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

Result code in 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>

Result on the web

<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>

Result language

angličtina

Original language name

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

Original language description

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.

Czech name

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Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

—

OECD FORD branch

10611 - Plant sciences, botany

Project

Result was created during the realization of more than one project. More information in the Projects tab.

Continuities

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

Publication year

2023

Confidentiality

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

Name of the periodical

Biochemical Journal

ISSN

0264-6021

e-ISSN

1470-8728

Volume of the periodical

480

Issue of the periodical within the volume

20

Country of publishing house

GB - UNITED KINGDOM

Number of pages

24

Pages from-to

1615-1638

UT code for WoS article

001087197200002

EID of the result in the Scopus database

2-s2.0-85174641175