Project Outline

The Xanthomonas genus includes diverse phytopathogens that collectively cause disease in more than 350 plant species. Xanthomonas campestris pv. campestris (Xcc), causes black rot on crucifers and 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. Xcc is not easy to study as 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!).

Objectives

(i) To understand dynamics of Xanthomonas infection, disease development and transmission (tools already available) and (ii) to engineer resistance Xcc using a novel approach by expressing bacteriocins specifically in specialised hydrathode cells (you develop this strategy).

To study Xcc dymamics we have developed a dual reporter vector that allows us to “tag” a highly virulent Xcs strain with the ilux bacterial bioluminescence reporter (has 7 higher emission intensity compared to conventional lux reporters; Gregor et al. 2018 PNAS doi/10.1073/pnas.1715946115) and the fluorescent protein mScarlet (Canty et al. 2018 PLoS One 13, e0208075) which has a strong quantum yield on the far-red spectrum yet imaging is not confounded by (i) interference from natural chlorophyll fluorescence and (ii) absorbance from the polysaccharide coat of Xcc which precludes the use of traditional GFP reporters.

The project aims to get spatial, temporal imaging data from both Brassica and the model plant Arabidopsis thaliana using different races of Xcc that are economically important; we have recently sequenced over 400 Xcc isolated and pathotested 5 key races on Brassica lines to identify resistance genes. A. thaliana will enable us to deploy key immune compromised mutants.  Collectively these tools will allow 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). Importantly, this objective aims to address the transmission of Xcc to the seed by imaging. Infected seed is the major issue in the Brassica seed industry, thus there exists potential to develop a non-destructive diagnostic platform to support UK industry.

The second part of the project is to develop in A, thaliana a proof of concept, by engineering the specialised hydrathode cells to specifically express bacteriocins – highly active antibacterial proteins that can specifically target Xcc. We have already identified a number of potential bacteriocins through our large scale Xcc sequencing.

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.

You will be exposed to a range of technique including whole plant real time imaging (chlorophyll fluorescence and ilux imaging), confocal imaging, microbiology, genetics, pant molecular biology and synthetic biology (GoldenGate Cloning) was well as quantitative image analysis.

See our review Greer et al. 2023 Front Microbiol doi: 10.3389/fmicb.2023.1209258 for an overview of the importance of Brassica’s in Food Security and the disease threats.

References

Contreras et al. 2023, EMBO Reports DOI 10.15252/embr.202357495

Chai et al. 2023 Current Opinion in Plant Biology DOI 10.1016/j.pbi.2022.102334

Techniques

• Microbiology & plant pathology
• Plant Molecular Biology
• Imaging, whole plant and confocal
• Synthetic biology – re-enginerring the hydrathode