Principal Supervisor: Murray Grant - SLS
Co-supervisor: Lorenzo Frigerio - SLS
PhD project title: Chloroplast immunity
University of Registration: Warwick
This project examines a newly emerging area of plant defence, chloroplast immunity, which the Grant lab is pioneering. Its objective is to develop totally novel and innovative approaches to improve plant protection strategies and hence enhance food security. Classically, pathogen recognition induces a rapid (~ 30 min) apoplastic reactive oxygen species (ROS) burst driven by plant pattern recognition receptors at the membrane recognising invariant pathogen associated molecular patterns (PAMPs). We recently discovered that the chloroplast plays a central, and previously unrecognised, role in plant basal immunity (de Torres et al. 2015, Nature Plants). Chloroplast immunity (CI) is characterised by a transient ROS burst ~5 h after activation of pattern recognition receptors that is indispensable for defence, suggesting sequential, compartmentalised ROS signalling underpins innate immunity. Pathogens deliver effector proteins into the chloroplast and suppress this ROS burst. Suppression of chloroplastic ROS proceeds in planta multiplication of bacteria. This is achieved by uncoupling electron flow from photosystem II to photosystem I by an unknown mechanism.
Our most recent data using a ROS dye (see Figure) shows that some, but not all, chloroplasts stain green (blue arrows) in the presence of ROS and reveals three striking features; (i) not all chloroplasts generate a ROS signal, (ii) chloroplasts are in direct contact with the nuclear membrane and, (iii) some stained chloroplasts produce stromules - membrane connections with the nucleus. Collectively these data suggest that during activated immunity there is direct physical communication between the chloroplast and nucleus. Because the dye is irreversibly oxidised by many ROS and lacks spatial resolution, it cannot be used to probe these exciting observations in more detail.
This project comprises two main components:
- Visualisation of ROS dynamics during innate immunity. Using “genetically encoded” reporters targeted to either the chloroplast, nucleus or peroxisomes to visualise in real time ROS dynamics during PAMP triggered immunity. These proteins report H2O2, (roGFP2-Orp1) the class of ROS generated during chloroplastic immune responses, or pH (pHRed). Using ratiometric imaging, combinations of reporters with different intracellular addresses will be used to precisely map the timing and source of both H2O2 and pH changes in different cellular compartments following pathogen challenges as a prerequisite to understanding the dynamics of basal immunity and how nucleo/chloroplastic exchange of cellular material drives plant basal immunity. This work will involve learning con-focal microscopy and training in image analysis.
- Investigating the post-transcriptional targets of chloroplast localised effectors. To identify chloroplast proteins targeted by effectors we will undertake a yeast-2-hybrid screen with HopC1, Hop R1 and HopO1-2; effectors for which we have evidence for chloroplast localisation. HopC1 is putatively localised to the chloroplast and interacts with two chloroplast associated kinesins. A knockout of one kinesin attenuates growth of DC3000. HopR1 is highly conserved and a knockout of a predicted chloroplast interactor, PFT1/TCP13 results in enhanced resistance to DC3000 interaction. HopO1-2, a predicted ADP-ribosyltransferase is imported into pea chloroplasts. Mutants of the ADP-riboslytransferase HopU1 have reduced virulence and HopU1 interacts with chloroplast ribonucleoproteins in vitro, thus HopO1-2 may be required for processing/stabilisation of specific chloroplast mRNAs. These effectors will be screened against 3 new MatchmakerGold yeast 2-hybrid libraries representing mRNA derived from early innate and late innate or susceptible responses. Putative interactors will be quantified by single read Illumina sequencing using bar-coded amplicons from yeast pools. An interaction map will be generated and highly ranked interactors tested, using a combination of available T-DNA knockout populations and CRISP/CAS9 multiplex editing. This component will provide training in molecular biology, yeast genetics and new genome editing techniques.
BBSRC Strategic Research Priority: Food security
Techniques that will be undertaken during the project:
Characterisation of genetic reporters, single and dual reporter confocal imaging, ratiometric imaging of ROS dynamics, image analysis and quantitation, yeast 2-hybrid screening and interactor characterisation, next-generation sequence data analysis and some basic bioinformatics, multiplex CRISPR/CAS9, pathogen infection and bacterial enumeration.
Contact: Professor Murray Grant, University of Warwick