Effector gene persistence in bacterial plant pathogens

Lead Research Organisation: University of Oxford
Department Name: Plant Sciences

Abstract

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.

Technical Summary

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

Planned Impact

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.

Publications

10 25 50
 
Description Recommendations for Augmenting Contact Tracing in the UK
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
URL http://www.newton.ac.uk/files/preprints/ni20001.pdf
 
Description Plant Health Undergraduate Studentship (awarded for 2020 - rescheduled to summer 2021)
Amount £4,000 (GBP)
Organisation Royal Society of Biology (RSB) 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2021 
End 09/2021
 
Description Royal Society International Exchanges Cost Share Award - Argentina
Amount £9,000 (GBP)
Funding ID IEC\R2\170201 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2018 
End 12/2019
 
Title Library of luminescence and dual fluorescence-luminescence biosensors 
Description Bacterial bioluminescence is widely used to study the spatiotemporal dynamics of bacterial populations and gene expression in vivo at a population level but cannot easily be used to study bacterial activity at the level of individual cells. We have developed a new library of mini-Tn7-lux and lux::eyfp reporter constructs that provide a wide range of lux expression levels, and which combine the advantages of both bacterial bioluminescence and fluorescent proteins to bridge the gap between macro- and micro-scale imaging techniques. 
Type Of Material Cell line 
Year Produced 2020 
Provided To Others? Yes  
Impact We have demonstrated that a dual bioluminescence-fluorescence approach using the lux operon and eYFP can be used to monitor bacterial movement in plants both macro- and microscopically and specifically shown that Pseudomonas syringae pv phaseolicola can colonize the leaf vascular system and systemically infect leaves of common bean (Phaseolus vulgaris). We have also shown that bacterial bioluminescence can be used to study the impact of plant immune responses on bacterial multiplication, viability and spread within plant tissues. These constructs can be used to study the spatiotemporal dynamics of bacterial colonization and to link population dynamics and cellular interactions in a wide range of biological contexts. 
 
Description Bean research network 
Organisation Pan African Bean Research Alliance
Country Uganda 
Sector Charity/Non Profit 
PI Contribution Organised workshop in February 2018 to develop collaborative research projects, and joint projects with the Pan-African Bean Research Alliance and South African Bean Research Network.
Collaborator Contribution Partners (Phillip Miklas (USDA-ARS), Timothy Porch (USDA-ARS), Rowland Chirwa (Coordinator Southern African Bean Research Network), Robin Buruchara (Coordinator Pan-African Bean Research Alliance), Deidre Fourie (ARS South Africa, PABRA/SABRN), Dawn Arnold (University of the West of England), Robert Jackson (University of Reading)) participated in a workshop in February 2018 to develop collaborative research projects, including joint projects with the Pan-African Bean Research Alliance and Southern African Bean Research Network.
Impact Research grant written, but no formal outcomes yet. This collaboration brings together researchers with expertise in plant pathology and plant breeding.
Start Year 2018
 
Description Bean research network 
Organisation U.S. Department of Agriculture USDA
Country United States 
Sector Public 
PI Contribution Organised workshop in February 2018 to develop collaborative research projects, and joint projects with the Pan-African Bean Research Alliance and South African Bean Research Network.
Collaborator Contribution Partners (Phillip Miklas (USDA-ARS), Timothy Porch (USDA-ARS), Rowland Chirwa (Coordinator Southern African Bean Research Network), Robin Buruchara (Coordinator Pan-African Bean Research Alliance), Deidre Fourie (ARS South Africa, PABRA/SABRN), Dawn Arnold (University of the West of England), Robert Jackson (University of Reading)) participated in a workshop in February 2018 to develop collaborative research projects, including joint projects with the Pan-African Bean Research Alliance and Southern African Bean Research Network.
Impact Research grant written, but no formal outcomes yet. This collaboration brings together researchers with expertise in plant pathology and plant breeding.
Start Year 2018
 
Description Royal Society Cost Share Award - Argentina 
Organisation National University of San Martin
Country Argentina 
Sector Academic/University 
PI Contribution We have initiated a new collaboration to study polyamine metabolism in endophytic bacterial plant pathogens and its significance in stress resistance and pathogenicity, funded by a Royal Society International Exchanges Cost Share Award.
Collaborator Contribution Participation in future research exchanges and collaborative work.
Impact This award has supported a research visit by a member of my group to Argentina to conduct collaborative research, and a visit by Dr. Andres Garriz to Oxford in 2018. There are no publications or further awards arising as yet.
Start Year 2018
 
Description Understanding how PTI limits bacterial growth in the plant apoplast 
Organisation University of Georgia
Department Department of Microbiology
Country United States 
Sector Academic/University 
PI Contribution Building on research and techniques developed in this award I have started a new collaboration with Prof. Brian Kvitko, University of Georgia, studying how changes in apoplast during induction of PAMP-triggered immunity (PTI) limit pathogen growth.
Collaborator Contribution We have shared ideas and data pre-publication, and have planned a research visit by a member of Prof. Kvitko's group to Oxford in 2019.
Impact The collaboration is still at an early stage, and thus there are no outputs or outcomes as yet.
Start Year 2017
 
Description Animation describing the evolution of bacterial effectors in response to host resistance 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Co-developed animation 'Evolution of plant pathogenic bacteria to defeat host resistance' - with Dawn Arnold, Helen Neale, Robert Jackson and Sci Ani (www.sciani.com) https://youtu.be/nCZZfXBU2Wg (2020). The video has been viewed over 8000 times to date, and is being used to enhance undergraduate teaching and interactions with schools.
Year(s) Of Engagement Activity 2020
URL https://www.youtube.com/watch?v=nCZZfXBU2Wg
 
Description UNIQ summer school 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Schools
Results and Impact The talk stimulated discussion and interests among the students, who had not previously been introduced to the science of plant disease.

The event resulted in an increase in students applying from the programme to study bioscience at University level, and specifically at the University of Oxford, with 75% of students applying for entry in 2014.
Year(s) Of Engagement Activity 2013,2014,2015,2016,2017,2018
URL http://www.uniq.ox.ac.uk/courses/mpls/biology
 
Description Webinar: Visualising how Plants and Microbes Function in British Sign Language 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact We organised a webinar with researchers from the Scottish Sensory Centre describing how scientific signs can be developed for use in teaching and communication, and specifically illustrated it in the context of plant diseases by creating a signed video with supplementary graphics. The webinar was well-attended, and the webinar and videos will be released on YouTube in the near future to reach a wider audience. We have been awarded funding from the British Society for Plant Pathology to take the project further in 2021 and develop a wide range of plant pathology-related signs and associated resources.
Year(s) Of Engagement Activity 2020
URL https://www.bspp.org.uk/conferences/bsl-webinar-visualising-how-plants-and-microbes-function-in-sign...