Biological and Biochemical Characterization of Atypical Receptor-Like Cytoplasmic Kinases in Arabidopsis Immune Signaling

In plants, immune responses are initiated upon perception of microbial signals and are rapidly transmitted through intracellular networks largely governed by phosphorylation and ubiquitination - post-translational protein modifications catalyzed by protein kinases and E3 ligases, respectively. Protein kinases are central regulators of cellular signaling, with receptor-like cytoplasmic kinases (RLCKs) playing key roles in plant immune and developmental processes. Proteins within the RLCK-VIII subfamily exhibit limited catalytic activity and are known to function in both immune and developmental pathways, particularly oxidative stress and abscisic acid signaling. In contrast, members of the RLCK-IXb subgroup possess a plant ubiquitin-box (PUB) type E3 ubiquitin ligase domain and have been identified as targets of diverse pathogen effectors in multiple plant species, including Arabidopsis thaliana, potato, tomato, and Nicotiana benthamiana. However, the biochemical functions and regulatory mechanisms of both RLCK-VIII and RLCK-IXb proteins remain poorly understood. In Chapter 2, I characterize the RLCK-VIII proteins MAZ, CARK6, CARK7, and CARK9 (MAZZA, CYTOSOLIC ABA RECEPTOR KINASE 6, 7 and 9). I found that MAZ and CARK7 associate with and are phosphorylated by the calcium-dependent protein kinase CPK28 in planta at residues important for kinase activation. Genetic analyses indicate that MAZ and CARK6 function redundantly as negative regulators of the immune-associated oxidative burst. Notably, neither MAZ nor CARK7 exhibit detectable kinase activity in vitro, and a kinase-inactive MAZ variant complements maz-1 loss-of-function mutants, supporting a noncatalytic role in vivo. In Chapter 3, I examine proteins within the RLCK-IXb subgroup, focusing on PUB32, PUB33, and PUB50 (so named because RLCK-IXb is also considered part of the PUB-VI subgroup). I demonstrate that PUB33 possesses both kinase and E3 ubiquitin ligase activities, which are inversely regulated by autophosphorylation at Thr333. PUB33 forms homo- and heteromeric complexes with PUB32 that attenuate its catalytic activity. Although loss-of-function mutants do not display strong immune phenotypes, PUB33 overexpression suppresses effector-triggered cell death and promotes ubiquitination and destabilization of the effector RipV1 from the vascular bacterial pathogen Ralstonia pseudosolanacearum. Together, my work identifies RLCK-VIII and RLCK-IXb proteins as important regulators of plant immunity and highlights the significance of both catalytic and noncatalytic mechanisms in signaling networks.

Department dc:contributor.department

Biology

Year dc:date.issued

2026

Author dc:creator

• Dharmasena, Uththama Dewage Thakshila Damayanthi

Advisor dc:contributor.supervisor

• Monaghan, Jacqueline

• Arabidopsis thaliana
• plant immune signaling
• protein kinase
• receptor-like cytoplasmic kinase
• Ralstonia pseudosolanacearum
• RipV1
• Plant U-box peoteins (PUBs)
• CARK6
• CARK7
• CARK9
• CPK28
• MAZZA
• PUB33
• PUB32

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1974/36499

OAI identifier oai:identifier

oai:queensu.scholaris.ca:1974/36499