Identification and characterization of a J-domain containing protein as a novel co-modifier of Self-incompatibility response in Brassica napus
Date
2021-08-20
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Self-incompatibility (SI) is a mechanism that plants utilize to prevent inbreeding and promote outcrossing. In Brassicaceae, this is a genetic mechanism that is controlled by S-locus in which haplotype-specific rejection of self-pollen is achieved through recognition of the pollen ligand SCR/SP11 by the S-receptor kinase of the papillary cells. The receptor-ligand activation converges on an E3 ligase, ARC1, which targets degradation of compatibility factors that result in blocking of delivery of resources required for pollen germination, leading to SI response. In this thesis, I have used the CRISPR-Cas9 gene editing platform to confirm the role of ARC1 during SI signaling and have also identified JDP1 as an interactor and modifier of ARC1, required for full manifestation of SI response.The role of ARC1 in the self-incompatibility pathway in Brassicaceae has remained a point of contention within the scientific community for over 20 years. Through gene-editing to create loss-of-function of alleles of ARC1 in Brassica napus, I was able to show that loss of ARC1 resulted in complete breakdown of SI in two different haplotype combinations proving the necessity of ARC1 for successful manifestation of SI.I next explored whether there are other positive regulators of SI at or downstream of ARC1 through a cytosolic interaction assay that identified J-domain containing protein (JDP1) as a potential cytosolic interactor of ARC1. This HSP-40 family protein interacted with ARC1 only when Tyr8 was modified or absent suggesting likely post-translational in-vivo regulation of this Tyr8 of JDP1. Through transgenic approaches to suppress JDP1, I was able to demonstrate that JDP1 is a positive regulator of SI and overexpression of the JDPY8F form led to constitutive rejection response irrespective of upstream signals demonstrating that the ARC1 interacting JDPY8F was sufficient to confer SI response.Further biochemical characterization of JDP1 revealed that the X-domain of JDP1 which is necessary for binding ARC1, harbored E3 ligase activity. Both JDPY8F and JDP?J (X-domain) were able to enhance ARC1-dependent ubiquitination of previously identified ARC1 substrates GLO1 and EXO70A1 suggesting that interaction of ARC1 with Tyr8 modified JDP1 may be essential for full functionality of ARC1.
Description
Keywords
Self-incompatibility (SI), S-locus, Brassicaceae, SCR/SP11, HSP-40, ARC1, E3 ligase, JDP1, ubiquitination, CRISPR-Cas9, Brassica napus, protein interaction, GLO1, EXO70A1
Citation
Kumar, A. (2021). Identification and characterization of a J-domain containing protein as a novel co-modifier of Self-incompatibility response in Brassica napus (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.