Understanding how plant antimicrobial "hot zones" can accelerate pathogen evolution

Gone are the days of food mountains whereby agricultural policies and crop production resulted in large stocks of food. Instead, the world is facing a major challenge to produce enough food to feed a growing population. Food security is a major global research priority and we know that we must double our food production within the next 20 years just to keep pace with population increases. To do this requires improvements in many aspects of food production. One of the major areas for improvement is preventing crop loss due to plant disease.

Most of the microorganisms that cause plant disease are engaged in a constant arms race with plants such that microorganisms are rapidly evolving to infect disease resistant plants while plants are evolving to resist pathogen attack. In an agricultural setting, plant breeders face the increasingly difficult challenge of developing new disease-resistant varieties to replace those rendered ineffective due to microbial evolution. To prolong the usefulness of disease resistant plant varieties, and to reduce the rate at which microorganisms overcome disease resistance, it is imperative that we fully understand how microorganisms evolve and the drivers of this evolution. We have developed a model system for understanding microbial evolution to overcome plant disease 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 represents an excellent system for studying both microbial evolution and the factors that increase or decrease the durability of plant disease resistance.

In the case of Pph and bean, the plant has developed mechanisms to recognise specific strains of Pph, and so resist invasion. In this dynamic system the bacterium has a number of ways of changing its genome, and therefore the proteins it expresses, in order to evade plant recognition. Alterations in the structure or production of bacterial proteins may prevent their detection by potential host plants and allow the bacteria to grow within the plant. We have shown that changes in the genome of Pph allow this bacterium to overcome plant disease resistance. Certain strains of Pph carry a gene that produces a protein the plant can detect as belonging to Pph, alerting it to trigger its defence systems and prevent Pph growth. The gene for this protein lies within a discrete region of the Pph genome known as a genomic island. To counter plant recognition, Pph removes the genomic island from its chromosome such that daughter cells no longer have the island. Interestingly, we observed that this dramatic change in Pph occurs most frequently in infection site "hot zones" in resistant varieties of bean. These hot zones generate highly antimicrobial conditions following bacterial invasion. Therefore the chemical changes that occur in resistant plants actually accelerate the evolution of a more virulent form of the pathogen.

More recently we have also shown that bacteria growing in plant tissue can become competent to take up foreign DNA in a process called transformation. This acquisition of new genes could allow Pph to gain an advantage in the fight between the plant and the bacterium. In this proposal we aim to study the chemical composition of the "hot zone" to understand which factors are responsible for inducing gene loss and gene gain in Pph. We also aim to identify the genes responsible for DNA uptake in the bacteria, and to understand how signals present in the "hot zone" cause increased DNA uptake in Pph. 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 mechanistic basis of bacterial pathogen evolution during the colonisation of resistant plant hosts, using the interaction of Pseudomonas syringae pv. phaseolicola (Pph) with bean as a model system. We hypothesise that identifiable factors from the plant apoplast trigger genomic island excision from the chromosome of Pph, and uptake of naked DNA through natural transformation competence, and that specific genes for competence are present in Pph. We thus aim to identify the signals that induce gene loss and gene gain in Pph, and to characterise the mechanism used by Pph to acquire DNA. This will be achieved through the following objectives:

Objective 1. Investigate the impact of host cultivar, pathogen genotype and environment on the excision and transformation competence-inducing activity of apoplast extracts.
Objective 2. Identify components present in apoplast extracts that induce excision and competence in Pph.
Objective 3. Identify genes required for competence in Pph.
Objective 4. Profile the transcriptional response of Pph to apoplast extracts and determine whether competence genes are expressed at a higher level from resistant plants.

We will first investigate the impact of host, pathogen and environment on the excision and competence-inducing activity of apoplastic extract. We will use this information to compare the composition of apoplast extracts with high or low levels of inducing activity and to identify components present in apoplastic extracts that induce excision and competence. Competence gene identification will be achieved using a variety of methods ranging from a targeted bioinformatics approach to a random gene knock-out method. Global RNA sequencing and targeted gene expression analyses will be used to study the expression of competence genes in response to environmental factors and to understand the physiological context of competence induction in Pph.

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 antimicrobial "hot zones" formed in response to pathogen attack trigger genetic changes within the pathogen and provide a foundation for selection of more virulent pathogens. We also aim to uncover the mechanisms by which pathogens can acquire DNA to evolve enhanced virulence. This proposal fits within the BBSRC strategic priority of Crop Science 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 plant factors that promote pathogen evolution and the breakdown of disease resistance. Ultimately, an understanding of the causes of pathogen evolution could lead to directed breeding for plants that do not trigger pathogen change as rapidly. For example, breeders may be able to breed plants that fail to produce a specific molecule that accelerates pathogen evolution. Alternatively, farmers and horticulturalists may be able to alter growth management practices to modify plant chemical composition to reduce the levels of inducing factors. A direct benefit of this project will be the development of quantitative bioassays for studying the impact of specific factors on pathogen evolution, which can be used by plant breeders and government research institutes, and which may also have broad applications in other fields of research such as drug discovery and clinical microbiology. Our long term goal of developing models of the loss and transfer of DNA in pathogen populations on plants will also have an impact by allowing researchers to make predictions regarding plant pathogen evolution and disease management, which can be experimentally tested in greenhouse and field trials.
2. Government organisations and policy makers will benefit by having more detailed information on the drivers of pathogen evolution and understanding how pathogens evolve. This will not only benefit the national 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 increased food supply, and improved economics resulting from it, thus directly addressing Food Security, which goes to the heart of BBSRC's priorities. The public will also benefit from our outreach programmes, which will present the data that 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: all the investigators associated with the project teach aspects of bacterial pathogen evolution. Students will also be able to participate directly in this research area by undertaking undergraduate and graduate research projects in our research groups.
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.