Hiding in wait – examining pathogen evolution via effector gene gain and loss

Project Outline

The co-evolution of bacterial plant pathogens and their hosts is a complex and dynamic process. Bacteria can rapidly evolve to overcome host resistance, leading to virulent pathogens with expanded host ranges. This is a major cause for concern because of the threat it poses to UK and global food security. It is therefore important that we understand the causes and consequences of pathogen evolution to deliver better strategies for plant protection. This project will investigate how virulence genes persist in a pathogen population even when they are selected against by the plant immune system. This project will build on our previous work studying the evolution of pathogenicity of the plant pathogenic bacterium Pseudomonas syringae pv. phaseolicola (Pph). We have shown that under the stress of encountering effector-triggered immunity in a resistant plant strain Pph 1302A excises a genomic island PPHGI-1, and with it an effector gene (avrPphB), which allows Pph to evade host recognition and cause disease. We will expand this previous work to investigate how Pph is transmitted within the plant and how this affects evolution of resistance and we will expand the study to investigate other P. syringae plant pathogens and how they overcome host resistance via genomic changes.

Objectives

1. Study how pathogen transmission mechanisms affect effector persistence

In a field setting Pph is commonly transmitted in aerosols or infects plants from seed. We will examine the impact of aerosol and seed mediated transmission on PPHGI-1 persistence. These experiments will show the impact of different transmission mechanisms on effector persistence and provide insight into whether PPHGI-1 confers a competitive advantage to epiphytic or seed-borne bacteria.

2. Investigate the persistence of other P. syringae effectors

We will use the well-defined Pph-bean and P. syringae pv. pisi-pea systems to investigate whether persistence is widely observed for effector genes in both host and non-host plants. These experiments will increase our understanding of the prevalence of effector persistence and provide insight into whether the mode of effector inheritance (chromosomal/plasmid/GI) and inactivation/loss affects persistence.

Methods

1. To quantify the effect of aerosol inoculation on PPHGI-1 persistence, spray and infiltration inoculations will be made into bean leaves at concentrations ranging from 8x104 to 5x108 cfu/ml, which are known to give a range of phenotypic symptoms. The bacteria will be re-isolated after a period of time and endophytic and epiphytic populations will be examined for PPHGI-1 loss. This will be done over successive passages through the plant.
2. To examine PPHGI-1 persistence following seed inoculation, bean seeds will be artificially inoculated by soaking in bacterial suspensions. The persistence of PPHGI-1 will be tested over 10-12 weeks by plating bacterial populations from seeds at regular intervals. Infested seeds will be germinated to monitor the persistence of PPHGI-1 in the fully expanded leaves of 3-4-week-old plants.
3. A number of effector genes from a range of Pph and Ppi strains have been identified for further investigation of their persistence during a resistance response from the host. The bacteria will be inoculated into various host and non-host plants, recovered weekly and screened for change in their pathogenicity over time.

References

Neale HC, Jackson RW, Preston GM, Arnold DL. (2018) Supercoiling of an excised genomic island represses effector gene expression to prevent activation of host resistance. Molecular Microbiology, 110(3):444-454

Neale HC, Laister R, Payne J, Preston G, Jackson RW, Arnold DL. (2016) A low frequency persistent reservoir of a genomic island in a pathogen population ensures island survival and improves pathogen fitness in a susceptible host. Environ Microbiol. 18(11):4144-4152.

Techniques

Microbiology (culturing, isolations, stock maintenance)

Plant pathology (plant inoculations and bacterial recovery from plants).

Molecular biology and genomics (DNA isolation, PCR, sequencing, bioinformatics)