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Out damn spot – multicellular behaviour of bacterial colonies during plant infection

Principal Supervisor: Professor Murray Grant - School of Life Sciences

Co-supervisors: Professor Lorenzo Frigerio - School of Life Sciences

PhD project title: Out damn spot – multicellular behaviour of bacterial colonies during plant infection

University of Registration: Warwick

Project outline:

Bacterial diseases of plants are increasing alarmingly across Europe, USA and Africa, threating food security. Unlike fungal pathogens there is currently no effective control measures. The UK landscape is being changed by bacterial infections of oak and horse chestnut trees. Understanding bacteria disease dynamics and colony establishment will provide answers to many fundamental questions about how bacterial infections establish as well as offering insight into new intervention strategies.

This project address a novel, previously unexplored aspect of plant-microbe interactions, how a colony evolves into a distinct community within the apoplast; in other words the sociobiology of bacteria. It will suit a candidate with a strong interest in microbiology, confocal imaging and image analysis. It will provide the opportunity to push technological boundaries to understand the inter-relationship between bacteria with different community roles using transcriptomics.

The hemi-biotrophic pathogen, Pseudomonas syringae infects the model plant, Arabidopsis thaliana by delivering 28 effector proteins into the plant cell via a type III secretion system (T3SS). These Type 3 effectors collectively suppress plant defences and reconfigure metabolism for pathogen nutrition. Bacteria subsequently multiply and establish discrete colonies within the apoplast of the infected leaf (Fig. 1). These colonies initially rapidly multiple, but remain spatially restricted, presumably through inter-colony communication that monitors and responds to nutrient availability. Remarkably, our understanding of how these bacterial colonies form in the apoplast is virtually non-existent.

1

This project combines scanning and confocal microscopy of a novel bacterial line carrying multiple reporter genes to quantify and describe the following; (i) how colonies form, (ii) whether colony composition is static or internally dynamic, (iii) how bacterial populations within colonies co-operate and distribute workload, and (iv) how communities evolve to be spatially distinct. This will be achieved using virulent P. syringae strains with multiple reporter constructs (see Fig. 2) recording effector delivery (virulence; pink cells), quorum sensing (purple-blue cells) or public goods catabolism (both carbohydrate and nitrogen; green cells). These reporters will serve to both map population dynamics in the colonies and enable reconstruction of metabolic virulence strategies, which may help identify agrochemical targets. Subsequently, studies will be extended to capture and model a range of heterogeneous colony dynamics and composition using e.g. mixed infections (Pseudomonas and Xanthomonas; Pseudomonas and fungi) or determine the spatial distribution of non-pathogenic P. syringae “cheaters” in colony composition. These studies will provide insight into how colonies develop, and how the populations within the colonies co-operate to maintain infection and metabolise public goods.

BBSRC Strategic Research Priority: Food security

Techniques that will be undertaken during the project:

The candidate will receive a very broad grounding in a range of techniques while exploring this novel area of research. Skills the will be developed include core skills in scanning and confocal microscopy, plant pathology, basic molecular cloning, microbiology, plant husbandry image acquisition, cell sorting, transcriptomics, colony data analysis and modelling (colony growth and spatial distribution within colonies). While a reporter strain will be provided, it is expected the candidate will modify/refine this as the project develops thus developing skills in gene-knockouts in bacteria.. A range of plant mutants with altered responses to Pseudomonas infection and a wealth of microarray data on the host response to the infection process itself underpin the project, thus providing training in reverse genetics and big data analysis.

Contact: Professor Murray Grant, University of Warwick