A cell-free approach to identify binding hotspots in plant immune receptors

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
George C. Markou ◽  
Casim A. Sarkar
Keyword(s):  
Ligand Binding ◽  
Hypersensitive Response ◽  
Polypeptide Chain ◽  
Trichoderma Viride ◽  
Leucine Rich Repeat ◽  
In Planta ◽  
Immune Receptors ◽  
A Cell ◽  
Modular Nature

AbstractPlant immune receptors are often difficult to express heterologously, hindering study of direct interactions between these receptors and their targets with traditional biochemical approaches. The cell-free method ribosome display (RD) enables expression of such recalcitrant proteins by keeping each nascent polypeptide chain tethered to its ribosome, which can enhance protein folding by virtue of its size and solubility. Moreover, in contrast to an in planta readout of receptor activity such as a hypersensitive response that conflates binding and signaling, RD enables direct probing of the interaction between plant immune receptors and their targets. Here, we demonstrate the utility of this approach using tomato recognition of Trichoderma viride ethylene-inducing xylanase (EIX) as a case study. Leveraging the modular nature of the tomato LeEIX2 and LeEIX1 leucine-rich repeat (LRR) receptors, we applied an entropy-informed algorithm to maximize the information content in our receptor segmentation RD experiments to identify segments implicated in EIX binding. Unexpectedly, two distinct EIX-binding hotspots were discovered on LeEIX2 and both hotspots are shared with decoy LeEIX1, suggesting that their contrasting receptor functions are not due to differential modes of ligand binding. Given that most plant immune receptors are thought to engage targets via their LRR sequences, this approach should be of broad utility in rapidly identifying their binding hotspots.

scholarly journals Novel Method for Quantifying AhR-Ligand Binding Affinities Using Microscale Thermophoresis

Biosensors ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 60
Author(s):  
Anne Stinn ◽  
Jens Furkert ◽  
Stefan H. E. Kaufmann ◽  
Pedro Moura-Alves ◽  
Michael Kolbe
Keyword(s):  
Ligand Binding ◽  
Environmental Pollutants ◽  
Screening Strategy ◽  
Pharmacological Intervention ◽  
Binding Affinities ◽  
Free System ◽  
Microscale Thermophoresis ◽  
Aryl Hydrocarbon ◽  
Promising Target ◽  
A Cell

The aryl hydrocarbon receptor (AhR) is a highly conserved cellular sensor of a variety of environmental pollutants and dietary-, cell- and microbiota-derived metabolites with important roles in fundamental biological processes. Deregulation of the AhR pathway is implicated in several diseases, including autoimmune diseases and cancer, rendering AhR a promising target for drug development and host-directed therapy. The pharmacological intervention of AhR processes requires detailed information about the ligand binding properties to allow specific targeting of a particular signaling process without affecting the remaining. Here, we present a novel microscale thermophoresis-based approach to monitoring the binding of purified recombinant human AhR to its natural ligands in a cell-free system. This approach facilitates a precise identification and characterization of unknown AhR ligands and represents a screening strategy for the discovery of potential selective AhR modulators.

scholarly journals Specific hypersensitive response-associated recognition of new apoplastic effectors fromCladosporium fulvumin wild tomato

2017 ◽  
Author(s):  
Carl H. Mesarich ◽  
Bilal Ökmen ◽  
Hanna Rovenich ◽  
Scott A. Griffiths ◽  
Changchun Wang ◽  
...  
Keyword(s):  
Hypersensitive Response ◽  
Expression System ◽  
Potato Virus X ◽  
Cladosporium Fulvum ◽  
In Planta ◽  
Antimicrobial Proteins ◽  
Wild Tomato ◽  
Microbe Interactions ◽  
Leaf Apoplast ◽  
Tomato Leaf Mould

ABSTRACTTomato leaf mould disease is caused by the biotrophic fungusCladosporium fulvum. During infection,C. fulvumproduces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed byCfimmune receptor genes that encode receptor-like proteins (RLPs). These RLPs recognize specific SSP effectors to initiate a hypersensitive response (HR) that renders the pathogen avirulent.C. fulvumstrains capable of overcoming one or more of all clonedCfgenes have now emerged. To combat these strains, newCfgenes are required. An effectoromics approach was employed to identify wild tomato accessions carrying newCfgenes. Proteomics and transcriptome sequencing were first used to identify 70 apoplasticin planta-inducedC. fulvumSSPs. Based on sequence homology, 61 of these SSPs were novel or lacked known functional domains. Seven, however, had predicted structural homology to antimicrobial proteins, suggesting a possible role in mediating antagonistic microbe−microbe interactionsin planta. Wild tomato accessions were then screened for HR-associated recognition of 41 SSPs using thePotato virus X-based transient expression system. Nine SSPs were recognized by one or more accessions, suggesting that these plants carry newCfgenes available for incorporation into cultivated tomato.

scholarly journals Lessons in effector and NLR biology of plant-microbe systems

2017 ◽  
Author(s):  
Aleksandra Białas ◽  
Erin K. Zess ◽  
Juan Carlos De la Concepcion ◽  
Marina Franceschetti ◽  
Helen G. Pennington ◽  
...  
Keyword(s):  
Plant Physiology ◽  
Magnaporthe Oryzae ◽  
Rice Blast ◽  
Molecular Mechanisms ◽  
Rice Blast Fungus ◽  
Leucine Rich Repeat ◽  
Nucleotide Binding Domain ◽  
Immune Receptors ◽  
Microbe Interactions ◽  
Host Infection

A diversity of plant-associated organisms secrete effectors—proteins and metabolites that modulate plant physiology to favor host infection and colonization. However, effectors can also activate plant immune receptors, notably nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins, enabling plants to fight off invading organisms. This interplay between effectors, their host targets, and the matching immune receptors is shaped by intricate molecular mechanisms and exceptionally dynamic coevolution. In this article, we focus on three effectors, AVR-Pik, AVR-Pia, and AVR-Pii, from the rice blast fungus Magnaporthe oryzae (syn. Pyricularia oryzae), and their corresponding rice NLR immune receptors, Pik, Pia, and Pii, to highlight general concepts of plant-microbe interactions. We draw 12 lessons in effector and NLR biology that have emerged from studying these three little effectors and are broadly applicable to other plant-microbe systems.

scholarly journals Identification of Immune Related LRR-Containing Genes in Maize (Zea maysL.) by Genome-Wide Sequence Analysis

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Wei Song ◽  
Baoqiang Wang ◽  
Xinghua Li ◽  
Jianfen Wei ◽  
Ling Chen ◽  
...  
Keyword(s):  
Zea Mays ◽  
Plant Disease Resistance ◽  
Interleukin 1 ◽  
Protein Structures ◽  
Gene Clusters ◽  
Amino Acid Sequences ◽  
Leucine Rich Repeat ◽  
Immune Receptors ◽  
Genome Wide ◽  
First Time

A large number of immune receptors consist of nucleotide binding site-leucine rich repeat (NBS-LRR) proteins and leucine rich repeat-receptor-like kinases (LRR-RLK) that play a crucial role in plant disease resistance. Although many NBS-LRR genes have been previously identified inZea mays, there are no reports on identifying NBS-LRR genes encoded in the N-terminal Toll/interleukin-1 receptor (TIR) motif and identifying genome-wide LRR-RLK genes. In the present study, 151 NBS-LRR genes and 226 LRR-RLK genes were identified after performing bioinformatics analysis of the entire maize genome. Of these identified genes, 64 NBS-LRR genes and four TIR-NBS-LRR genes were identified for the first time. The NBS-LRR genes are unevenly distributed on each chromosome with gene clusters located at the distal end of each chromosome, while LRR-RLK genes have a random chromosomal distribution with more paired genes. Additionally, six LRR-RLK/RLPs including FLS2, PSY1R, PSKR1, BIR1, SERK3, and Cf5 were characterized inZea maysfor the first time. Their predicted amino acid sequences have similar protein structures with their respective homologues in other plants, indicating that these maize LRR-RLK/RLPs have the same functions as their homologues act as immune receptors. The identified gene sequences would assist in the study of their functions in maize.

New insights into ligand binding by plant lipid transfer proteins: A case study of the lentil Lc-LTP2

2020 ◽  
Vol 528 (1) ◽  
pp. 39-45 ◽  
Author(s):  
D.N. Melnikova ◽  
I.V. Bogdanov ◽  
A.A. Ignatova ◽  
T.V. Ovchinnikova ◽  
E.I. Finkina
Keyword(s):  
Ligand Binding ◽  
Lipid Transfer ◽  
Lipid Transfer Proteins ◽  
Plant Lipid

scholarly journals Alien domains shaped the modular structure of plant NLR proteins

2019 ◽  
Author(s):  
Giuseppe Andolfo ◽  
Antimo Di Donato ◽  
Pasquale Chiaiese ◽  
Antonino De Natale ◽  
Antonino Pollio ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Cell Surface ◽  
Surveillance System ◽  
Genomic Structure ◽  
Defense Responses ◽  
Leucine Rich Repeat ◽  
Unicellular Alga ◽  
Unicellular Algae ◽  
A Cell ◽  
Chromochloris Zofingiensis

Abstract Plant innate immunity mostly relies on nucleotide-binding (NB) and leucine-rich repeat (LRR) intracellular receptors to detect pathogen-derived molecules and to induce defense responses. A multi-taxa reconstruction of NB-domain associations allowed us to identify the first NB-LRR arrangement in the Chlorophyta division of the Viridiplantae. Our analysis points out that the basic NOD-like receptor (NLR) unit emerged in Chlorophytes by horizontal transfer and its diversification started from TIR-NB-LRR (TNL) members. The operon-based genomic structure of Chromochloris zofingiensis NLR copies suggests a functional origin of NLR clusters. Moreover, the transmembrane signatures of NLR proteins in the unicellular alga C. zofingiensis supports the hypothesis that the NLR-based immunity system of plants derives from a cell-surface surveillance system. Taken together, our findings suggest that NLRs originated in unicellular algae and may have a common origin with cell surface LRR receptors.

Application of Multivariate Analysis toward Biotech Processes: Case Study of a Cell-Culture Unit Operation

2007 ◽  
Vol 23 (1) ◽  
pp. 61-67 ◽  
Author(s):  
A.O. Kirdar ◽  
J.S. Conner ◽  
J. Baclaski ◽  
A.S. Rathore
Keyword(s):  
Cell Culture ◽  
Multivariate Analysis ◽  
Unit Operation ◽  
A Cell

scholarly journals Characterization, expression, and evolution of the mouse embryonic zeta-globin gene.

1985 ◽  
Vol 5 (5) ◽  
pp. 1025-1033 ◽  
Author(s):  
A Leder ◽  
L Weir ◽  
P Leder
Keyword(s):  
Amino Acid ◽  
Polypeptide Chain ◽  
Globin Gene ◽  
Complete Sequence ◽  
Globin Genes ◽  
Globin Mrna ◽  
A Cell ◽  
Erythroleukemic Cell ◽  
Zeta Globin Gene ◽  
Cloned Gene

We have determined the complete sequence of the embryonic alpha-like, zeta (zeta)-globin gene of the BALB/c mouse. The structure of this gene establishes the amino acid sequence of the mouse embryonic zeta-globin polypeptide chain and allows us to identify sequences within the gene that may be important for its expression. One of these is a 300-base segment that is tightly conserved between mice and humans and is located at the 5' end of the zeta-globin gene. By introducing the cloned gene into permanently transfected mouse erythroleukemic cell lines and comparing its transcript with that of zeta-globin mRNA derived from embryonic yolk sac erythrocytes, we are able to show that the cloned gene is transcriptionally active and that its transcript is correctly initiated and processed. Interestingly, the zeta-globin gene is also active when permanently transfected into an immunoglobulin-producing B-cell, a cell that presumably has tissue-specific requirements for gene expression. Further, a comparison of the amino acid coding sequence of the mouse zeta-globin gene to that of zeta-like globin genes of other species supports a revised evolutionary lineage in which goats and humans are closely related, whereas mice are further removed.

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