Effector gene persistence in bacterial plant pathogens

The co-evolution of plant pathogens and their hosts is a complex and dynamic process. Pathogens can rapidly evolve to overcome host resistance to become virulent pathogens. 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.

In this project we aim to study the ability of plant pathogenic bacteria to overcome plant disease resistance. One of the ways that bacterial plant pathogens cause disease is to inject proteins into plant cells that inactivate plant defence mechanisms and allow them to grow inside plant tissues. These proteins are known as effector proteins. One way in which plants can protect themselves against infection is to recognise the effector proteins as they are being injected into the plant cells. If the protein is recognised the plants cells deliberately die, releasing anti-microbial chemicals, thus cutting off the source of nutrients for the bacteria and making the plant resistant to attack. However bacteria can evolve to overcome host plant resistance by losing or changing their effector genes so that the proteins they produce are not recognised by the plant.

We have worked with a model bacteria-plant system that has allowed us to study in more detail how the bacteria can evolve to overcome host resistance. This system uses a bacterium called Pseudomonas syringae pv. phaseolicola (Pph), which causes an important disease of bean plants known as halo blight, and has allowed us to study both microbial evolution and the factors that increase or decrease the durability of plant disease resistance.

In the case of the Pph-bean system we have so far concentrated on the fate of one particular effector gene called avrPphB. This effector gene is interesting because it is carried on a mobile piece of DNA known as a genomic island, which can be acquired and lost by the bacteria. When the bacteria carrying this island infect a plant that is resistant, because the plant recognises avrPphB, the bacteria loses the island and can therefore go onto infect the plant without being recognised. This is an excellent example of the evolution of a pathogen to overcome host resistance. However, surprisingly, we have observed the complete loss of this effector over many experiments.

Recently, using a combination of mathematical modelling and laboratory experiments, we have shown that over the course of many weeks in the resistant plant, the bacterial population will still maintain a low level of the effector gene, below the level that can be recognised by the plant, and if conditions change so the effector gene is no longer recognized, its frequency can increase.

In this proposal we now aim to look in more detail at why this 'effector persistence' occurs. We will specifically study whether island and effector persistence confer any additional benefits to the bacteria. We will develop our mathematical model to provide additional insight into the basis of pathogen evolution and effector retention, and investigate whether this phenomenon is widespread. This research will help to elucidate the fundamental mechanisms underpinning the evolution of bacterial pathogenicity and the breakdown of disease resistance in crop plants, providing knowledge that, in the future, may be used to improve the disease management strategies used against disease-causing microorganisms.

This project will focus on understanding the mechanisms of effector gene persistence in the plant pathogenic bacterium Pseudomonas syringae pv. phaseolicola when it is exposed to a resistant bean host plant. We hypothesise that there may be an advantage to the bacteria maintaining the effector gene even in the resistant host and this effector gene persistence phenomenon is a wider spread mechanism for maintaining genetic diversity within bacterial populations. We thus aim to understand the mechanistic basis of effector persistence, the role it plays in plant pathogen interactions and if the phenomenon is wider than the model system we have used so far.

This will be achieved through the following objectives:
1. Identify the genetic factors affecting PPHGI-1 persistence in resistant hosts
2. Identify the benefit to Pph of PPHGI-1 persistance in resistant hosts
3. Evaluate how pathogen transmission mechanisms affect effector persistence
4. To investigate the persistence of other P. syringae effectors
5. Using mathematical modelling in combination with experimental evolution to understand the dynamics of pathogen evolution to polygenic resistance

The main methods to be used are:
1. Microbiological methods for culturing bacteria, analysing growth curves, examining competition
2. Plant inoculations to study the evolution of virulence: infiltrations, sprays, seed soaks, examining symptoms and removing bacteria from inoculated sites
3. Molecular biology techniques to clone genes, delete genes, carry out site-directed mutagenesis, examine island excision and integration (e.g. by Q-PCR)
4. Plant physiology and chemical analysis using Raman spectroscopy, GC-MS, AAS to examine apoplastic changes such as ROS production, callose deposition, ion and nutrient composition
5. Mathematical modelling to predict the outcomes of different effectors in different genomic contexts

The long term aim of our research is to understand the interaction between plant pathogens and plants with the goal of being able to use this information to develop control strategies in the field or glasshouse. This project specifically aims to understand how plant pathogenic bacteria maintain reservoir of effector genes in their population that may have an advantage to the bacteria when conditions change. We will investigate in more detail the avrPphB-bean model system but we will also widen this to include other effectors. This proposal fits within the BBSRC strategic priority of Sustainably Enhancing Agricultural Production as it focuses on a serious problem for crop performance i.e. loss of crop yield or quality though plant disease, and therefore has relevance to Global Food Security. A number of groups, aside from academics, will also benefit from this work, although it should be stressed that further research may be required to realise the benefits to some of these users.
1. Agriculture and the private sector will benefit because this work will lead to a better understanding of the persistence of potential pathogenicity determinants in plant pathogenic populations. This is of relevance for plant breeders who are targeting effector genes to breed against, as in some cases these genes may be effectively 'hiding' in the bacteria and not actually being eliminated from the bacterial population. In the long term results from this study could enhance the ability of plant breeders to predict whether specific combinations of resistance genes are likely to confer durable resistance based on knowledge of the potential evolution of the effectors recognised by these resistance genes. This phenomenon may also be of importance for seed certification, as it could underpin the spread of pathogen genotypes that are present in seed, but not detected by infestation assays.
2. Government organisations and policy makers will benefit by having more detailed information on the drivers of pathogen evolution and greater understanding of how pathogens evolve to overcome host plant resistance. A key point will be communicating the observation that effectors can persist, therefore highlighting that pathogens are not necessarily eliminated from the host through the use of resistant cultivars. This will not only benefit the global agenda for food security, but can be disseminated through a variety of agencies to the international agriculture arena.
3. The public will ultimately benefit through improved disease management practices that reduce yield losses and therefore increase food supply, and the stability of agricultural economies resulting from it. The public will also benefit from our public engagement and outreach activities, which will present the data we generate and highlight the impact of plant disease on food security and the research that is on-going to protect our crops.
4. Undergraduate and postgraduate students will benefit from progressive developments in teaching curricula that will be underpinned by the research outputs from the investigators: three of the five investigators associated with the project teach aspects of bacterial pathogen evolution and regularly give seminars on this topic. Students will also be able to participate directly in this research area by undertaking undergraduate summer projects and final year projects as well as graduate research projects or internships linked to this area of research.
5. The staff who are involved in the project, both investigators and research associates, will benefit from the research through learning new research skills and techniques. The RAs will also benefit from the research in terms of developing generic career skills, for example through attendance of the BBSRC media training workshop; presentations to both the scientific community and the public; preparation of manuscripts and grant applications; student supervision and participation in public engagement events.