About the Project
While we can generate copious amounts of transcriptomic and proteomic data, these all invariable lack any spatial and subcellular context. During plant-microbe interactions there are rapid dynamical changes in both the infected and uninfected host cells that we just don’t understand. However, researchers are taking a more holistic approach to understanding infection biology. A nice example is our paradigm changing discovery that the chloroplast is a key battlefield in determining the eventual outcome of plant-microbe interactions (de Torres et al. 2015). 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 – yet until recently has been largely ignored.
We now have evidence that the mitochondria, peroxisomes and endoplasmic reticulum are key players in establishing whether the outcome of an infection is disease or defence. This project seeks to understand the temporal spatial dynamics of plant-microbe interactions at the sub-cellular level, specifically now organelles communicate, and more specifically the mechanistic basis of why they communicate.
For example, we have shown that the chloroplast responds to recognition of conserved pathogen motifs (non-self) by generating a burst of reactive oxygen species (ROS) that acts as a defensive signal. Successful pathogens deliver proteins and small molecules known as effectors – to intervene in this process; bacterial, oomycete and fungal, achieve this by reconfiguring expression of nuclear encoded plant genes and some effectors even enter the chloroplast, abrogating the ROS burst by suppressing photosynthesis - arguably one of the most important reactions on this planet.
This multidisciplinary project will use plants with labelled organelle markers, novel “genetically encoded reporters” capable of registering changes in different physiological parameters (such as changes in ATP, reactive oxygen and pH), proteomics, cell biology and reverse genetic approaches to address the mechanistic basis of organelle mediated immunity, and how this is modified by pathogen virulence strategies. The initial focus will be the chloroplast, endoplasmic reticulum and nucleus behaviour during the infection for which all necessary tools are available.
These include novel Arabidopsis lines expressing reporter genes localised to different plant organelles which are specifically induced by delivery of pathogen receptors, thus providing a cell specific context to discriminate responses in infected cells and those immediately adjacent to the infected cell. The candidate will ultimately be expected to develop computational models and simulations of the different infection outcomes.
Techniques that will be undertaken during the project:
- Bioimaging (primarily confocal) temporal and spatial infection dynamics (organellular) during plant disease and defense responses. This includes quantitative imaging and dynamic imaging.
- Microbiology; host infection, targeted modification of the effector complement of the pathogen Pseudomonas syringae(microbial genetics).
- Organelle isolation (e.g. chloroplast isolation) and fluorescence activated sorting of infected leaf organelles for downstream “omic” approaches.
- Plant molecular biology, including generating novel plant reporter lines.
- Yeast 2-hybrid screening of existing libraries
Wagner, et al. (2019) Multiparametric real-time sensing of cytosolic physiology links hypoxia responses to mitochondrial electron transport New Phytologist doi: 10.1111/np.16093
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