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Imaging infection dynamics of Xanthomonas on brassica; from whole plant to tissue specificity and host subcellular changes.

Primary Supervisor: Professor Murray Grant, SLS

Secondary supervisor: Dr Vardis Ntoukakis

PhD project title: Imaging infection dynamics of Xanthomonas on brassica; from whole plant to tissue specificity and host subcellular changes.

University of Registration: University of Warwick

Project outline:

The Xanthomonas genus includes diverse phytopathogens that collectively cause disease in more than 350 plant species. Xanthomonas campestris pv. campestris (Xcc) for example, causes black rot on crucifers is now considered the most important disease of brassicas worldwide. This seed-borne pathogen has been spread around the world via the seed trade. Black rot of crucifers is a regular occurrence in the main brassica production areas of the UK, notably Lincolnshire and Cornwall, and can cause significant economic losses.

Objective: To understand dynamics of Xanthomonas infection, disease development and transmission. Xc is a bit tricky to study. It is a hydathode-infecting vascular pathogen meaning it enters through specialised cells in the leaf then invades the vasculature (plumbing) of the plant causing wilting (“clogging – think of a fat berg!).

So to study Xc dymamics at the whole pant level we want to “tag” a highly virulent Xc strain with the recently described ilux bacterial bioluminescence reporter which has a 7 fold increase in intensity compared to conventional lux reporters (Gregor et al. 2018 PNAS doi/10.1073/pnas.1715946115).

To complement whole plant imaging, we will also generate Xc strains with the newer fluorescent proteins mScarlet and mCardinal (Canty et al. 2018 PLoS One 13, e0208075) which show strong quantum yield on the far-red spectrum yet imaging is not confounded by interference from natural chlorophyll fluorescence.

Collectively these tools will enable high resolution imaging and assessment of infection dynamic and will be complemented by chlorophyll fluorescence imaging that we have pioneered for bacterial infection studies (de Torres Zabala et al. 2015 Nature Plants 1, 15074). So, while you may not be a fan of Brussel sprouts, the tools and technologies developed here are applicable to kale, rocket, watercress and, strawberries.

Importantly, we aim to address for the first time, the transmission of Xc to the seed by imaging. Infected seed is the route of transmission and a major issue in the Brassica seed industry. Thus there exists potential to develop a non-destructive diagnostic platform to support UK industry.

Environment: You will join a team recently funded by BBSRC to address the dearth of research and expertise on plant bacterial diseases – in Warwick’s case Xanthomonas. We benefit form state-of the-art controlled environment growth facilities at Gibbet Hill to excellent glasshouse and quarantine fields at Wellesbourne. The School of Life sciences has excellent imaging facilities and the group has access to multiple whole plant imaging platforms.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Plant Science. Sustainable Agriculture and Food: Plant and Crop Science.

      Techniques that will be undertaken during the project:

      • Microbiology: pathogen maintenance, transformation and genetic characterisation of a range of Xanthomonas strains.

      • Molecular biology: Developing new state-of-the-art reporter lines for whole plant (iLUX) and fluorescence imaging (mCardinal, mScarlet).

      • Image analysis: Whole plant; chlorophyll fluorescence & iLux (photon counting): tissue & subcellular (pathogen dynamics in vasculature, adjacent cells and organellular perturbations): Seed infection and transmission – spatial and temporal dynamics.

      • Plant pathology: Infection studies. Assessment of pathogen growth and load in infected tissues.

      Contact: Professor Murray Grant, University of Warwick