Understanding the mechanism of chloroplast immunity.

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This multidisciplinary proposal uses proteomics, cell biology, mass spectrometry, biophysical and genetic approaches to address the mechanistic basis of chloroplast mediated immunity. In Postdam, using state-of-the-art techniques, we measure the biophysical changes in the chloroplast during MTI and suppression of MTI and will include both PRR mutants and pharmacological challenge.
Secondly, we will undertake an untargeted proteomics screen on chloroplasts isolated from the same challenges and sampling times as used in Postdam, using Progenesis label free quantitation. Both these work programmes will be complemented by comparative analysis of the chloroplast metabolome by GC-QToF during virulent and non-pathogenic bacterial challenges. These biophysical, proteomic and metabolomics data will be interrogated collectively and targets selected for reverse genetic screens.

To accurately quantify the number of DC3000 effectors that target the chloroplast we will generate transplastomic tomato plants expressing the C-terminus of a self-assembling GFP construct in the chloroplast. Chloroplast localization will be tested using Pseudomonas derived effectors expressing the N-terminal self-assembling GFP. In addition targeted proteomics approach will be undertaken using selected, chloroplast localised effectors, HopO1-2.

The third major component examines the dynamics of ROS production and inter-organelle communication using transgenic Arabidopsis lines expressing (i) the novel genetically encoded marker roGFP2-Orp1 targeted to report changes in chloroplast and nuclear H2O2 and (ii) marked lines carrying GFP, NEON and YFP targeted to the perixosome, nucleus and chloroplast respectively. These studies will provide the first comprehensive insight into the temporal spatial dynamics of intracellular ROS generation and inter-organellar dynamics during MTI and suppression of MTI.

Who might benefit from this research?
The research has broad economic, social impact and industrial impact in two areas;
(a) gaining a new understanding novel link between organelle (chloroplast) redox-signalling and plant immunity which can be exploited to develop enhanced resistance to pathogens - given the current concerns about global food insecurity, novel approaches to improving crop resilience to biotic stress have tremendous potential to increase productivity.
(b) directly linked to productivity, identification of the mechanism(s) by which plant pathogen effector proteins inhibit photosynthesis can be exploited in two ways (i) enhanced crop productivity, (ii) development of new herbicides - therefore this work will be of wide interest to agrotech companies, farmers and breeders.

How might they benefit from this research?
Outputs from this project could lead to significant potentially exploitable impact, including; .
Health and ecological impacts: Reduced waste. Reduced pollution of the environment as a result of decreased application of pesticides.
Socio-economic impacts: Increased resilience to pathogens means enhanced food security for the UK and global population. Increased public trust of genetically modified transgenic crops and synthetic biology (linked to Pathways to Impact activities) e.g. the application of CRISPR-Cas9 dual gene editing will help illustrate to the general public the advantages of using synthetically engineered crops for enhanced resilience to biotic stresses.
Economic benefits: Minimizing agricultural losses from pathogens due to enhanced pathogen resistance of engineered crops will substantially increase crop production, minimise use of pesticides and reduce waste.
Agrochemical Industry
Targeting suppression of chloroplast ROS generation offers opportunities to identify novel targets for chemical intervention. This has a number of attractions. 1, the chloroplast is a reduced complexity system. 2, many agrochemicals target the chloroplast thus companies already have expertise that can be focussed on identifying chemicals could enhance resistance to pathogens. This work will also help inform on the cross-talk between biotic and abiotic stress networks modified by pathogen induced ABA.
Farmers/Crop Producers
If successful, uptake of knowledge will be beneficial to farmers and agricultural systems globally. As we have shown that pathogens suppress photosynthesis, solutions will directly increase productivity, enabling greater yields as well as addressing increasing threats from pests and pathogens. This impact will be downstream of this project but stakeholders will be kept informed through agricultural shows and the Warwick Crop Centre open days, and MGs public engagement role as Elizabeth Creak Chair in Food Security.

Plant breeders/genetic modification
Identification of susceptibility targets offers potential for genetic editing approaches to rewire pathogen virulence strategies to prevent suppression of photosynthesis and ROS. Diseases where there no natural resistance to emerging pathogens, for example Xanthomonas Banana Wilt in Africa will particularly benefit.

Environment, public and policy
Photosynthesis is part of the national curriculum, our system lends itself to exciting real time imaging of whole plants and subcellular compartments -generating educational resources that capture the public's imagination. We anticipate that this will also be of interest to the public throughout and have implemented measures in our Pathways to Impact to exploit this.

Finally, the PDRA and Technician will both receive full and relevant training across a range of disciplines, thus increasing the skills base of UK science. Importantly, the training in plant pathology and imaging/image analysis are two skills areas identified by BBSRC/MRC as vulnerable or deficient areas.