Page 170 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

*[O132] EXPLORING SPECIFICITY OF PLANT RLCK-VII SIGNALLING. Eleanor Khochaba and
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Thomas A. DeFalco . Department of Biology, Western University, 1151 Richmond St, London, ON,
Canada N6A3K7
Correspondence to: ekhochab@uwo.ca

Receptor kinases (RKs) are receptors at the cell membrane that perceive exogenous or endogenous
ligands to initiate essential signalling cascades vital for various aspects of plant growth, development, and
immunity. Though many RKs have been identified, the molecular mechanisms downstream of receptor
activation are not fully understood. Receptor-like cytoplasmic kinases (RLCKs) are key components of
downstream RK signalling. RLCKs associate with receptor complexes to execute downstream signal
transduction via substrate phosphorylation. Despite apparently similar mechanisms of activation and
substrate interaction, individual RLCKs can exhibit distinct functionalities and involvement in specific RK
pathways, sometimes even exerting opposing effects. For instance, within the RLCK-VII family of kinases,
both BIK1 and PBL13 interact with RBOHD, an NADPH oxidase, to regulate immune responses;
however, their phosphorylation either promotes or inhibits RBOHD function, respectively. Other RLCK-
VIIs, such as PBL34 and PBL15, have also been found to function in RK pathways regulating specific
aspects of development. Our research focuses on understanding the specificity within RLCK-mediated
signalling, using both genetic and biochemical approaches. By generating chimeric RLCK constructs,
wherein the N-terminus, kinase domain, and C-terminus are interchanged, we aim to decipher the roles of
individual protein domains in dictating RLCK signalling specificity. The results of this project will contribute
to our understanding of the signalling mechanisms governing diverse aspects of plant biology, as well as
how kinases target their protein substrates.

*[O133a] DOWNSTREAM SIGNALING RESULTING FROM DAMAGED RIBOSOMAL RNA BY

POKEWEED ANTIVIRAL PROTEIN (PAP). Tanya Prashar and Katalin A. Hudak . Department of
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Biology, York University, 4700 Keele St, Toronto, ON, Canada, M3J 1P3
Correspondence to: tanya97@my.yorku.ca

In addition to their role in translation, ribosomes serve as a central hub for detecting mRNA damage,
which could cause ribosome stalling, collisions, or slowed translation elongation. Ribosome surveillance
pathways, such as the integrated stress response (ISR) leading to GCN2-dependent eIF2α
phosphorylation, or ribosome quality control (RQC) causing E3 ubiquitin ligase Hel2-dependent
ubiquitination of 40S ribosomal subunit, are triggered depending on the extent and nature of mRNA
damage. However, the mechanisms of sensing and signaling ribosomal RNA (rRNA) damage are not fully
understood. We investigated downstream signaling pathways triggered by depurinated 25S rRNA caused
by pokeweed antiviral protein (PAP) expression in Saccharomyces cerevisiae. PAP is a ribosome-
inactivating protein (RIP) found in Phytolacca americana, a native North American plant. PAP's antiviral
activity arises from its inhibition of protein translation resulting from the removal of an adenine residue
from the sarcin-ricin loop (SRL) in 25S rRNA. Depurinated rRNA prevents the binding of elongation
factors, thus limiting both viral and cellular protein production. In this study, our findings demonstrate that
the expression of PAP in yeast leads to depurination of 25S rRNA causing inhibited cell growth and
reduced viability. Additionally, our results reveal a low-level increase in GCN2-dependent GCN4
translation levels indicating minimal ISR activation upon PAP expression. Furthermore, we examined
whether PAP expression activates RQC and observed a Hel2-dependent increase in ubiquitination levels,
suggesting potential ubiquitination of 60S or 40S ribosomal subunits. These findings offer initial evidence
supporting the idea that ribosomes with depurinated rRNA might be subject to proteasomal degradation.
Our future work involves studying whether depurinated ribosomes are engaged in active translation and
quantifying their elongation rate, which may indicate when cells detect damage to rRNA. The sarcin-ricin
loop is a critical component of the ribosomal GTPase center required for translation elongation, and our
research will link effects of its depurination to activation of stress response pathways to understand how
cells detect and manage damage to rRNA.

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