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Title: Role of redox regulated E3-ligase 1 in plant immunity at the cell surface
Author: Lubega, Jibril
ISNI:       0000 0004 7969 2133
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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S-nitrosylation, the addition of a nitric oxide moiety to a cysteine thiol to form an S-nitrosothiol (SNO) is emerging as a key redox-based post-translational modification in the control of plant immunity. Redox Regulated E3 ligase 1 (RRE1) was initially identified following transcriptome analysis of pathogen challenged Arabidopsis S-nitrosylation and Salicylic acid induction deficient mutants. RRE1 has two conserved domains, an ankyrin repeat known to mediate protein-protein interaction and a RING finger motif driving E3 ligase activity. The expression of RRE1 correlated with expression level of Arabidopsis thaliana Penetration 1 protein (AtPEN1), a protein integral to surface level resistance against Blumeria pathogens. AtPEN1 recycling at the plasma membrane is a key feature of this immune response. Mutation in either RRE1 or AtPEN1 enhanced increased penetration rates of the non-adapted pathogen Blumeria graminis f. sp. hordei (Bgh). Previous data indicated that, XA21 binding protein Hordeum vulgare 35 (XBHV35) a barley homologue of RRE1 interacted with Candidate secreted effector protein 0443 (CSEP0443). CSEP0443 is highly expressed during early stages of fungal invasion likely aiding penetration and haustoria formation, thus promoting fungal aggressiveness. I investigated the functional relevance of RRE1 in pathogen-plant interaction with respect to AtPEN1 and CSEP0443. Initially I investigated the contribution of CSEP0443 to Bgh pathogenicity in both host plant barley and non-host plant Arabidopsis. By applying BSMV-VIGS mediated HIGS to barley the expression levels of CSEP0443 was significantly reduced to 43%. The plants displayed low levels of Bgh susceptibility, reduced by 61% relative to wild-type. In non-host systems, expression of CSEP0443 enhanced Bgh penetration rates by 30% relative to wild-type. However, further growth of the pathogen was attenuated by cells undergoing programmed cell death. The tight co-expression of RRE1 and AtPEN1 during Bgh challenge prompted to investigate the possible interaction of the two proteins and the consequences of the interactions. My results displayed interactions of RRE1 and AtPEN1. RRE1 further, mediated the ubiquitination of AtPEN1, underpinning AtPEN1 as a substrate for RRE1. By mutating lysines 48 and 63 to arginine in ubiquitin, I revealed that AtPEN1 is sufficiently polyubiquitinated along the two ubiquitin sites. As the two types of polyubiquitinations target protein to different fates, I investigated the effect of these modifications. AtPEN1 conjugated with K48 linkages was degraded by 26S proteasome. AtPEN1 conjugated with K63 linkage was not targeted to vacuolar degradation, likely targeted for other functions like activation for relocalization. By applying mutagenesis to probable lysine residues in AtPEN1, lysine 103 and 281 were identified as the ubiquitination sites. My findings reveal the first syntaxin in plant systems whose ubiquitination sites have been disclosed. Further, I investigated the possible interaction of RRE1 and CSEP0443. CSEP0443 exhibited an interaction with RRE1. Interaction was significantly observed with the RING domain likely aimed to compromise E3 ligase activity of RRE1. Indeed RRE1-RING-C340S, with the loss of E3 ligase activity due to disruption of zinc coordination, exhibited reduced interaction with CSEP0443. I investigated the consequences of the interaction by determining if RRE1 could ubiquitinate CSEP0443 or CSEP0443 could affect the ability of RRE1 to ubiquitinate AtPEN1. In my findings CSEP0443 was not ubiquitinated by RRE1 however, CSEP0443 compromised the ability of RRE1 to ubiquitinate AtPEN1. The results reveal RRE1 as a target for CSEP0443 in Arabidopsis adding to few effectors from Bgh proteome whose targets are disclosed. My findings support the hypothesis that RRE mediate the relocalizations of AtPEN1 to the site of attempted Bgh penetration by driving AtPEN1 polyubiquitination with K63 chain linkages. Bgh secrete CSEP0443 that hinder the E3 ligase ability of RRE1 hence curtailing the accumulation of AtPEN1 to fungal ingress sites.
Supervisor: Loake, Gary ; Hoebe, Peter Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: S-nitrosylation ; Redox Regulated E3 ligase 1 ; RRE1 ; Blumeria pathogens ; AtPEN1 ; Hordeum vulgare 35 ; CSEP0443