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RCD1 nuclear body dynamics and IDR2 phosphorylation in Arabidopsis thaliana

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Title: RCD1 nuclear body dynamics and IDR2 phosphorylation in Arabidopsis thaliana
Author(s): Reda, Shaimaa Roshdy Abdullah
Contributor: University of Helsinki, Faculty of Agriculture and Forestry
Degree program: Erasmus-Mundus Master's Programme in Plant Breeding (EMPLANT)
Specialisation: Plant production sciences
Language: English
Acceptance year: 2022
Abstract:
Abstract Nuclear bodies (NB) have been studied for their importance of being one of the sites for gene regulation activities. RADICAL INDUCED CELL DEATH1 (RCD1) has proven to be a potential nuclear protein in A.thaliana that localizes to NB. It can interact with different transcription regulators responsible for many physiological functions. One of which is light signaling. Hence, it shares mutual functions with some phytochrome photoreceptors (PHYs), e.g., PHYB. It also contains intrinsically disordered regions (IDRs) that stabilize RCD1 protein upon phosphorylation. This study aims to examine the colocalization events of RCD1 that coexpressed with PHYB in full-length RCD1 complementation line and domain deletion lines, given the fact that both RCD1 and PHYB have previously shown mutual interaction with some of PHYTOCHROME INTERACTING FACTORS (PIFs). To achieve this aim, the colocalization of Venus-tagged RCD1 and Green fluorescent protein (GFP)-tagged PHYB using confocal Microscopy was performed. Another objective is to study the phosphorylation effect of one of the IDRs between WWE and PARP-like domain -IDR2- on RCD1 NB localization. Two phosphomutants -non-phosphorylatable and phosphomimetic- constructs were transiently and stably expressed in the rcd1-4 background. Furthermore, they were screened using Confocal Microscopy. We were able to see the colocalization events in 2 domain deletion lines, RCD1-∆WWE-3xVenus and RCD1 ∆PARP 3xVenus. On the other hand, we could not see any colocalization in the RCD1 ∆RST 3xVenus, which indicates the importance of the RST domain in the colocalization. In addition, phosphorylation was found to affect the abundance of RCD1 protein in both transiently and stably expressed lines. Our study showed non-phosphorylatable forms of IDR2 having a higher abundance of RCD1 NB than the control line RCD1-3xVenus, whereas phosphomimetic IDR2 showed no signal. Collectively, Our experiments showed the effect of phosphorylation on RCD1 NB localization and the importance of the RST domain in the colocalization of RCD1 with PHYB.
Keyword(s): Nuclear bodies - RCD1– PHYs– phyB-5 – PIFs- Phosphorylation - IDR2- Non-phosphorylatable IDR – Phosphomimmitic IDR - Confocal Microscopy


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