Transgenerational immune priming in plants

Lead Research Organisation: Lancaster University
Department Name: Lancaster Environment Centre

Abstract

In response to stress, plants and animals can exhibit rapid changes in their biology, enabling them to maximise their fitness. Exposure to stress can also generate long-term immunological 'memory', which enables the individual to develop faster and stronger defence responses to future exposures. Over recent years, evidence has accumulated that exposure of an individual to stress can also influence future stress responses in its offspring. Such effects are referred to as 'transgenerational', and are assumed to have evolved to maximise survival of an individual's gene pool in future generations, which are likely to encounter similar stresses.

We recently made the important discovery that progeny from diseased plants are more resistant than genetically identical offspring from healthy plants. This increased resistance persisted over at least two stress-free generations, suggesting epigenetic inheritance. Resistant progeny did not show increased defence activity in the absence of pathogens, but instead exhibited an increased responsiveness of defence genes to infection. We therefore refer to this phenomenon as 'transgenerational immune priming' (TGIP), since progeny plants are 'primed' to respond more rapidly to infection. Importantly, we have begun to uncover the mechanisms underpinning this immunological plant memory, and discovered that TGIP is based on DNA methylation, a reversible DNA modification that can have a profound impact on gene activity without changes in DNA sequence.

Transgenerational immune responses have important implications for natural plant populations, and present an opportunity for exploitation in sustainable agriculture. The ability to improve resistance to pests and diseases through epigenetic manipulation provides a new mechanism by which reliance on chemicals can be reduced without having to change the genetic make-up of our elite crop varieties.

In this project, we will investigate the biological significance of TGIP and the molecular mechanisms behind it. Firstly, we will examine whether TGIP can be established when disease stress is experienced only by the maternal plant, or whether paternal tissues can also transmit priming information. This will provide clues as to how TGIP evolved and the mechanism by which it is established. As well as measuring the benefits of TGIP in terms of increased stress resistance in offspring, we will also identify where there are trade-offs: when priming against one form of stress has a negative impact on tolerance to another. Thus, we will employ different forms of stress (disease, herbivory, salinity) to induce TGIP in different parental lines, and then measure the degree of resistance to each of these different stresses in their progeny. These costs and benefits will be measured initially in terms of stress resistance phenotypes, but to add depth of understanding, we will follow up with assays to measure expression of a defined set of stress-responsive marker genes and use mass spectrometry to generate metabolite profiles. Finally, we will grow progeny lines from primed populations in competition to test for overall reproductive fitness benefits for TGIP.

In parallel with these phenomenological studies, we will exploit next-generation sequencing technologies to identify the mechanisms involved in the establishment and maintenance of TGIP. Since long-term epigenetic changes in gene responsiveness are associated with DNA methylation, we will profile genome-wide DNA methylation patterns in control and primed plants. Thus, we will identify differentially-methylated regions linked to enhanced stress resistance. We have evidence that certain forms of small RNA molecule (siRNAs) known to regulate DNA methylation are important in establishing TGIP. Therefore, we will also profile siRNAs in control and primed plants to identify correlations between differentially expressed siRNAs and DNA methylation patterns.

Technical Summary

Transgenerational immune priming (TGIP) is a phenomenon whereby the offspring of parents who have been exposed to stress are themselves more resistant to that same stress than offspring from healthy parents. This project is designed to uncover the biological significance of TGIP and the mechanisms by which it operates. We will measure the costs and benefits of TGIP by assessing stress resistance phenotypes and associated effects on molecular and biochemical responses as a result of stress in previous generations. These experiments will include an examination of trade-offs of TGIP by measuring the impacts of priming with one stress type on responses to the same and different kinds of stress in subsequent generations. In addition to phenotypic outcomes, we will also monitor metabolite profiles and the expression of stress-specific marker genes.

To investigate mechanisms of TGIP, we aim to identify epigenetically-regulated loci involved in the establishment and maintenance of priming. We will employ next generation sequencing approaches to test the model that primed defence is inherited via changes in DNA methylation. We will use whole genome bisulphite sequencing to map methylated cytosine bases in control and primed plants to identify differentially-methylated loci associated with maintenance of TGIP. In parallel, we will examine establishment of TGIP by sequencing ARGONAUTE-associated RNAs, a class of small interfering RNA (siRNA) molecules implicated in triggering differential methylation. We will then be able to identify loci which respond to stress by the production of siRNAs and which are subsequently subject to heritable changes in methylation status.

Finally, to measure evolutionary benefits of TGIP, we will test how priming responses to different stress types interact when plants are grown in competition, and determine whether TGIP increases seed production of primed offspring relative to offspring of naïve plants when grown under stress.

Planned Impact

Safeguarding food security is one of the most urgent challenges this century, and one which is aggravated by changes in climate that render land less suitable for agriculture. Consequently, there is a pressing need to improve the sustainability of food production, including intensification of crop protection.

In addition to its broad range of effectiveness, induced resistance by means of defence priming is durable. Once induced, priming can be maintained throughout the life of a plant and inherited epigenetically to following generations. Consequently, primed crops should require fewer pesticide applications in order to reach similar levels of protection. By reducing pesticide inputs, integration of transgenerational immune priming (TGIP) into existing crop protection schemes could provide multiple benefits to the environment. First, lower pesticide usage would reduce impacts on non-pest species, enhancing biodiversity and ecosystem service provision. Second, reduced chemical inputs equate to lower energy consumption, reducing the carbon footprint of agriculture. This would provide a perfect example of the kind of sustainable intensification called for by the Royal Society and the UK Foresight report.

We believe that with an appropriate mechanistic understanding, as offered by this project, TGIP represents an attractive concept for exploitation by agribusiness. For instance, the identification of heritable epialleles associated with TGIP could lead to the development of molecular markers, that could be used to assist in the optimisation of resistance-inducing seed treatments of crops. Such treatments would not require alteration of the genetic make-up of elite crop varieties, and would offer an attractive alternative to the time-consuming introgression of new genes by traditional breeding. The exploitation of epiallele variation for the selection of agronomically-important traits has already been demonstrated. High-yielding lines of Brassica napus were selected from an isogenic population on the basis of high energy use efficiency as a consequence of changes in DNA methylation (i.e. epialleles) that were stably-inherited for at least 8 generations (PNAS 106: 20109-20114). Although our experiments are based on the model plant Arabidopsis to facilitate rapid scientific progress, it is likely that we would be able to apply the same knowledge to crop plants relatively quickly. Our research will therefore have a stimulatory impact on agricultural companies that aim to improve the efficiency of IPM in sustainable agriculture.

Our project will also generate valuable knowledge for aid programmes in the developing world, where poor infrastructure and limited financial capacity demand a small-scale and self-sustaining mode of agriculture. Under these circumstances, crop seed stocks are commonly maintained by farmers themselves. An efficient induction of TGIP would allow poor farmers to collect their own seed stocks of more resistant crop varieties, thereby making their food production less vulnerable to outbreaks of pests and disease.

To ensure maximum impact, sequencing data will be deposited in public databases, and manuscripts describing our results submitted to journals that allow for open-access publication. At present, there are no commercial partners or proprietary issues related to this project, which allows for quick public data sharing. However, to allow exploitation, we will of course seek to protect commercially valuable IP where it arises.

A wider impact with other stakeholders and the general public will be reached by presentations at events targeted at growers and agribusinesses, through educational outreach activities, public press releases of scientific findings, and through presentations during public events, such as open days. Thus, the work outlined in this project proposal will bring about impact at different levels, ranging from specialist scientific communities to commercial and public communities.

Publications

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Description Transgenerational immune priming, the subject covered by this grant, refers to the enhanced resistance to biotic stress observed in the offspring of parent plants exposed to herbivory or disease. In this project, we are investigating the role of 'epigenetic' chromosome modifications, such as DNA methylation, that may be responsible for the inheritance of altered defence states. We have learned that defence priming is transmitted maternally (i.e. by stressed mother, but not by stressed fathers) and is associated with altered genome-wide DNA methylation patterns.
Mutants affected in genome-wide DNA methylation patterns (the hypo-methylated nrpe1 mutant and the hyper-methylated ros1 mutant) failed to develop transgenerational acquired resistance against the biotrophic oomycete pathogen, Hyaloperonospora arabidopsidis. nrpe1 mutants show elevated basal disease resistance, whereas ros1 mutants are more susceptible to infection than wild type plants. Our results suggest that in wild type plants, hypo-methylation of DNA is required for transgenerational immune priming against biotrophic pathogens. We are currently analysing whole genome bisulphite sequencing data in order to map patterns of DNA methylation during the establishment, transmission and expression of primed immunity in the parental and offspring generations of plants infected with the bacterial pathogen, Pseudomonas syringae.
We have also discovered that plants that are infected with disease, or attacked by herbivorous insects, produce seed with altered levels of dormancy. Seed dormancy is imposed by seasonal cues such as day length and temperature, to synchronise life cycles with seasons. We have found that stress caused by disease or herbivory interferes with this process. Seeds from herbivore-attacked parents possess altered levels of various defence and dormancy-regulating hormones, and germinate precociously. Such seeds are insensitive to the dormancy-inducing hormone abscisic acid, which is a consequence of elevated levels of the herbivore defence hormone, jasmonic acid. In contrast, seeds from diseased parents show enhanced dormancy, which is linked to salicylic acid signalling pathways that are responsible for disease resistance.
Exploitation Route The demonstration that transgenerational immune priming is a consistent, reproducible phenomenon opens up possibilities for its use in crop protection. We have initiated a new project, building on the work done funded by this grant, to work with a major UK tomato grower to test whether transgenerational priming might be exploited for crop protection. If successful, this could reduce the need for chemical control of pests and diseases. Our discovery of the maternal effects of biotic stress on seed dormancy is of relevance to the seed industry. We have discussed possibilities for exploitation with UK seed companies, for whom seed control of seed dormancy is a significant issue for some crops.
Sectors Agriculture, Food and Drink

 
Description Faculty Studetnship linked to Waitrose CTP
Amount £70,000 (GBP)
Organisation Lancaster University 
Sector Academic/University
Country United Kingdom
Start 10/2018 
End 03/2022
 
Description Graham lab 
Organisation University of York
Country United Kingdom 
Sector Academic/University 
PI Contribution Series of experiments based around the transgenerational immune priming grant that resulted in acceptance of a paper in a high-impact journal.
Collaborator Contribution Contributed scientific materials, experimental work and analytical capability (plant hormone profiling by mass spectrometry)
Impact Research article: Singh P., Dave A., Vaistij F.E., Worrall D., Holroyd G.H., Wells J.G., Kaminski F., Graham I.A., Roberts M.R. (2017) Jasmonic acid-dependent regulation of seed dormancy following maternal herbivory in Arabidopsis. New Phytologist (in press as of 21/02/17)
Start Year 2015
 
Description Raul 
Organisation Centre for Research and Advanced Studies of the National Polytechnic Institute (CINVESTAV)
Country Mexico 
Sector Academic/University 
PI Contribution PI acted as host for sabbatical visit, and provided research direction/supervision for the visitor, Dr. Raúl Alvarez Venegas.
Collaborator Contribution Dr. Alvarez Venegas worked on the epigenetic basis of defence priming whilst in Lancaster, contributing to the overall research programme of which this grant is a major part.
Impact None to date.
Start Year 2016
 
Description SUMO 
Organisation Durham University
Department School of Biological and Biomedical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Conducted experiments on materials provided by collaborator.
Collaborator Contribution A major funded research project for this group, to which we have made a minor contribution.
Impact None yet - scientific article manuscript in preparation.
Start Year 2014
 
Description Septoria 
Organisation Agrii
Country United Kingdom 
Sector Private 
PI Contribution Contribution to workshop on septoria disease in wheat, and subsequent involvement in a funding bid to Innovate UK
Collaborator Contribution Contribution to workshop on septoria disease in wheat, and subsequent involvement in a funding bid to Innovate UK
Impact Bid for ~£500,000 project submitted to Innovate UK Agri-Tech Catalyst programme.
Start Year 2015
 
Description Septoria 
Organisation Croda International
Country United Kingdom 
Sector Private 
PI Contribution Contribution to workshop on septoria disease in wheat, and subsequent involvement in a funding bid to Innovate UK
Collaborator Contribution Contribution to workshop on septoria disease in wheat, and subsequent involvement in a funding bid to Innovate UK
Impact Bid for ~£500,000 project submitted to Innovate UK Agri-Tech Catalyst programme.
Start Year 2015
 
Description Septoria 
Organisation Durham University
Department School of Biological and Biomedical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Contribution to workshop on septoria disease in wheat, and subsequent involvement in a funding bid to Innovate UK
Collaborator Contribution Contribution to workshop on septoria disease in wheat, and subsequent involvement in a funding bid to Innovate UK
Impact Bid for ~£500,000 project submitted to Innovate UK Agri-Tech Catalyst programme.
Start Year 2015
 
Description Septoria 
Organisation Velcourt Ltd
Country United Kingdom 
Sector Private 
PI Contribution Contribution to workshop on septoria disease in wheat, and subsequent involvement in a funding bid to Innovate UK
Collaborator Contribution Contribution to workshop on septoria disease in wheat, and subsequent involvement in a funding bid to Innovate UK
Impact Bid for ~£500,000 project submitted to Innovate UK Agri-Tech Catalyst programme.
Start Year 2015
 
Description Alpha Galileo press release 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release to publicize new research grant award.
Story was taken up by several online news carriers.
Year(s) Of Engagement Activity 2014
URL http://www.alphagalileo.org/ViewItem.aspx?ItemId=145991&CultureCode=en
 
Description EPQ 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Via his participation in the Leadership Programme for Early Career Researchers, the PDRA Prashant Singh, has been a member of an A-level Extended Project Qualification student monitoring network, developed as partnership between Lancaster University and the South Lakes Teaching School Alliance. This allows researchers to use their experience to assist A-level school students in generating independent literature-based research projects.
Year(s) Of Engagement Activity 2015,2016
 
Description GPC blog 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited blog article for Global Plant Council web site.
Year(s) Of Engagement Activity 2016
URL http://blog.globalplantcouncil.org/2016/12/
 
Description LEC Grad school 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Summary of project presented at launch event for the Lancaster Environment Centre Graduate School. Event attended by staff and students from Lancaster and Rothamsted Research, and representatives from RCUK and businesses.
Year(s) Of Engagement Activity 2016
 
Description Lancs farmers 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact The Lancaster Farmer Network helped their AGM at the Lancaster Environment Centre, during which, LEC staff gave presentations about science that relates to farming practice, and the farmers were given tours of the research facilities. Attended by around 20 members of the network.
Year(s) Of Engagement Activity 2018
 
Description UGRG 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact The PI and PDRA have acted as tutors in a new initiative at Lancaster called the Undergraduate Research Group, which aims to introduce high-achieving biology students to a research environment from the beginning of their degrees. Selected students are introduced to laboratory techniques, experimental design, and participate in a small plant science research project. We have run student projects based on transgenerational immune priming in both 2016 and 2017.
Some of these students have now changed their career plans to include plant sciences as possible destinations.
Year(s) Of Engagement Activity 2016,2017
 
Description UGRG 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact We ran a research club for undergraduate students to introduce then to real scientific research skills. Several students undertook projects in my lab directly related to the funded BBSRC project. Two students are now looking for plant science PhDs as a result of their new-found interest in plants.
Year(s) Of Engagement Activity 2016,2017