Understanding the mechanism of chloroplast immunity.

One of the "big challenges" for our next generation is to ensure global food security. This can be achieved through a combination of increasing productivity and selecting for plants which respond robustly to changing environmental conditions. Increasing productivity is challenging without bespoke local breeding solutions and this is reflected in the ever decreasing average annual crop yields achieved through conventional breeding. Crop losses due to biotic stress contribute disproportionately to yield losses, often around one quarter but in extreme cases in excess of three quarters of a crop. Thus developing novel approaches to restricting pathogen infections of crops and consequently yields must be a primary objective if we are to realistically ensure we can feed the estimated 9 billion people by 2050.
We have recently shown that the chloroplast is a key battlefield in determining the eventual outcome of plant-microbe interactions. Aside from its ability to fix carbon, chloroplasts play a central role in integrating multiple environmental stimuli and sensing the metabolic status of the plant. As a principal source of reactive oxygen species, the site of a significant amount of primary carbon metabolism and synthesis of the majority of hormone metabolic precursors, the chloroplast represents a prime target for pathogen manipulation.
Our pioneering work has shown that the chloroplast responds to recognition of conserved pathogen motifs (non-self) by generating a burst of reactive oxygen species (ROS) that we believe act as a defensive signal. It is not surprising therefore that successful pathogens deliver proteins and small molecules known as effectors - to intervene in this process. Our data indicate that pathogens, both bacterial and fungal, achieve this by reconfiguring expression of nuclear encoded plant genes and some effectors actually even enter the chloroplast. These effectors stop the ROS burst by suppressing photosynthesis - arguably one of the most important reactions on this planet - but we don't know how. What we do know is that effectors increase the production of a hormone called abscisic acid (ABA), and stopping ABA production makes the plant more resistant. Conversely, adding ABA stops the chloroplast ROS burst, enabling pathogen growth.
Here our primary objective is to understand how recognition of non-self activates chloroplast immunity and how pathogen effector proteins have evolved to suppress this immunity. One major objective is to undertake detailed studies of the biophysical changes in the chloroplast during treatments that cause disease or induce defence. We will look at the changes in proteins within chloroplasts during these treatments and changes in the small molecules as well. Merging these data we will predict proteins that contribute to these processes. To access their role in defence we will change their abundance and looking at how those plants behave to pathogens. We will also work out how many effectors, and the functional nature of those effectors, enter the chloroplast.
A second major strand of work is to visualise the dynamics of ROS production in the chloroplast and the nucleus during the transition from healthy to diseased plants. We are also interested in how organelles within the cell behave during disease and defence promoting challenges. To visualise this we have labelled different organelles in the cell with fluorescent markers and we will use these to monitor their behaviours during the infection process.
As chloroplast immunity appears conserved, our longer term goal is to use the knowledge gained from these studies in novel re-engineering or intervention strategies that will provide plants with broad spectrum resistance against pathogens.

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.