A cysteine-rich receptor-like kinase NCRK and a pathogen-induced protein kinase RBK1 are Rop GTPase interactors.
In plants, Rop/Rac GTPases have emerged as central regulators of diverse signalling pathways in plant growth and pathogen defence. When active, they interact with a wide range of downstream effectors. Using yeast two-hybrid screening we have found three previously uncharacterized receptor-like protein kinases to be Rop GTPase-interacting molecules: a cysteine-rich receptor kinase, named NCRK, and two receptor-like cytosolic kinases from the Arabidopsis RLCK-VIb family, named RBK1 and RBK2. Uniquely for Rho-family small GTPases, plant Rop GTPases were found to interact directly with the protein kinase domains. Rop4 bound NCRK preferentially in the GTP-bound conformation as determined by flow cytometric fluorescence resonance energy transfer measurements in insect cells. The kinase RBK1 did not phosphorylate Rop4 in vitro, suggesting that the protein kinases are targets for Rop signalling. Bimolecular fluorescence complementation assays demonstrated that Rop4 interacted in vivo with NCRK and RBK1 at the plant plasma membrane. In Arabidopsis protoplasts, NCRK was hyperphosphorylated and partially co-localized with the small GTPase RabF2a in endosomes. Gene expression analysis indicated that the single-copy NCRK gene was relatively upregulated in vasculature, especially in developing tracheary elements. The seven Arabidopsis RLCK-VIb genes are ubiquitously expressed in plant development, and highly so in pollen, as in case of RBK2. We show that the developmental context of RBK1 gene expression is predominantly associated with vasculature and is also locally upregulated in leaves exposed to Phytophthora infestans and Botrytis cinerea pathogens. Our data indicate the existence of cross-talk between Rop GTPases and specific receptor-like kinases through direct molecular interaction.
Molendijk
Arthur J
AJ
Institute for Biologie II/Botany, Faculty of Biology, Albert-Ludwigs-University Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany.
Ruperti
Benedetto
B
Singh
Manoj K
MK
Dovzhenko
Alexander
A
Ditengou
Franck A
FA
Milia
Mattia
M
Westphal
Lore
L
Rosahl
Sabine
S
Soellick
Tim-Robert
TR
Uhrig
Joachim
J
Weingarten
Lars
L
Huber
Michael
M
Palme
Klaus
K
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000032:analysis*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary
D029681:Arabidopsis Proteins; D011494:Protein Kinases; C528916:NCRK protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases; C528915:RBK1 protein, Arabidopsis; D020558:GTP Phosphohydrolases; D003545:Cysteine