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Plant Physiology Preview. Published on June 26, 2017, as DOI:10.1104/pp.17.00441 1 Short title: Immune complex interactions 2 Corresponding author information: J.D. Lewis; Email: [email protected]; Telephone: 3 510-559-5909; Fax: 510-559-5678 4 Title: Analysis of the ZAR1 immune complex reveals determinants for immunity and 5 molecular interactions 6 Authors: Maël Baudin1, Jana A. Hassan1, Karl J. Schreiber1 and Jennifer D. Lewis1,2*. 7 8 9 1 Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA 2 Plant Gene Expression Center, United States Department of Agriculture, 800 Buchanan St., 10 Albany, CA, 94710, USA 11 One sentence summary: Deciphering the molecular interactions within the ZAR1-ZED1 12 immune complex that lead to the induction of plant immunity. 13 Author contributions: M.B., J.A.H., K.J.S., and J.D.L. performed the experiments, analyzed 14 the data, and wrote the manuscript. 15 Funding: Research on plant immunity in the Lewis laboratory was supported by United 16 States Department of Agriculture Agricultural Research Service 5335-21000-040-00D 17 (J.D.L), NSF IOS-1557661 (J.D.L.), and an INRA postdoctoral fellowship (M.B.). 18 Corresponding author email: [email protected] 19 Abstract: 20 Plants depend on innate immunity to prevent disease. Plant pathogenic bacteria, like 21 Pseudomonas syringae and Xanthomonas campestris, use the type III secretion system as a 22 molecular syringe to inject type III secreted effector (T3SE) proteins in plants. The primary 23 function of most T3SEs is to suppress immunity, however the plant can evolve NBD-LRR 24 domain-containing (NLR) proteins to recognize specific T3SEs. The AtZAR1 NLR induces 25 strong defense responses against P. syringae and X. campestris. The P. syringae T3SE 1 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. Copyright 2017 by the American Society of Plant Biologists 26 HopZ1a is an acetyltransferase that acetylates the pseudokinase AtZED1, and triggers 27 recognition by AtZAR1. However, little is known about the molecular mechanisms that lead 28 to AtZAR1-induced immunity in response to HopZ1a. We established a transient expression 29 system in Nicotiana benthamiana to study detailed interactions among HopZ1a, AtZED1 and 30 AtZAR1. We show that the AtZAR1 immune pathway is conserved in N. benthamiana, and 31 identify AtZAR1 domains, and residues in AtZAR1 and AtZED1, that are important for 32 immunity and protein-protein interactions in planta and in yeast. We show that the coiled-coil 33 domain of AtZAR1 oligomerizes, and this domain acts as a signal to induce immunity. This 34 detailed analysis of the AtZAR1-AtZED1 protein complex provides a better understanding of 35 the immune signaling hub controlled by AtZAR1. 36 37 38 Introduction To successfully colonize their hosts, plant pathogens must overcome several layers of 39 plant innate immunity (Jones and Dangl, 2006; Dou and Zhou, 2012). Many pathogenic 40 bacteria, like Pseudomonas syringae, use a needle-like structure called the Type III Secretion 41 System (T3SS) to directly inject type III effector proteins (T3SE) into the host cell (Galán 42 and Wolf-Watz, 2006). Bacterial effectors are structurally and biochemically very diverse 43 and are able to manipulate the host cell machinery to promote disease (Mudgett, 2005; Grant 44 et al., 2006; Göhre and Robatzek, 2008; Lewis et al., 2009; Deslandes and Rivas, 2012; Xin 45 and He, 2013; Macho and Zipfel, 2015). To counter the activity of these effectors, plants may 46 evolve a new layer of immunity called Effector Triggered Immunity (ETI) that directly or 47 indirectly detects the activity of effector proteins (Jones and Dangl, 2006; Schreiber et al., 48 2016a). This recognition induces strong defense mechanisms, often resulting in a form of 49 localized programmed cell death called the hypersensitive response (HR) (Heath, 2000; Cui 50 et al., 2015). 2 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 51 ETI is typically regulated by Nucleotide-Binding Domain-Leucine-Rich Repeat (NBD- 52 LRR) containing proteins (NLRs, also called NOD-like receptors in the mammalian 53 literature) that act as a molecular switch (Van Der Biezen and Jones, 1998; Ting et al., 2008; 54 Jones et al., 2016). In the absence of the pathogen, NLR proteins are maintained in an “off” 55 state by intra- and inter-molecular interactions. Upon detection of the effector or its activity, 56 NLRs undergo conformational changes and switch to an “on” state resulting in the induction 57 of the downstream signaling (Takken and Goverse, 2012; Sukarta et al., 2016). Plant 58 genomes encode large numbers of NLR proteins, including 151 members in A. thaliana, and 59 are relatively specific for the recognition of effectors (Meyers et al., 2003; Jones et al., 2016). 60 In some cases, the detection of an effector requires another plant protein (the “guardee”) that 61 is guarded by the NLR (Van Der Biezen and Jones, 1998; Dangl and Jones, 2001; Khan et al., 62 2016; Schreiber et al., 2016a). Modification of the “guardee” by the effector is sensed, 63 presumably by conformational changes, and leads to the activation of the NLR. The 64 “guardee” can be a virulence target of the effector; its modification in the absence of the 65 cognate NLR promotes susceptibility. In other cases the NLR monitors a decoy protein that 66 looks like the true virulence target of the effector but does not play a role in immunity beside 67 trapping the effector (van der Hoorn and Kamoun, 2008; Khan et al., 2016). 68 Plant NLR proteins typically contain a Nucleotide-Binding (NB) domain, followed by a 69 LRR domain at the C-terminal end of the NLR (Meyers et al., 2003; Sukarta et al., 2016). 70 The LRR domain plays a role in maintaining the “off” state of the NLR by intra-molecular 71 interactions, and is involved in extra-molecular interaction with the T3SE or the “guardee” 72 (Collier and Moffett, 2009; Qi et al., 2012; Wang et al., 2015b; Schreiber et al., 2016a). The 73 NB domain is believed to act as a molecular switch by binding ADP in the inactive state and 74 ATP in the active state (Takken and Goverse, 2012). In addition to these two domains, plant 75 NLRs possess another N-terminal domain that is commonly a Coiled-coil (CC) or 3 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 76 Toll/interleukin-1 receptor (TIR). The CC or TIR domains are the signaling part of the NLR, 77 and can cause constitutive ETI when they are overexpressed by themselves (Frost et al., 78 2004; Swiderski et al., 2009; Williams et al., 2014). In some cases, the signaling activity 79 involves oligomerization of the CC and TIR domains (Bernoux et al., 2011; Maekawa et al., 80 2011; Williams et al., 2014). Downstream signaling that results after activation of the NLR is 81 still poorly understood. However, the identification of some genetic regulators such as NDR1 82 (NON-RACE-SPECIFIC DISEASE RESISTANCE 1) (Century et al., 1997) and EDS1 83 (ENHANCED DISEASE SUSCEPTIBILITY 1) (Parker et al., 1996), suggest that downstream 84 NLR activation is transduced through several signaling pathways. 85 ZAR1 (HOPZ-ACTIVATED RESISTANCE 1) is a canonical CC-type NLR protein from 86 Arabidopsis thaliana. AtZAR1 was first identified as being required for the recognition of the 87 T3SE HopZ1a from the pathogenic bacteria Pseudomonas syringae (Lewis et al., 2010). 88 HopZ1a is part of the YopJ superfamily of T3SEs, that are found in animal and plant 89 pathogenic bacteria, and is an acetyltransferase (Lewis et al., 2008; Lewis et al., 2011; Lee et 90 al., 2012; Ma and Ma, 2016). Detection of HopZ1a by AtZAR1 requires AtZED1 (HOPZ- 91 ETI-DEFICIENT 1) a Receptor-Like Cytoplasmic Kinase (RLCK) belonging to the RLCK 92 XII-2 family (Lewis et al., 2013). AtZED1 interacts with AtZAR1 and HopZ1a, and is 93 acetylated by HopZ1a, which is hypothesized to trigger the activation of AtZAR1 (Lewis et 94 al., 2013). AtZED1 is believed to be a decoy guarded by AtZAR1, and senses the activity of 95 HopZ1a in the plant cell. More recently, AtZAR1 has been described to be required for the 96 resistance induced by the T3SE AvrAC from Xanthomonas campestris pv. campestris (Wang 97 et al., 2015b), and the recognition of HopF2a, a T3SE with ADP-ribosyltransferase activity, 98 from P. syringae pv. aceris M302273PT (Seto et al., 2017). AvrAC uridylylates PBL2, a 99 RLCK in the VII family, which triggers recognition by the AtZED1-related kinase (ZRK) 100 RKS1 (Resistance related KinaSe 1, also called ZRK1), and AtZAR1 (Feng et al., 2012; 4 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 101 Huard-Chauveau et al., 2013; Wang et al., 2015b). Recognition of HopF2a requires ZRK3 102 and AtZAR1, but HopF2a does not ADP-ribosylate ZRK3, suggesting that ZRK3 may act as 103 an adaptor between AtZAR1 and an unidentified kinase that is modified by HopF2a (Seto et 104 al., 2017). AtZED1, ZRK3 and RKS1 are found in the same genomic cluster of kinase genes, 105 and all three proteins interact with AtZAR1. Interestingly AtZAR1 appears to be a 106 recognition hub that is able to use RLCK XII-2 proteins (AtZED1, ZRK3 and RKS1) to sense 107 three T3SEs that have different enzymatic activities, and are from different bacteria (Lewis et 108 al., 2014a; Roux et al., 2014; Seto et al., 2017). 109 Here, we established a system to study HopZ1a-induced immune responses, and the 110 molecular interactions between HopZ1a, AtZED1 and AtZAR1 in Nicotiana benthamiana. 111 We demonstrate that recognition of HopZ1a requires co-expression with AtZED1, and the N. 112 benthamiana homolog of AtZAR1. We identified essential residues in AtZAR1 or AtZED1 113 for immune induction and protein-protein interactions, and characterized the functions of 114 AtZAR1 domains, in planta and in yeast. This work provides a better understanding of the 115 molecular interactions between HopZ1a, AtZED1 and AtZAR1 that contribute to innate 116 immunity. 117 118 Results 119 ZAR1-dependent recognition of HopZ1a is conserved in Nicotiana benthamiana 120 We sought to establish an Agrobacterium tumefaciens-mediated transient expression 121 system for HopZ1a recognition in N. benthamiana. In order to carefully regulate the 122 expression of HopZ1a, AtZED1, or AtZAR1, we cloned these genes under the control of the 123 dexamethasone-inducible promoter. The expression of HopZ1a or the catalytic mutant 124 HopZ1aC216A (hereafter HopZ1aC/A) (Lewis et al., 2008) did not induce an HR in leaves of 5- 125 week-old plants (Figure 1A). We detected HopZ1a and HopZ1aC/A by Western blot analysis, 5 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 126 which demonstrated that the absence of HR is not due to a lack of protein (Figure S1). When 127 we co-expressed AtZED1 from Arabidopsis thaliana with HopZ1a in N. benthamiana, we 128 observed a strong and rapid HR detectable about 8 hours post-induction (hpi) that was 129 dependent on the catalytic cysteine residue of HopZ1a (Figure 1A). To quantitatively 130 measure the HR, we monitored ion leakage into the medium over time. At 24 hpi (T21), co- 131 expression of HopZ1a and AtZED1 led to a strong and significant increase in conductivity 132 compared to HopZ1a alone (Figure 1A). As expected, the HopZ1aC/A catalytic mutant did not 133 induce ion leakage whether or not it was co-expressed with AtZED1 (Figure 1A). 134 We hypothesized that the HR was dependent on the presence of a functional AtZAR1 135 immune pathway in N. benthamiana. We therefore looked for AtZAR1 homologs in the N. 136 benthamiana draft genome using the blastp tool on the Sol Genomics Network website 137 (Fernandez-Pozo et al., 2015). To specifically identify AtZAR1 homologs, we searched using 138 the AtZAR1CC domain (amino acids 1-144), which is more similar to NLR proteins in other 139 species than it is to NLRs in Arabidopsis (Lewis et al., 2010). We identified two proteins 140 Niben101Scf17398g00012 (hereafter NbZAR1) and Niben101Scf00383g03003 (hereafter 141 NbZAR2), that are 89% identical to each other. When we aligned AtZAR1 protein sequences 142 to NbZAR1 and NbZAR2, we could identify CC, NB and LRR domains in NbZAR1 while 143 NbZAR2 only contained the CC domain and most of the NB domain. NbZAR1 covered 99% 144 of AtZAR1 with 58% identity and NbZAR2 covered 47% of AtZAR1 with 57% identity 145 (Figure S2A). At the mRNA level, NbZAR1 and NbZAR2 were almost identical (95%) and 146 NbZAR2 covered approximately the first half of NbZAR1. We then employed Virus Induced 147 Gene Silencing (VIGS) to knock down the expression of NbZAR1 and NbZAR2 by targeting 148 the first ~700 base pairs of the two genes. As NbZAR1 and NbZAR2 were so similar to each 149 other, we were unable to monitor the mRNA level of each gene independently. Therefore, we 150 measured the pool of mRNA of both genes together (hereafter NbZAR). Using semi- 6 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 151 quantitative RT-PCR, we showed that NbZAR mRNA was strongly reduced in NbZAR-VIGS 152 leaves compared to GUS-VIGS leaves. In addition, the expression of an unrelated N. 153 benthaminana NLR gene Niben101Scf00383g03004 was not affected by the VIGS construct 154 (Figure S2B). These results confirmed the specificity and efficiency of the NbZAR-VIGS 155 construct. In the NbZAR silenced plants, we did not observe an HR when HopZ1a was 156 expressed, or when HopZ1a and AtZED1 were co-expressed (Figure 1B). In the control 157 GUS-silenced VIGS plant, co-expression of HopZ1a and AtZED1 led to a strong HR as 158 observed in wild type N. benthamiana, and similar levels of ion leakage were observed in 159 GUS-silenced VIGS plants and wild type N. benthamiana (Figure 1). Interestingly we were 160 able to partially complement the silencing of NbZAR1 by delivering AtZAR1 with 161 Agrobacterium-mediated transient expression (Figure 1B). Conductivity measurements 162 confirmed the partial complementation, with increases in ion leakage detectable around 24 163 hpi (T21) (Figure 1B). To determine whether silencing of NbZAR was specific, we tested a 164 highly expressed AtRPS2 construct, under a double 35S promoter. RPS2 is normally required 165 for the recognition of AvrRpt2, and does not play a role in HopZ1a recognition (Bent et al., 166 1994; Mindrinos et al., 1994; Lewis et al., 2008). Overexpression of AtRPS2 causes an HR in 167 the absence of its cognate effector in N. benthamiana (Jin et al., 2002). In NbZAR-VIGS 168 plants, AtRPS2 overexpression still causes an HR, confirming the specificity of the NbZAR- 169 VIGS construct (Figure S2C). 170 These results suggest that NbZAR proteins are able to interact with AtZED1 and monitor 171 its acetylation by HopZ1a. To test this hypothesis, we cloned the full-length NbZAR1 and a 172 shorter form of NbZAR1 that encodes the CC and most of the NB domains (hereafter 173 NbZAR1CC-NB). We cloned NbZAR1CC-NB, as this part of the protein is included in NbZAR2, 174 and because we were unable to specifically amplify NbZAR2 due to its high similarity to 175 NbZAR1 (Figure S2). We co-expressed ZAR1 proteins tagged with different epitopes in N. 7 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 176 benthamiana, and then conducted co-immunoprecipitation assays (CoIP). We first co- 177 expressed AtZED1-3xFlag with AtZAR1-5xMyc, and immunoprecipitated (IP) protein 178 complexes using agarose beads coupled with α-Myc antibody. AtZED1-3xFlag only CoIP 179 with AtZAR1-5xMyc, not the 5xMyc tag alone (Figure 1C), which confirms the previously 180 observed AtZAR1-AtZED1 interaction (Lewis et al., 2013; Wang et al., 2015b). 181 Interestingly, AtZED1 was able to interact with NbZAR1 but not NbZAR1CC-NB. This 182 suggests that NbZAR1 is functional in N. benthamiana, and that the C-terminal part of the 183 NB domain and/or the LRR domain is critical for coimmunoprecipitation of ZAR1 and ZED1 184 in planta. Taken together, these results show that HopZ1a recognition depends on a 185 functional ZAR1 immune pathway that is conserved between Arabidopsis thaliana and N. 186 benthamiana. The transient assay system can therefore be used to probe the determinants for 187 HopZ1a recognition. 188 189 AtZED1 interacts with the CC and LRR domains of AtZAR1 190 Previous bioinformatic analysis of AtZAR1 indicated that it was composed of three 191 domains: CC (amino acids 1-144), NB (amino acids 145-391) and LRR (amino acids 610- 192 852) with a linker of unknown function (amino acids 391-609). We reanalyzed the AtZAR1 193 protein by generating a structural model in the Phyre2 web portal (Kelley et al., 2015), and 194 searching for domains in the Conserved Domain database (Marchler-Bauer et al., 2015) and 195 InterPro (Mitchell et al., 2015) (Figure 2A). In this analysis, the CC domain (amino acids 1- 196 144) was the same, while the NB (amino acids 145-504) and LRR (amino acids 526-852) 197 domains comprised the rest of the protein with a short linker domain (amino acids 505-525) 198 (Figure 2A). The Phyre2 analysis identified 13 LRR domains, which is typical of NLR 199 proteins. 8 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 200 Based on our bioinformatic analysis, we sought to investigate the interaction between 201 AtZED1 and AtZAR1 in planta, and refine the regions of AtZAR1 required for this 202 interaction. We generated a series of AtZAR1 deletions from the C-terminal part of the 203 protein, to maintain the CC and NB domains while varying the number of LRRs. These 204 constructs encoded the CC-NB plus 12 LRRs (ZAR1Δ1), 9 LRRs (ZAR1Δ2), 5 LRRs 205 (ZAR1Δ3), or 2 LRRs (ZAR1Δ4) and a 5xMyc tag (Figure 2A). We co-expressed proteins 206 tagged with different epitopes in N. benthamiana, and conducted CoIP assays. AtZED1- 207 3xFlag CoIP with AtZAR1-5xMyc, but not with any of the C-terminal truncations of 208 AtZAR1 (AtZAR1Δ1 to AtZAR1Δ4) (Figure 2B). This indicates that the last 29 amino acids of 209 the LRR domain were required for the interaction. These data, along with our previous yeast 210 two-hybrid interactions between AtZAR1CC and AtZED1 (Lewis et al., 2013), suggest that 211 multiple domains of AtZAR1 interact with AtZED1. Since AtZAR1 was able to partially 212 complement the NbZAR knock down for the induction of HR caused by HopZ1a and AtZED1 213 (Figure 1B), we tested whether the AtZAR∆1 mutant could complement the NbZAR knock 214 down in this system. AtZAR1Δ1 was unable to complement NbZAR-VIGS which validates the 215 importance of these residues (Figure 2C, 2D). 216 We previously demonstrated that the AtZAR1CC domain was sufficient to interact with 217 AtZED1 in the LexA yeast two-hybrid system (Lewis et al., 2013), and wondered whether we 218 might be missing interactions between AtZAR1 domains and AtZED1 due to steric 219 hindrance. We therefore independently tested AtZAR1CC, AtZAR1NB or AtZAR1LRR domains 220 for interaction with AtZED1 (Figure 2A). Interestingly, AtZED1 co-precipitated only with 221 full length AtZAR1 and AtZAR1LRR in planta (Figure 3A). We were unable to recapitulate 222 the interaction between AtZED1 and AtZAR1CC previously observed in yeast (Lewis et al., 223 2013), however this is likely due to a weak or transient interaction in planta. We also tested a 224 series of AtZAR1 truncations which contained different domains, and tested them for CoIP 9 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 225 with AtZED1 (Figure S3A, S3B). AtZED1 only co-precipitated with AtZAR1LRR and 226 AtZAR1LRR2, but not with AtZAR1CC2, AtZAR1CC-NB, AtZAR1CC-NB2, AtZAR1CC-NB3 or 227 AtZAR1NB2 (Figure S3A, S3B). To determine whether the LRR region was sufficient to 228 interact with AtZED1, we compared the AtZED1 interaction with full length AtZAR1, 229 AtZAR1LRR (containing all 13 LRRs), or a shorter AtZAR1LRR2 (containing the last 8 LRRs) 230 (Figure S3C). AtZAR1LRR2 was sufficient for the interaction with AtZED1 in planta, 231 however it did not interact as strongly as AtZAR1LRR or full length AtZAR1. This suggests 232 that other parts of the AtZAR1 protein stabilize the interaction with AtZED1. 233 In previous yeast-two hybrid assays, AtZED1 was tested against AtZAR1CC, AtZAR1NB2, 234 AtZAR1CC-NB2 and AtZAR1 lacking the last 8 LRRs (Lewis et al., 2013). We therefore tested 235 the new AtZAR1 truncations, as a fusion to the activation domain, for interaction with 236 AtZED1, as a fusion to the DNA-binding domain, in the LexA yeast two-hybrid system 237 (Figures 2A, 3B). For each combination, the same optical density of yeast was spotted on the 238 plates in order to quantitate the interaction strength. We observed the same strong interaction 239 between AtZED1 and AtZAR1CC, but we did not observe any interaction between AtZED1 240 and AtZAR1LRR in yeast (Figure 3B) (Lewis et al., 2013). All of these constructs were 241 expressed in yeast, indicating that the lack of interaction was not due to a lack of protein 242 expression (Figure S4). We also tested a shorter version AtZAR1CC2 lacking residues 122- 243 144, and found that it displayed a weaker interaction with AtZED1. This suggests that the last 244 23 residues of AtZAR1CC contribute to the interaction with AtZED1. 245 To confirm the AtZAR1CC-AtZED1 interaction in planta, we used Agrobacterium- 246 mediated transient expression and Bimolecular Fluorescence Complementation (BiFC) in N. 247 benthamiana (Figure 2A). We generated chimeric fusion proteins of AtZAR1CC or AtZED1 248 with the N-terminus of YFP (nYFP) or the C-terminus of YFP (cYFP), expressed under a 249 dexamethasone-inducible promoter (Lewis et al., 2012; Lewis et al., 2014b). We observed 10 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 250 fluorescence mostly in the nucleus when AtZED1-nYFP and AtZAR1CC-cYFP, or 251 AtZAR1CC-nYFP and AtZED1-cYFP were co-expressed, but no fluorescence when the 252 negative controls were expressed (Figure 3C). Taken together, these data demonstrate that 253 multiple domains of AtZAR1 interact with AtZED1 in planta. 254 255 256 Overexpression of AtZAR1CC-YFP induces an HR in N. benthamiana Previous studies have reported that the overexpression of NLR domains can induce auto- 257 activity and spontaneous HR in the absence of the cognate effector (Frost et al., 2004; 258 Bernoux et al., 2011; Maekawa et al., 2011). The autoimmune phenotype can also be 259 enhanced when NLR domains are fused to a GFP or YFP tag, which itself can dimerize 260 (Swiderski et al., 2009; Wang et al., 2015a). We observed an HR when a chimeric construct 261 of AtZAR1CC fused to YFP, was expressed in N. benthamiana. Overexpression of the 262 AtZAR1CC-YFP protein induced an HR in 80% of the infiltrated leaves, of which 20% 263 displayed a strong HR (Figure 4A). The ZARCC-YFP induced HR was associated with a 264 significant increase in ion leakage (Figure 4B, 4C). However, we did not observe a strong HR 265 or an increase in ion leakage when any of the other AtZAR1 domains were expressed (Figure 266 4). Interestingly, ZAR1CC-NB2-YFP did not cause a strong HR or rapid ion leakage (Figure 267 4A, 4B), indicating that the NB domain suppresses the autoactivity of the ZAR1CC domain. 268 As AtZAR1CC-YFP caused a range of HR phenotypes, we measured ion leakage from 269 independent leaves, and binned the data into four groups by the HR phenotype (no HR, weak 270 HR, medium HR, or strong HR). As expected, leaves that did not show an HR had similar 271 levels of ion leakage to EV-YFP whereas leaves with weak, medium or strong HRs displayed 272 increasing levels of conductivity (Figure 4C). 273 The CC or TIR domains of NLRs are known to oligomerize, and to act as a signal for 274 immunity (Krasileva et al., 2010; Bernoux et al., 2011; Maekawa et al., 2011; Williams et al., 11 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 275 2014). We hypothesized that the YFP epitope stabilized the dimerization of AtZAR1CC which 276 then triggered the HR. In order to test this hypothesis, we tested the self-association of 277 AtZAR1CC and AtZAR1CC2 in the LexA yeast two-hybrid system. We observed strong 278 homodimerization of AtZAR1CC and AtZAR1CC2, as well as heterodimerization between 279 AtZAR1CC and AtZAR1CC2 (Figure 5A). This indicated that the first 122 residues of 280 AtZAR1CC are sufficient for homodimerization. 281 To confirm the CC interaction in planta, we used Agrobacterium-mediated transient 282 expression and a BiFC assay in N. benthamiana. We tested chimeric fusion proteins of 283 AtZAR1CC with full-length YFP, nYFP or cYFP, expressed under a dexamethasone-inducible 284 promoter. We observed a strong fluorescent signal in the epidermal cells of N. benthamiana 285 expressing AtZAR1CC-cYFP and AtZAR1CC-nYFP, but not in the cells expressing the 286 negative controls (Figure 5B). The florescence signal was localized to the nucleus and to the 287 cytoplasm, as observed for AtZAR1CC-YFP. We also tested whether co-expression of 288 HopZ1a, or AtZED1 and HopZ1a would affect dimerization of AtZAR1CC. However, we did 289 not observe any effect of the co-expression of AtZED1 and/or HopZ1a on the 290 oligomerization of AtZAR1CC (Figure S5). We attempted to validate the AtZAR1CC 291 interaction using CoIP experiments but were not able to observe a stable interaction. This is 292 likely due to a labile interaction between the CC domains in the absence of the YFP epitope. 293 Taken together, this suggests that dimerization of the CC domain may contribute to the 294 AtZAR1 HR. 295 296 Specific AtZAR1 residues are required for its function in A. thaliana, and contribute to 297 AtZED1 interactions in N. benthamiana 298 In the genetic screen that identified AtZED1 (Lewis et al., 2013), we recovered five EMS 299 mutations in AtZAR1 that abolished the HopZ1a-induced HR. Sequencing of AtZAR1 in each 12 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 300 mutant revealed five amino acid substitutions: Atzar1V202M, Atzar1S291N and Atzar1L465F in the 301 NB domain, and Atzar1G645E and Atzar1S831F in the LRR domain (Figure 6A). We carried out 302 macroscopic HR assays with Pseudomonas syringae pv. tomato DC3000 (hereafter 303 PtoDC3000) carrying hopZ1a under the control of its native promoter (Figure 6B). All of the 304 mutants displayed an abrogated HR, demonstrating that these amino acids are essential for 305 HopZ1a recognition and likely AtZAR1 function. To quantify the degree of HopZ1a-induced 306 immunity in these mutants, we carried out bacterial growth assays with PtoDC3000 carrying 307 HopZ1a. The growth of PtoDC3000 carrying HopZ1a was 1-2 logs lower in Col-0 compared 308 to Atzar1-1 at 3 days post-infection (Figure 6C), as previously observed (Lewis et al., 2010). 309 Atzar1S291N, Atzar1L465F, Atzar1G645E and Atzar1S831F supported similar levels of bacterial 310 growth as Atzar1-1 (Figure 6C). This indicates that the loss of HR in Atzar1S291N, Atzar1L465F, 311 Atzar1G645E and Atzar1S831F is accompanied by a loss of resistance. We were unable to test the 312 Atzar1V202M line, as the seed quality was very poor. 313 We took advantage of our transient assay in N. benthamiana to investigate how these 314 mutations impacted the interaction with AtZED1. We specifically tested for the interaction 315 between the AtZAR1 mutants and AtZED1 by CoIP, as none of the mutations were found in 316 the AtZAR1CC domain. We also included the amino acid substitution Atzar1P816L, that is 317 unable to interact with AtZED1 and was identified in a genetic screen for the loss of AvrAC- 318 induced growth arrest (Wang et al., 2015b). The Atzar1G645E and Atzar1P816L substitutions 319 completely abolished the interaction between AtZAR1 and AtZED1, while the Atzar1S291N 320 substitution strongly reduced the interaction affinity with AtZED1 (Figure 6D). The 321 Atzar1V202M, Atzar1L465F or Atzar1S831F substitutions did not affect the interaction with 322 AtZED1 (Figure 6D). Interestingly, V202, L465, G645, and S831 (Figure S2A), and P359 323 and P816 (from Wang et al., 2015b) are all conserved between AtZAR1 and NbZAR1, while 324 S291 is changed to a threonine in N. benthamiana (Figure S2A). These data suggest that 13 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 325 these residues are particularly important for ZAR1 function, and support a model where 326 multiple domains of AtZAR1 interact with AtZED1. 327 328 329 AtZED1D231 is required for HopZ1a recognition and for interaction with AtZAR1LRR Our genetic screen in A. thaliana for the loss of the HopZ1a-induced HR identified four 330 ethylmethanesulfonate (EMS)-induced mutations in AtZED1 (Lewis et al., 2013). We also 331 identified two threonines (T125 and T177) in AtZED1 that are specifically acetylated by 332 HopZ1a (Lewis et al., 2013). We employed the N. benthamiana system to test the four EMS 333 mutants and the double acetylation mutant for their role in HopZ1a recognition. Surprisingly, 334 co-expression of AtZED1G66S, AtZED1G128E, AtZED1A305T and AtZED1T125A/T177A with 335 HopZ1a led to a macroscopic HR and similar levels of ion leakage compared to HopZ1a co- 336 expressed with wild type AtZED1 (Figure 7). However, co-expression of AtZED1D231N with 337 HopZ1a resulted in a completely abrogated HR, and conductivity levels were comparable to 338 HopZ1a expressed by itself. When we co-expressed HopZ1aC/A with the AtZED1 mutants, 339 none of the AtZED1 mutants showed an HR. This demonstrated that the HR phenotype in N. 340 benthamiana is dependent on the catalytic activity of HopZ1a (Figure 7), as we have 341 previously observed in Arabidopsis (Lewis et al., 2008). 342 To further explore the functions of the AtZED1 point mutants in HopZ1a recognition, we 343 tested these proteins for physical interactions with AtZAR1. We co-expressed AtZED1- 344 3xFlag, the four AtZED1 EMS mutants, or the double acetylation mutant of AtZED1, with 345 AtZAR1-5xMyc in N. benthamiana, and then conducted CoIP assays (Figure 8A). All of the 346 AtZED1 mutants were still able to interact with AtZAR1-5xMyc with the same affinity as 347 wild type AtZED1. Since AtZED1 can interact with multiple domains of AtZAR1 (Figure 3), 348 we tested whether the AtZED1 mutations affected their ability to interact with AtZAR1 349 domains using several different assays. We first tested for an interaction between the AtZED1 14 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 350 mutants and AtZAR1LRR by CoIP. Interestingly, the interaction between AtZED1D231N and 351 AtZAR1LRR was completely abrogated (Figure 8B). AtZED1G128E interacted more weakly 352 with AtZAR1LRR, while AtZED1G66S, AtZED1A305T, and AtZED1T125A/T177A were still able to 353 interact with AtZAR1LRR. In addition, we tested whether the AtZED1 mutations affected in 354 planta interactions with AtZAR1CC by BiFC. When AtZAR1CC-nYFP was co-expressed with 355 AtZED1G66S, AtZED1D231N or AtZED1A305T as cYFP fusions, all displayed reduced nuclear 356 fluorescence, compared to AtZAR1CC-nYFP and AtZED1-cYFP (Figure 8C). The acetylation 357 mutant AtZED1T125A/T177A-cYFP or AtZED1G128E, co-expressed with AtZAR1CC-nYFP, 358 displayed nuclear-localized fluorescence that was indistinguishable from that of AtZAR1CC- 359 nYFP and AtZED1-cYFP (Figure 8C). Lastly, we tested for interactions between AtZAR1CC 360 and AtZED1 or the AtZED1 mutants in the yeast two-hybrid system, as previously described 361 (Figure 3B). We observed an interaction between AtZAR1CC and AtZED1D231N that was 362 similar to the interaction between AtZAR1CC and wild type AtZED1, in multiple experiments 363 (Figure S6A). However, AtZED1G66S, AtZED1G128E and AtZED1A305T showed a weaker 364 interaction with AtZAR1CC compared to the native AtZED1. The weaker interaction was not 365 due to differing levels of protein expression, as all proteins were expressed to a similar level 366 (Figure S4). 367 In addition to its physical association with AtZAR1, AtZED1 has been shown to directly 368 interact with HopZ1a (Lewis et al., 2013). We therefore tested whether the mutations in 369 AtZED1 compromised the interaction with HopZ1a by BiFC in N. benthamiana (Figure 8D). 370 We used the catalytic mutant of HopZ1a (HopZ1aC/A) in these assays, as co-expression of 371 HopZ1a and AtZED1 causes a rapid HR (Figure 1). Co-expression of HopZ1aC/A-nYFP and 372 AtZED1-cYFP resulted in strong fluorescence, primarily in the vicinity of the plasma 373 membrane and in the nucleus, as we have previously observed (Lewis et al., 2013). When 374 HopZ1aC/A-nYFP was co-expressed with AtZED1G66S, AtZED1G128E, AtZED1D231N or 15 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 375 AtZED1A305T as cYFP fusions, we observed very little or no fluorescence (Figure 8D). 376 Although the level of fluorescence was weaker, AtZED1T125A/T177A-cYFP co-expressed with 377 HopZ1aC/A-nYFP displayed similar patterns of fluorescence as AtZED1-cYFP and 378 HopZ1aC/A-nYFP, indicating that it could still interact (Figure 8D). We attempted to confirm 379 the loss of these interactions in planta using the CoIP system but we were unable to observe 380 any interaction between HopZ1a and AtZED1, or between HopZ1aC/A and AtZED1. This is 381 likely due to a transient or weak interaction between these two proteins in planta. We also 382 employed the LexA yeast two-hybrid system to further explore the effect of AtZED1 383 mutations on their interaction with HopZ1a. AtZED1 showed an interaction with HopZ1a as 384 we have previously observed (Lewis et al., 2013) (Figure S6B). However, the AtZED1G66S, 385 AtZED1G128E and AtZED1D231N mutations strongly reduced the interaction with HopZ1a, 386 while the AtZED1A305T mutation totally abolished the interaction with HopZ1a (Figure S6B). 387 The lack of interaction is not due to a lack of protein expression (Figure S4). 388 Taken together, our data show that AtZED1G66S, AtZED1G128E, and AtZED1A305T are still 389 able to contribute to an HR when overexpressed with HopZ1a in N. benthamiana, despite 390 their weaker interactions with HopZ1a, AtZAR1CC and AtZAR1LRR. AtZED1T125A/T177A is 391 able to interact with HopZ1a, AtZAR1CC and AtZAR1LRR, and triggers an HR when 392 overexpressed with HopZ1a in N. benthamiana, suggesting there are additional acetylated 393 sites on AtZED1. AtZED1D231N is unable to interact with AtZAR1LRR, and has weaker 394 interactions with HopZ1a and AtZAR1CC. Importantly, this mutation impairs the HR when 395 AtZED1D231N is co-expressed with HopZ1a, suggesting that an interaction between AtZED1 396 and the LRR domain is critical to trigger an HR. 397 398 Discussion 16 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 399 Here we established a transient expression system in N. benthamiana to decipher the 400 molecular mechanisms leading to the defense responses activated by AtZAR1. We 401 demonstrate that N. benthamiana contains a conserved ZAR1-dependent immune signaling 402 pathway that induces a strong and rapid HR in the presence of HopZ1a and AtZED1 (Figure 403 1). We used this system and yeast to investigate interactions among HopZ1a, AtZED1 and 404 AtZAR1, and demonstrate roles for specific domains (Figures 2 and 3). We show that 405 overexpression of AtZAR1CC-YFP results in an HR (Figure 4), and that AtZAR1CC is able to 406 self-associate both in yeast and in planta (Figure 5). Lastly, we used our transient assay 407 system to dissect the functions of specific AtZAR1 and AtZED1 point mutations (Figures 6, 408 7 and 8). We demonstrate that multiple domains of AtZAR1 interact with AtZED1, and that 409 specific residues in AtZAR1 and AtZED1 contribute to immunity and/or protein-protein 410 interactions. 411 Co-expression of HopZ1a and AtZED1 leads to a strong immune response that is 412 dependent on a pair of N. benthamiana NLR proteins closely related to AtZAR1 (Figure 1A, 413 1B). NbZAR1 is a CC-NB-LRR protein with 845 amino acids, compared to 852 amino acids 414 in AtZAR1, while NbZAR2 is only 374 amino acids long and lacks a LRR domain (Figure 415 S2A). We identified five new amino acid substitutions in AtZAR1 that lead to a complete 416 loss of recognition of HopZ1a in A. thaliana (Figure 6A). Four of the five residues identified 417 in this study and the two residues identified in Wang and colleagues (2015b) (Figure 6A) are 418 conserved in NbZAR1 (Figure S2A). We were able to partially complement (~65% of the ion 419 leakage phenotype) the down-regulation of NbZAR genes by transiently expressing AtZAR1 420 under the control of a dexamethasone-inducible promoter (Figure 1B). We may have 421 observed partial complementation as we used an inducible promoter instead of the native 422 promoter, which can result in an unbalanced stoichiometry of the different proteins. NbZAR 423 and AtZAR1 are dissimilar at the nucleotide level; they share <70% of identity, and lack a 17 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 424 20bp stretch of common nucleotides, which would be necessary for silencing. Thus we do not 425 think it is likely that the NbZAR-VIGS construct affects the expression of AtZAR1. 426 Previous phylogenetic analysis showed that ZAR1 was an ancient NLR, with putative 427 homologs in a wide range of plant species (Lewis et al., 2010). Here, we show that the 428 endogenous NbZAR1 protein is able to interact with transiently expressed AtZED1 to 429 activate the HopZ1a-induced HR (Figure 1). Our data demonstrate that ZAR1 recognition is 430 functionally conserved from the Brassicaceae to the Solanaceae, and the ZAR1-signaling 431 pathway is at least partially conserved between A. thaliana and N. benthamiana. To our 432 knowledge, this is the first example of conserved bacterial effector recognition across plant 433 species. Recognition of AvrRpt2 in N. benthamiana requires co-expression of the RPS2 NLR 434 and its guardee RIN4 (Day et al., 2005). Similarly, recognition of AvrPphB in N. 435 benthamiana requires co-expression of the RPS5 NLR and its decoy PBS1 (DeYoung et al., 436 2012). Our data also suggest that ZED1 is not conserved, which is perhaps not surprising as 437 ZAR1 guards multiple kinases in Arabidopsis (Lewis et al., 2013; Seto et al., 2017; Wang et 438 al., 2015b). 439 We demonstrated that AtZAR1LRR2 interacts with AtZED1 by CoIP in planta (Figure 440 3A), and that the last 29 amino acids of AtZAR1 are required for this interaction (Figure 2B). 441 We also demonstrated that the AtZAR1CC domain interacts with AtZED1 by BiFC in planta 442 (Figure 3C) and in yeast two-hybrid assays (Figure 3B), indicating that multiple domains of 443 AtZAR1 are involved in the interaction with AtZED1. This conclusion is also reinforced by 444 our data showing that AtZAR1LRR2 (with the last 8 LRRs) displays a significantly weaker 445 interaction compared to AtZAR1LRR (with all 13 LRRs) or full length AtZAR1. In addition, 446 AtZAR1G645E (in the 6th LRR repeat), and AtZAR1P816L (in the 12th LRR repeat) (Wang et al., 447 2015b) do not interact with AtZED1, and AtZAR1S291N (in the NB domain) had a weaker 448 interaction with AtZED1 (Figure 6D). Wang and colleagues (2015b) demonstrated that 18 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 449 AtZAR1LRR2 interacted with the RKS1 pseudokinase in planta, and that the AtZAR1P816L 450 substitution abolished interactions with AtZED1 (Figure 6D), as well as RKS1, ZRK3, ZRK6 451 and ZRK15. This suggests that the AtZAR1–pseudokinase interaction surfaces at least 452 partially overlap. Interestingly, the AtZAR1S831F substitution (in the 13th LRR) abrogates the 453 HopZ1a-induced HR in A. thaliana, but is not affected in the interaction with AtZED1. This 454 substitution might contribute to transducing HopZ1a recognition into effective immunity. The 455 AtZAR1S291N, AtZAR1L465F, AtZAR1G645E and AtZAR1S831F substitutions all result in a loss 456 of HR and a loss of resistance, unlike the HR-independent resistance described for AvrAC, 457 HopF2a, HopZ5, HopA1 (formerly HopPsyA) or AvrRps4 (Gassmann et al., 1999; 458 Gassmann, 2005; Jayaraman et al., 2017; Seto et al., 2017; Wang et al., 2015b). 459 The yeast two-hybrid system provides a complementary approach to dissect immune 460 complex interactions, as it may reveal interactions that are weak or transient in planta and 461 missed by CoIP (Xing et al., 2016). We previously showed that AtZAR1CC was sufficient for 462 the interaction with AtZED1 in yeast (Lewis et al., 2013) (Figure 3B). Interestingly, this 463 interaction was strongly reduced when the shorter AtZAR1CC2 was used (Figure 3B), 464 suggesting that the CC region between amino acids 122-144 contributes to the AtZED1 465 interaction. Taken together, our data indicate that the CC and LRR domains of AtZAR1 are 466 involved in the interaction with AtZED1. Different domains of NLRs have previously been 467 showed to interact with their guarded targets (Collier and Moffett, 2009). The RPS5 NLR 468 guards the PBS1 kinase, and triggers resistance when the cysteine protease AvrPphB cleaves 469 PBS1 (Shao et al., 2002; Shao et al., 2003; DeYoung et al., 2012). The CC domain of RPS5 470 interacts with PBS1, and the LRR domain is required to sense the cleavage of PBS1 by 471 AvrPphB (Ade et al., 2007; Qi et al., 2012). 472 We showed that AtZAR1CC as a YFP protein fusion induces an HR when overexpressed 473 in N. benthamiana leaves. Although we observed some variability in the strength of the HR 19 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 474 (Figure 4), we found there was a strong correlation between the strength of the macroscopic 475 HR and the level of ion leakage (Figure 4C). Such variability in the intensity of HR induced 476 by auto-active NLRs has been shown for several other NLRs, including Rx, I-2 from tomato, 477 L6 from flax, Rp1-D21 from maize, and SNC1 from Arabidopsis (Bendahmane et al., 2002; 478 Shirano et al., 2002; De La Fuente Van Bentem et al., 2005; Tameling et al., 2006; Wang et 479 al., 2015a; Bernoux et al., 2016). We also observed that AtZAR1CC oligomerizes in both 480 yeast and in planta (Figure 5). GFP or YFP fluorescent proteins are known to form weak 481 homodimers (Zacharias, 2002). However in yeast, the CC domain was constructed as a DNA- 482 binding domain or activation domain fusion, indicating that dimerization does not require the 483 YFP moiety. We hypothesize that the formation of this dimer is linked to the induction of the 484 HR, and that the YFP tag artificially stabilizes the formation of AtZAR1CC dimers which 485 result in the induction of HR in planta. Interestingly, inclusion of part of the NB domain 486 (AtZARCC-NB2-YFP) is sufficient to suppress the AtZAR1CC auto-active HR (Figure 4), 487 suggesting that conformational changes to AtZAR1 may be important in triggering the HR. 488 Other NLRs have been shown to be auto-active as GFP or YFP fusions, including the CC 489 domain of maize Rp1-D21 and Rp1-D (Wang et al., 2015a), the TIR domain of RPS4 490 (Swiderski et al., 2009), and the CC domain of barley MLA10 (Maekawa et al., 2011). 491 Solving the structures of the CC domain for Rx (Hao et al., 2013), MLA10 (Maekawa et al., 492 2011) and the wheat NLR Sr33 (Casey et al., 2016) revealed the protein surfaces involved in 493 oligomerization, and strongly suggested that these interactions are required for the induction 494 of the HR. In Sr33 and MLA10, the region between the amino acids 120 and 142 is required 495 for both oligomerization and the induction of HR (Casey et al., 2016; Césari et al., 2016). 496 AtZAR1CC also contains an acidic EDVID motif (amino acids 73-77) that was first identified 497 in the potato Rx NLR, which recognizes Potato virus X coat protein (Bendahmane et al., 498 1999; Rairdan et al., 2008). The EDVID motif is necessary for Rx intramolecular 20 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 499 interactions, while other regions of the Rx CC domain contribute to interactions with 500 RanGAP2, which regulates nucleocytoplasmic trafficking of Rx (Sacco et al., 2007; 501 Tameling and Baulcombe, 2007; Rairdan et al., 2008; Tameling et al., 2010; Hao et al., 502 2013). Our CC truncations suggest that the EDVID motif does not contribute to AtZAR1- 503 AtZED1 interactions, but could contribute to dimerization of the CC domain. The crystal 504 structure of AtZAR1CC has not yet been solved, however a fine structure-function analysis 505 may reveal the residues required for AtZAR1CC oligomerization and signaling. 506 We previously identified four point mutations in AtZED1 that abolish the HopZ1a- 507 induced HR in A. thaliana when delivered by PtoDC3000 (Lewis et al., 2013). When we 508 tested these mutants in our N. benthamiana system, co-expression of HopZ1a with 509 AtZED1G66S, AtZED1G128E or AtZED1A305T induced an HR, while AtZED1D231N lacked an 510 HR (Figure 7). This suggests that the modification induced by the AtZED1D231N mutation has 511 a more severe effect on the protein and cannot be compensated by overexpression in the N. 512 benthamiana system. We also tested the four AtZED1 mutants for interactions with AtZAR1 513 in planta, AtZAR1CC in planta and in yeast, and HopZ1a in yeast. AtZED1G66S, and 514 AtZED1A305T had weaker interactions with AtZAR1CC in planta and in yeast, compared to 515 AtZED1, while AtZED1G128E and AtZED1D231N were still able to interact with AtZAR1CC 516 (Figure 8C and S6A). Although all four AtZED1 mutants were able to interact with full- 517 length AtZAR1 in planta (Figure 8A), AtZED1D231N was unable to interact with AtZAR1LRR, 518 and AtZED1G128E had a weaker interaction with AtZAR1LRR (Figure 8B). AtZED1G66S, 519 AtZED1G128E, AtZED1D231N, and AtZED1A305T displayed weaker or no interactions with 520 HopZ1a in planta and in yeast (Figure 8D, S6B). We speculate that overexpression of 521 AtZED1G66S, AtZED1G128E, and AtZED1A305T might compensate for weaker interactions with 522 HopZ1a and AtZAR1CC, and consequently trigger the immune response. The AtZED1D231N 523 mutation displays a loss of interaction with AtZAR1LRR (Figure 8B) and an abrogated HR 21 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 524 when co-expressed with HopZ1a (Figure 7). This suggests that the AtZED1-AtZAR1LRR 525 interaction is required for an HR. Interestingly, the AtZED1G66, AtZED1D231 and AtZED1A305 526 amino acids are conserved among the ZED1-RELATED KINASEs (ZRKs), RKS1, ZRK3, 527 ZRK6 and ZRK15, that have been shown to interact with AtZAR1 (Wang et al., 2015b). We 528 infer that G66, D231 and A305 contribute to core functions of AtZAR1-interacting 529 pseudokinases, and that D231 is necessary for the LRR interaction. 530 The N. benthamiana system also provided us with the opportunity to test the double 531 acetylation mutant AtZED1T125A/T177A for its ability to activate the HopZ1a-induced HR, and 532 for its interactions with AtZAR1 and HopZ1a. AtZED1T125A/T177A was still able to interact 533 with full-length AtZAR1, AtZAR1CC and AtZAR1LRR, and HopZ1aC/A in planta (Figure 8), 534 suggesting these residues are not necessary for interaction. Co-expression of HopZ1a with 535 AtZED1T125A/T177A or native AtZED1 induced similarly strong HRs in N. benthamiana 536 (Figure 7), suggesting that AtZED1 is acetylated on additional residues and that acetylation 537 of these unknown residues is sufficient for the recognition of HopZ1a in N. benthamiana 538 (Figure 7). Our previous data support the possibility of additional acetylated sites, as 539 ZED1T177A still exhibited 15% acetylation in vitro while AtZED1T125A/T177A showed 68% 540 acetylation in vitro compared to AtZED1 (Lewis et al., 2013). 541 AtZAR1 appears to be a platform for the ZRK pseudokinases, and allows the host to 542 perceive at least two unrelated bacteria, P. syringae and X. campestris (Lewis et al., 2014a; 543 Roux et al., 2014; Wang et al., 2015b; Seto et al., 2017). Interestingly, AtZAR1 seems to 544 activate two levels of immunity depending on the T3SE that is sensed. AvrAC induces a 545 weak ETI that is described as broad spectrum immunity (Xu et al., 2008; Huard-Chauveau et 546 al., 2013), and HopF2a induces a HR-independent ETI (Seto et al., 2017). In contrast, 547 HopZ1a induces a strong ETI with a typical macroscopic HR (Lewis et al., 2008; Lewis et al., 548 2010). HopF2a may behave more like AvrAC, and modify a different kinase that interacts 22 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 549 with a ZRK3-AtZAR1 complex (Seto et al., 2017). We propose that for at least HopZ1a, 550 recognition occurs as a multi-step process. First, HopZ1a acetylates AtZED1 on at least two 551 residues (Lewis et al., 2013), which cause conformational changes that are detected by 552 AtZAR1. AtZAR1 is proposed to then unfold, allowing the exchange of ADP to ATP 553 (Lukasik and Takken, 2009; Takken and Goverse, 2012). AtZAR1CC might then dimerize and 554 trigger downstream immune signaling. Alternatively, AtZAR1 might exist as a dimer through 555 interactions between the CC domain (Figure 5) prior to effector recognition, but not be 556 competent for signaling due to intramolecular interactions (Figure 4A). In either case, 557 intramolecular interactions are likely to help stabilize CC dimerization, as has been shown for 558 RPP1 and RPS5 (Ade et al., 2007; Schreiber et al., 2016b). However, our data do not allow 559 us to distinguish between pre-activation or post-activation interactions of the NLR. 560 Regardless, the CC domain can induce an effector-independent HR (Figure 4). The 561 downstream pathway activated by AtZAR1 in response to HopZ1a is still uncharacterized 562 and does not involve any of the classic ETI regulators identified so far (Lewis et al., 2010; 563 Macho et al., 2010). Our identification of an auto-active form of AtZAR1 will help to identify 564 new components of this signaling pathway. Finally, we still have much to learn regarding the 565 conformational changes and molecular interactions that lead to the activation of AtZAR1 566 upon perception of the effector. 567 568 Materials and Methods 569 Cloning 570 Phusion polymerase (New England Biolabs) was used for all cloning, and all constructs were 571 confirmed by sequencing. Sequence analysis was performed with CLC Main Workbench. All 572 constructs for Pseudomonas expression were expressed under their native promoters, and 573 contained an in-frame HA tag at the C terminus, as described in Lewis and colleagues (2008). 23 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 574 For the VIGS constructs, the genomic fragment of NbZAR1 gene was amplified to 575 contain a 5’EcoRI site and a 3’XhoI site and then cloned into pYL156 vector (Hayward et al., 576 2011). To construct 5xMyc fusions for our proteins of interest for coimmunoprecipitation, the 577 genes were amplified by PCR to add a 5’XhoI site, and cloned into the pMac15 vector to 578 maintain the frame for the vector-encoded C-terminal 5xMyc tag. The pMac15 vector was 579 modified from pBD to contain a 5xMyc tag between the StuI and SpeI sites. The 3xFlag 580 constructs were cloned by a cross-over PCR approach to add the 3xFlag tag in frame at the 3’ 581 end. The PCR products were then cloned into the pMac14 vector (modified from pBD) 582 (Aoyama and Chua, 1997; Lewis et al., 2008). The AtZAR1 point mutants were generated 583 using the Q5 Site-directed mutagenesis kit (New England Biolabs). For the split YFP 584 experiment, the genes were amplified by PCR to contain a 5′ XhoI restriction site. The 3′ 585 primers were designed to maintain the reading frame of the C-terminal fusion. pBD-YFP, 586 pBD-nYFP and pBD-cYFP were modified from pTA7002 (Aoyama and Chua, 1997) to add 587 an HA tag and the full-length YFP, the N-terminus of YFP (nYFP, residues 1–155), or the C- 588 terminus of YFP (cYFP, residues 156 to the stop codon) between the StuI and SpeI sites 589 (Lewis et al., 2014b). 590 For yeast two-hybrid experiments, we cloned AtZAR1, AtZED1 or HopZ1a as in- 591 frame fusions to the B42 activation domain and HA tag in the pJG4-5 vector, under the 592 control of the GAL1 promoter (DupLEXA Yeast Two Hybrid System; OriGene 593 Technologies). We amplified AtZAR1 by PCR using primers to add XhoI sites at the 5’ and 594 3’ ends, and constructed two AtZAR1 clones. AtZAR1CC (containing residues 1-144) 595 includes the N-terminal coiled-coil domain, including the EVILVD motif. AtZAR1CC2 596 (containing residues 1-122) includes the EVILVD motif, and lacks the last 22 residues in 597 AtZAR1CC. We amplified AtZED1 and zed1 point mutants, AtZED1G66S, AtZED1G128E, 598 AtZED1D231N and AtZED1A305T, by PCR using primers to add unique XhoI and EcoRI sites at 24 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 599 the 5’ and 3’ ends respectively. HopZ1a and HopZ1aC/A were amplified by PCR, using 600 primers to add unique BamHI and NotI at the 5’ and 3’ ends, and a C-terminal HA tag to the 601 3’ end. To clone AtZAR1 domain truncations into the pEG202 bait vector with a LexA 602 binding domain, we amplified AtZAR1CC by PCR using primers to add NotI to the 5’ and 3’ 603 ends. For AtZARCC2, AtZAR1NB3, and AtZAR1LRR, we amplified the genes by PCR using 604 primers to add XhoI to the 5’ and 3’ ends. AtZAR1NB3 includes the NB domain (residues 122- 605 504). AtZAR1LRR contains residues 526-852, with the 13 LRRs but not the linker region 606 between the NB and the LRR domain. HopF2 was amplified by PCR, cloned into Gateway- 607 compatible vector, pDONR207 through the BP reaction, and then cloned into pEG202 using 608 the LR reaction. 609 610 Plant material and growth conditions 611 Plants were grown in a growth chamber at 22°C with 9 h light (~130 µEinsteins m-2 s-1) and 612 15 h dark cycles. For Nicotiana benthamiana, seeds were sterilized in 10% bleach for 10 min 613 and then washed 6 to 7 times with sterile nanopure H2O. After germination on soil, seedlings 614 were transplanted onto individual pots, and used 3 to 5 weeks after transplanting. For 615 Arabidopsis thaliana, the seeds were vapor-sterilized (Clough and Bent, 1998), and then 616 germinated on 0.5X Murashige and Skoog and 0.8% agar media. After germination, the 617 seedlings were transplanted on Sunshine #1 soil supplemented with 20:20:20 fertilizer, and 618 tested 3 to 4 weeks after transplanting. 619 620 Agrobacterium-mediated transient expression 621 A. tumefaciens GV2260 cultures were grown overnight at 28°C in LB broth with kanamycin 622 and rifampicin. The next day, the cultures were resuspended in 10 mL of induction medium 623 (50 mM MES pH 5.6, 0.5% (wt/vol) glucose, 1.7 mM NaH2PO4, 20mM NH4Cl, 1.2 mM 25 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 624 MgSO4, 2mM KCl, 17 μM FeSO4, 70 μM CaCl2, and 200 μM acetosyringone) and incubated 625 at 28°C for ~4 hours. Cultures were spun down and resuspended in 10 mM MES pH 5.6 with 626 200 μM acetosyringone to an optical density of 600 nm (OD600) of 1.0. The cultures 627 containing each plasmid were mixed in equal volumes to a final OD600 of 0.25 per construct. 628 The underside of the leaves of 5- to 7-week-old Nicotiana benthamiana plants were 629 infiltrated by hand with a needleless syringe. For dexamethasone-inducible constructs, the 630 plants were sprayed with 20 μM dexamethasone (Sigma) 6–24 h after inoculation. Tissue was 631 collected 3–24 h after dexamethasone induction. For the VIGS experiments, the plants were 632 used 1 to 2 weeks after inoculation. 633 634 RT-PCR 635 Total RNA was extracted from N. benthamiana leaves using Trizol reagent (Ambion) 636 according to the manufacturer’s protocol. Reverse transcription (RT) was performed on 1 µg 637 of RNA using SuperScript III Reverse Transcriptase (Invitrogen) following the 638 manufacturer’s protocol. RT-PCR was performed on 1 µL of 1/20 cDNA dilution using 639 GoTaq DNA polymerase (Promega) following the manufacturer’s protocol. We used specific 640 primers targeting NbZAR genes or the NLR Niben101Scf00383g03004. The N. benthamiana 641 TUBULIN (NbTUB) gene was used as an internal control. 642 643 Plant infection assays 644 For ion leakage assays in N. benthamiana, 2 disks (1.5 cm2) were harvested, soaked in 645 nanopure H20 for 1 hour and transferred to 6 mL of nanopure H2O. Readings were taken with 646 an Orion 3 Star conductivity meter (Thermo Electron Corporation, Beverly, MA). For 647 infiltrations in Arabidopsis thaliana, PtoDC3000 was resuspended to an OD600 = 0.1 (∼5 × 648 107 cfu/mL) in 10 mM MgCl2 for HR assays, or to 1 × 105 cfu/mL in 10 mM MgCl2 for 26 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 649 bacterial growth assays. Diluted inocula were hand-infiltrated by using a needleless syringe 650 as described in Katagiri and colleagues (2002). The HR was scored at 16–24 hpi. For growth 651 assays, 4 disks of 1.5 cm2 were harvested, ground in 10 mM MgCl2, and plated on KB with 652 rifampicin and cycloheximide on day 0 and day 3. 653 654 Co-immunopurification 655 In planta co-immunopurification were performed using 3 cm2 of N. benthamiana leaves 656 transiently expressing our genes of interest. Tissue was homogenized in liquid nitrogen and 657 protein complexes were extracted using 1mL of IP1 buffer (50 mM HEPES, 50 mM NaCl, 10 658 mM EDTA, 0.2% Triton X-100, 0.1 mg/mL Dextran (Sigma D1037) pH 7.5). The clarified 659 extract was then mixed with 100 µL of protein G agarose beads coupled with anti c-Myc 660 antibody (Abcam ab32072) and incubated in a rocking shaker for 2.5 hours at 4°C. After 661 centrifugation at 5000 x g at 4°C, the co-immunoprecipitated proteins were washed twice 662 with IP2 buffer (50 mM HEPES, 50 mM NaCl, 10 mM EDTA, 0.1% Triton X-100, pH 7.5) 663 and twice with IP3 buffer (50 mM HEPES, 150 mM NaCl, 10 mM EDTA, 0.1% Triton X- 664 100, pH 7.5). IP1, IP2 and IP3 buffers were supplemented with proteinase inhibitor cocktail 665 (1 mM PMSF, 1 µg/mL leupeptin (Sigma L2023), 1 µg/mL aprotinin (Sigma A6191), 1 666 µg/mL antipain (Sigma A1153), 1 µg/mL chymostatin (Sigma C7268), 1 µg/mL pepstatin 667 (Sigma P5315)). The immunopurified fraction was eluted by boiling the beads in 50 µL of 668 Laemmli buffer for 5 mins. Finally, the input and immunopurified fractions were separated 669 on 10% acrylamide gels by SDS-PAGE and detected by immunoblot using α-Hemagglutinin 670 (HA) antibody (Roche 12CA5), α-c-Myc antibody (Abcam ab32072) or α-Flag antibody 671 (Sigma F1804) followed by the appropriate secondary antibody coupled with horseradish 672 peroxidase (HRP). 673 27 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 674 Confocal microscopy 675 N. benthamiana leaf disks were infiltrated with water and mounted on microscope slides. 676 Samples were imaged using a ZEISS LSM710 confocal laser-scanning microscope equipped 677 with a 40x oil-immersion objective (Apochromat 40x/1.0 DIC M27). The 514 nm argon laser 678 line was used to excite YFP and florescence was observed using the specific emission 679 window of 520-625 nm. Images were processed using the ZEN 2.3 software. 680 681 Yeast interaction 682 The EGY48 MATα strain (DupLEX-A Yeast Two Hybrid System; OriGene Technologies) 683 was transformed with pJG4-5-AtZED1 wild type or mutants (pJG4-5-AtZED1G66S, pJG4-5- 684 AtZED1G128E, pJG4-5-AtZED1D231N, pJG4-5-AtZED1A305T), pJG4-5-AtZARCC, pJG4-5- 685 AtZARCC2, pJG4-5-EV (empty vector), or pJG4-5-ShcF2. Transformants were selected on 686 synthetic defined (SD) Glucose-Trp media. pEG202-HopZ1a, pEG202-HopZ1aC/A, pEG202- 687 ZAR1CC, pEG202-ZAR1CC2, pEG202-ZAR1NB3, pEG202-ZAR1LRR, pEG202-EV or 688 pEG202-HopF2 were transformed into the MAT A strain, RFY206 (DupLEX-A Yeast Two 689 Hybrid System; OriGene Technologies). Transformants were selected on SD Glucose-HisUra 690 media. Standard yeast mating was performed in yeast extract/peptone/dextone/adenine sulfate 691 (YPAD) overnight at 30˚C. To select diploids, matings were selected twice on SD Glucose- 692 HisUraTrp selective media at 30˚C for 1-2 days each time. Diploid yeast samples were 693 resuspended and diluted to a starting OD600 of 10, allowing the same amount of yeast to be 694 tested for interaction in the assay for each construct. 50 (OD600 = 10), 5-1, 5-2 and 5-3 695 dilutions were plated onto SD Glucose-UraLeuHisTrp and SD Galactose-UraLeuHisTrp to 696 assay for protein-protein interactions. The reporter used to identify protein interactions was 697 LEU2. 698 28 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 699 Yeast Western Blots 700 The RFY206A strain carrying pEG202 constructs was grown overnight in liquid SD glucose- 701 UraHis media. The EGY48α strain carrying pJG4-5 constructs was grown overnight in SD 702 raffinose-Trp media. Overnight cultures containing the pEG202 constructs in the RFY206A 703 strain were used to inoculate new cultures in YPAD at an OD600 of 0.15. Overnight cultures 704 containing the pJG4-5 constructs in the EGY48α strain were used to inoculate new cultures in 705 yeast extract/peptone (YP) galactose at an OD600 of 0.15. Subcultures were grown for 5-7 706 hours. The cultures were then pelleted and resuspended in 250 µL of cracking buffer (1% β- 707 mercaptoethanol and 0.25 M NaOH). After incubating on ice for 10 minutes, 160 µL of 50% 708 TCA (diluted in nanopure H20 and pre-chilled on ice) was added to the cultures. Cultures 709 were incubated on ice for another 10 minutes, centrifuged at 4˚C for 10 minutes at 16,000 x g 710 and resuspended in 50 µL of Thorner buffer [8 M urea, 5% (wt/vol) SDS, 40 mM Tris-HCl at 711 pH 6.8, 0.1 mM EDTA, 0.4 mg/mL bromophenol blue, and 1% (vol/vol) β-mercaptoethanol]. 712 2M unbuffered Tris was added dropwise until the samples turned blue, in order to adjust the 713 pH. Samples were loaded on 10% SDS-PAGE gels, transferred to nitrocellulose membranes 714 and analyzed by Western blot. α-HA and LexA antibodies were used to detect HA-tagged 715 proteins and LexA fusion proteins, respectively. 716 717 Statistical analysis 718 The conductivity data were analyzed with a one way ANOVA at a significance level of 719 p<0.05 followed by a multiple comparisons of means using Tukey’s post hoc test. The 720 bacterial growth assay data were analyzed using a one-factor ANOVA using a general linear 721 model (GLM) procedure followed by a multiple comparisons of means using Tukey’s post 722 hoc test. A significance level of α = 0.05 was chosen for the statistical analyses. Data were 723 statistically analyzed using Minitab 17 software (Minitab Inc.). 29 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 724 Supplemental materials 725 Figure S1. HopZ1aC/A-HA, HopZ1a-HA and AtZED1-5xMyc proteins are expressed in 726 Nicotiana benthamiana. 727 Figure S2. NbZAR-VIGS construct specifically targets NbZAR1 and NbZAR2. 728 Figure S3. AtZED1 interacts with AtZAR1LRR in planta. 729 Figure S4. Protein expression of yeast-two hybrid constructs. 730 Figure S5. AtZAR1CC dimerization is not affected by co-expression of HopZ1a, or AtZED1 731 and HopZ1a. 732 Figure S6. AtZED1 point mutants have impaired interactions with AtZAR1CC and HopZ1a in 733 yeast. 734 735 Acknowledgements 736 We are grateful to Tess Scavuzzo-Duggan for assisting with some of the CoIPs and Juan 737 Hernandez for his help in cloning AtZAR1 mutants. We thank Dr. Jacob Brunkard for the 738 kind gifts of pYL156 and pYL192 for the VIGS experiments in N. benthamiana. Confocal 739 imaging was supported in part by the National Institutes of Health S10 program award 740 #1S10RR026866-01 to the CNR Biological Imaging Facility. 741 742 Figure Legends 743 Figure 1. Co-expression of HopZ1a and AtZED1 in Nicotiana benthamiana leads to a strong 744 HR dependent on NbZAR. Agrobacterium tumefaciens carrying constructs expressing Empty 745 Vector (EV), HopZ1a, HopZ1aC/A, AtZED1 and/or AtZAR1 were syringe infiltrated into N. 746 benthamiana leaves. Leaves with an HR are indicated to the left of the red boxes, and ion 747 leakage was measured 3 hours (T0) and 24 hours (T21) after dexamethasone induction. The 748 error bars indicate the standard error from 6 repetitions. The letters beside the bars indicate 30 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 749 significance groups, as determined by a one way ANOVA comparison followed by a Tukey’s 750 post-hoc test (pvalue ≤ 0.05). The experiment was performed at least 3 times with similar 751 results. A, HopZ1a-HA or HopZ1aC/A-HA was co-expressed with EV-5xMyc or AtZED1- 752 5xMyc in N. benthamiana leaves. B, HopZ1a-HA or HopZ1aC/A-HA was co-expressed with 753 AtZED1-3xFlag, and EV-5xMyc or AtZAR1-5xMyc in N. benthamiana leaves silenced for 754 GUS or NbZAR genes. C, Immunoblots of co-immunoprecipitation assays for the interaction 755 between AtZAR1 or NbZAR1 proteins with AtZED1. AtZAR1, NbZAR1 or NbZAR1CC-NB 756 with a 5xMyc tag were co-expressed with AtZED1 containing a 3xFlag tag in N. 757 benthamiana leaves. Western blot analysis (WB) was performed on the crude extract (Input) 758 or the immunopurified fractions (IP) from anti Myc beads. An asterisk (*) indicates the band 759 corresponding to the protein of interest, if multiple bands were observed. EV is Empty 760 Vector. The molecular masses of the proteins are: AtZED1-3xFlag 42 kDa; AtZAR1-5xMyc 761 107 kDa; NbZAR1 5xMyc 106 kDa; and NbZAR1CC-NB-5xMyc 55 kDa. The experiments 762 were repeated two times with similar results. 763 764 Figure 2. The AtZAR1LRR region is necessary for interaction and HR in Nicotiana 765 benthamiana. A, Schematic representation of AtZAR1 protein domains and truncations. The 766 colored boxes correspond to the following domains: yellow is the coiled-coil (CC) domain, 767 blue is the nucleotide-binding-Apaf1-R-CED4 (NB-ARC) domain, grey is the linker region, 768 and green is the leucine-rich repeat (LRR) domain. All constructs were expressed under the 769 control of a dexamethasone-inducible promoter and with a C-terminal 5xMyc tag. The 770 molecular masses are shown for the fusion with a 5xMyc tag. B, Immunoblots of co- 771 immunoprecipitation assays for the interaction between AtZAR1 deletions and AtZED1. 772 AtZAR1 or the AtZAR1Δ1 to AtZAR1Δ4 deletions with a 5xMyc tag were co-expressed with 773 AtZED1 containing a 3xFlag tag in N. benthamiana leaves. Western blot analysis (WB) was 31 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 774 performed on the crude extract (Input) or the immunopurified fractions (IP) from anti Myc 775 beads. An asterisk (*) indicates the band corresponding to the protein of interest, if multiple 776 bands were observed. EV is Empty Vector. The experiments were repeated at least 3 times 777 with similar results. C, AtZAR1Δ1 is unable to complement NbZAR1-VIGS. Agrobacterium 778 tumefaciens carrying constructs expressing HopZ1a-HA or HopZ1aC/A-HA was co-expressed 779 with AtZED1-3xFlag, and EV-5xMyc, AtZAR1-5xMyc or AtZAR1Δ1 in Nicotiana 780 benthamiana leaves silenced for GUS or NbZAR genes. The HR is shown 24 hours after 781 dexamethasone induction, and is indicated with an asterisk. The scale bar is 1cm. D, 782 AtZAR1Δ1 is unable to complement NbZAR1-VIGS. The electrolyte leakage level for a set of 783 co-infiltrations in NbZAR-VIGS plants was measured 3 hours (T0) and 24 hours (T21) after 784 dexamethasone induction. The error bars indicate the standard error from 6 repetitions. The 785 letters beside the bars indicate significance groups, as determined by a one way ANOVA 786 comparison followed by a Tukey’s post-hoc test (pvalue ≤ 0.05). The experiment was 787 performed at least 3 times with similar results. 788 789 Figure 3. Multiple domains of AtZAR1 interact with AtZED1. A, Immunoblots of co- 790 immunoprecipitation assays for interactions between AtZAR1 domains and AtZED1. 791 AtZAR1, AtZAR1CC, AtZAR1NB or AtZAR1LRR with a 5xMyc were co-expressed with 792 AtZED1 containing a 3xFlag tag, in Nicotiana benthamiana leaves. Western blot analysis 793 (WB) was performed on the crude extract (Input) or the immunopurified fractions (IP) from 794 anti Myc beads. An asterisk (*) indicates the band corresponding to the protein of interest, if 795 multiple bands were observed. EV is Empty Vector. The experiments were repeated at least 3 796 times with similar results. B, AtZED1 was constructed as a fusion with the DNA-binding 797 domain, and tested for interaction against AtZAR1CC2, AtZAR1CC, AtZAR1CC-NB3, 798 AtZAR1LRR or EV as a fusion to the activation domain, in the LexA yeast two-hybrid system. 32 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 799 HopF2PtoDC3000 and its chaperone ShcF2PtoDC3000 were used as positive controls because they 800 are known to strongly interact (Shan et al., 2004). The experiment was performed twice with 801 similar results. C, Bimolecular Fluorescence Complementation assay of AtZED1-AtZAR1CC 802 interactions. Agrobacterium tumefaciens carrying AtZAR1CC, AtZED1 or EV as a fusion to 803 the C-terminus of YFP (cYFP) or the N-terminus of YFP (nYFP) were mixed in equivalent 804 optical densities and pressure-infiltrated into the leaves of N. benthamiana. The top panel 805 shows the YFP channel alone, and the bottom panel shows a merge of the YFP channel and 806 bright field (BF). Leaf sections were imaged using a Zeiss LSM710 confocal scanning 807 microscope 24–48 h after induction. The red arrowheads show the nuclei. The scale bar is 50 808 μm. The experiment was performed twice with similar results. 809 810 Figure 4. AtZAR1CC-YFP induces constitutive HR when inducibly expressed in Nicotiana 811 benthamiana. Agrobacterium tumefaciens carrying constructs expressing Empty Vector (EV) 812 or AtZAR1 domains with a C-terminal YFP fusion were syringe infiltrated into N. 813 benthamiana leaves. The experiments were performed at least 3 times with similar results. 814 A, For each construct, the percentage of leaves with a strong HR (black bars), medium HR 815 (hatched bars), weak HR (dotted bars) or no HR (white bars) is shown. B and C, Electrolyte 816 leakage was measured 15 (T0) and 36 (T21) hours after dexamethasone induction. The error 817 bars indicate the standard error from 6 repetitions. The letters beside the bars indicate 818 significance groups, as determined by a one way ANOVA comparison followed by a Tukey’s 819 post-hoc test (pvalue ≤ 0.05). C, Electrolyte leakage for AtZAR1CC-YFP or EV-YFP was 820 grouped into 4 phenotypic categories based on the macroscopic HR (none, weak, medium 821 and strong) observed 36 hours after dexamethasone induction. 822 33 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 823 Figure 5. AtZAR1CC oligomerizes in yeast and in planta. A, AtZAR1CC or AtZAR1CC2 was 824 constructed as a fusion with the DNA-binding domain and the activation domain, and tested 825 for interaction against themselves or Empty Vector (EV) in the LexA yeast two-hybrid assay. 826 HopF2PtoDC3000 and its chaperone ShcF2PtoDC3000 were used as positive controls because they 827 are known to strongly interact (Shan et al., 2004). The experiment was performed twice with 828 similar results. B, Bimolecular Fluorescence-Complementation assay of AtZAR1CC 829 interactions. Agrobacterium tumefaciens carrying AtZAR1CC-YFP, AtZAR1CC and EV as a 830 fusion to the C-terminus of YFP (cYFP) or N-terminus of YFP (nYFP) were mixed in 831 equivalent optical densities and pressure-infiltrated into the leaves of Nicotiana benthamiana. 832 The left panel shows the YFP channel alone, and the right panel shows a merge of the YFP 833 channel and bright field (BF). Leaf sections were imaged using a Zeiss LSM710 confocal 834 scanning microscope 24–48 h after induction. The red arrowheads show the nuclei. The scale 835 bar is 50 μm. The experiment was performed twice with similar results. 836 837 Figure 6. Atzar1 mutants are susceptible to P. syringae carrying HopZ1a, and AtZAR1G645E 838 is unable to interact with AtZED1. A, AtZAR1 sequence schematic showing the 3 main 839 domains. The colored boxes correspond to the following domains: yellow is the coiled-coil 840 (CC) domain, blue is the nucleotide-binding-Apaf1-R-CED4 (NB-ARC) domain, grey is the 841 linker region, and green is the leucine-rich repeat (LRR) domain. The amino acid changes 842 induced by the mutation are indicated above the schematic. The P816L substitution was 843 previously described in Wang and colleagues (2015b). B, Half-leaves of Arabidopsis Col-0, 844 Atzar1-1, Atzar1V202M, Atzar1S291N, Atzar1L465F, Atzar1G645E or Atzar1S831F were infiltrated 845 with 10 mM MgCl2 or PtoDC3000 carrying empty vector (EV) or HopZ1a. The bacteria were 846 pressure-infiltrated into the leaves at 5 × 107 cfu/mL. Photos were taken 20 h after 847 infiltration. The number of leaves showing an HR is indicated below the leaf. HRs are 34 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 848 marked with an asterisk. The scale bar is 1 cm. C, PtoDC3000 carrying HopZ1a was syringe- 849 infiltrated at ~1 x 105 cfu/mL into the leaves of Arabidopsis Col-0, Atzar1-1 or Atzar1 point 850 mutants, and bacterial counts were determined one hour post-infiltration (Day 0) and 3 days 851 post-infection (Day 3). One-factor ANOVA using a general linear model (GLM) followed by 852 multiple comparisons of means using Tukey’s post hoc test was performed to determine 853 significant differences between plant genotypes, and significant differences are indicated by 854 the letters above the bars. Error bars indicate standard deviation from the mean. The 855 experiment was repeated 3 times with similar results. D, Immunoblot showing the co- 856 immunopurification of AtZAR1 with AtZED1 from Nicotiana benthamiana tissues. AtZAR1 857 with a 5xMyc epitope tag was co-expressed with AtZED1 or AtZED1 with a 3xFlag epitope 858 tag in N. benthamiana. Western blot analysis (WB) was performed on the crude extract 859 (Input) or the immunopurified fractions (IP) from anti Myc beads. An asterisk (*) indicates 860 the band corresponding to the protein of interest, if multiple bands were observed. This 861 experiment was repeated 3 times with similar results. 862 863 Figure 7. AtZED1D231N is impaired in the HopZ1a-induced HR. Agrobacterium tumefaciens 864 carrying constructs expressing empty vector (EV) or HopZ1a (A) or HopZ1aC/A (B) co- 865 expressed with AtZED1 or AtZED1 point mutants were syringe infiltrated into Nicotiana 866 benthamiana leaves. Leaves with an HR are indicated to the left of the red boxes, and ion 867 leakage was measured 3 hours (T0) and 24 hours (T21) after dexamethasone induction. The 868 error bars indicate the standard error from 6 repetitions. The letters beside the bars indicate 869 significance groups, as determined by a one way ANOVA comparison followed by a Tukey’s 870 post-hoc test (pvalue ≤ 0.05). The experiment was performed at least 3 times with similar 871 results. 872 35 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 873 Figure 8. AtZED1 point mutants are impaired in their interactions with AtZAR1 and 874 HopZ1a. A, Immunoblot showing the co-immunopurification of AtZAR1 with AtZED1 875 transiently expressed in Nicotiana benthamiana tissues. AtZAR1 or Empty vector (EV) 876 tagged with 5xMyc epitope was co-expressed with AtZED1 or AtZED1 tagged with 3xFlag 877 tag epitope in N. benthamiana. Western blot analysis (WB) was performed on the crude 878 extract (Input) or the immunopurified (IP) fractions from anti Myc beads. An asterisk (*) 879 indicates the band corresponding to the protein of interest, if multiple bands were observed. 880 The experiment was repeated 3 times with similar results. B, Immunoblot showing the co- 881 immunopurification of AtZAR1LRR with AtZED1 transiently expressed in N. benthamiana 882 tissues. AtZAR1LRR or Empty vector (EV) tagged with 5xMyc epitope was co-expressed with 883 AtZED1 or AtZED1 tagged with 3xFlag tag epitope in N. benthamiana. Western blot 884 analysis (WB) was performed on the crude extract (Input) or the immunopurified (IP) 885 fractions from anti Myc beads. An asterisk (*) indicates the band corresponding to the protein 886 of interest, if multiple bands were observed. The experiment was repeated 2 times with 887 similar results. C and D, Bimolecular Fluorescence Complementation assay of AtZED1 or 888 AtZED1 mutants with AtZAR1CC (C) or HopZ1aC/A (D). Agrobacterium tumefaciens 889 carrying AtZAR1CC (C), or HopZ1a (D) as a fusion to the N-terminus of YFP (nYFP) was 890 mixed in equivalent optical densities with A. tumefaciens carrying AtZED1, AtZED1 891 mutants, or EV as a fusion to the C-terminus of YFP (cYFP), and pressure-infiltrated into the 892 leaves of N. benthamiana. The top panel shows the YFP channel alone, and the bottom panel 893 shows a merge of the YFP channel and bright field (BF). Leaf sections were imaged using a 894 Zeiss LSM710 confocal scanning microscope 24–48 h after induction. The red arrowheads 895 show the nuclei. The scale bar is 50 μm. The experiment was performed twice with similar 896 results. 897 36 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 898 Figure S1. HopZ1aC/A-HA, HopZ1a-HA and AtZED1-5xMyc proteins are expressed in 899 Nicotiana benthamiana. Anti-HA (top panel) and anti-Myc (middle panel) show protein 900 expression of HopZ1aC/A-HA, HopZ1a-HA and AtZED1-5xMyc in N. benthamiana leaves 12 901 hours after dexamethasone induction. The Ponceau stained membrane (bottom panel) is the 902 loading control. 903 904 Figure S2. NbZAR-VIGS construct specifically targets NbZAR1 and NbZAR2. A, Protein 905 alignment of AtZAR1, NbZAR1 and NbZAR2. The alignment was performed with Clustal 906 Omega (Sievers et al., 2011), and depicted using Boxshade. The 3 main domains of AtZAR1 907 and small linker region are shown by colored lines above the alignment and correspond to the 908 following domains: yellow is the coiled-coil (CC) domain, blue is the nucleotide-binding- 909 Apaf1-R-CED4 (NB-ARC) domain, grey is the linker region, and green is the leucine-rich 910 repeat (LRR) domain. The asterisk (*) indicates the residues identified in genetic screens in 911 Arabidopsis thaliana (Lewis et al., 2013; Wang et al., 2015b). B, Semi-quantitative RT-PCR 912 analysis of the VIGS efficiency. Total mRNA of 6-week old Nicotiana benthamiana plants 913 expressing GUS-VIGS or NbZAR-VIGS constructs was tested for NbZAR and 914 Niben101Scf00383g03004 (Nb3004) gene expression. TUBULIN (NbTUB) was used to 915 normalize the expression. C, Agrobacterium tumefaciens carrying constructs expressing 916 empty vector (EV), AtRPS2, AtZED1 + HopZ1a, or AtZED1 + HopZ1aC/A were syringe 917 infiltrated into N. benthamiana leaves expressing GUS-VIGS or NbZAR-VIGS. The HR is 918 indicated with an asterisk and was observed 24 hours after dexamethasone induction. The 919 scale bar is 1 cm. 920 921 Figure S3. AtZED1 interacts with AtZAR1LRR in planta. A and B, Immunoblots of co- 922 immunoprecipitation assays. All constructs were transiently co-expressed in Nicotiana 37 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 923 benthamiana leaves. Western blot analysis (WB) was performed on the crude extract (Input) 924 or the immunopurified fractions (IP) from anti Myc beads. An asterisk (*) indicates the band 925 corresponding to the protein of interest, if multiple bands were observed. EV is Empty 926 Vector. The experiments were repeated at least 3 times with similar results. A, AtZAR1, 927 AtZAR1CC, AtZAR1CC-NB2, AtZAR1NB2 or AtZAR1LRR2 with a 5xMyc tag were co-expressed 928 with AtZED1 with a 3xFlag tag. B, AtZAR1, AtZAR1CC2, AtZAR1CC-NB, AtZAR1CC-NB3 or 929 AtZAR1LRR with a 5xMyc tag were co-expressed with AtZED1 with a 3xFlag tag. C, 930 AtZAR1, AtZAR1LRR2 or AtZAR1LRR with a 5xMyc tag were co-expressed with AtZED1 931 containing a 3xFlag tag. 932 933 Figure S4. Protein expression of yeast-two hybrid constructs. Immunoblot analysis of empty 934 vector (Ev) or binding-domain (BD) fusions in the RFY206A yeast strain (right panel), and 935 empty vector or activation-domain fusions with an HA tag in the EGY48α yeast strain (left 936 panel). Equal amounts of proteins were resolved on 10% SDS-PAGE gels, blotted onto 937 nitrocellulose, and probed with HA or LexA antibodies. The Ponceau red stained blot was 938 used as a loading control (bottom panel). 939 940 Figure S5. AtZAR1CC dimerization is not affected by co-expression of HopZ1a, or AtZED1 941 and HopZ1a. Bimolecular Fluorescence-Complementation assay of AtZAR1CC interactions 942 with co-expression of HopZ1a, or HopZ1a and AtZED1. Agrobacterium tumefaciens carrying 943 AtZAR1CC as a fusion to the C-terminus of YFP (cYFP) or N-terminus of YFP (nYFP) were 944 mixed in equivalent optical densities with A. tumefaciens carrying HopZ1a, HopZ1aC/A, 945 HopZ1a and AtZED1, or HopZ1aC/A and AtZED1, and pressure-infiltrated into the leaves of 946 Nicotiana benthamiana. The top panel shows the YFP channel alone, and the bottom panel 947 shows a merge of the YFP channel and bright field (BF). Leaf sections were imaged using a 38 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 948 Zeiss LSM710 confocal scanning microscope 24–48 h after induction. The red arrowheads 949 show the nuclei. The scale bar is 50 μm. The experiment was performed twice with similar 950 results. 951 952 Figure S6. AtZED1 point mutants have impaired interactions with AtZAR1CC and HopZ1a in 953 yeast. AtZED1 or the AtZED1 point mutants were constructed as a fusion with the DNA- 954 binding domain and tested for interaction against AtZAR1CC (A) or HopZ1a (B) constructed 955 as a fusion to the activation domain, in the LexA yeast two-hybrid system. HopF2PtoDC3000 and 956 its chaperone ShcF2PtoDC3000 were used as positive controls because they are known to 957 strongly interact (Shan et al., 2004). EV is empty vector. The experiment was performed 958 twice with similar results. 959 960 961 References 962 Ade J, DeYoung BJ, Golstein C, Innes RW (2007) Indirect activation of a plant nucleotide 963 binding site-leucine-rich repeat protein by a bacterial protease. Proc Natl Acad Sci U S 964 A 104: 2531–2536 965 966 967 Aoyama T, Chua N-H (1997) A glucocorticoid-mediated transcriptional induction system in transgenic plants. 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Parsed Citations Ade J, DeYoung BJ, Golstein C, Innes RW (2007) Indirect activation of a plant nucleotide binding site-leucine-rich repeat protein by a bacterial protease. Proc Natl Acad Sci U S A 104: 2531-2536 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Aoyama T, Chua N-H (1997) A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J 11: 605-612 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Bendahmane A, Farnham G, Moffett P, Baulcombe DC (2002) Constitutive gain-of-function mutants in a nucleotide binding siteleucine rich repeat protein encoded at the Rx locus of potato. Plant J 32: 195-204 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Bendahmane A, Kanyuka K, Baulcombe DC (1999) The Rx gene from potato controls separate virus resistance and cell death responses. Plant Cell 11: 781-92 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Bent AF, Kunkel BN, Dahlbeck D, Brown KL, Schmidt R, Giraudat J, Leung J, Staskawicz BJ (1994) RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance genes. Science 265: 1856-1860 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Bernoux M, Burdett H, Williams SJ, Zhang X, Chen C, Newell K, Lawrence GJ, Kobe B, Ellis JG, Anderson PA, et al (2016) Comparative analysis of the Flax immune receptors L6 and L7 suggests an equilibrium-based switch activation model. 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Science 278: 1963-1965 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Césari S, Moore J, Chen C, Webb D, Periyannan S, Mago R, Bernoux M, Lagudah ES, Dodds PN (2016) Cytosolic activation of cell death and stem rust resistance by cereal MLA-family CC-NLR proteins. Proc Natl Acad Sci 113: 10204-10209 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Clough SJ, Bent AF (1998) Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Collier SM, Moffett P (2009) NB-LRRs work a "bait and switch" on pathogens. 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