Transgenerational immune priming in plants

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.

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.

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.