Determining the molecular mechanism of plant disease resistance following pathogen effector perception by the resistance gene pair RPS4/RRS1

Plant and animal cells have an assortment of receptors employed for pathogen surveillance. The Nucleotide-binding domain and Leucine-rich Repeat-containing (NLR) family of proteins are important receptors involved in cell autonomous surveillance. In animals, upon perception of virulence molecules or conserved molecular patterns from pathogens several NLRs initiate immune signalling by the induced proximity of signalling domains. In order to do this, these NLRs form oligomeric wheel-shaped complexes, called inflammasomes, that bring the N-terminal signalling domains of the component NLRs into close proximity and initiates downstream signalling. In plants, much less is known about signal initiation and transduction during NLR-mediated immunity. Plant NLRs recognise virulence factors that are often race specific, called effectors. I used the model NLR pair RRS1/RPS4 to investigate effector recognition by NLRs. RRS1 and RPS4 have the archetypal TIR-NB-LRR architecture of a large subset of plant NLRs, but also contain non-canonical domains. Of particular note, RRS1 contains a C-terminal WRKY DNA-binding domain. RRS1 and RPS4 act in concert to recognise at least three effectors: AvrRps4 secreted by Pseudomonas syringae; PopP2, an acetyltransferase secreted by Ralstonia sotanacearum; and an unidentified effector from Cottetotrichum higginsianum. In this thesis, I present evidence that supports a new conceptual framework for effector recognition. In this model, the virulence target of PopP2 and AvrRps4 has fused to an NLR (in this case the WRKY domain in RRS1) and acts as a decoy, baiting the effector to attack the NLR and trigger immunity, instead of enhancing susceptibility by interfering with the function of its intended target. This mechanism of effector perception may be widespread in plants and I discuss other examples of NLRs with integrated atypical domains. NLRs exert intramolecular inhibition of immune signalling in the absence of effectors in order to prevent autoimmunity. The domains of RRS1 C-terminal to the WRKY exert negative regulation of activation. Upon establishing the method of effector recognition by RRS1 and RPS4, I leverage this knowledge of the mechanism of RRS1 autoinhibition to engineer recognition of viral proteases. In the final chapter of this thesis engineer a mammalian inflammasomeforming NLR system, fused to the signalling domain of RPS4, into plants to 1) introduce into plants an intracellular receptor that recognises conserved pathogen associated molecular patterns and 2) test if inducing the proximity of plant NLR signalling domains is sufficient for activation of NLR-mediated immunity

Glucan, Water Dikinase Exerts Little Control over Starch Degradation in Arabidopsis Leaves at Night

The first step on the pathway of starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night is the phosphorylation of starch polymers, catalyzed by glucan, water dikinase (GWD). It has been suggested that GWD is important for the control of starch degradation, because its transcript levels undergo strong diel fluctuations, its activity is subject to redox regulation in vitro, and starch degradation is strongly decreased in gwd mutant plants. To test this suggestion, we analyzed changes in GWD protein abundance in relation to starch levels in wild-type plants, in transgenic plants in which GWD transcripts were strongly reduced by induction of RNA interference, and in transgenic plants overexpressing GWD. We found that GWD protein levels do not vary over the diel cycle and that the protein has a half-life of 2 d. Overexpression of GWD does not accelerate starch degradation in leaves, and starch degradation is not inhibited until GWD levels are reduced by 70%. Surprisingly, this degree of reduction also inhibits starch synthesis in the light. To discover the importance of redox regulation, we generated transgenic plants expressing constitutively active GWD. These plants retained normal control of degradation. We conclude that GWD exerts only a low level of control over starch degradation in Arabidopsis leaves

Distinct modes of derepression of an Arabidopsis immune receptor complex by two different bacterial effectors

Plant intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors often function in pairs to detect pathogen effectors and activate defense. The Arabidopsis RRS1-R–RPS4 NLR pair recognizes the bacterial effectors AvrRps4 and PopP2 via an integrated WRKY transcription factor domain in RRS1-R that mimics the effector’s authentic targets. How the complex activates defense upon effector recognition is unknown. Deletion of the WRKY domain results in an RRS1 allele that triggers constitutive RPS4-dependent defense activation, suggesting that in the absence of effector, the WRKY domain contributes to maintaining the complex in an inactive state. We show the WRKY domain interacts with the adjacent domain 4, and that the inactive state of RRS1 is maintained by WRKY–domain 4 interactions before ligand detection. AvrRps4 interaction with the WRKY domain disrupts WRKY–domain 4 association, thus derepressing the complex. PopP2-triggered activation is less easily explained by such disruption and involves the longer C-terminal extension of RRS1-R. Furthermore, some mutations in RPS4 and RRS1 compromise PopP2 but not AvrRps4 recognition, suggesting that AvrRps4 and PopP2 derepress the complex differently. Consistent with this, a “reversibly closed” conformation of RRS1-R, engineered in a method exploiting the high affinity of colicin E9 and Im9 domains, reversibly loses AvrRps4, but not PopP2 responsiveness. Following RRS1 derepression, interactions between domain 4 and the RPS4 C-terminal domain likely contribute to activation. Simultaneous relief of autoinhibition and activation may contribute to defense activation in many immune receptors

Induced proximity of a TIR signaling domain on a plant-mammalian NLR chimera activates defense in plants

Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants

Protein-protein interactions in the RPS4/RRS1 immune receptor complex

Plant NLR (Nucleotide-binding domain and Leucine-rich Repeat) immune receptor proteins are encoded by Resistance (R) genes and confer specific resistance to pathogen races that carry the corresponding recognized effectors. Some NLR proteins function in pairs, forming receptor complexes for the perception of specific effectors. We show here that the Arabidopsis RPS4 and RRS1 NLR proteins are both required to make an authentic immune complex. Over-expression of RPS4 in tobacco or in Arabidopsis results in constitutive defense activation; this phenotype is suppressed in the presence of RRS1. RRS1 protein co-immunoprecipitates (co-IPs) with itself in the presence or absence of RPS4, but in contrast, RPS4 does not associate with itself in the absence of RRS1. In the presence of RRS1, RPS4 associates with defense signaling regulator EDS1 solely in the nucleus, in contrast to the extra-nuclear location found in the absence of RRS1. The AvrRps4 effector does not disrupt RPS4-EDS1 association in the presence of RRS1. In the absence of RRS1, AvrRps4 interacts with EDS1, forming nucleocytoplasmic aggregates, the formation of which is disturbed by the co-expression of PAD4 but not by SAG101. These data indicate that the study of an immune receptor protein complex in the absence of all components can result in misleading inferences, and reveals an NLR complex that dynamically interacts with the immune regulators EDS1/PAD4 or EDS1/SAG101, and with effectors, during the process by which effector recognition is converted to defense activation

Spectral properties of bacteriophytochrome AM1_5894 in the chlorophyll d-containing cyanobacterium Acaryochloris marina

Acaryochloris marina, a unicellular oxygenic photosynthetic cyanobacterium, has uniquely adapted to far-red light-enriched environments using red-shifted chlorophyll d. To understand red-light use in Acaryochloris, the genome of this cyanobacterium was searched for red/far-red light photoreceptors from the phytochrome family, resulting in identification of a putative bacteriophytochrome AM1_5894. AM1_5894 contains three standard domains of photosensory components as well as a putative C-terminal signal transduction component consisting of a histidine kinase and receiver domain. The photosensory domains of AM1_5894 autocatalytically assemble with biliverdin in a covalent fashion. This assembled AM1_5894 shows the typical photoreversible conversion of bacterial phytochromes with a ground-state red-light absorbing (Pr) form with ⋋BV max[Pr] 705 nm, and a red-light inducible far-red light absorbing (Pfr) form with ⋋BV max[Pfr] 758 nm. Surprisingly, AM1_5894 also autocatalytically assembles with phycocyanobilin, involving photoreversible conversion of ⋋PCB max[Pr] 682 nm and ⋋PCB max[Pfr] 734 nm, respectively. Our results suggest phycocyanobilin is also covalently bound to AM1_5894, while mutation of a cysteine residue (Cys11Ser) abolishes this covalent binding. The physiological function of AM1_5894 in cyanobacteria containing red-shifted chlorophylls is discussed.12 page(s

Induced proximity of a TIR signaling domain on a plant-mammalian NLR chimera activates defense in plants

Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants..D., S.U.H., S.W.,H.G., and L.Hu were supported on European Research Council (ERC) Grant“Immunity by pair design”Project ID 669926 (to J.D.G.J.). Y.M. was supported onBiotechnology and Biological Sciences Research Council (BBSRC) Grant BB/M008193/1 (to J.D.G.J.). S.U.H. was supported on Next-Generation BioGreen 21 Program (Project No. PJ01365301), Rural Development Administration, Republic of Korea. J.C. was supported by a Chinese Scholarship Council Postgraduate Fellowship. P.D. was supported by the European Union’s Horizon 2020 Research and Innovation Program under Marie Skłodowska-Curie Individual Fellowship (Project ID 656243) and a Future Leader Fellowship from BBSRC (Grant Agreement BB/R012172/1). P.N.M. was supported by a Marie Skłodowska-Curie Action Individual Fellowship (Project ID 656011

RRS1 promotes association of RPS4 and EDS1 in the nucleus.

<p>(A) In the presence of RRS1, the RPS4/EDS1 are predominantly localized to the nucleus. BiFC assays with the co-expression of <i>nVenus-RPS4</i>/<i>cCFP-EDS1</i>/<i>GUS-HF/mCherry</i> reveal reconstruction of YFP signal in the cytoplasmic aggregations and in the nucleus (arrows). In the presence of RRS1-HF, nVenus-RPS4/cCFP-EDS1 association revealed a YFP signal in the nucleus. Scale bar = 10 μm. (B) EDS1 associates with RPS4/RRS1. Upon transient co-delivery of <i>RPS4-HA</i> and <i>RRS1-HF</i> with <i>GFP-EDS1</i> or <i>GFP</i> in <i>N</i>. <i>benthamiana</i> leaves, samples were harvested at 2 dpi and total extracts were immunoprecipitated with anti-GFP beads. Specific protein-protein interactions were detected by immunoblotting with the indicated antibodies. All the experiments were repeated at least three times with similar results.</p

RPS4 homodimerization is dependent on RRS1.

<p>(A) BiFC assays using nVenus- and cCFP-tagged RPS4 reveal that RPS4 self-association in the nucleus is RRS1-dependent. The <i>nVenus-RPS4</i>, <i>cCFP-RPS4</i>, and <i>mCherry</i> were transiently co-expressed in the presence of <i>RRS1-HF</i> or <i>GUS-HF</i> in <i>N</i>. <i>benthamiana</i> leaves. At 2 dpi, the reconstruction YFP signal is observed with confocal microscope (Leica SP5). <i>mCherry</i> was used as a nuclear and cytoplasmic marker. Scale bar = 10 μm. (B) Co-immunoprecipitation (co-IP) assays reveal that RPS4 self-associates only in the presence of RRS1. <i>Agrobacterium</i>-mediated transient co-expression of <i>RRS1-GFP</i>/<i>RPS4-HF</i>/<i>RPS4-HA</i> or <i>GFP</i>/ <i>RPS4-HF</i>/<i>RPS4-HA</i> was performed in <i>N</i>. <i>benthamiana</i> leaves. Anti-FLAG co-IPs were performed with total protein extracts and probed with anti-GFP, -FLAG, and -HA antibodies. (C) Co-IPs show that RRS1 self-associates and forms a heteromeric complex with RPS4. Transient co-expression assays of <i>RRS1-GFP</i>/<i>RRS1-HF</i>, <i>RRS1-GFP</i>/<i>RPS4-HF</i> or <i>GFP</i>/<i>RRS1-HF</i> were performed in <i>N</i>. <i>benthamiana</i> leaves. Immunoblots show the presence of proteins in total extracts (input) and after immunoprecipitation with anti-GFP beads (IP-GFP). All the experiments were repeated at least three times with similar results.</p

AvrRps4 and PopP2 do not disrupt the EDS1/PAD4/RPS4/RRS1 complex.

<p>(A) Anti-FLAG immunoprecipitation of RRS1-HF, RPS4, EDS1 and PAD4 in the presence and absence of AvrRps4 or PopP2. Samples were prepared from transiently co-expressed <i>RRS1-HF</i>, <i>RPS4-HA</i>, <i>EDS1-V5</i> and <i>PAD4-HA</i> in the presence of <i>AvrRps4-GFP</i>, <i>PopP2-GFP</i> or <i>GFP</i> in <i>N</i>. <i>benthamiana</i>. (B) Both AvrRps4 and PopP2 associate with RPS4/RRS1/EDS1/PAD4. To confirm effector protein association with a putative RPS4/RRS1/EDS1/PAD4 complex, samples were prepared from <i>N</i>. <i>benthamiana</i> leaves transiently co-expressing <i>RRS1-HF</i>, <i>RPS4-Myc</i>, <i>EDS1-V5</i> and <i>PAD4-HA</i> in presence of <i>AvrRps4-GFP</i>, <i>PopP2-GFP</i> or <i>GFP</i>. Total extracts were immunoprecipitated with anti-GFP beads followed by immunoblotting with the indicated antibodies. AvrRps4^C represents processed AvrRps4 C-terminus. All the experiments were repeated at least three times with similar results.</p