During this summer, I was working on a project under the supervision of Dr Tolga Bozkurt at Imperial College London for which I was awarded the BSPP undergraduate vacation bursary. The aim was to investigate the functionality of the different domains of the Chloroplast Unusual Positioning Protein 1 (CHUP1), a chloroplast outer membrane protein. It has been previously shown that chloroplasts are involved in plant immunity. More recently, chloroplasts were found to accumulate at the haustorial interface during infection by Phytophthora infestans. I specifically pursued the following objectives: (i) dissecting the role of CHUP1 in plant immunity during P. infestans infections; (ii) generate CHUP1 mutants that lack different domains and assessing the actin- and profilin- binding activities of the mutants by performing co-immunoprecipitation assays.
During my placement, I first focused on conducing co-immunoprecipitation assays to functionally validate the actin binding domain mutant of CHUP1. A previous experiment has suggested that a CHUP1 mutant lacking the actin-binding domain results in the loss of CHUP1 ability to bind actin. However, this observation could be also attributed to the loss of dimerization capacity of the mutant. I have designed and performed a co-immunoprecipitation assay to investigate whether this is the case; however, it is still unclear whether the mutant forms a dimer or not, as the experiments were unsuccessful. In addition to actin-binding domain, CHUP1 also carries a profilin-binding domain, and both proteins (actin and profilin) have been shown to be required for chloroplast photorelocation. To determine which domains are important for chloroplast movement towards the haustorial interface, I also generated a CHUP1 mutant that lacks the profilin-binding domain. The mutant has been validated by sequencing and the work will be continued in the future in Bozkurt lab.
Part of the work I’ve been performing was to assess the impact of CHUP1 on plant immunity. This work is a follow-up on one of an earlier report from the lab, which will be included as part of a future publication. In three independent biological repeats using an independent virus induced gene silencing (VIGS) constructs for CHUP1, P. infestans showed significantly more hyphal growth (pictured below) than control plants.
This work improves understanding the mechanisms by which chloroplasts contribute to plant immunity. The emerging view is that chloroplast movement to pathogen interface is necessary for plant immunity. However, it is unclear whether chloroplast movement during pathogen attacks is regulated in the same way as light induced chloroplast movement. My project represented the first steps in understanding the role of CHUP1, as well as the ways in which this protein’s domains are involved in chloroplast movement.
Andrada Ioana Balmez
Imperial College London
P. infestans haustorial growth is significantly larger in CHUP1 silenced via VIGS plants (B), when compared to control plants (A).