Comprehensive comparative assessment of the Arabidopsis thaliana MLO2–CALMODULIN2 interaction by various in vitro and in vivo protein–protein interaction assays

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216208%3A11310%2F23%3A10470806" target="_blank" >RIV/00216208:11310/23:10470806 - isvavai.cz</a>

Výsledek na webu

<a href="https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=aze-elKQbw" target="_blank" >https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=aze-elKQbw</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1042/BCJ20230255" target="_blank" >10.1042/BCJ20230255</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Comprehensive comparative assessment of the Arabidopsis thaliana MLO2–CALMODULIN2 interaction by various in vitro and in vivo protein–protein interaction assays

Popis výsledku v původním jazyce

Mildew resistance locus o (MLO) proteins are heptahelical integral membrane proteins of which some isoforms act as susceptibility factors for the powdery mildew pathogen. In many angiosperm plant species, loss-of-function mlo mutants confer durable broadspectrum resistance against the fungal disease. Barley Mlo is known to interact via a cytosolic carboxyl-terminal domain with the intracellular calcium sensor calmodulin (CAM) in a calcium-dependent manner. Site-directed mutagenesis has revealed key amino acid residues in the barley Mlo calmodulin-binding domain (CAMBD) that, when mutated, affect the MLO-CAM association. We here tested the respective interaction between Arabidopsis thaliana MLO2 and CAM2 using seven different types of in vitro and in vivo protein-protein interaction assays. In each assay, we deployed a wild-type version of either the MLO2 carboxyl terminus (MLO2(CT)), harboring the CAMBD, or the MLO2 fulllength protein and corresponding mutant variants in which two key residues within the CAMBD were substituted by non-functional amino acids. We focused in particular on the substitution of two hydrophobic amino acids (LW/RR mutant) and found in most protein- protein interaction experiments reduced binding of CAM2 to the corresponding MLO2/ MLO2(CT-LW/RR) mutant variants in comparison with the respective wild-type versions. However, the Ura3-based yeast split-ubiquitin system and in planta bimolecular fluorescence complementation (BiFC) assays failed to indicate reduced CAM2 binding to the mutated CAMBD. Our data shed further light on the interaction of MLO and CAM proteins and provide a comprehensive comparative assessment of different types of protein- protein interaction assays with wild-type and mutant versions of an integral membrane protein.

Název v anglickém jazyce

Comprehensive comparative assessment of the Arabidopsis thaliana MLO2–CALMODULIN2 interaction by various in vitro and in vivo protein–protein interaction assays

Popis výsledku anglicky

Mildew resistance locus o (MLO) proteins are heptahelical integral membrane proteins of which some isoforms act as susceptibility factors for the powdery mildew pathogen. In many angiosperm plant species, loss-of-function mlo mutants confer durable broadspectrum resistance against the fungal disease. Barley Mlo is known to interact via a cytosolic carboxyl-terminal domain with the intracellular calcium sensor calmodulin (CAM) in a calcium-dependent manner. Site-directed mutagenesis has revealed key amino acid residues in the barley Mlo calmodulin-binding domain (CAMBD) that, when mutated, affect the MLO-CAM association. We here tested the respective interaction between Arabidopsis thaliana MLO2 and CAM2 using seven different types of in vitro and in vivo protein-protein interaction assays. In each assay, we deployed a wild-type version of either the MLO2 carboxyl terminus (MLO2(CT)), harboring the CAMBD, or the MLO2 fulllength protein and corresponding mutant variants in which two key residues within the CAMBD were substituted by non-functional amino acids. We focused in particular on the substitution of two hydrophobic amino acids (LW/RR mutant) and found in most protein- protein interaction experiments reduced binding of CAM2 to the corresponding MLO2/ MLO2(CT-LW/RR) mutant variants in comparison with the respective wild-type versions. However, the Ura3-based yeast split-ubiquitin system and in planta bimolecular fluorescence complementation (BiFC) assays failed to indicate reduced CAM2 binding to the mutated CAMBD. Our data shed further light on the interaction of MLO and CAM proteins and provide a comprehensive comparative assessment of different types of protein- protein interaction assays with wild-type and mutant versions of an integral membrane protein.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10611 - Plant sciences, botany

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2023

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Biochemical Journal

ISSN

0264-6021

e-ISSN

1470-8728

Svazek periodika

480

Číslo periodika v rámci svazku

20

Stát vydavatele periodika

GB - Spojené království Velké Británie a Severního Irska

Počet stran výsledku

24

Strana od-do

1615-1638

Kód UT WoS článku

001087197200002

EID výsledku v databázi Scopus

2-s2.0-85174641175