scholarly journals Protein engineering expands the effector recognition profile of a rice NLR immune receptor

2019 ◽  
Author(s):  
JC De la Concepcion ◽  
M Franceschetti ◽  
R Terauchi ◽  
S Kamoun ◽  
MJ Banfield
Keyword(s):  
Protein Engineering ◽  
Direct Interaction ◽  
In Planta ◽  
Pathogen Effectors ◽  
Binding Interface ◽  
Pathogen Effector ◽  
Integrated Domains ◽  
Effector Recognition

AbstractPlant NLR receptors detect pathogen effectors and initiate an immune response. Since their discovery, NLRs have been the focus of protein engineering to improve disease resistance. However, this has proven challenging, in part due to their narrow response specificity. Here, we used structure-guided engineering to expand the response profile of the rice NLR Pikp to variants of the rice blast pathogen effector AVR-Pik. A mutation located within an effector binding interface of the integrated Pikp-HMA domain increased the binding affinity for AVR-Pik variants in vitro and in vivo. This translates to an expanded cell death response to AVR-Pik variants previously unrecognized by Pikp in planta. Structures of the engineered Pikp-HMA in complex with AVR-Pik variants revealed the mechanism of expanded recognition. These results provide a proof-of-concept that protein engineering can improve the utility of plant NLR receptors where direct interaction between effectors and NLRs is established, particularly via integrated domains.

scholarly journals Protein engineering expands the effector recognition profile of a rice NLR immune receptor

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Juan Carlos De la Concepcion ◽  
Marina Franceschetti ◽  
Dan MacLean ◽  
Ryohei Terauchi ◽  
Sophien Kamoun ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Direct Interaction ◽  
Structural Basis ◽  
In Planta ◽  
Pathogen Effectors ◽  
Binding Interface ◽  
Pathogen Effector ◽  
Integrated Domains

Plant nucleotide binding, leucine-rich repeat (NLR) receptors detect pathogen effectors and initiate an immune response. Since their discovery, NLRs have been the focus of protein engineering to improve disease resistance. However, this approach has proven challenging, in part due to their narrow response specificity. Previously, we revealed the structural basis of pathogen recognition by the integrated heavy metal associated (HMA) domain of the rice NLR Pikp (Maqbool et al., 2015). Here, we used structure-guided engineering to expand the response profile of Pikp to variants of the rice blast pathogen effector AVR-Pik. A mutation located within an effector-binding interface of the integrated Pikp–HMA domain increased the binding affinity for AVR-Pik variants in vitro and in vivo. This translates to an expanded cell-death response to AVR-Pik variants previously unrecognized by Pikp in planta. The structures of the engineered Pikp–HMA in complex with AVR-Pik variants revealed the mechanism of expanded recognition. These results provide a proof-of-concept that protein engineering can improve the utility of plant NLR receptors where direct interaction between effectors and NLRs is established, particularly where this interaction occurs via integrated domains.

scholarly journals Specific recognition of two MAX effectors by integrated HMA domains in plant immune receptors involves distinct binding surfaces

2018 ◽  
Vol 115 (45) ◽  
pp. 11637-11642 ◽  
Author(s):  
Liwei Guo ◽  
Stella Cesari ◽  
Karine de Guillen ◽  
Véronique Chalvon ◽  
Léa Mammri ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Rice Cultivar ◽  
Target Proteins ◽  
Binding Interface ◽  
Binding Surface ◽  
Effector Recognition ◽  
Host Target ◽  
Effector Binding

The structurally conserved but sequence-unrelated MAX (Magnaporthe oryzaeavirulence and ToxB-like) effectors AVR1-CO39 and AVR-PikD from the blast fungusM. oryzaeare recognized by the rice nucleotide-binding domain and leucine-rich repeat proteins (NLRs) RGA5 and Pikp-1, respectively. This involves, in both cases, direct interaction of the effector with a heavy metal-associated (HMA) integrated domain (ID) in the NLR. Here, we solved the crystal structures of a C-terminal fragment of RGA5 carrying the HMA ID (RGA5_S), alone, and in complex with AVR1-CO39 and compared it to the structure of the Pikp1HMA/AVR-PikD complex. In both complexes, HMA ID/MAX effector interactions involve antiparallel alignment of β-sheets from each partner. However, effector-binding occurs at different surfaces in Pikp1HMAand RGA5HMA, indicating that these interactions evolved independently by convergence of these two MAX effectors to the same type of plant target proteins. Interestingly, the effector-binding surface in RGA5HMAoverlaps with the surface that mediates RGA5HMAself-interaction. Mutations in the HMA-binding interface of AVR1-CO39 perturb RGA5HMA-binding, in vitro and in vivo, and affect the recognition ofM. oryzaein a rice cultivar containingPi-CO39. Our study provides detailed insight into the mechanisms of effector recognition by NLRs, which has substantial implications for future engineering of NLRs to expand their recognition specificities. In addition, we propose, as a hypothesis for the understanding of effector diversity, that in the structurally conserved MAX effectors the molecular mechanism of host target protein-binding is conserved rather than the host target proteins themselves.

scholarly journals Cross-reactivity of a rice NLR immune receptor to distinct effectors from the blast pathogen leads to partial disease resistance

2019 ◽  
Author(s):  
Freya A. Varden ◽  
Hiromasa Saitoh ◽  
Kae Yoshino ◽  
Marina Franceschetti ◽  
Sophien Kamoun ◽  
...  
Keyword(s):  
Immune Response ◽  
Disease Resistance ◽  
Effector Proteins ◽  
In Planta ◽  
Immune Receptor ◽  
Immune Receptors ◽  
Binding Interface ◽  
Pathogen Effector ◽  
Integrated Domains

ABSTRACTUnconventional integrated domains in plant intracellular immune receptors (NLRs) can directly bind translocated pathogen effector proteins to initiate an immune response. The rice immune receptor pairs Pik-1/Pik-2 and RGA5/RGA4 both use integrated heavy metal-associated (HMA) domains to bind the Magnaporthe oryzae effectors AVR-Pik and AVR-Pia, respectively. These effectors both belong to the MAX effector family and share a core structural fold, despite being divergent in sequence. How integrated domains maintain specificity of recognition, even for structurally similar effectors, has implications for understanding plant immune receptor evolution and function. Here we show that the rice NLR pair Pikp-1/Pikp-2 triggers an immune response leading to partial disease resistance towards the “mismatched” effector AVR-Pia in planta, and that the Pikp-HMA domain binds AVR-Pia in vitro. The HMA domain from another Pik-1 allele, Pikm, is unable to bind AVR-Pia, and does not trigger a response in plants. The crystal structure of Pikp-HMA bound to AVR-Pia reveals a different binding interface compared to AVR-Pik effectors, suggesting plasticity in integrated domain/effector interactions. This work shows how a single NLR can bait multiple pathogen effectors via an integrated domain, and may enable engineering immune receptors with extended disease resistance profiles.

Two chloroplastic PLGG1 isoforms function together to transport photorespiratory glycolate and glycerate in rice

2021 ◽  
Author(s):  
Lili Cui ◽  
Chuanling Zhang ◽  
Zhichao Li ◽  
Tuxiu Xian ◽  
Limin Wang ◽  
...  
Keyword(s):  
Rice Genome ◽  
Direct Interaction ◽  
Crop Productivity ◽  
Chloroplast Envelope ◽  
Starch Accumulation ◽  
Loss Of Function ◽  
In Planta ◽  
In Vivo Experiments

Abstract The photorespiratory pathway is highly compartmentalized. As such, metabolite shuttles between organelles are critical to ensure efficient photorespiratory carbon flux. Arabidopsis PLGG1 has been reported as a key chloroplastic glycolate/glycerate transporter. Two homologous genes OsPLGG1a and OsPLGG1b have been identified in the rice genome, although their distinct functions and relationships remain unknown. Herein, our analysis of exogenous expression in oocytes and yeast shows that both OsPLGG1a and OsPLGG1b have the ability to transport glycolate and glycerate. Furthermore, we demonstrate in planta, that the perturbation of OsPLGG1a or OsPLGG1b expression leads to extensive accumulation of photorespiratory metabolites, especially glycolate and glycerate. Under ambient CO2 conditions, loss-of-function osplgg1a or osplgg1b mutant plants exhibited significant decreases in photosynthesis efficiency, starch accumulation, plant height, and crop productivity. These morphological defects were almost entirely recovered when the mutant plants were grown under elevated CO2 conditions instead. In contrast to osplgg1a, osplgg1b mutant alleles produced a mild photorespiratory phenotype and had reduced accumulation of photorespiratory metabolites. Subcellular localization analysis showed that OsPLGG1a and OsPLGG1b are located in the inner and outer membranes of the chloroplast envelope, respectively. In vitro and in vivo experiments revealed that OsPLGG1a and OsPLGG1b have a direct interaction. Our results indicate that both OsPLGG1a and OsPLGG1b are chloroplastic glycolate/glycerate transporters required for photorespiratory metabolism and plant growth, and that they may function as a singular complex.

scholarly journals An unconventional NOI/RIN4 domain of a rice NLR protein binds host EXO70 protein to confer fungal immunity

2017 ◽  
Author(s):  
Koki Fujisaki ◽  
Yoshiko Abe ◽  
Eiko Kanzaki ◽  
Kazue Ito ◽  
Hiroe Utsushi ◽  
...  
Keyword(s):  
Host Protein ◽  
Current View ◽  
Nucleotide Binding Domain ◽  
Host Proteins ◽  
Core Motif ◽  
Pathogen Effectors ◽  
Pathogen Effector ◽  
Integrated Domains ◽  
Effector Recognition ◽  
Fungal Immunity

A subset of plant nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins carry extraneous integrated domains that have been proposed to mediate pathogen effector recognition. The current view is that these unconventional domains function by directly binding or serving as substrates for pathogen effectors, yet only a few domains have been functionally characterized to date. Here we report that the integrated NOI domain of the rice NLR protein Pii-2, together with its partner Pii-1, mediates immunity to the rice blast fungus Magnaporthe oryzae by indirect recognition of the AVR-Pii effector. We discovered that the Pii-2 NOI domain does not physically interact with the effector itself but instead binds the host protein OsExo70-F3, which is a target of AVR-Pii. We further identified mutations within the NOI core motif (PxFGxW) of Pii-2 that abolish both OsExo70-F3 binding and Pii-mediated resistance to M. oryzae expressing AVR-Pii. This led us to propose a novel conceptual model in which an NLR-integrated domain functions to detect host proteins targeted by pathogen effectors, in a framework that extends classical indirect recognition models.

Defibrotide Inhibits Platelet Activation by Cathepsin G Released From Stimulated Polymorphonuclear Leukocytes

1992 ◽  
Vol 67 (06) ◽  
pp. 660-664 ◽  
Author(s):  
Virgilio Evangelista ◽  
Paola Piccardoni ◽  
Giovanni de Gaetano ◽  
Chiara Cerletti
Keyword(s):  
Platelet Activation ◽  
Polymorphonuclear Leukocytes ◽  
Direct Interaction ◽  
Cathepsin G ◽  
In Vivo Test ◽  
Cell Functions ◽  
Test Models ◽  
Antithrombotic Effects

SummaryDefibrotide is a polydeoxyribonucleotide with antithrombotic effects in experimental animal models. Most of the actions of this drug have been observed in in vivo test models but no effects have been reported in in vitro systems. In this paper we demonstrate that defibrotide interferes with polymorphonuclear leukocyte-induced human platelet activation in vitro. This effect was not related to any direct interaction with polymorphonuclear leukocytes or platelets, but was due to the inhibition of cathepsin G, the main biochemical mediator of this cell-cell cooperation. Since cathepsin G not only induces platelet activation but also affects some endothelial cell functions, the anticathepsin G activity of defibrotide could help to explain the antithrombotic effect of this drug.

scholarly journals Pathogen effector recognition-dependent association of NRG1 with EDS1 and SAG101 in TNL receptor immunity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xinhua Sun ◽  
Dmitry Lapin ◽  
Joanna M. Feehan ◽  
Sara C. Stolze ◽  
Katharina Kramer ◽  
...  
Keyword(s):  
Disease Susceptibility ◽  
Nucleotide Binding ◽  
Molecular Evidence ◽  
Pathogen Effectors ◽  
Pathogen Effector ◽  
Family Protein ◽  
Activated State ◽  
Defence Signalling ◽  
Effector Recognition ◽  
Dependent Interaction

AbstractPlants utilise intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors to detect pathogen effectors and activate local and systemic defence. NRG1 and ADR1 “helper” NLRs (RNLs) cooperate with enhanced disease susceptibility 1 (EDS1), senescence-associated gene 101 (SAG101) and phytoalexin-deficient 4 (PAD4) lipase-like proteins to mediate signalling from TIR domain NLR receptors (TNLs). The mechanism of RNL/EDS1 family protein cooperation is not understood. Here, we present genetic and molecular evidence for exclusive EDS1/SAG101/NRG1 and EDS1/PAD4/ADR1 co-functions in TNL immunity. Using immunoprecipitation and mass spectrometry, we show effector recognition-dependent interaction of NRG1 with EDS1 and SAG101, but not PAD4. An EDS1-SAG101 complex interacts with NRG1, and EDS1-PAD4 with ADR1, in an immune-activated state. NRG1 requires an intact nucleotide-binding P-loop motif, and EDS1 a functional EP domain and its partner SAG101, for induced association and immunity. Thus, two distinct modules (NRG1/EDS1/SAG101 and ADR1/EDS1/PAD4) mediate TNL receptor defence signalling.

scholarly journals Localization matters: nuclear-trapped Survivin sensitizes glioblastoma cells to temozolomide by elevating cellular senescence and impairing homologous recombination

2021 ◽  
Author(s):  
Thomas R. Reich ◽  
Christian Schwarzenbach ◽  
Juliana Brandstetter Vilar ◽  
Sven Unger ◽  
Fabian Mühlhäusler ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Nuclear Export ◽  
Cell Model ◽  
Chromosome Instability ◽  
Direct Interaction ◽  
High Grade Glioma ◽  
Strand Break ◽  
Γh2ax Foci

AbstractTo clarify whether differential compartmentalization of Survivin impacts temozolomide (TMZ)-triggered end points, we established a well-defined glioblastoma cell model in vitro (LN229 and A172) and in vivo, distinguishing between its nuclear and cytoplasmic localization. Expression of nuclear export sequence (NES)-mutated Survivin (SurvNESmut-GFP) led to impaired colony formation upon TMZ. This was not due to enhanced cell death but rather due to increased senescence. Nuclear-trapped Survivin reduced homologous recombination (HR)-mediated double-strand break (DSB) repair, as evaluated by γH2AX foci formation and qPCR-based HR assay leading to pronounced induction of chromosome aberrations. Opposite, clones, expressing free-shuttling cytoplasmic but not nuclear-trapped Survivin, could repair TMZ-induced DSBs and evaded senescence. Mass spectrometry-based interactomics revealed, however, no direct interaction of Survivin with any of the repair factors. The improved TMZ-triggered HR activity in Surv-GFP was associated with enhanced mRNA and stabilized RAD51 protein expression, opposite to diminished RAD51 expression in SurvNESmut cells. Notably, cytoplasmic Survivin could significantly compensate for the viability under RAD51 knockdown. Differential Survivin localization also resulted in distinctive TMZ-triggered transcriptional pathways, associated with senescence and chromosome instability as shown by global transcriptome analysis. Orthotopic LN229 xenografts, expressing SurvNESmut exhibited diminished growth and increased DNA damage upon TMZ, as manifested by PCNA and γH2AX foci expression, respectively, in brain tissue sections. Consequently, those mice lived longer. Although tumors of high-grade glioma patients expressed majorly nuclear Survivin, they exhibited rarely NES mutations which did not correlate with survival. Based on our in vitro and xenograft data, Survivin nuclear trapping would facilitate glioma response to TMZ.

scholarly journals Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I

2002 ◽  
Vol 364 (2) ◽  
pp. 343-347 ◽  
Author(s):  
Gareth J.O. EVANS ◽  
Alan MORGAN
Keyword(s):  
Rat Brain ◽  
Direct Interaction ◽  
Secretory Vesicle ◽  
Brain Membrane ◽  
Synaptotagmin I ◽  
Phosphorylated Form ◽  
Order Of Magnitude ◽  
And Function

The secretory vesicle cysteine string proteins (CSPs) are members of the DnaJ family of chaperones, and function at late stages of Ca2+-regulated exocytosis by an unknown mechanism. To determine novel binding partners of CSPs, we employed a pull-down strategy from purified rat brain membrane or cytosolic proteins using recombinant hexahistidine-tagged (His6-)CSP. Western blotting of the CSP-binding proteins identified synaptotagmin I to be a putative binding partner. Furthermore, pull-down assays using cAMP-dependent protein kinase (PKA)-phosphorylated CSP recovered significantly less synaptotagmin. Complexes containing CSP and synaptotagmin were immunoprecipitated from rat brain membranes, further suggesting that these proteins interact in vivo. Binding assays in vitro using recombinant proteins confirmed a direct interaction between the two proteins and demonstrated that the PKA-phosphorylated form of CSP binds synaptotagmin with approximately an order of magnitude lower affinity than the non-phosphorylated form. Genetic studies have implicated each of these proteins in the Ca2+-dependency of exocytosis and, since CSP does not bind Ca2+, this novel interaction might explain the Ca2+-dependent actions of CSP.

scholarly journals SpoVID Guides SafA to the Spore Coat inBacillus subtilis

2001 ◽  
Vol 183 (10) ◽  
pp. 3041-3049 ◽  
Author(s):  
Amanda J. Ozin ◽  
Craig S. Samford ◽  
Adriano O. Henriques ◽  
Charles P. Moran
Keyword(s):  
Direct Interaction ◽  
Spore Coat ◽  
Coat Proteins ◽  
Yeast Two Hybrid System ◽  
Spore Coat Proteins ◽  
Basement Layer ◽  
Two Hybrid ◽  
Spore Coat Protein

ABSTRACT Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encasesBacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.

Sign in / Sign up

Export Citation Format

Share Document