

Tissues were collected from 3 to 48 h after dexamethasone induction depending on the 657

experiment. For the VIGS experiments, the plants were used 1 to 2 weeks after inoculation. 658

659

Phenotyping and conductivity 660

To phenotype the HR-like phenotype induced by ZAR1CC-YFP autoactivity, infiltrated 661

leaves were observed ~40 h after dexamethasone induction. The severity of HR was rated on the 662

following phenotypic scale: no HR, weak HR, medium HR and strong HR. For ion leakage 663

assays in N. benthamiana, two disks (1.5 cm2) were harvested 20 h after infiltration (3 h after 664

dexamethasone induction) floated in nanopure water for 1 h and transferred to 6 mL of nanopure 665


https://doi.org/10.1101/592824

water. Readings were taken with an Orion 3 Star conductivity meter (Thermo Electron) at 24 h 666

(T0) and 48 h (T24) after infiltration. 667

668

Confocal microscopy 669

N. benthamiana leaves transiently expressing the constructs of interest were infiltrated 670

with water and mounted on microscope slides. Samples were imaged using a Leica SP8 confocal 671

laser- scanning microscope equipped with a 40x water-immersion objective (HC PL APO CS2 672

40x/1.10 WATER). The 514 nm argon laser line was used to excite YFP, and florescence was 673

observed using the specific emission window of 520 to 600 nm. The laser power, gain, zoom and 674

average settings were kept consistent over the same image series to allow fluorescence intensity 675

comparison across samples. Images were processed using the Leica Application Suite X 676

software. 677

678

Western blots 679

1 cm2 of leaf tissue was collected 24 h after dexamethasone induction and frozen in liquid 680

nitrogen. The frozen tissue was then homogenized and proteins were extracted in 100 µL of 1X 681

Laemmli buffer and boiled for 5 min. Proteins were then separated on 10% acrylamide SDS-682

PAGE gels. After transferring onto nitrocellulose membranes, tagged proteins were detected 683

using α-c-Myc antibody (Abcam ab32072), α-GFP antibody (Roche 11814460001) or α-Flag 684

antibody (Sigma-Aldrich F1804), followed by the appropriate secondary antibody coupled to 685

horseradish peroxidase. Protein expression in yeast was evaluated following shaking incubation 686

of cultures at 30°C overnight in SD+Glucose-HisUra (pEG202 constructs) or 687

SD+Galactose+Raffinose-Trp (pJG4-5) media. Cultures were processed for protein extraction 688

according to a lithium acetate/sodium hydroxide pre-treatment protocol described by (Zhang et 689

al., 2011). Samples were resolved on 8% discontinuous SDS-PAGE gels, transferred to 690

nitrocellulose membranes, and detected by either α-HA (Roche 12CA5) or α-LexA antibodies 691

(Santa Cruz Biotechnology SC7544). 692

693

Statistical analyses 694

The conductivity data were analyzed using Minitab 17 software (Minitab). For each 695

conductivity assay, the T24 data were analyzed by a one-way ANOVA statistical test at a 696


https://doi.org/10.1101/592824

significance level of p = 0.05 followed by a multiple comparisons of means using Tukey’s post 697

hoc test. 698

699

Accession numbers 700

Sequence data from this article can be found in the GenBank/EMBL data 701

libraries under accession numbers At3g50950 (AtZAR1), At3g57750 (AtZED1), and 702

Niben101Scf17398g00012 (NbZAR1). 703

704

Supplemental Data 705

The following supplemental materials are available. 706

Supplemental Figure S1. Alignment and modeling of ZAR1CC and CC domain mutants. 707

Supplemental Figure S2. Protein expression in yeast strains used for two-hybrid analyses. 708

Supplemental Figure S3. ZARCC autoactivity in N. benthamiana is independent from NbZAR1. 709

Supplemental Figure S4. BiFC analysis of the ZAR1CC – ZAR1NBARC interaction and protein 710

expression of ZAR1 domain constructs. 711

Supplemental Figure S5. Residues associated with autoactive NLRs do not activate immunity in 712

ZAR1. 713

Supplemental Figure S6. The NBARC and LRR domains interact in planta by bimolecular 714

fluorescence complementation. 715

Supplemental Figure S7. ZAR1NBARC alignments used to build the closed and open conformation 716

of ZAR1NBARC 3D models starting from human Apaf1 crystal structures. 717

Supplemental Figure S8. ZAR1LRR alignments and predicted/crystal secondary structure used in 718

modeling the LRR domain. 719

720

ACKNOWLEDGMENTS 721

722

We thank Jana A. Hassan from the Lewis Lab for providing constructive comments and 723

suggestions on the manuscript. 724

725

REFERENCES 726

727


https://doi.org/10.1101/592824

Ade, J., DeYoung, B.J., Golstein, C., and Innes, R.W. (2007). Indirect activation of a plant 728

nucleotide binding site-leucine-rich repeat protein by a bacterial protease. Proc. Natl. Acad. 729

Sci. U. S. A. 104: 2531–2536. 730

Aoyama, T. and Chua, N.-H. (1997). A glucocorticoid-mediated transcriptional induction 731

system in transgenic plants. Plant J. 11: 605–612. 732

Baggs, E., Dagdas, G., and Krasileva, K.V.K. (2017). NLR diversity, helpers and integrated 733

domains: making sense of the NLR IDentity. Curr. Opin. Plant Biol. 38: 59–67. 734

Bai, S., Liu, J., Chang, C., Zhang, L., Maekawa, T., Wang, Q., Xiao, W., Liu, Y., Chai, J., 735

Takken, F.L.W., Schulze-Lefert, P., and Shen, Q.H. (2012). Structure-function analysis 736

of barley NLR immune receptor MLA10 reveals its cell compartment specific activity in 737

cell death and disease resistance. PLoS Pathog. 8: 21–25. 738

Baudin, M., Hassan, J.A., Schreiber, K., and Lewis, J.D. (2017). Analysis of the ZAR1 739

immune complex reveals determinants for immunity and molecular interactions. Plant 740

Physiol. 174: 2038–2053. 741

Bawono, P. and Heringa, J. (2014). PRALINE: a versatile multiple sequence alignment toolkit. 742

Methods Mol. Biol. 1079: 245–62. 743

Bendahmane, A., Farnham, G., Moffett, P., and Baulcombe, D.C. (2002). Constitutive gain-744

of-function mutants in a nucleotide binding site-leucine rich repeat protein encoded at the 745

Rx locus of potato. Plant J. 32: 195–204. 746

Bendahmane, A., Kanyuka, K., and Baulcombe, D.C. (1999). The Rx gene from potato 747

controls separate virus resistance and cell death responses. Plant Cell 11: 781–792. 748

Bentham, A., Burdett, H., Anderson, P.A., Williams, S.J., and Kobe, B. (2017). Animal 749

NLRs provide structural insights into plant NLR function. Ann. Bot. 119: 689–702. 750

Bernoux, M., Burdett, H., Williams, S.J., Zhang, X., Chen, C., Newell, K., Lawrence, G.J., 751

Kobe, B., Ellis, J.G., Anderson, P.A., and Dodds, P.N. (2016). Comparative analysis of 752

the flax immune receptors L6 and L7 suggests an equilibrium-based switch activation 753

model. Plant Cell 28: 146–159. 754

Bernoux, M., Ve, T., Williams, S., Warren, C., Hatters, D., Valkov, E., Zhang, X., Ellis, 755

J.G., Kobe, B., and Dodds, P.N. (2011). Structural and functional analysis of a plant 756

resistance protein TIR domain reveals interfaces for self-association, signaling, and 757

autoregulation. Cell Host Microbe 9: 200–211. 758


https://doi.org/10.1101/592824

Van Der Biezen, E.A. and Jones, J.D.G. (1998). Plant disease-resistance proteins and the gene-759

for-gene concept. Trends Biochem. Sci. 23: 454–456. 760

Block, A., Li, G., Fu, Z.Q., and Alfano, J.R. (2008). Phytopathogen type III effector weaponry 761

and their plant targets. Curr. Opin. Plant Biol. 11: 396–403. 762

Bonardi, V. and Dangl, J.L. (2012). How complex are intracellular immune receptor signaling 763

complexes? Front. Plant Sci. 3: 1–9. 764

Casey, L.W. et al. (2016). The CC domain structure from the wheat stem rust resistance protein 765

Sr33 challenges paradigms for dimerization in plant NLR proteins. Proc. Natl. Acad. Sci. U. 766

S. A. 113: 12856–12861. 767

Cesari, S. (2018). Multiple strategies for pathogen perception by plant immune receptors. New 768

Phytol. 219: 17–24. 769

Cheng, T.C., Akey, I. V., Yuan, S., Yu, Z., Ludtke, S.J., and Akey, C.W. (2017). A near-770

atomic structure of the dark apoptosome provides insight into assembly and activation. 771

Structure 25: 40–52. 772

Cheng, T.C., Hong, C., Akey, I. V., Yuan, S., and Akey, C.W. (2016). A near atomic structure 773

of the active human apoptosome. Elife 5: 1–28. 774

Collier, S.M. and Moffett, P. (2009). NB-LRRs work a “bait and switch” on pathogens. Trends 775

Plant Sci. 14: 521–529. 776

Dangl, J.L. and Jones, J.D.G. (2001). Plant pathogens and integrated defence responses to 777

infection. Nature 411: 826–833. 778

Davis, I.W., Leaver-Fay, A., Chen, V.B., Block, J.N., Kapral, G.J., Wang, X., Murray, 779

L.W., Arendall, W.B., Snoeyink, J., Richardson, J.S., and Richardson, D.C. (2007). 780

MolProbity: All-atom contacts and structure validation for proteins and nucleic acids. 781

Nucleic Acids Res. 35: 375–383. 782

Deslandes, L. and Rivas, S. (2012). Catch me if you can: bacterial effectors and plant targets. 783

Trends Plant Sci. 17: 644–655. 784

DeYoung, B.J. and Innes, R.W. (2006). Plant NBS-LRR proteins in pathogen sensing and host 785

defense. Nat. Immunol. 7: 1243–1249. 786

Dodds, P.N., Lawrence, G.J., Catanzariti, A.-M., Teh, T., Wang, C.-I.A., Ayliffe, M.A., 787

Kobe, B., and Ellis, J.G. (2006). Direct protein interaction underlies gene-for-gene 788

specificity and coevolution of the flax resistance genes and flax rust avirulence genes. Proc. 789


https://doi.org/10.1101/592824

Natl. Acad. Sci. 103: 8888–8893. 790

Essuman, K., Summers, D.W., Sasaki, Y., Mao, X., Yim, A.K.Y., DiAntonio, A., and 791

Milbrandt, J. (2018). TIR domain proteins are an ancient family of NAD+-consuming 792

enzymes. Curr. Biol. 28: 421–430.e4. 793

Frost, D., Way, H., Howles, P., Luck, J., Manners, J., Hardham, A., Finnegan, J., and Ellis, 794

J. (2004). Tobacco transgenic for the flax rust resistance gene L expresses allele-specific 795

activation of defense responses. Mol. Plant-Microbe Interact. 17: 224–232. 796

Galán, J.E. and Wolf-Watz, H. (2006). Protein delivery into eukaryotic cells by type III 797

secretion machines. Nature 444: 567–573. 798

Grant, S.R., Fisher, E.J., Chang, J.H., Mole, B.M., and Dangl, J.L. (2006). Subterfuge and 799

manipulation: type III effector proteins of phytopathogenic bacteria. Annu. Rev. Microbiol. 800

60: 425–449. 801

Gutierrez, J.R., Balmuth, A.L., Ntoukakis, V., Mucyn, T.S., Gimenez-Ibanez, S., Jones, 802

A.M.E., and Rathjen, J.P. (2010). Prf immune complexes of tomato are oligomeric and 803

contain multiple Pto-like kinases that diversify effector recognition. Plant J. 61: 507–18. 804

Hao, W., Collier, S.M., Moffett, P., and Chai, J. (2013). Structural basis for the interaction 805

between the Potato Virus X resistance protein (Rx) and its cofactor ran GTPase-activating 806

protein 2 (RanGAP2). J. Biol. Chem. 288: 35868–35876. 807

Heath, M.C. (2000). Hypersensitive response-related death. Plant Mol. Biol. 44: 321–334. 808

van der Hoorn, R.A.L. and Kamoun, S. (2008). From Guard to Decoy: a new model for 809

perception of plant pathogen effectors. Plant Cell 20: 2009–17. 810

Howles, P., Lawrence, G., Finnegan, J., McFadden, H., Ayliffe, M., Dodds, P., and Ellis, J. 811

(2005). Autoactive alleles of the flax L6 rust resistance gene induce non-race-specific rust 812

resistance associated with the hypersensitive response. Mol. Plant-Microbe Interact. 18: 813

570–582. 814

Hu, Z. et al. (2013). Crystal structure of NLRC4 reveals its autoinhibition mechanism. Science 815

(80-. ). 341: 172–175. 816

Humphrey, W., Dalke, A., and Schulten, K. (1996). VMD: Visual molecular dynamics. J. 817

Mol. Graph. 14: 33–38. 818

Jones, J.D.G. and Dangl, J.L. (2006). The plant immune system. Nature 444: 323–329. 819

Jones, J.D.G., Vance, R.E., and Dangl, J.L. (2016). Intracellular innate immune surveillance 820


https://doi.org/10.1101/592824

devices in plants and animals. Science 354: aaf6395-aaf6395. 821

El Kasmi, F., Chung, E.-H., Anderson, R.G., Li, J., Wan, L., Eitas, T.K., Gao, Z., and 822

Dangl, J.L. (2017). Signaling from the plasma-membrane localized plant immune receptor 823

RPM1 requires self-association of the full-length protein. Proc. Natl. Acad. Sci. 114: 824

E7385–E7394. 825

Khan, M., Seto, D., Subramaniam, R., and Desveaux, D. (2017). Oh, the places they’ll go! A 826

survey of phytopathogen effectors and their host targets. Plant J. 12: 3218–3221. 827

Khan, M., Subramaniam, R., and Desveaux, D. (2016). Of guards, decoys, baits and traps: 828

Pathogen perception in plants by type III effector sensors. Curr. Opin. Microbiol. 29: 49–55. 829

Kourelis, J. and van der Hoorn, R.A.L. (2018). Defended to the nines: 25 years of resistance 830

gene cloning identifies nine mechanisms for R protein function. Plant Cell 30: 285–299. 831

Kroj, T., Chanclud, E., Michel-Romiti, C., Grand, X., and Morel, J.B. (2016). Integration of 832

decoy domains derived from protein targets of pathogen effectors into plant immune 833

receptors is widespread. New Phytol. 210: 618–626. 834

De La Fuente Van Bentem, S., Vossen, J.H., De Vries, K.J., Van Wees, S., Tameling, 835

W.I.L., Dekker, H.L., De Koster, C.G., Haring, M.A., Takken, F.L.W., and 836

Cornelissen, B.J.C. (2005). Heat shock protein 90 and its co-chaperone protein 837

phosphatase 5 interact with distinct regions of the tomato I-2 disease resistance protein. 838

Plant J. 43: 284–298. 839

Laskowski, R.A., Moss, D.S., and Thornton, J.M. (1993). Main-chain bond lengths and bond 840

angles in protein structures. J. Mol. Biol. 231: 1049–1067. 841

Lechtenberg, B.C., Mace, P.D., and Riedl, S.J. (2014). Structural mechanisms in NLR 842

inflammasome signaling. Curr. Opin. Struct. Biol. 29: 17–25. 843

Leister, R.T., Dahlbeck, D., Day, B., Li, Y., Chesnokova, O., and Staskawicz, B.J. (2005). 844

Molecular genetic evidence for the role of SGT1 in the intramolecular complementation of 845

Bs2 protein activity in Nicotiana benthamiana. Plant Cell 17: 1268–78. 846

Lewis, J.D., Abada, W., Ma, W., Guttman, D.S., and Desveaux, D. (2008). The HopZ family 847

of Pseudomonas syringae type III effectors require myristoylation for virulence and 848

avirulence functions in Arabidopsis thaliana. J. Bacteriol. 190: 2880–2891. 849

Lewis, J.D., Guttman, D.S., and Desveaux, D. (2009). The targeting of plant cellular systems 850

by injected type III effector proteins. Semin. Cell Dev. Biol. 20: 1055–1063. 851


https://doi.org/10.1101/592824

Lewis, J.D., Lee, A.H.-Y., Hassan, J.A., Wan, J., Hurley, B., Jhingree, J.R., Wang, P.W., 852

Lo, T., Youn, J.-Y., Guttman, D.S., and Desveaux, D. (2013). The Arabidopsis ZED1 853

pseudokinase is required for ZAR1-mediated immunity induced by the Pseudomonas 854

syringae type III effector HopZ1a. Proc. Natl. Acad. Sci. U. S. A. 110: 18722–18727. 855

Lewis, J.D., Wilton, M., Mott, G.A., Lu, W., Hassan, J.A., Guttman, D.S., and Desveaux, D. 856

(2014). Immunomodulation by the Pseudomonas syringae HopZ type III effector family in 857

Arabidopsis. PLoS One 9: e116152. 858

Lewis, J.D., Wu, R., Guttman, D.S., and Desveaux, D. (2010). Allele-specific virulence 859

attenuation of the Pseudomonas syringae HopZ1a type III effector via the Arabidopsis 860

ZAR1 resistance protein. PLoS Genet. 6: e1000894. 861

Liu, L., Zhang, Y., Tang, S., Zhao, Q., Zhang, Z., Zhang, H., Dong, L., Guo, H., and Xie, Q. 862

(2010). An efficient system to detect protein ubiquitination by agroinfiltration in Nicotiana 863

benthamiana. Plant J. 61: 893–903. 864

Lolle, S., Greeff, C., Petersen, K., Roux, M., Jensen, M.K., Bressendorff, S., Rodriguez, E., 865

Sømark, K., Mundy, J., and Petersen, M. (2017). Matching NLR immune receptors to 866

autoimmunity in camta3 mutants using antimorphic NLR alleles. Cell Host Microbe 21: 867

518–529.e4. 868

Macho, A.P. (2016). Subversion of plant cellular functions by bacterial type-III effectors: 869

Beyond suppression of immunity. New Phytol. 210: 51–57. 870

Macho, A.P., Guevara, C.M., Tornero, P., Ruiz-Albert, J., and Beuzón, C.R. (2010). The 871

Pseudomonas syringae effector protein HopZ1a suppresses effector-triggered immunity. 872

New Phytol. 187: 1018–1033. 873

Maekawa, T. et al. (2011). Coiled-coil domain-dependent homodimerization of intracellular 874

barley immune receptors defines a minimal functional module for triggering cell death. Cell 875

Host Microbe 9: 187–199. 876

Di Matteo, A., Federici, L., Mattei, B., Salvi, G., Johnson, K.A., Savino, C., De Lorenzo, G., 877

Tsernoglou, D., and Cervone, F. (2003). The crystal structure of polygalacturonase-878

inhibiting protein (PGIP), a leucine-rich repeat protein involved in plant defense. Proc. Natl. 879

Acad. Sci. 100: 10124–10128. 880

McHale, L., Tan, X., Koehl, P., and Michelmore, R.W. (2006). Plant NBS-LRR proteins: 881

adaptable guards. Genome Biol. 7: 212. 882


https://doi.org/10.1101/592824

Meyers, B.C., Kozik, A., Griego, A., Kuang, H., and Michelmore, R.W. (2003). Genome-883

wide analysis of NBS-LRR-encoding genes in Arabidopsis. Plant Cell 15: 809–834. 884

Miras, I., Saul, F., Nowakowski, M., Weber, P., Haouz, A., Shepard, W., and Picardeau, M. 885

(2015). Structural characterization of a novel subfamily of leucine-rich repeat proteins from 886

the human pathogen Leptospira interrogans. Acta Crystallogr. D. Biol. Crystallogr. 71: 887

1351–9. 888

Moffett, P., Farnham, G., Peart, J., and Baulcombe, D.C. (2002). Interaction between 889

domains of a plant NBS-LRR protein in disease resistance-related cell death. EMBO J. 21: 890

4511–9. 891

Monaghan, J. and Zipfel, C. (2012). Plant pattern recognition receptor complexes at the plasma 892

membrane. Curr. Opin. Plant Biol. 15: 349–357. 893

Mudgett, M.B. (2005). New insights to the function of phytopathogenic bacterial type III 894

effectors in plants. Annu. Rev. Plant Biol. 56: 509–531. 895

Myers, E.W. and Miller, W. (1988). Optimal alignments in linear space. Bioinformatics 4: 11–896

17. 897

Pang, Y., Bai, X.C., Yan, C., Hao, Q., Chen, Z., Wang, J.W., Scheres, S.H.W., and Shi, Y. 898

(2015). Structure of the apoptosome: Mechanistic insights into activation of an initiator 899

caspase from Drosophila. Genes Dev. 29: 277–287. 900

Phillips, J.C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., 901

Skeel, R.D., Kalé, L., and Schulten, K. (2005). Scalable molecular dynamics with NAMD. 902

J. Comput. Chem. 26: 1781–802. 903

Qi, D., DeYoung, B.J., and Innes, R.W. (2012). Structure-function analysis of the coiled-coil 904

and leucine-rich repeat domains of the RPS5 disease resistance protein. Plant Physiol. 158: 905

1819–1832. 906

Qi, S. et al. (2010). Crystal structure of the Caenorhabditis elegans apoptosome reveals an 907

octameric assembly of CED-4. Cell 141: 446–457. 908

Rairdan, G.J., Collier, S.M., Sacco, M. a, Baldwin, T.T., Boettrich, T., and Moffett, P. 909

(2008). The coiled-coil and nucleotide binding domains of the potato Rx disease resistance 910

protein function in pathogen recognition and signaling. Plant Cell 20: 739–751. 911

Ravensdale, M., Bernoux, M., Ve, T., Kobe, B., Thrall, P.H., Ellis, J.G., and Dodds, P.N. 912

(2012). Intramolecular interaction influences binding of the flax L5 and L6 resistance 913


https://doi.org/10.1101/592824

proteins to their AvrL567 ligands. PLoS Pathog. 8: e1003004. 914

Reubold, T.F., Wohlgemuth, S., and Eschenburg, S. (2011). Crystal structure of full-length 915

Apaf-1: How the death signal is relayed in the mitochondrial pathway of apoptosis. 916

Structure 19: 1074–1083. 917

Riedl, S.J., Li, W., Chao, Y., Schwarzenbacher, R., and Shi, Y. (2005). Structure of the 918

apoptotic protease-activating factor 1 bound to ADP. Nature 434: 926–33. 919

Roberts, M., Tang, S., Stallmann, A., Dangl, J.L., and Bonardi, V. (2013). Genetic 920

requirements for signaling from an autoactive plant NB-LRR intracellular innate immune 921

receptor. PLoS Genet. 9: e1003465. 922

Schreiber, K.J., Baudin, M., Hassan, J.A., and Lewis, J.D. (2016a). Die another day: 923

Molecular mechanisms of effector-triggered immunity elicited by type III secreted effector 924

proteins. Semin. Cell Dev. Biol. 56: 124–33. 925

Schreiber, K.J., Bentham, A., Williams, S.J., Kobe, B., and Staskawicz, B.J. (2016b). 926

Multiple domain associations within the Arabidopsis immune receptor RPP1 regulate the 927

activation of programmed cell death. PLOS Pathog. 12: e1005769. 928

Schultink, A., Qi, T., Bally, J., and Staskawicz, B. (2019). Using forward genetics in 929

Nicotiana benthamiana to uncover the immune signaling pathway mediating recognition of 930

the Xanthomonas perforans effector XopJ4. New Phytol. 221: 1001–1009. 931

Segonzac, C. and Zipfel, C. (2011). Activation of plant pattern-recognition receptors by 932

bacteria. Curr. Opin. Microbiol. 14: 54–61. 933

Sela, H., Spiridon, L.N., Ashkenazi, H., Bhullar, N.K., Brunner, S., Petrescu, A.-J., Fahima, 934

T., Keller, B., and Jordan, T. (2014). Three-dimensional modeling and diversity analysis 935

reveals distinct Avr recognition sites and evolutionary pathways in wild and domesticated 936

wheat Pm3 R genes. Mol. Plant-Microbe Interact. 27: 835–845. 937

Sela, H., Spiridon, L.N., Petrescu, A.J., Akerman, M., Mandel-Gutfreund, Y., Nevo, E., 938

Loutre, C., Keller, B., Schulman, A.H., and Fahima, T. (2012). Ancient diversity of 939

splicing motifs and protein surfaces in the wild emmer wheat (Triticum dicoccoides) LR10 940

coiled coil (CC) and leucine-rich repeat (LRR) domains. Mol. Plant Pathol. 13: 276–287. 941

Seto, D., Koulena, N., Lo, T., Menna, A., Guttman, D.S., and Desveaux, D. (2017). Expanded 942

type III effector recognition by the ZAR1 NLR protein using ZED1-related kinases. Nat. 943

plants 3: 1–4. 944


https://doi.org/10.1101/592824

Shan, L., Oh, H., Chen, J., Guo, M., Zhou, J.-M., Alfano, J.R., Collmer, A., Jia, X., and 945

Tang, X. (2004). The HopPtoF locus of Pseudomonas syringae pv. tomato DC3000 946

encodes a type III chaperone and a cognate effector. Mol. Plant-Microbe Interact. 17: 447–947

455. 948

Slootweg, E.J. et al. (2018). Distinct Roles of Non-Overlapping Surface Regions of the Coiled-949

Coil Domain in the Potato Immune Receptor Rx1. Plant Physiol. 178: 1310–1331. 950

Slootweg, E.J., Spiridon, L.N., Roosien, J., Butterbach, P., Pomp, R., Westerhof, L., 951

Wilbers, R., Bakker, E., Bakker, J., Petrescu, A.-J., Smant, G., and Goverse, A. (2013). 952

Structural determinants at the interface of the ARC2 and leucine-rich repeat domains 953

control the activation of the plant immune receptors Rx1 and Gpa2. Plant Physiol. 162: 954

1510–28. 955

Sueldo, D.J., Shimels, M., Spiridon, L.N., Caldararu, O., Petrescu, A.-J., Joosten, M.H.A.J., 956

and Tameling, W.I.L. (2015). Random mutagenesis of the nucleotide-binding domain of 957

NRC1 (NB-LRR Required for Hypersensitive Response-Associated Cell Death-1), a 958

downstream signalling nucleotide-binding, leucine-rich repeat (NB-LRR) protein, identifies 959

gain-of-function mutations in t. New Phytol. 208: 210–23. 960

Sukarta, O.C.A., Slootweg, E.J., and Goverse, A. (2016). Structure-informed insights for NLR 961

functioning in plant immunity. Semin. Cell Dev. Biol. 56: 134–149. 962

Swiderski, M.R., Birker, D., and Jones, J.D.G. (2009). The TIR domain of TIR-NB-LRR 963

resistance proteins is a signaling domain involved in cell death induction. Mol. Plant-964

Microbe Interact. 22: 157–165. 965

Takken, F.L.W. and Goverse, A. (2012). How to build a pathogen detector: Structural basis of 966

NB-LRR function. Curr. Opin. Plant Biol. 15: 375–384. 967

Tameling, W.I.L., Elzinga, S.D.J., Darmin, P.S., Vossen, J.H., Takken, F.L.W., Haring, 968

M.A., and Cornelissen, B.J.C. (2002). The tomato R gene products I-2 and MI-1 are 969

functional ATP binding proteins with ATPase activity. Plant Cell 14: 2929–39. 970

Tameling, W.I.L., Vossen, J.H., Albrecht, M., Lengauer, T., Berden, J.A., Haring, M.A., 971

Cornelissen, B.J.C., and Takken, F.L.W. (2006). Mutations in the NB-ARC domain of I-972

2 that impair ATP hydrolysis cause autoactivation. Plant Physiol. 140: 1233–45. 973

Urbach, J.M. and Ausubel, F.M. (2017). The NBS-LRR architectures of plant R-proteins and 974

metazoan NLRs evolved in independent events. Proc. Natl. Acad. Sci. 114: 1063–1068. 975


https://doi.org/10.1101/592824

Vajjhala, P.R., Ve, T., Bentham, A., Stacey, K.J., and Kobe, B. (2017). The molecular 976

mechanisms of signaling by cooperative assembly formation in innate immunity pathways. 977

Mol. Immunol. 86: 23–37. 978

Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003). An enhanced transient 979

expression system in plants based on suppression of gene silencing by the p19 protein of 980

Tomato Bushy Stunt Virus. Plant J. 33: 949–56. 981

Vorobiev, S.M., Neely, H., Seetharaman, J., Ma, L.C., Xiao, R., Acton, T.B., Montelione, 982

G.T., and Tong, L. (2007). Crystal structure of AGR_C_4470p from Agrobacterium 983

tumefaciens. Protein Sci 16: 535–538. 984

Wang, G.-F., Ji, J., EI-Kasmi, F., Dangl, J.L., Johal, G., and Balint-Kurti, P.J. (2015a). 985

Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise 986

structural requirements for activity. PLOS Pathog. 11: e1004674. 987

Wang, G. et al. (2015b). The decoy substrate of a pathogen effector and a pseudokinase specify 988

pathogen-induced modified-self recognition and immunity in plants. Cell Host Microbe 18: 989

285–295. 990

Webb, B. and Sali, A. (2014). Comparative protein structure modeling using MODELLER. 991

Curr. Protoc. Bioinforma. 47: 5.6.1-32. 992

Wendler, P., Ciniawsky, S., Kock, M., and Kube, S. (2012). Structure and function of the 993

AAA+ nucleotide binding pocket. Biochim. Biophys. Acta - Mol. Cell Res. 1823: 2–14. 994

Williams, S.J. et al. (2014). Structural basis for assembly and function of a heterodimeric plant 995

immune receptor. Science 344: 299–303. 996

Williams, S.J., Sornaraj, P., DeCourcy-Ireland, E., Menz, R.I., Kobe, B., Ellis, J.G., Dodds, 997

P.N., and Anderson, P.A. (2011). An autoactive mutant of the M flax rust resistance 998

protein has a preference for binding ATP, whereas wild-type M protein binds ADP. Mol. 999

Plant-Microbe Interact. 24: 897–906. 1000

Wróblewski, T., Spiridon, L., Martin, E.C., Petrescu, A.-J., Cavanaugh, K., Truco, M.J., 1001

Xu, H., Gozdowski, D., Pawłowski, K., Michelmore, R.W., and Takken, F.L.W. (2018). 1002

Genome-wide functional analyses of plant coiled–coil NLR-type pathogen receptors reveal 1003

essential roles of their N-terminal domain in oligomerization, networking, and immunity. 1004

PLOS Biol. 16: e2005821. 1005

Wu, C.-H., Derevnina, L., and Kamoun, S. (2018). Receptor networks underpin plant 1006


https://doi.org/10.1101/592824

immunity. Science 360: 1300–1301. 1007

Xin, X.-F. and He, S.Y. (2013). Pseudomonas syringae pv. tomato, DC3000: A model pathogen 1008

for probing disease susceptibility and hormone signaling in plants. Annu. Rev. Phytopathol. 1009

51: 473–498. 1010

Zhang, L. et al. (2015). Cryo-EM structure of the activated NAIP2-NLRC4 inflammasome 1011

reveals nucleated polymerization. Science 350: 404–9. 1012

Zhang, T., Lei, J., Yang, H., Xu, K., Wang, R., and Zhang, Z. (2011). An improved method 1013

for whole protein extraction from yeast Saccharomyces cerevisiae. Yeast 28: 795–798. 1014

Zhang, X., Dodds, P.N., and Bernoux, M. (2017). What do we know about NOD-like receptors 1015

in plant immunity? Annu. Rev. Phytopathol. 55: 205–229. 1016

1017

1018


https://doi.org/10.1101/592824

FIGURE LEGENDS 1019

1020

Figure 1. Effect of motifs in ZAR1CC on dimerization. 1021

A, Predicted structural representation of a four-helices bundle monomer, a two-helices bundle 1022

monomer and a hairpin dimer. The different helical segments are indicated as α1a, α1b, α2 and 1023

α3. The 4 consecutive helices are colored from bright orange to dark red, and the second 1024

molecule in the dimer is shown in grey. B, Sequence alignment of ZAR1CC and mutants in the 1025

CC domain are shown with a schematic representation of the helical domains. Colored residues 1026

were mutated to the residues shown in each construct. The EDVID motif is boxed. C, Yeast two-1027

hybrid analysis of ZAR1CC dimerization. Sequences in the pEG202 vector are constitutively 1028

expressed as fusions to a LexA DNA-binding domain, while pJG4-5 confers galactose-inducible 1029

expression of sequences as fusions to a B42 transcriptional activation domain. The CC domain 1030

from an unrelated Arabidopsis NLR (At5g48620) was used as a negative control, while the genes 1031

HopF2 and ShcF from Pseudomonas syringae pv. tomato DC3000 are a positive control, having 1032

been previously shown to strongly interact (Shan et al., 2004). 1033

1034

Figure 2. ZAR1CC motifs contribute to HR when inducibly expressed in N. benthamiana. 1035

Agrobacterium tumefaciens carrying the indicated constructs were pressure-infiltrated into N. 1036

benthamiana leaves. Constructs were expressed under a dexamethasone-inducible promoter. A, 1037

CC domain mutants do not trigger a HR-like phenotype. ZAR1CC, ZAR1CC mutants or a control 1038

protein (At3g46600) were expressed as fusions to YFP. Phenotypes were observed and 1039

photographs were taken 48 h after dexamethasone induction. The percentage of leaves showing 1040

the HR phenotype is shown after being scored on a scale from no HR, weak HR, medium HR to 1041

strong HR. A representative leaf with the HR phenotype is shown. The scale bar is 1 cm. The 1042

experiment was repeated 3 times with similar results. B, Immunoblot analysis of ZAR1CC-YFP 1043

protein expression. Samples were harvested 24 h after dexamethasone induction. Equal amounts 1044

of proteins were resolved on 10% SDS-PAGE gels, blotted onto nitrocellulose, and probed with 1045

GFP antibodies in Western blot analysis (WB). The Ponceau red stained blot was used as the 1046

loading control. C, Full-length ZAR1 with mutations in the CC domain exhibit partial or no 1047

complementation of NbZAR1-VIGS. Levels of ion leakage induced by ZAR1, ZAR1 mutants, or 1048

EV were measured at 4 h (T0) and 28 h (T24) after dexamethasone application. Error bars 1049


https://doi.org/10.1101/592824

indicate the standard error from the mean of 6 samples. The letters beside the bars indicate 1050

significance groups, as determined by a one- way ANOVA comparison followed by a Tukey’s 1051

post hoc test (Pvalue ≤ 0.05). The experiment was repeated 3 times with similar results. D, 1052

Immunoblot analysis of ZAR1 protein expression. Samples were harvested 24 h after 1053

dexamethasone induction. Equal amounts of proteins were resolved on 10% SDS-PAGE gels, 1054

blotted onto nitrocellulose, and probed with Myc antibodies in Western blot analysis (WB). The 1055

Ponceau red stained blot was used as the loading control. 1056

1057

Figure 3. The CC and NBARC domains interact and the NBARC domain suppresses 1058

autoactivity. 1059

A, Yeast two-hybrid analysis of ZAR1CC interactions with varying regions of the NBARC 1060

domain. NB indicates that only the NB subdomain is present. NBARC1 indicates that the NB 1061

and ARC1 subdomains are present. NBARC1+2 indicates that the NB, ARC1 and ARC2 1062

subdomains are present. Sequences in the pEG202 vector are constitutively expressed as fusions 1063

to a LexA DNA-binding domain, while pJG4-5 confers galactose-inducible expression of 1064

sequences as fusions to a B42 transcriptional activation domain. The CC domain from an 1065

unrelated Arabidopsis NLR (At5g48620) was used as a negative control, while the genes HopF2 1066

and ShcF from Pseudomonas syringae pv. tomato DC3000 provided a positive control, having 1067

been previously shown to strongly interact (Shan et al., 2004). B, Immunoblot showing the 1068

coimmunopurification of ZAR1CC with ZAR1NB, ZAR1NBARC1 and ZAR1NBARC1+2 from N. 1069

benthamiana tissues. ZAR1CC with a 5xMyc epitope tag was coexpressed with empty vector 1070

(EV) or NBARC variants with a 3xFlag epitope tag in N. benthamiana. Western-blot analysis 1071

(WB) was performed on the crude extract (input) or the immunopurified fractions (IP) from anti 1072

Flag beads. An asterisk indicates the band corresponding to the protein of interest, if multiple 1073

bands were observed. This experiment was repeated three times with similar results. C, Yeast 1074

two-hybrid analysis of interactions between ZAR1CC mutants and ZAR1NBARC1+2, as in A. D, The 1075

NB domain of ZAR1 is sufficient to suppress autoactivity of the CC domain. ZAR1CC, 1076

ZAR1CCNBARC truncations, ZAR1 or a control protein (At3g46600) were expressed as fusions to 1077

YFP. The colored boxes correspond to the following domains: yellow is the CC domain, medium 1078

blue is the nucleotide-binding (NB) subdomain, dark blue is the Apaf1-R protein-CED4 1 1079

(ARC1) subdomain, purple is the ARC2 subdomain, and green is the leucine-rich repeat (LRR) 1080


https://doi.org/10.1101/592824

domain. The percentage of leaves showing the HR phenotype is shown after being scored on a 1081

scale from no HR, weak HR, medium HR to strong HR. Phenotypes were observed 48 h after 1082

dexamethasone induction. The experiment was repeated 3 times with similar results. 1083

1084

Figure 4. Mutations in the NBARC domain do not impair interactions with ZAR1CC or ZAR1CC-1085

YFP autoactivity. 1086

A, ZAR1 sequence schematic showing the three main domains. The colored boxes correspond to 1087

the following domains: yellow is the CC domain, medium blue is the nucleotide-binding (NB) 1088

subdomain, dark blue is the Apaf1-R protein-CED4 1 (ARC1) subdomain, purple is the ARC2 1089

subdomain, and green is the leucine-rich repeat (LRR) domain. The amino acid changes induced 1090

by the mutation are indicated above the schematic. 1091

B, Immunoblot showing the coimmunopurification of ZAR1CC with ZAR1NBARC1+2 wild type 1092

(WT) or carrying mutations from N. benthamiana tissues. ZAR1CC with a 5xMyc epitope tag was 1093

coexpressed with empty vector (EV) or NBARC variants with a 3xFlag epitope tag in N. 1094

benthamiana. Western-blot analysis (WB) was performed on the crude extract (input) or the 1095

immunopurified fractions (IP) from anti Flag beads. An asterisk indicates the band 1096

corresponding to the protein of interest, if multiple bands were observed. This experiment was 1097

repeated three times with similar results. 1098

C, ZAR1CC, ZAR1CCNBARC truncations carrying mutations, or a control protein (At3g46600) 1099

were expressed as fusions to YFP. The percentage of leaves showing the HR phenotype is shown 1100

after being scored on a scale from no HR, weak HR, medium HR to strong HR. Phenotypes were 1101

observed 48 h after dexamethasone induction. The experiment was repeated twice with similar 1102

results. 1103

1104

Figure 5. The LRR and NBARC domain of ZAR1 interact. 1105

A, Immunoblot showing the coimmunopurification of ZAR1LRR with ZAR1NB, ZAR1NBARC1 and 1106

ZAR1NBARC1+2 from N. benthamiana tissues. ZAR1LRR with a 5xMyc epitope tag was 1107


benthamiana. NB indicates that only the NB subdomain is present. NBARC1 indicates that the 1109

NB and ARC1 subdomains are present. NBARC1+2 indicates that the NB, ARC1 and ARC2 1110

subdomains are present. Western-blot analysis (WB) was performed on the crude extract (input) 1111


https://doi.org/10.1101/592824

or the immunopurified fractions (IP) from anti Flag beads. This experiment was repeated three 1112

times with similar results. 1113

B, ZAR1 sequence schematic showing the three main domains. The colored boxes correspond to 1114

the following domains: yellow is the CC domain, medium blue is the nucleotide-binding (NB) 1115

subdomain, dark blue is the Apaf1-R protein-CED4 1 (ARC1) subdomain, purple is the ARC2 1116

subdomain, and green is the leucine-rich repeat (LRR) domain. The amino acid changes induced 1117

by the mutation are indicated above the schematic. 1118

C, Immunoblot showing the coimmunopurification of ZAR1LRR with ZAR1NBARC1+2 wild type 1119

(WT) or carrying mutations from N. benthamiana tissues. ZAR1CC with a 5xMyc epitope tag was 1120


benthamiana. Western-blot analysis (WB) was performed on the crude extract (input) or the 1122

immunopurified fractions (IP) from anti Flag beads. An asterisk indicates the band 1123


repeated twice with similar results. 1125

D, Immunoblot showing the coimmunopurification of ZAR1LRR wild type (WT) or carrying 1126

mutations with ZAR1NBARC1+2 from N. benthamiana tissues. ZAR1LRR or mutants with a 5xMyc 1127

epitope tag were coexpressed with empty vector (EV) or ZAR1NBARC1+2 with a 3xFlag epitope 1128

tag in N. benthamiana. Western-blot analysis (WB) was performed on the crude extract (input) 1129

or the immunopurified fractions (IP) from anti Flag beads. An asterisk indicates the band 1130


repeated twice with similar results. 1132

1133

Figure 6. Predicted models of ZAR1NBARC and ZAR1LRR, with mutations mapped onto the 1134

proposed 3D model. 1135

A, Models of ZAR1NBARC in the closed ADP-bound conformation (left) and open ATP-bound 1136

conformation (right). NB and ARC1 were superimposed showing the rotation of ARC2 in this 1137

transition. Domains are colored as follows: medium blue is the nucleotide-binding (NB) 1138

subdomain, dark blue is the Apaf1-R protein-CED4 1 (ARC1) subdomain, and purple is the 1139

ARC2 subdomain. ADP and ATP are represented as red spheres and mutated amino acids are 1140

colored in magenta. B, The ZAR1LRR domain is shown in the top view with the characteristic 1141

horseshoe shape, and as a side view for the beta sheets. Mutations are colored in magenta, 1142


https://doi.org/10.1101/592824

hydrophobic amino acids are yellow, positively charged amino acid are blue, and negatively 1143

charged amino acids are red. 1144

1145

Figure 7. Model for ZAR1 activation. 1146

In the absence of the pathogen, ZAR1 is present in an equilibrium between inactive complexes 1147

lacking or containing ZED1 or ZRKs. We propose that inactive ZAR1 versus inactive 1148

ZAR1/ZED1/ZRKs complexes would be necessary to allow for interactions of ZAR1 with 1149

diverse RLCKs. We propose that acetylation of ZED1 (shown with stars) by HopZ1a leads to 1150

conformational changes in ZAR1 that make the CC domain more accessible, allow the exchange 1151

of ADP for ATP, and cause rotation of the ARC2 domain. Although dimerization of ZAR1CC has 1152

been shown, it is unknown whether the rest of the protein also self-associates. Intramolecular and 1153

intermolecular conformational changes trigger activation of ZAR1 and ETI. 1154

1155

SUPPLEMENTAL FIGURE LEGENDS 1156

1157

Supplemental Figure S1. Alignment and modeling of ZAR1CC. 1158

A, The Sr33 and Rx structures were used to generate the 1CC4α monomeric model, while 1159

MLA10 structure was used to generate the 2CC2α dimer model of ZAR1CC. The alignment of 1160

ZAR1CC to RPM1 is also shown. Conserved hydrophobic residues are shown in black boxes, and 1161

the EDVID motif is shown in a brown box. Amino acids are colored based on their properties as 1162

follows: hydrophobic (yellow), positively charged (blue), negatively charged (red), proline and 1163

glycine (green), and polar (grey). 1164

B, Schematic (representation of a four-helices bundle monomer, a two-helices bundle monomer 1165

and a hairpin dimer. The different helical segments are indicated as α1a, α1b, α2 and α3.The 4 1166

consecutive helices are colored from bright orange to dark red. N indicates the N-terminus of the 1167

CC domain and C indicates the C-terminus of the CC domain. 1168

C, Mutations were mapped onto the 3D models 1CC4α (CC monomer, left) and 2CC2α (CC 1169

dimer, right) respectively. The 4 consecutive helices are colored as in Figure 1A and 1B, from 1170

bright orange to dark red. The region corresponding to the first turn in monomeric 1CC4α, which 1171

breaks the long α1 helix of the dimer in α1a & α1b, is shown in green. Mutated amino acids are 1172

shown with side chains in sticks&dots representation. 1173


https://doi.org/10.1101/592824

1174

Supplemental Figure S2. Protein expression in yeast strains used for two-hybrid analyses. 1175

Yeast cultures were incubated with shaking at 30°C overnight in SD+Glucose-HisUra (for 1176

pEG202 constructs) or SD+Galactose+Raffinose-Trp (for pJG4-5 constructs media. Proteins 1177

expressed from pEG202 were detected with an α -LexA antibody, while an α -HA antibody was 1178

used to detect proteins expressed from pJG4-5. 1179

1180

Supplemental Figure S3. ZARCC autoactivity in N. benthamiana is independent from NbZAR1. 1181

A. tumefaciens carrying constructs expressing ZAR1CC-YFP, HopZ1a + ZED1, HopZ1a + ZED1 1182

+ ZAR1 or Control-YFP (At3g46600) were infiltrated into N. benthamiana leaves silenced for 1183

GUS or NbZAR1 genes. The HR is shown 48 h after dexamethasone induction (top). The scale 1184

bar is 1 cm. The graph (bottom) quantitates the HR strength for each construct. The experiment 1185

was repeated three times with similar results. 1186

1187

Supplemental Figure S4. BiFC analysis of the ZAR1CC – ZAR1NBARC interaction and protein 1188

expression of constructs. 1189

A, Bimolecular fluorescence complementation of ZAR1CC and ZAR1NBARC interactions. Equal 1190

amounts of A. tumefaciens carrying ZAR1CC or empty vector (EV) as a fusion to cYFP, and A. 1191

tumefaciens carrying ZAR1NBARC truncations as a fusion to nYFP, were mixed and infiltrated. 1192

Leaf sections were imaged 24 h after dexamethasone induction using a Leica SP8 confocal 1193

scanning microscope. The YFP channel is shown for all images. The scale bar is 50 µm. The 1194

experiment was repeated 3 times with similar results. 1195

B, Immunoblot analysis of ZAR1, ZAR1 truncations or control (At3g46600) proteins as fusions 1196

to YFP. Samples were harvested 24 h after dexamethasone induction. Equal amounts of proteins 1197

were resolved on 10% SDS-PAGE gels, blotted onto nitrocellulose, and probed with GFP 1198

antibodies in Western blot analysis (WB). The Ponceau red stained blot was used as the loading 1199

control. 1200

C, Immunoblot analysis of ZAR1CC, ZAR1 truncations carrying mutations, or control 1201

(At3g46600) proteins as fusions to YFP, as in B. 1202

1203


https://doi.org/10.1101/592824

Supplemental Figure S5. Residues associated with autoactive NLRs do not activate immunity 1204

in ZAR1. 1205

A, Schematic representation of putative autoactive mutations in ZAR1. Residues in located in the 1206

following motifs as follows: G194A/K195A in the P-loop, D268E in the kinase 2 motif, and 1207

D489V in the MHD motif. B, HR assay of putative ZAR1 autoactivation mutants. A. tumefaciens 1208

carrying wild type ZAR1 with a myc tag, ZAR1 mutants with a myc tag, or empty vector (EV) 1209

were pressure-infiltrated into N. benthamiana leaves. A. tumefaciens carrying HopZ1a with a HA 1210

tag, and A. tumefaciens carrying ZED1 with a flag tag, were mixed to the same optical density 1211

and pressure-infiltrated into N. benthamiana leaves. Constructs were expressed under a 1212

dexamethasone-inducible promoter. Photographs were taken 48 h after dexamethasone 1213

application. The experiment was repeated 3 times with similar results. C, Immunoblot analysis of 1214

ZAR1 or ZAR1 mutant proteins as fusions to the Myc tag. Samples were harvested 24 h after 1215

dexamethasone induction. Equal amounts of proteins were resolved on 10% SDS-PAGE gels, 1216

blotted onto nitrocellulose, and probed with Myc antibodies in Western blot analysis (WB). The 1217

Ponceau red stained blot was used as the loading control. 1218

1219

Supplemental Figure S6. The NBARC and LRR domains interact in planta by bimolecular 1220

fluorescence complementation. 1221

Equal amounts of A. tumefaciens carrying ZAR1NBARC truncations or mutants, or empty vector 1222

(EV) as a fusion to cYFP, and A. tumefaciens carrying ZAR1LRR as a fusion to nYFP, were 1223

mixed and pressure-infiltrated into N. benthamiana leaves. Constructs were expressed under a 1224

dexamethasone-inducible promoter. Leaf sections were imaged 24 h after dexamethasone 1225

induction using a Leica SP8 confocal scanning microscope. The YFP channel is shown for all 1226

images. The scale bar is 50 µm. The colored boxes correspond to the following domains: 1227

medium blue is the nucleotide-binding (NB) subdomain, dark blue is the Apaf1-R protein-CED4 1228

1 (ARC1) subdomain, purple is the ARC2 subdomain, and green is the leucine-rich repeat (LRR) 1229

domain. NBARC1 indicates that the NB and ARC1 subdomains are present. NBARC1+2 1230

indicates that the NB, ARC1 and ARC2 subdomains are present. The experiment was repeated 3 1231

times with similar results. 1232

1233


https://doi.org/10.1101/592824

Supplemental Figure S7. ZAR1NBARC alignments used to build the closed and open 1234

conformation of ZAR1NBARC 3D models starting from human Apaf1 crystal structures. 1235

Despite the rearrangement of the ARC2 domain which is rotated with respect to NB and ARC1, 1236

the internal structure of each subdomain remains almost unchanged in the two conformational 1237

states. The locations of K195N, V202M, S291N, P359L and L465F are shown with magenta 1238

arrows. Amino acids are colored based on their properties as follows: hydrophobic (yellow), 1239

positively charged (blue), negatively charged (red), proline and glycine (green), and polar (grey). 1240

1241

Supplemental Figure S8. ZAR1LRR alignments and predicted/crystal secondary structure used in 1242

modeling the LRR domain. 1243

LRR motifs are shown in blue boxes. The locations of G645E, P816Q and S831F are shown with 1244

magenta arrows. Amino acids are colored based on their properties as follows: hydrophobic 1245

(yellow), positively charged (blue), negatively charged (red), proline and glycine (green), and 1246

polar (grey). 1247


https://doi.org/10.1101/592824


https://doi.org/10.1101/592824

structure-function analysis of zar1 immune …structure-function analysis of zar1 immune receptor...

Documents