Download Analysis of the ZAR1 immune complex reveals

Plant Physiology Preview. Published on June 26, 2017, as DOI:10.1104/pp.17.00441
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Short title: Immune complex interactions
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Corresponding author information: J.D. Lewis; Email: [email protected]; Telephone:
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510-559-5909; Fax: 510-559-5678
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Title: Analysis of the ZAR1 immune complex reveals determinants for immunity and
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molecular interactions
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Authors: Maël Baudin1, Jana A. Hassan1, Karl J. Schreiber1 and Jennifer D. Lewis1,2*.
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Department of Plant and Microbial Biology, University of California Berkeley, Berkeley,
CA
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Plant Gene Expression Center, United States Department of Agriculture, 800 Buchanan St.,
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Albany, CA, 94710, USA
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One sentence summary: Deciphering the molecular interactions within the ZAR1-ZED1
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immune complex that lead to the induction of plant immunity.
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Author contributions: M.B., J.A.H., K.J.S., and J.D.L. performed the experiments, analyzed
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the data, and wrote the manuscript.
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Funding: Research on plant immunity in the Lewis laboratory was supported by United
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States Department of Agriculture Agricultural Research Service 5335-21000-040-00D
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(J.D.L), NSF IOS-1557661 (J.D.L.), and an INRA postdoctoral fellowship (M.B.).
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Corresponding author email: [email protected]
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Abstract:
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Plants depend on innate immunity to prevent disease. Plant pathogenic bacteria, like
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Pseudomonas syringae and Xanthomonas campestris, use the type III secretion system as a
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molecular syringe to inject type III secreted effector (T3SE) proteins in plants. The primary
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function of most T3SEs is to suppress immunity, however the plant can evolve NBD-LRR
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domain-containing (NLR) proteins to recognize specific T3SEs. The AtZAR1 NLR induces
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strong defense responses against P. syringae and X. campestris. The P. syringae T3SE
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HopZ1a is an acetyltransferase that acetylates the pseudokinase AtZED1, and triggers
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recognition by AtZAR1. However, little is known about the molecular mechanisms that lead
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to AtZAR1-induced immunity in response to HopZ1a. We established a transient expression
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system in Nicotiana benthamiana to study detailed interactions among HopZ1a, AtZED1 and
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AtZAR1. We show that the AtZAR1 immune pathway is conserved in N. benthamiana, and
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identify AtZAR1 domains, and residues in AtZAR1 and AtZED1, that are important for
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immunity and protein-protein interactions in planta and in yeast. We show that the coiled-coil
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domain of AtZAR1 oligomerizes, and this domain acts as a signal to induce immunity. This
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detailed analysis of the AtZAR1-AtZED1 protein complex provides a better understanding of
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the immune signaling hub controlled by AtZAR1.
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Introduction
To successfully colonize their hosts, plant pathogens must overcome several layers of
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plant innate immunity (Jones and Dangl, 2006; Dou and Zhou, 2012). Many pathogenic
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bacteria, like Pseudomonas syringae, use a needle-like structure called the Type III Secretion
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System (T3SS) to directly inject type III effector proteins (T3SE) into the host cell (Galán
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and Wolf-Watz, 2006). Bacterial effectors are structurally and biochemically very diverse
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and are able to manipulate the host cell machinery to promote disease (Mudgett, 2005; Grant
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et al., 2006; Göhre and Robatzek, 2008; Lewis et al., 2009; Deslandes and Rivas, 2012; Xin
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and He, 2013; Macho and Zipfel, 2015). To counter the activity of these effectors, plants may
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evolve a new layer of immunity called Effector Triggered Immunity (ETI) that directly or
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indirectly detects the activity of effector proteins (Jones and Dangl, 2006; Schreiber et al.,
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2016a). This recognition induces strong defense mechanisms, often resulting in a form of
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localized programmed cell death called the hypersensitive response (HR) (Heath, 2000; Cui
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et al., 2015).
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ETI is typically regulated by Nucleotide-Binding Domain-Leucine-Rich Repeat (NBD-
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LRR) containing proteins (NLRs, also called NOD-like receptors in the mammalian
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literature) that act as a molecular switch (Van Der Biezen and Jones, 1998; Ting et al., 2008;
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Jones et al., 2016). In the absence of the pathogen, NLR proteins are maintained in an “off”
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state by intra- and inter-molecular interactions. Upon detection of the effector or its activity,
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NLRs undergo conformational changes and switch to an “on” state resulting in the induction
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of the downstream signaling (Takken and Goverse, 2012; Sukarta et al., 2016). Plant
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genomes encode large numbers of NLR proteins, including 151 members in A. thaliana, and
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are relatively specific for the recognition of effectors (Meyers et al., 2003; Jones et al., 2016).
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In some cases, the detection of an effector requires another plant protein (the “guardee”) that
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is guarded by the NLR (Van Der Biezen and Jones, 1998; Dangl and Jones, 2001; Khan et al.,
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2016; Schreiber et al., 2016a). Modification of the “guardee” by the effector is sensed,
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presumably by conformational changes, and leads to the activation of the NLR. The
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“guardee” can be a virulence target of the effector; its modification in the absence of the
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cognate NLR promotes susceptibility. In other cases the NLR monitors a decoy protein that
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looks like the true virulence target of the effector but does not play a role in immunity beside
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trapping the effector (van der Hoorn and Kamoun, 2008; Khan et al., 2016).
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Plant NLR proteins typically contain a Nucleotide-Binding (NB) domain, followed by a
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LRR domain at the C-terminal end of the NLR (Meyers et al., 2003; Sukarta et al., 2016).
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The LRR domain plays a role in maintaining the “off” state of the NLR by intra-molecular
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interactions, and is involved in extra-molecular interaction with the T3SE or the “guardee”
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(Collier and Moffett, 2009; Qi et al., 2012; Wang et al., 2015b; Schreiber et al., 2016a). The
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NB domain is believed to act as a molecular switch by binding ADP in the inactive state and
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ATP in the active state (Takken and Goverse, 2012). In addition to these two domains, plant
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NLRs possess another N-terminal domain that is commonly a Coiled-coil (CC) or
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Toll/interleukin-1 receptor (TIR). The CC or TIR domains are the signaling part of the NLR,
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and can cause constitutive ETI when they are overexpressed by themselves (Frost et al.,
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2004; Swiderski et al., 2009; Williams et al., 2014). In some cases, the signaling activity
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involves oligomerization of the CC and TIR domains (Bernoux et al., 2011; Maekawa et al.,
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2011; Williams et al., 2014). Downstream signaling that results after activation of the NLR is
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still poorly understood. However, the identification of some genetic regulators such as NDR1
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(NON-RACE-SPECIFIC DISEASE RESISTANCE 1) (Century et al., 1997) and EDS1
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(ENHANCED DISEASE SUSCEPTIBILITY 1) (Parker et al., 1996), suggest that downstream
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NLR activation is transduced through several signaling pathways.
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ZAR1 (HOPZ-ACTIVATED RESISTANCE 1) is a canonical CC-type NLR protein from
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Arabidopsis thaliana. AtZAR1 was first identified as being required for the recognition of the
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T3SE HopZ1a from the pathogenic bacteria Pseudomonas syringae (Lewis et al., 2010).
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HopZ1a is part of the YopJ superfamily of T3SEs, that are found in animal and plant
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pathogenic bacteria, and is an acetyltransferase (Lewis et al., 2008; Lewis et al., 2011; Lee et
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al., 2012; Ma and Ma, 2016). Detection of HopZ1a by AtZAR1 requires AtZED1 (HOPZ-
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ETI-DEFICIENT 1) a Receptor-Like Cytoplasmic Kinase (RLCK) belonging to the RLCK
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XII-2 family (Lewis et al., 2013). AtZED1 interacts with AtZAR1 and HopZ1a, and is
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acetylated by HopZ1a, which is hypothesized to trigger the activation of AtZAR1 (Lewis et
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al., 2013). AtZED1 is believed to be a decoy guarded by AtZAR1, and senses the activity of
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HopZ1a in the plant cell. More recently, AtZAR1 has been described to be required for the
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resistance induced by the T3SE AvrAC from Xanthomonas campestris pv. campestris (Wang
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et al., 2015b), and the recognition of HopF2a, a T3SE with ADP-ribosyltransferase activity,
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from P. syringae pv. aceris M302273PT (Seto et al., 2017). AvrAC uridylylates PBL2, a
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RLCK in the VII family, which triggers recognition by the AtZED1-related kinase (ZRK)
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RKS1 (Resistance related KinaSe 1, also called ZRK1), and AtZAR1 (Feng et al., 2012;
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Huard-Chauveau et al., 2013; Wang et al., 2015b). Recognition of HopF2a requires ZRK3
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and AtZAR1, but HopF2a does not ADP-ribosylate ZRK3, suggesting that ZRK3 may act as
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an adaptor between AtZAR1 and an unidentified kinase that is modified by HopF2a (Seto et
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al., 2017). AtZED1, ZRK3 and RKS1 are found in the same genomic cluster of kinase genes,
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and all three proteins interact with AtZAR1. Interestingly AtZAR1 appears to be a
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recognition hub that is able to use RLCK XII-2 proteins (AtZED1, ZRK3 and RKS1) to sense
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three T3SEs that have different enzymatic activities, and are from different bacteria (Lewis et
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al., 2014a; Roux et al., 2014; Seto et al., 2017).
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Here, we established a system to study HopZ1a-induced immune responses, and the
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molecular interactions between HopZ1a, AtZED1 and AtZAR1 in Nicotiana benthamiana.
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We demonstrate that recognition of HopZ1a requires co-expression with AtZED1, and the N.
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benthamiana homolog of AtZAR1. We identified essential residues in AtZAR1 or AtZED1
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for immune induction and protein-protein interactions, and characterized the functions of
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AtZAR1 domains, in planta and in yeast. This work provides a better understanding of the
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molecular interactions between HopZ1a, AtZED1 and AtZAR1 that contribute to innate
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immunity.
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Results
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ZAR1-dependent recognition of HopZ1a is conserved in Nicotiana benthamiana
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We sought to establish an Agrobacterium tumefaciens-mediated transient expression
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system for HopZ1a recognition in N. benthamiana. In order to carefully regulate the
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expression of HopZ1a, AtZED1, or AtZAR1, we cloned these genes under the control of the
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dexamethasone-inducible promoter. The expression of HopZ1a or the catalytic mutant
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HopZ1aC216A (hereafter HopZ1aC/A) (Lewis et al., 2008) did not induce an HR in leaves of 5-
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week-old plants (Figure 1A). We detected HopZ1a and HopZ1aC/A by Western blot analysis,
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which demonstrated that the absence of HR is not due to a lack of protein (Figure S1). When
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we co-expressed AtZED1 from Arabidopsis thaliana with HopZ1a in N. benthamiana, we
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observed a strong and rapid HR detectable about 8 hours post-induction (hpi) that was
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dependent on the catalytic cysteine residue of HopZ1a (Figure 1A). To quantitatively
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measure the HR, we monitored ion leakage into the medium over time. At 24 hpi (T21), co-
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expression of HopZ1a and AtZED1 led to a strong and significant increase in conductivity
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compared to HopZ1a alone (Figure 1A). As expected, the HopZ1aC/A catalytic mutant did not
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induce ion leakage whether or not it was co-expressed with AtZED1 (Figure 1A).
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We hypothesized that the HR was dependent on the presence of a functional AtZAR1
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immune pathway in N. benthamiana. We therefore looked for AtZAR1 homologs in the N.
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benthamiana draft genome using the blastp tool on the Sol Genomics Network website
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(Fernandez-Pozo et al., 2015). To specifically identify AtZAR1 homologs, we searched using
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the AtZAR1CC domain (amino acids 1-144), which is more similar to NLR proteins in other
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species than it is to NLRs in Arabidopsis (Lewis et al., 2010). We identified two proteins
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Niben101Scf17398g00012 (hereafter NbZAR1) and Niben101Scf00383g03003 (hereafter
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NbZAR2), that are 89% identical to each other. When we aligned AtZAR1 protein sequences
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to NbZAR1 and NbZAR2, we could identify CC, NB and LRR domains in NbZAR1 while
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NbZAR2 only contained the CC domain and most of the NB domain. NbZAR1 covered 99%
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of AtZAR1 with 58% identity and NbZAR2 covered 47% of AtZAR1 with 57% identity
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(Figure S2A). At the mRNA level, NbZAR1 and NbZAR2 were almost identical (95%) and
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NbZAR2 covered approximately the first half of NbZAR1. We then employed Virus Induced
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Gene Silencing (VIGS) to knock down the expression of NbZAR1 and NbZAR2 by targeting
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the first ~700 base pairs of the two genes. As NbZAR1 and NbZAR2 were so similar to each
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other, we were unable to monitor the mRNA level of each gene independently. Therefore, we
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measured the pool of mRNA of both genes together (hereafter NbZAR). Using semi-
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quantitative RT-PCR, we showed that NbZAR mRNA was strongly reduced in NbZAR-VIGS
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leaves compared to GUS-VIGS leaves. In addition, the expression of an unrelated N.
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benthaminana NLR gene Niben101Scf00383g03004 was not affected by the VIGS construct
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(Figure S2B). These results confirmed the specificity and efficiency of the NbZAR-VIGS
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construct. In the NbZAR silenced plants, we did not observe an HR when HopZ1a was
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expressed, or when HopZ1a and AtZED1 were co-expressed (Figure 1B). In the control
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GUS-silenced VIGS plant, co-expression of HopZ1a and AtZED1 led to a strong HR as
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observed in wild type N. benthamiana, and similar levels of ion leakage were observed in
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GUS-silenced VIGS plants and wild type N. benthamiana (Figure 1). Interestingly we were
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able to partially complement the silencing of NbZAR1 by delivering AtZAR1 with
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Agrobacterium-mediated transient expression (Figure 1B). Conductivity measurements
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confirmed the partial complementation, with increases in ion leakage detectable around 24
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hpi (T21) (Figure 1B). To determine whether silencing of NbZAR was specific, we tested a
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highly expressed AtRPS2 construct, under a double 35S promoter. RPS2 is normally required
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for the recognition of AvrRpt2, and does not play a role in HopZ1a recognition (Bent et al.,
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1994; Mindrinos et al., 1994; Lewis et al., 2008). Overexpression of AtRPS2 causes an HR in
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the absence of its cognate effector in N. benthamiana (Jin et al., 2002). In NbZAR-VIGS
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plants, AtRPS2 overexpression still causes an HR, confirming the specificity of the NbZAR-
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VIGS construct (Figure S2C).
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These results suggest that NbZAR proteins are able to interact with AtZED1 and monitor
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its acetylation by HopZ1a. To test this hypothesis, we cloned the full-length NbZAR1 and a
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shorter form of NbZAR1 that encodes the CC and most of the NB domains (hereafter
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NbZAR1CC-NB). We cloned NbZAR1CC-NB, as this part of the protein is included in NbZAR2,
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and because we were unable to specifically amplify NbZAR2 due to its high similarity to
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NbZAR1 (Figure S2). We co-expressed ZAR1 proteins tagged with different epitopes in N.
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benthamiana, and then conducted co-immunoprecipitation assays (CoIP). We first co-
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expressed AtZED1-3xFlag with AtZAR1-5xMyc, and immunoprecipitated (IP) protein
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complexes using agarose beads coupled with α-Myc antibody. AtZED1-3xFlag only CoIP
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with AtZAR1-5xMyc, not the 5xMyc tag alone (Figure 1C), which confirms the previously
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observed AtZAR1-AtZED1 interaction (Lewis et al., 2013; Wang et al., 2015b).
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Interestingly, AtZED1 was able to interact with NbZAR1 but not NbZAR1CC-NB. This
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suggests that NbZAR1 is functional in N. benthamiana, and that the C-terminal part of the
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NB domain and/or the LRR domain is critical for coimmunoprecipitation of ZAR1 and ZED1
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in planta. Taken together, these results show that HopZ1a recognition depends on a
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functional ZAR1 immune pathway that is conserved between Arabidopsis thaliana and N.
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benthamiana. The transient assay system can therefore be used to probe the determinants for
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HopZ1a recognition.
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AtZED1 interacts with the CC and LRR domains of AtZAR1
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Previous bioinformatic analysis of AtZAR1 indicated that it was composed of three
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domains: CC (amino acids 1-144), NB (amino acids 145-391) and LRR (amino acids 610-
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852) with a linker of unknown function (amino acids 391-609). We reanalyzed the AtZAR1
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protein by generating a structural model in the Phyre2 web portal (Kelley et al., 2015), and
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searching for domains in the Conserved Domain database (Marchler-Bauer et al., 2015) and
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InterPro (Mitchell et al., 2015) (Figure 2A). In this analysis, the CC domain (amino acids 1-
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144) was the same, while the NB (amino acids 145-504) and LRR (amino acids 526-852)
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domains comprised the rest of the protein with a short linker domain (amino acids 505-525)
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(Figure 2A). The Phyre2 analysis identified 13 LRR domains, which is typical of NLR
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proteins.
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Based on our bioinformatic analysis, we sought to investigate the interaction between
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AtZED1 and AtZAR1 in planta, and refine the regions of AtZAR1 required for this
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interaction. We generated a series of AtZAR1 deletions from the C-terminal part of the
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protein, to maintain the CC and NB domains while varying the number of LRRs. These
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constructs encoded the CC-NB plus 12 LRRs (ZAR1Δ1), 9 LRRs (ZAR1Δ2), 5 LRRs
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(ZAR1Δ3), or 2 LRRs (ZAR1Δ4) and a 5xMyc tag (Figure 2A). We co-expressed proteins
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tagged with different epitopes in N. benthamiana, and conducted CoIP assays. AtZED1-
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3xFlag CoIP with AtZAR1-5xMyc, but not with any of the C-terminal truncations of
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AtZAR1 (AtZAR1Δ1 to AtZAR1Δ4) (Figure 2B). This indicates that the last 29 amino acids of
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the LRR domain were required for the interaction. These data, along with our previous yeast
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two-hybrid interactions between AtZAR1CC and AtZED1 (Lewis et al., 2013), suggest that
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multiple domains of AtZAR1 interact with AtZED1. Since AtZAR1 was able to partially
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complement the NbZAR knock down for the induction of HR caused by HopZ1a and AtZED1
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(Figure 1B), we tested whether the AtZAR∆1 mutant could complement the NbZAR knock
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down in this system. AtZAR1Δ1 was unable to complement NbZAR-VIGS which validates the
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importance of these residues (Figure 2C, 2D).
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We previously demonstrated that the AtZAR1CC domain was sufficient to interact with
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AtZED1 in the LexA yeast two-hybrid system (Lewis et al., 2013), and wondered whether we
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might be missing interactions between AtZAR1 domains and AtZED1 due to steric
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hindrance. We therefore independently tested AtZAR1CC, AtZAR1NB or AtZAR1LRR domains
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for interaction with AtZED1 (Figure 2A). Interestingly, AtZED1 co-precipitated only with
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full length AtZAR1 and AtZAR1LRR in planta (Figure 3A). We were unable to recapitulate
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the interaction between AtZED1 and AtZAR1CC previously observed in yeast (Lewis et al.,
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2013), however this is likely due to a weak or transient interaction in planta. We also tested a
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series of AtZAR1 truncations which contained different domains, and tested them for CoIP
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with AtZED1 (Figure S3A, S3B). AtZED1 only co-precipitated with AtZAR1LRR and
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AtZAR1LRR2, but not with AtZAR1CC2, AtZAR1CC-NB, AtZAR1CC-NB2, AtZAR1CC-NB3 or
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AtZAR1NB2 (Figure S3A, S3B). To determine whether the LRR region was sufficient to
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interact with AtZED1, we compared the AtZED1 interaction with full length AtZAR1,
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AtZAR1LRR (containing all 13 LRRs), or a shorter AtZAR1LRR2 (containing the last 8 LRRs)
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(Figure S3C). AtZAR1LRR2 was sufficient for the interaction with AtZED1 in planta,
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however it did not interact as strongly as AtZAR1LRR or full length AtZAR1. This suggests
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that other parts of the AtZAR1 protein stabilize the interaction with AtZED1.
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In previous yeast-two hybrid assays, AtZED1 was tested against AtZAR1CC, AtZAR1NB2,
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AtZAR1CC-NB2 and AtZAR1 lacking the last 8 LRRs (Lewis et al., 2013). We therefore tested
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the new AtZAR1 truncations, as a fusion to the activation domain, for interaction with
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AtZED1, as a fusion to the DNA-binding domain, in the LexA yeast two-hybrid system
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(Figures 2A, 3B). For each combination, the same optical density of yeast was spotted on the
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plates in order to quantitate the interaction strength. We observed the same strong interaction
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between AtZED1 and AtZAR1CC, but we did not observe any interaction between AtZED1
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and AtZAR1LRR in yeast (Figure 3B) (Lewis et al., 2013). All of these constructs were
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expressed in yeast, indicating that the lack of interaction was not due to a lack of protein
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expression (Figure S4). We also tested a shorter version AtZAR1CC2 lacking residues 122-
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144, and found that it displayed a weaker interaction with AtZED1. This suggests that the last
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23 residues of AtZAR1CC contribute to the interaction with AtZED1.
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To confirm the AtZAR1CC-AtZED1 interaction in planta, we used Agrobacterium-
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mediated transient expression and Bimolecular Fluorescence Complementation (BiFC) in N.
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benthamiana (Figure 2A). We generated chimeric fusion proteins of AtZAR1CC or AtZED1
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with the N-terminus of YFP (nYFP) or the C-terminus of YFP (cYFP), expressed under a
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dexamethasone-inducible promoter (Lewis et al., 2012; Lewis et al., 2014b). We observed
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fluorescence mostly in the nucleus when AtZED1-nYFP and AtZAR1CC-cYFP, or
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AtZAR1CC-nYFP and AtZED1-cYFP were co-expressed, but no fluorescence when the
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negative controls were expressed (Figure 3C). Taken together, these data demonstrate that
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multiple domains of AtZAR1 interact with AtZED1 in planta.
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Overexpression of AtZAR1CC-YFP induces an HR in N. benthamiana
Previous studies have reported that the overexpression of NLR domains can induce auto-
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activity and spontaneous HR in the absence of the cognate effector (Frost et al., 2004;
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Bernoux et al., 2011; Maekawa et al., 2011). The autoimmune phenotype can also be
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enhanced when NLR domains are fused to a GFP or YFP tag, which itself can dimerize
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(Swiderski et al., 2009; Wang et al., 2015a). We observed an HR when a chimeric construct
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of AtZAR1CC fused to YFP, was expressed in N. benthamiana. Overexpression of the
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AtZAR1CC-YFP protein induced an HR in 80% of the infiltrated leaves, of which 20%
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displayed a strong HR (Figure 4A). The ZARCC-YFP induced HR was associated with a
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significant increase in ion leakage (Figure 4B, 4C). However, we did not observe a strong HR
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or an increase in ion leakage when any of the other AtZAR1 domains were expressed (Figure
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4). Interestingly, ZAR1CC-NB2-YFP did not cause a strong HR or rapid ion leakage (Figure
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4A, 4B), indicating that the NB domain suppresses the autoactivity of the ZAR1CC domain.
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As AtZAR1CC-YFP caused a range of HR phenotypes, we measured ion leakage from
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independent leaves, and binned the data into four groups by the HR phenotype (no HR, weak
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HR, medium HR, or strong HR). As expected, leaves that did not show an HR had similar
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levels of ion leakage to EV-YFP whereas leaves with weak, medium or strong HRs displayed
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increasing levels of conductivity (Figure 4C).
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The CC or TIR domains of NLRs are known to oligomerize, and to act as a signal for
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immunity (Krasileva et al., 2010; Bernoux et al., 2011; Maekawa et al., 2011; Williams et al.,
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2014). We hypothesized that the YFP epitope stabilized the dimerization of AtZAR1CC which
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then triggered the HR. In order to test this hypothesis, we tested the self-association of
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AtZAR1CC and AtZAR1CC2 in the LexA yeast two-hybrid system. We observed strong
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homodimerization of AtZAR1CC and AtZAR1CC2, as well as heterodimerization between
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AtZAR1CC and AtZAR1CC2 (Figure 5A). This indicated that the first 122 residues of
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AtZAR1CC are sufficient for homodimerization.
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To confirm the CC interaction in planta, we used Agrobacterium-mediated transient
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expression and a BiFC assay in N. benthamiana. We tested chimeric fusion proteins of
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AtZAR1CC with full-length YFP, nYFP or cYFP, expressed under a dexamethasone-inducible
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promoter. We observed a strong fluorescent signal in the epidermal cells of N. benthamiana
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expressing AtZAR1CC-cYFP and AtZAR1CC-nYFP, but not in the cells expressing the
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negative controls (Figure 5B). The florescence signal was localized to the nucleus and to the
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cytoplasm, as observed for AtZAR1CC-YFP. We also tested whether co-expression of
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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. Plant J 11: 605–612
Bendahmane A, Farnham G, Moffett P, Baulcombe DC (2002) Constitutive gain-of-
968
function mutants in a nucleotide binding site-leucine rich repeat protein encoded at the
969
Rx locus of potato. Plant J 32: 195–204
970
971
972
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
Bent AF, Kunkel BN, Dahlbeck D, Brown KL, Schmidt R, Giraudat J, Leung J,
39
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
973
Staskawicz BJ (1994) RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant
974
disease resistance genes. Science 265: 1856–1860
975
Bernoux M, Burdett H, Williams SJ, Zhang X, Chen C, Newell K, Lawrence GJ, Kobe
976
B, Ellis JG, Anderson PA, et al (2016) Comparative analysis of the Flax immune
977
receptors L6 and L7 suggests an equilibrium-based switch activation model. Plant Cell
978
28: 146–159
979
Bernoux M, Ve T, Williams S, Warren C, Hatters D, Valkov E, Zhang X, Ellis JG, Kobe
980
B, Dodds PN (2011) Structural and functional analysis of a plant resistance protein TIR
981
domain reveals interfaces for self-association, signaling, and autoregulation. Cell Host
982
Microbe 9: 200–211
983
984
985
Van Der Biezen EA, Jones JDG (1998) Plant disease-resistance proteins and the gene-forgene concept. Trends Biochem Sci 23: 454–456
Casey LW, Lavrencic P, Bentham AR, Cesari S, Ericsson DJ, Croll T, Turk D,
986
Anderson PA, Mark AE, Dodds PN, et al (2016) The CC domain structure from the
987
wheat stem rust resistance protein Sr33 challenges paradigms for dimerization in plant
988
NLR proteins. Proc Natl Acad Sci U S A 113: 12856–12861
989
Century KS, Shapiro AD, Repetti PP, Dahlbeck D, Holub E, Staskawicz BJ (1997)
990
NDR1, a pathogen-induced component required for Arabidopsis disease resistance.
991
Science 278: 1963–1965
992
Césari S, Moore J, Chen C, Webb D, Periyannan S, Mago R, Bernoux M, Lagudah ES,
993
Dodds PN (2016) Cytosolic activation of cell death and stem rust resistance by cereal
994
MLA-family CC–NLR proteins. Proc Natl Acad Sci 113: 10204–10209
995
996
997
Clough SJ, Bent AF (1998) Floral dip: A simplified method for Agrobacterium-mediated
transformation of Arabidopsis thaliana. Plant J 16: 735–743
Collier SM, Moffett P (2009) NB-LRRs work a “bait and switch” on pathogens. Trends
40
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
998
999
1000
1001
1002
1003
Plant Sci 14: 521–529
Cui H, Tsuda K, Parker JE (2015) Effector-Triggered Immunity: from pathogen perception
to robust defense. Annu Rev Plant Biol 66: 487–511
Dangl JL, Jones JD (2001) Plant pathogens and integrated defence responses to infection.
Nature 411: 826–833
Day B, Dahlbeck D, Huang J, Chisholm ST, Li DH, Staskawicz BJ (2005) Molecular
1004
basis for the RIN4 negative regulation of RPS2 disease resistance. Plant Cell 17: 1292-
1005
1305
1006
1007
1008
Deslandes L, Rivas S (2012) Catch me if you can: bacterial effectors and plant targets.
Trends Plant Sci 17: 644–655
DeYoung BJ, Qi D, Kim SH, Burke TP, Innes RW (2012) Activation of a plant nucleotide
1009
binding-leucine rich repeat disease resistance protein by a modified self protein. Cell
1010
Microbiol 14: 1071–1084
1011
1012
1013
Dou D, Zhou J-M (2012) Phytopathogen effectors subverting host immunity: Different foes,
similar battleground. Cell Host Microbe 12: 484–495
Feng F, Yang F, Rong W, Wu X, Zhang J, Chen S, He C, Zhou J-M (2012) A
1014
Xanthomonas uridine 5′-monophosphate transferase inhibits plant immune kinases.
1015
Nature 485: 114–118
1016
Fernandez-Pozo N, Menda N, Edwards JD, Saha S, Tecle IY, Strickler SR, Bombarely
1017
A, Fisher-York T, Pujar A, Foerster H, et al (2015) The Sol Genomics Network
1018
(SGN)-from genotype to phenotype to breeding. Nucleic Acids Res 43: D1036–D1041
1019
Frost D, Way H, Howles P, Luck J, Manners J, Hardham A, Finnegan J, Ellis J (2004)
1020
Tobacco transgenic for the flax rust resistance gene L expresses allele-specific activation
1021
of defense responses. Mol Plant-Microbe Interact 17: 224–232
1022
Galán JE, Wolf-Watz H (2006) Protein delivery into eukaryotic cells by type III secretion
41
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1023
1024
machines. Nature 444: 567–573
Gassmann W, Hinsch ME, Staskawicz BJ (1999) The Arabidopsis RPS4 bacterial
1025
resistance gene is a member of the TIR-NBS-LRR family of disease resistance genes.
1026
Plant J 20: 265–277
1027
Gassmann, W (2005) Natural variation in the Arabidopsis response to the avirulence gene
1028
hopPsyA uncouples the hypersensitive response from disease resistance Mol Plant-
1029
Microbe Interact 18: 1054–1060
1030
1031
1032
Göhre V, Robatzek S (2008) Breaking the barriers: microbial effector molecules subvert
plant immunity. Annu Rev Phytopathol 46: 189–215
Grant SR, Fisher EJ, Chang JH, Mole BM, Dangl JL (2006) Subterfuge and
1033
manipulation: type III effector proteins of phytopathogenic bacteria. Annu Rev
1034
Microbiol 60: 425–449
1035
Hao W, Collier SM, Moffett P, Chai J (2013) Structural basis for the interaction between
1036
the Potato virus X resistance protein (Rx) and its cofactor ran GTPase-activating protein
1037
2 (RanGAP2). J Biol Chem 288: 35868–35876
1038
1039
Hayward A, Padmanabhan M, Dinesh-Kumar SP (2011) Virus-induced gene silencing in
Nicotiana benthamiana and other plant species. Methods Mol Biol 678: 55–63
1040
Heath MC (2000) Hypersensitive response-related death. Plant Mol Biol 44: 321–334
1041
van der Hoorn R a L, Kamoun S (2008) From Guard to Decoy: a new model for perception
1042
1043
of plant pathogen effectors. Plant Cell 20: 2009–2017
Huard-Chauveau C, Perchepied L, Debieu M, Rivas S, Kroj T, Kars I, Bergelson J,
1044
Roux F, Roby D (2013) An atypical kinase under balancing selection confers broad-
1045
spectrum disease resistance in Arabidopsis. PLoS Genet 9: e1003766
1046
1047
Jayaraman J, Choi S, Prokchorchik M, Choi DS, Spiandore A, Rikkerink EH,
Templeton MD, Segonzac C, Sohn KH (2017) A bacterial acetyltransferase triggers
42
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1048
immunity in Arabidopsis thaliana independent of hypersensitive response. Sci Rep 7:
1049
3557
1050
Jin H, Axtell MJ, Dahlbeck D, Ekwenna O, Zhang S, Staskawicz B, Baker B (2002)
1051
NPK1, an MEKK1-like mitogen-activated protein kinase kinase kinase, regulates innate
1052
immunity and development in plants. Dev Cell 3: 291–297
1053
Jones JDG, Dangl JL (2006) The plant immune system. Nature 444: 323–329
1054
Jones JDG, Vance RE, Dangl JL (2016) Intracellular innate immune surveillance devices in
1055
1056
plants and animals. Science 354: aaf6395-aaf6395
Katagiri F, Thilmony R, He SY (2002) The Arabidopsis thaliana-Pseudomonas syringae
1057
interaction. The Arabidopsis Book, eds Somerville CR, Meyerowitz EM (Am Soc Plant
1058
Biologists, Rockville, MD), pp 1–35.
1059
1060
1061
Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE (2015) The Phyre2 web
portal for protein modeling, prediction and analysis. Nat Protoc 10: 845–858
Khan M, Subramaniam R, Desveaux D (2016) Of guards, decoys, baits and traps:
1062
Pathogen perception in plants by type III effector sensors. Curr Opin Microbiol 29: 49–
1063
55
1064
Krasileva K V., Dahlbeck D, Staskawicz BJ (2010) Activation of an Arabidopsis resistance
1065
protein is specified by the in Planta association of its leucine-rich repeat domain with
1066
the cognate oomycete effector. Plant Cell 22: 2444–2458
1067
De La Fuente Van Bentem S, Vossen JH, De Vries KJ, Van Wees S, Tameling WIL,
1068
Dekker HL, De Koster CG, Haring MA, Takken FLW, Cornelissen BJC (2005)
1069
Heat shock protein 90 and its co-chaperone protein phosphatase 5 interact with distinct
1070
regions of the tomato I-2 disease resistance protein. Plant J 43: 284–298
1071
1072
Lee AH-Y, Hurley B, Felsensteiner C, Yea C, Ckurshumova W, Bartetzko V, Wang
PW, Quach V, Lewis JD, Liu YC, et al (2012) A bacterial acetyltransferase destroys
43
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1073
plant microtubule networks and blocks secretion. PLoS Pathog 8: e1002523
1074
Lewis JD, Abada W, Ma W, Guttman DS, Desveaux D (2008) The HopZ family of
1075
Pseudomonas syringae type III effectors require myristoylation for virulence and
1076
avirulence functions in Arabidopsis thaliana. J Bacteriol 190: 2880–2891
1077
1078
1079
1080
1081
Lewis JD, Guttman DS, Desveaux D (2009) The targeting of plant cellular systems by
injected type III effector proteins. Semin Cell Dev Biol 20: 1055–1063
Lewis JD, Lee A, Ma W, Zhou H, Guttman DS, Desveaux D (2011) The YopJ superfamily
in plant-associated bacteria. Mol Plant Pathol 12: 928–937
Lewis JD, Lee AH-Y, Hassan JA, Wan J, Hurley B, Jhingree JR, Wang PW, Lo T,
1082
Youn J-Y, Guttman DS, et al (2013) The Arabidopsis ZED1 pseudokinase is required
1083
for ZAR1-mediated immunity induced by the Pseudomonas syringae type III effector
1084
HopZ1a. Proc Natl Acad Sci U S A 110: 18722–18727
1085
Lewis JD, Lo T, Bastedo P, Guttman DS, Desveaux D (2014a) The rise of the undead:
1086
pseudokinases as mediators of effector-triggered immunity. Plant Signal Behav 9:
1087
e27563
1088
Lewis JD, Wan J, Ford R, Gong Y, Fung P, Nahal H, Wang PW, Desveaux D, Guttman
1089
DS (2012) Quantitative Interactor Screening with next-generation Sequencing (QIS-Seq)
1090
identifies Arabidopsis thaliana MLO2 as a target of the Pseudomonas syringae type III
1091
effector HopZ2. BMC Genomics 13: 8
1092
Lewis JD, Wilton M, Mott GA, Lu W, Hassan JA, Guttman DS, Desveaux D (2014b)
1093
Immunomodulation by the Pseudomonas syringae HopZ type III effector family in
1094
Arabidopsis. PLoS One 9: e116152
1095
Lewis JD, Wu R, Guttman DS, Desveaux D (2010) Allele-specific virulence attenuation of
1096
the Pseudomonas syringae HopZ1a type III effector via the Arabidopsis ZAR1
1097
resistance protein. PLoS Genet 6: e1000894
44
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1098
1099
1100
1101
1102
Lukasik E, Takken FLW (2009) STANDing strong, resistance proteins instigators of plant
defence. Curr Opin Plant Biol 12: 427–436
Ma K, Ma W (2016) YopJ family effectors promote bacterial infection through a
acetyltransferase activity. Microbiol Mol Biol Rev 80: 1011–1027
Macho AP, Guevara CM, Tornero P, Ruiz-Albert J, Beuzón CR (2010) The
1103
Pseudomonas syringae effector protein HopZ1a suppresses effector-triggered immunity.
1104
New Phytol 187: 1018–1033
1105
Macho AP, Zipfel C (2015) Targeting of plant pattern recognition receptor-triggered
1106
immunity by bacterial type-III secretion system effectors. Curr Opin Microbiol 23: 14–
1107
22
1108
Maekawa T, Cheng W, Spiridon LN, Töller A, Lukasik E, Saijo Y, Liu P, Shen QH,
1109
Micluta MA, Somssich IE, et al (2011) Coiled-coil domain-dependent
1110
homodimerization of intracellular barley immune receptors defines a minimal functional
1111
module for triggering cell death. Cell Host Microbe 9: 187–199
1112
Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, Geer RC,
1113
He J, Gwadz M, Hurwitz DI, et al (2015) CDD: NCBI’s conserved domain database.
1114
Nucleic Acids Res 43: D222–D226
1115
1116
Meyers BC, Kozik A, Griego A, Kuang H, Michelmore RW (2003) Genome-wide analysis
of NBS-LRR-encoding genes in Arabidopsis. Plant Cell 15: 809–834
1117
Mindrinos M, Katagiri F, Yu G-L, Ausubel FM (1994) The A. thaliana disease resistance
1118
gene RPS2 encodes a protein containing a nucleotide-binding site and leucine-rich
1119
repeats. Cell 78: 1089–1099
1120
Mitchell A, Chang H-Y, Daugherty L, Fraser M, Hunter S, Lopez R, McAnulla C,
1121
McMenamin C, Nuka G, Pesseat S, et al (2015) The InterPro protein families
1122
database: the classification resource after 15 years. Nucleic Acids Res 43: D213–D221
45
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1123
1124
1125
Mudgett MB (2005) New insights to the function of phytopathogenic bacterial type III
effectors in plants. Annu Rev Plant Biol 56: 509–531
Parker JE, Holub EB, Frost LN, Falk A, Gunn ND, Daniels MJ (1996) Characterization
1126
of eds1, a mutation in Arabidopsis suppressing resistance to Peronospora parasitica
1127
specified by several different RPP genes. Plant Cell 8: 2033–2046
1128
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin
1129
TE (2004) UCSF Chimera-A visualization system for exploratory research and analysis.
1130
J Comput Chem 25: 1605–1612
1131
Qi D, DeYoung BJ, Innes RW (2012) Structure-function analysis of the coiled-coil and
1132
leucine-rich repeat domains of the RPS5 disease resistance protein. Plant Physiol 158:
1133
1819–1832
1134
Rairdan GJ, Collier SM, Sacco M a, Baldwin TT, Boettrich T, Moffett P (2008) The
1135
coiled-coil and nucleotide binding domains of the Potato Rx disease resistance protein
1136
function in pathogen recognition and signaling. Plant Cell 20: 739–751
1137
1138
1139
1140
1141
Roux F, Noël L, Rivas S, Roby D (2014) ZRK atypical kinases: emerging signaling
components of plant immunity. New Phytol 203: 713–716
Sacco MA, Mansoor S, Moffett P (2007) A RanGAP protein physically interacts with the
NB-LRR protein Rx, and is required for Rx-mediated viral resistance. Plant J 52: 82–93
Schreiber KJ, Baudin M, Hassan JA, Lewis JD (2016a) Die Another Day: molecular
1142
mechanisms of effector-triggered immunity elicited by type III secreted effector
1143
proteins. Semin Cell Dev Biol 56: 124–133
1144
Schreiber KJ, Bentham A, Williams SJ, Kobe B, Staskawicz BJ (2016b) Multiple domain
1145
associations within the Arabidopsis immune receptor RPP1 regulate the activation of
1146
programmed cell death. PLOS Pathog 12: e1005769
1147
Seto D, Koulena N, Lo T, Menna A, Guttman DS, Desveaux D (2017) Expanded type III
46
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1148
effector recognition by the ZAR1 NLR protein using ZED1-related kinases. Nat Plants
1149
3: 1–4
1150
Shan L, Oh H, Chen J, Guo M, Zhou J-M, Alfano JR, Collmer A, Jia X, Tang X (2004)
1151
The HopPtoF locus of Pseudomonas syringae pv. tomato DC3000 encodes a type III
1152
chaperone and a cognate effector. Mol Plant-Microbe Interact 17: 447–455
1153
Shao F, Golstein C, Ade J, Stoutemyer M, Dixon JE, Innes RW (2003) Cleavage of
1154
Arabidopsis PBS1 by a bacterial type III effector. Science 301: 1230–1233
1155
Shao F, Merritt PM, Bao Z, Innes RW, Dixon JE (2002) A Yersinia effector and a
1156
Pseudomonas avirulence protein define a family of cysteine proteases functioning in
1157
bacterial pathogenesis. Cell 109: 575–588
1158
Shirano Y, Kachroo P, Shah J, Klessig DF (2002) A gain-of-function mutation in an
1159
Arabidopsis Toll Interleukin1 receptor-nucleotide binding site-leucine-rich repeat type R
1160
gene triggers defense responses and results in enhanced disease resistance. Plant Cell 14:
1161
3149–62
1162
Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H,
1163
Remmert M, Söding J, et al (2011) Fast, scalable generation of high-quality protein
1164
multiple sequence alignments using Clustal Omega. Mol Syst Biol 7: 539
1165
1166
Sukarta OCA, Slootweg EJ, Goverse A (2016) Structure-informed insights for NLR
functioning in plant immunity. Semin Cell Dev Biol 56: 134–149
1167
Swiderski MR, Birker D, Jones JDG (2009) The TIR domain of TIR-NB-LRR resistance
1168
proteins is a signaling domain involved in cell death induction. Mol Plant-Microbe
1169
Interact 22: 157–165
1170
1171
1172
Takken FLW, Goverse A (2012) How to build a pathogen detector: Structural basis of NBLRR function. Curr Opin Plant Biol 15: 375–384
Tameling WIL, Baulcombe DC (2007) Physical association of the NB-LRR resistance
47
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1173
protein Rx with a Ran GTPase-activating protein is required for extreme resistance to
1174
Potato virus X. Plant Cell 19: 1682–1694
1175
Tameling WIL, Nooijen C, Ludwig N, Boter M, Slootweg E, Goverse A, Shirasu K,
1176
Joosten MH a J (2010) RanGAP2 mediates nucleocytoplasmic partitioning of the NB-
1177
LRR immune receptor Rx in the Solanaceae, thereby dictating Rx function. Plant Cell
1178
22: 4176–4194
1179
Tameling WIL, Vossen JH, Albrecht M, Lengauer T, Berden JA, Haring MA,
1180
Cornelissen BJC, Takken FLW (2006) Mutations in the NB-ARC domain of I-2 that
1181
impair ATP hydrolysis cause autoactivation. Plant Physiol 140: 1233–45
1182
1183
1184
Ting JP, Willingham SB, Bergstralh DT (2008) NLRs at the intersection of cell death and
immunity. Nat Rev Immunol 8: 372–379
Wang G-F, Ji J, EI-Kasmi F, Dangl JL, Johal G, Balint-Kurti PJ (2015a) Molecular and
1185
functional analyses of a maize autoactive NB-LRR protein identify precise structural
1186
requirements for activity. PLOS Pathog 11: e1004674
1187
Wang G, Roux B, Feng F, Guy E, Li L, Li N, Zhang X, Lautier M, Jardinaud M-F,
1188
Chabannes M, et al (2015b) The decoy substrate of a pathogen effector and a
1189
pseudokinase specify pathogen-induced modified-self recognition and immunity in
1190
plants. Cell Host Microbe 18: 285–295
1191
Williams SJ, Sohn KH, Wan L, Bernoux M, Sarris PF, Segonzac C, Ve T, Ma Y, Saucet
1192
SB, Ericsson DJ, et al (2014) Structural basis for assembly and function of a
1193
heterodimeric plant immune receptor. Science 344: 299–303
1194
Xin X-F, He SY (2013) Pseudomonas syringae pv. tomato, DC3000: A model pathogen for
1195
probing disease susceptibility and hormone signaling in plants. Annu Rev Phytopathol
1196
51: 473–498
1197
Xing S, Wallmeroth N, Berendzen KW, Grefen C (2016) Techniques for the analysis of
48
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1198
protein-protein interactions in vivo. Plant Physiol 171: 727–758
1199
Xu RQ, Blanvillain S, Feng JX, Jiang B Le, Li XZ, Wei HY, Kroj T, Lauber E, Roby D,
1200
Chen B, et al (2008) AvrACXcc8004, a type III effector with a leucine-rich repeat domain
1201
from Xanthomonas campestris pathovar campestris confers avirulence in vascular
1202
tissues of Arabidopsis thaliana ecotype Col-0. J Bacteriol 190: 343–355
1203
1204
Zacharias DA (2002) Sticky caveats in an otherwise glowing report: oligomerizing
fluorescent proteins and their use in cell biology. Sci Signal 2002: 1–3
1205
1206
49
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
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. Plant Cell
28: 146-159
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Bernoux M, Ve T, Williams S, Warren C, Hatters D, Valkov E, Zhang X, Ellis JG, Kobe B, Dodds PN (2011) Structural and functional
analysis of a plant resistance protein TIR domain reveals interfaces for self-association, signaling, and autoregulation. Cell Host
Microbe 9: 200-211
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Van Der Biezen EA, Jones JDG (1998) Plant disease-resistance proteins and the gene-for-gene concept. Trends Biochem Sci 23:
454-456
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Casey LW, Lavrencic P, Bentham AR, Cesari S, Ericsson DJ, Croll T, Turk D, Anderson PA, Mark AE, Dodds PN, et al (2016) The CC
domain structure from the wheat stem rust resistance protein Sr33 challenges paradigms for dimerization in plant NLR proteins.
Proc Natl Acad Sci U S A 113: 12856-12861
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Century KS, Shapiro AD, Repetti PP, Dahlbeck D, Holub E, Staskawicz BJ (1997) NDR1, a pathogen-induced component required
for Arabidopsis disease resistance. 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. Trends Plant Sci 14: 521-529
Pubmed: Author and Title
CrossRef: Author and Title
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Google Scholar: Author Only Title Only Author and Title
Cui H, Tsuda K, Parker JE (2015) Effector-Triggered Immunity: from pathogen perception to robust defense. Annu Rev Plant Biol
66: 487-511
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Dangl JL, Jones JD (2001) Plant pathogens and integrated defence responses to infection. Nature 411: 826-833
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Day B, Dahlbeck D, Huang J, Chisholm ST, Li DH, Staskawicz BJ (2005) Molecular basis for the RIN4 negative regulation of RPS2
disease resistance. Plant Cell 17: 1292-1305
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Deslandes L, Rivas S (2012) Catch me if you can: bacterial effectors and plant targets. Trends Plant Sci 17: 644-655
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
DeYoung BJ, Qi D, Kim SH, Burke TP, Innes RW (2012) Activation of a plant nucleotide binding-leucine rich repeat disease
resistance protein by a modified self protein. Cell Microbiol 14: 1071-1084
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Dou D, Zhou J-M (2012) Phytopathogen effectors subverting host immunity: Different foes, similar battleground. Cell Host Microbe
12: 484-495
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Feng F, Yang F, Rong W, Wu X, Zhang J, Chen S, He C, Zhou J-M (2012) A Xanthomonas uridine 5'-monophosphate transferase
inhibits plant immune kinases. Nature 485: 114-118
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Fernandez-Pozo N, Menda N, Edwards JD, Saha S, Tecle IY, Strickler SR, Bombarely A, Fisher-York T, Pujar A, Foerster H, et al
(2015) The Sol Genomics Network (SGN)-from genotype to phenotype to breeding. Nucleic Acids Res 43: D1036-D1041
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Frost D, Way H, Howles P, Luck J, Manners J, Hardham A, Finnegan J, Ellis J (2004) Tobacco transgenic for the flax rust resistance
gene L expresses allele-specific activation of defense responses. Mol Plant-Microbe Interact 17: 224-232
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Galán JE, Wolf-Watz H (2006) Protein delivery into eukaryotic cells by type III secretion machines. Nature 444: 567-573
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gassmann W, Hinsch ME, Staskawicz BJ (1999) The Arabidopsis RPS4 bacterial resistance gene is a member of the TIR-NBS-LRR
family of disease resistance genes. Plant J 20: 265-277
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gassmann, W (2005) Natural variation in the Arabidopsis response to the avirulence gene hopPsyA uncouples the hypersensitive
response from disease resistance Mol Plant-Microbe Interact 18: 1054-1060
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Göhre V, Robatzek S (2008) Breaking the barriers: microbial effector molecules subvert plant immunity. Annu Rev Phytopathol 46:
189-215
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Grant SR, Fisher EJ, Chang JH, Mole BM, Dangl JL (2006) Subterfuge and manipulation: type III effector proteins of
phytopathogenic bacteria. Annu Rev Microbiol 60: 425-449
Pubmed: Author and Title
CrossRef: Author and Title
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Google Scholar: Author Only Title Only Author and Title
Hao W, Collier SM, Moffett P, Chai J (2013) Structural basis for the interaction between the Potato virus X resistance protein (Rx)
and its cofactor ran GTPase-activating protein 2 (RanGAP2). J Biol Chem 288: 35868-35876
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hayward A, Padmanabhan M, Dinesh-Kumar SP (2011) Virus-induced gene silencing in Nicotiana benthamiana and other plant
species. Methods Mol Biol 678: 55-63
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Heath MC (2000) Hypersensitive response-related death. Plant Mol Biol 44: 321-334
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
van der Hoorn R a L, Kamoun S (2008) From Guard to Decoy: a new model for perception of plant pathogen effectors. Plant Cell
20: 2009-2017
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Huard-Chauveau C, Perchepied L, Debieu M, Rivas S, Kroj T, Kars I, Bergelson J, Roux F, Roby D (2013) An atypical kinase under
balancing selection confers broad-spectrum disease resistance in Arabidopsis. PLoS Genet 9: e1003766
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jayaraman J, Choi S, Prokchorchik M, Choi DS, Spiandore A, Rikkerink EH, Templeton MD, Segonzac C, Sohn KH (2017) A
bacterial acetyltransferase triggers immunity in Arabidopsis thaliana independent of hypersensitive response. Sci Rep 7: 3557
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jin H, Axtell MJ, Dahlbeck D, Ekwenna O, Zhang S, Staskawicz B, Baker B (2002) NPK1, an MEKK1-like mitogen-activated protein
kinase kinase kinase, regulates innate immunity and development in plants. Dev Cell 3: 291-297
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jones JDG, Dangl JL (2006) The plant immune system. Nature 444: 323-329
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jones JDG, Vance RE, Dangl JL (2016) Intracellular innate immune surveillance devices in plants and animals. Science 354:
aaf6395-aaf6395
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Katagiri F, Thilmony R, He SY (2002) The Arabidopsis thaliana-Pseudomonas syringae interaction. The Arabidopsis Book, eds
Somerville CR, Meyerowitz EM (Am Soc Plant Biologists, Rockville, MD), pp 1-35.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE (2015) The Phyre2 web portal for protein modeling, prediction and
analysis. Nat Protoc 10: 845-858
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Khan M, Subramaniam R, Desveaux D (2016) Of guards, decoys, baits and traps: Pathogen perception in plants by type III effector
sensors. Curr Opin Microbiol 29: 49-55
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Krasileva K V., Dahlbeck D, Staskawicz BJ (2010) Activation of an Arabidopsis resistance protein is specified by the in Planta
association of its leucine-rich repeat domain with the cognate oomycete effector. Plant Cell 22: 2444-2458
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
De La Fuente Van Bentem S, Vossen JH, De Vries KJ, Van Wees S, Tameling WIL, Dekker HL, De Koster CG, Haring MA, Takken
FLW, Cornelissen BJC (2005) Heat shock protein 90 and its co-chaperone protein phosphatase 5 interact with distinct regions of
fromJon
11, 2017 - Published by www.plantphysiol.org
the tomato I-2 disease resistanceDownloaded
protein. Plant
43:August
284-298
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lee AH-Y, Hurley B, Felsensteiner C, Yea C, Ckurshumova W, Bartetzko V, Wang PW, Quach V, Lewis JD, Liu YC, et al (2012) A
bacterial acetyltransferase destroys plant microtubule networks and blocks secretion. PLoS Pathog 8: e1002523
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lewis JD, Abada W, Ma W, Guttman DS, Desveaux D (2008) The HopZ family of Pseudomonas syringae type III effectors require
myristoylation for virulence and avirulence functions in Arabidopsis thaliana. J Bacteriol 190: 2880-2891
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lewis JD, Guttman DS, Desveaux D (2009) The targeting of plant cellular systems by injected type III effector proteins. Semin Cell
Dev Biol 20: 1055-1063
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lewis JD, Lee A, Ma W, Zhou H, Guttman DS, Desveaux D (2011) The YopJ superfamily in plant-associated bacteria. Mol Plant
Pathol 12: 928-937
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lewis JD, Lee AH-Y, Hassan JA, Wan J, Hurley B, Jhingree JR, Wang PW, Lo T, Youn J-Y, Guttman DS, et al (2013) The Arabidopsis
ZED1 pseudokinase is required for ZAR1-mediated immunity induced by the Pseudomonas syringae type III effector HopZ1a. Proc
Natl Acad Sci U S A 110: 18722-18727
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lewis JD, Lo T, Bastedo P, Guttman DS, Desveaux D (2014a) The rise of the undead: pseudokinases as mediators of effectortriggered immunity. Plant Signal Behav 9: e27563
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lewis JD, Wan J, Ford R, Gong Y, Fung P, Nahal H, Wang PW, Desveaux D, Guttman DS (2012) Quantitative Interactor Screening
with next-generation Sequencing (QIS-Seq) identifies Arabidopsis thaliana MLO2 as a target of the Pseudomonas syringae type III
effector HopZ2. BMC Genomics 13: 8
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lewis JD, Wilton M, Mott GA, Lu W, Hassan JA, Guttman DS, Desveaux D (2014b) Immunomodulation by the Pseudomonas syringae
HopZ type III effector family in Arabidopsis. PLoS One 9: e116152
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lewis JD, Wu R, Guttman DS, Desveaux D (2010) Allele-specific virulence attenuation of the Pseudomonas syringae HopZ1a type
III effector via the Arabidopsis ZAR1 resistance protein. PLoS Genet 6: e1000894
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lukasik E, Takken FLW (2009) STANDing strong, resistance proteins instigators of plant defence. Curr Opin Plant Biol 12: 427-436
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ma K, Ma W (2016) YopJ family effectors promote bacterial infection through a acetyltransferase activity. Microbiol Mol Biol Rev
80: 1011-1027
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Macho AP, Guevara CM, Tornero P, Ruiz-Albert J, Beuzón CR (2010) The Pseudomonas syringae effector protein HopZ1a
suppresses effector-triggered immunity. New Phytol 187: 1018-1033
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Macho AP, Zipfel C (2015) Targeting of plant pattern recognition receptor-triggered immunity by bacterial type-III secretion system
effectors. Curr Opin Microbiol 23: 14-22
Pubmed: Author and Title
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Maekawa T, Cheng W, Spiridon LN, Töller A, Lukasik E, Saijo Y, Liu P, Shen QH, Micluta MA, Somssich IE, et al (2011) Coiled-coil
domain-dependent homodimerization of intracellular barley immune receptors defines a minimal functional module for triggering
cell death. Cell Host Microbe 9: 187-199
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, Geer RC, He J, Gwadz M, Hurwitz DI, et al (2015) CDD:
NCBI's conserved domain database. Nucleic Acids Res 43: D222-D226
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Meyers BC, Kozik A, Griego A, Kuang H, Michelmore RW (2003) Genome-wide analysis of NBS-LRR-encoding genes in
Arabidopsis. Plant Cell 15: 809-834
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Mindrinos M, Katagiri F, Yu G-L, Ausubel FM (1994) The A. thaliana disease resistance gene RPS2 encodes a protein containing a
nucleotide-binding site and leucine-rich repeats. Cell 78: 1089-1099
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Mitchell A, Chang H-Y, Daugherty L, Fraser M, Hunter S, Lopez R, McAnulla C, McMenamin C, Nuka G, Pesseat S, et al (2015) The
InterPro protein families database: the classification resource after 15 years. Nucleic Acids Res 43: D213-D221
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Mudgett MB (2005) New insights to the function of phytopathogenic bacterial type III effectors in plants. Annu Rev Plant Biol 56:
509-531
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Parker JE, Holub EB, Frost LN, Falk A, Gunn ND, Daniels MJ (1996) Characterization of eds1, a mutation in Arabidopsis
suppressing resistance to Peronospora parasitica specified by several different RPP genes. Plant Cell 8: 2033-2046
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera-A visualization
system for exploratory research and analysis. J Comput Chem 25: 1605-1612
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Qi D, DeYoung BJ, Innes RW (2012) Structure-function analysis of the coiled-coil and leucine-rich repeat domains of the RPS5
disease resistance protein. Plant Physiol 158: 1819-1832
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Rairdan GJ, Collier SM, Sacco M a, Baldwin TT, Boettrich T, Moffett P (2008) The coiled-coil and nucleotide binding domains of
the Potato Rx disease resistance protein function in pathogen recognition and signaling. Plant Cell 20: 739-751
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Roux F, Noël L, Rivas S, Roby D (2014) ZRK atypical kinases: emerging signaling components of plant immunity. New Phytol 203:
713-716
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Sacco MA, Mansoor S, Moffett P (2007) A RanGAP protein physically interacts with the NB-LRR protein Rx, and is required for Rxmediated viral resistance. Plant J 52: 82-93
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Schreiber KJ, Baudin M, Hassan JA, Lewis JD (2016a) Die Another Day: molecular mechanisms of effector-triggered immunity
elicited by type III secreted effector proteins. Semin Cell Dev Biol 56: 124-133
Pubmed: Author and Title
CrossRef: Author and Title
from on August 11, 2017 - Published by www.plantphysiol.org
Author and Title
Google Scholar: Author Only Title OnlyDownloaded
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Schreiber KJ, Bentham A, Williams SJ, Kobe B, Staskawicz BJ (2016b) Multiple domain associations within the Arabidopsis immune
receptor RPP1 regulate the activation of programmed cell death. PLOS Pathog 12: e1005769
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Seto D, Koulena N, Lo T, Menna A, Guttman DS, Desveaux D (2017) Expanded type III effector recognition by the ZAR1 NLR
protein using ZED1-related kinases. Nat Plants 3: 1-4
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Shan L, Oh H, Chen J, Guo M, Zhou J-M, Alfano JR, Collmer A, Jia X, Tang X (2004) The HopPtoF locus of Pseudomonas syringae
pv. tomato DC3000 encodes a type III chaperone and a cognate effector. Mol Plant-Microbe Interact 17: 447-455
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Shao F, Golstein C, Ade J, Stoutemyer M, Dixon JE, Innes RW (2003) Cleavage of Arabidopsis PBS1 by a bacterial type III effector.
Science 301: 1230-1233
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Shao F, Merritt PM, Bao Z, Innes RW, Dixon JE (2002) A Yersinia effector and a Pseudomonas avirulence protein define a family of
cysteine proteases functioning in bacterial pathogenesis. Cell 109: 575-588
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Shirano Y, Kachroo P, Shah J, Klessig DF (2002) A gain-of-function mutation in an Arabidopsis Toll Interleukin1 receptornucleotide binding site-leucine-rich repeat type R gene triggers defense responses and results in enhanced disease resistance.
Plant Cell 14: 3149-62
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, et al (2011) Fast, scalable
generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7: 539
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Sukarta OCA, Slootweg EJ, Goverse A (2016) Structure-informed insights for NLR functioning in plant immunity. Semin Cell Dev
Biol 56: 134-149
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Swiderski MR, Birker D, Jones JDG (2009) The TIR domain of TIR-NB-LRR resistance proteins is a signaling domain involved in
cell death induction. Mol Plant-Microbe Interact 22: 157-165
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Takken FLW, Goverse A (2012) How to build a pathogen detector: Structural basis of NB-LRR function. Curr Opin Plant Biol 15:
375-384
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tameling WIL, Baulcombe DC (2007) Physical association of the NB-LRR resistance protein Rx with a Ran GTPase-activating
protein is required for extreme resistance to Potato virus X. Plant Cell 19: 1682-1694
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tameling WIL, Nooijen C, Ludwig N, Boter M, Slootweg E, Goverse A, Shirasu K, Joosten MH a J (2010) RanGAP2 mediates
nucleocytoplasmic partitioning of the NB-LRR immune receptor Rx in the Solanaceae, thereby dictating Rx function. Plant Cell 22:
4176-4194
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tameling WIL, Vossen JH, Albrecht M, Lengauer T, Berden JA, Haring MA, Cornelissen BJC, Takken FLW (2006) Mutations in the
NB-ARC domain of I-2 that impair ATP hydrolysis cause autoactivation. Plant Physiol 140: 1233-45
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Ting JP, Willingham SB, Bergstralh DT (2008) NLRs at the intersection of cell death and immunity. Nat Rev Immunol 8: 372-379
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang G-F, Ji J, EI-Kasmi F, Dangl JL, Johal G, Balint-Kurti PJ (2015a) Molecular and functional analyses of a maize autoactive NBLRR protein identify precise structural requirements for activity. PLOS Pathog 11: e1004674
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang G, Roux B, Feng F, Guy E, Li L, Li N, Zhang X, Lautier M, Jardinaud M-F, Chabannes M, et al (2015b) The decoy substrate of a
pathogen effector and a pseudokinase specify pathogen-induced modified-self recognition and immunity in plants. Cell Host
Microbe 18: 285-295
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Williams SJ, Sohn KH, Wan L, Bernoux M, Sarris PF, Segonzac C, Ve T, Ma Y, Saucet SB, Ericsson DJ, et al (2014) Structural basis
for assembly and function of a heterodimeric plant immune receptor. Science 344: 299-303
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Xin X-F, He SY (2013) Pseudomonas syringae pv. tomato, DC3000: A model pathogen for probing disease susceptibility and
hormone signaling in plants. Annu Rev Phytopathol 51: 473-498
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Xing S, Wallmeroth N, Berendzen KW, Grefen C (2016) Techniques for the analysis of protein-protein interactions in vivo. Plant
Physiol 171: 727-758
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Xu RQ, Blanvillain S, Feng JX, Jiang B Le, Li XZ, Wei HY, Kroj T, Lauber E, Roby D, Chen B, et al (2008) AvrACXcc8004, a type III
effector with a leucine-rich repeat domain from Xanthomonas campestris pathovar campestris confers avirulence in vascular
tissues of Arabidopsis thaliana ecotype Col-0. J Bacteriol 190: 343-355
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zacharias DA (2002) Sticky caveats in an otherwise glowing report: oligomerizing fluorescent proteins and their use in cell biology.
Sci Signal 2002: 1-3
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded from on August 11, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.