An RNA cascade and disease resistance in tomato

Growth, development and responses to the environment of all organisms - life - depends on regulated activity of genes. If this regulation does not occur the consequence is death or disease but, in some examples, if it is perturbed there is evolutionary advance. In crops and agriculture, for example, there are many examples of important traits due to mutation of regulatory pathways that have persisted because farmers selected the seed from these mutant plants. More recently plant breeders have carried out the selection. In this project we will illustrate a modern variation on this process in which we will engineer mutant plants with enhanced disease resistance.

In the historical situations the mutants arose randomly and they would have been selected because they performed better or gave higher quality produce than the non-mutant progenitor. In this project the difference from the historical plant breeding is that the mutations will be targeted by an emerging technology known as gene editing. These mutations will be introduced at genetic regulatory sites in the genome that normally reduce the activity of disease resistance pathways in the plant. The consequence of the mutation will, therefore, be enhanced disease resistance.

The experimental approach is based on preliminary experiments in which we disrupted a regulatory cascade affecting the innate immune system of tomato. The nodes in this cascade are the mRNAs encoding defense proteins and the connecting edges are small regulatory RNAs known as miRNAs. At present we do not know the full extent of this cascade or whether the different branches have overlapping or non-overlapping functions but we know that when the cascade system is blocked the plants exhibit enhanced resistance against late blight caused by a filamentous pathogen responsible for the potato famine in Ireland in the 1840s. This pathogen is often a problem for tomato growers. The plants were also partially resistant against a bacterium and, because these are such different pathogens, we infer that disrupting this miRNA cascade may confer resistance against a broad spectrum of other pathogens.

In the proposed work we aim to understand the miRNA cascade regulation of disease resistance in more detail. In getting this information, we will learn more about the ways that miRNAs act and so the information from this project will have more general relevance to understanding of genetic regulation.

The project will test the spectrum of pest and pathogen resistance that can be achieved through the disruption the miRNA cascade and we will have set up later translational work that will use gene edited plants with enhanced disease resistance as part of a new approach to sustainable agriculture. The experiments will all be carried out in tomato but the miRNA cascade regulation of disease resistance features in many plants and so our findings will be more generally relevant to a wide range of crops.

The mRNAs for defense proteins including those with nucleotide binding site/leucine rich repeats (NB-LRR) are targeted by an RNA silencing cascade involving miR482, 2118b and 6026 (collectively miR(N)). These miRNAs target the mRNAs and mediate either cleavage, a block in translation or they trigger secondary siRNAs with the potential to target additional members of the NB-LRR family in trans. Other defense mRNAs including DCL2 are also targeted in this cascade.

Target mimic RNAs (mimRNAs) of the miR(N) family cause the level of the defense protein mRNAs to increase, they block secondary siRNA production and they confer partial broad spectrum resistance to the oomycete Phytophthora infestans and the biotrophic bacterial pathogen Pseudomonas syringae pv tomato.

The aim of this proposed project is to use a gene-editing approach in which disease resistance will be enhanced by knocking out components of the miR(N) cascade. We will use the mimRNA and gene edited plants to characterize this miR(N) silencing cascade in more detail using ribosome profiling so that we can find out how much miRNAs regulate gene expression at the level of translation. We will also determine whether the level of the broad spectrum resistance can be enhanced by blocking multiple branches of the cascade simultaneously.

In parallel with this molecular analysis we will test other bacteria, fungi and viruses and investigate the potential for a growth or yield penalty associated with the enhanced resistance.

The outcome of this project will be advanced fundamental understanding of RNA silencing cascades and the mechanisms by which expression of the defense systems of plants are fine-tuned. We will also have advanced understanding of disease resistance mechanisms in tomato and novel germ plasm and know-how that can be used for protection of tomato and other crops against a broad spectrum of diseases.

The pathways to impact will describe how this project can be a vehicle for illustrating to general audiences some principles of genetic regulation and how gene editing can be used for crop improvement as a complement to conventional breeding.
The genetic regulation aspect of this project will be centred around miRNAs and the impact activities will illustrate the concept that gene expression is regulated, that RNA can be a regulator and that loss of this regulation has profound effects on the plants.
For the gene-editing based impact activities I will use the disease resistance effects in this project to illustrate how this new technology could facilitate crop improvement and sustainable agriculture. A feature of this project is that the quantitative resistance could be integrated with other forms of pest management including those used by organic practioners. It will illustrate the potential compatibility of organic and biotechnology approaches to sustainable agriculture.
The targets of our impact activities will be:
- the general public and educators so that they can develop informed opinions about genetic regulation gene editing .
- regulators who will need good understanding of the basic concepts associated with gene editing if any new regulatory framework is to have a rational and evidence base.
- investors and industry so that they can be helped to identify and support new gene edited traits as they emerge.
- the postdoctoral researcher so that they can develop their career with experience of driving impact in science

1. Public engagement in gene editing
The initial engagement will involve the internet and a display in the Cambridge University Botanic Garden. The Garden has a large audience (around 250000 pa) who will receive passive exposure to the display.
In the later stages of the project we will use publications from our work as an opportunity to broaden the public engagement.
Milestone I1 - 12 months - website presentation of basic concepts in genetic regulation gene editing.
Milestone I2 - 24 months - demonstration display in Cambridge University Botanic Garden to explain genetic regulation and gene editing.
Milestone I3 - 36 months - presentation to popular media of the concepts in gene editing.

2. Industry engagement in the importance of gene editing in crop plant breeding.
To ensure that industry, government, policy and regulatory bodies are engaged and familiar with the potential of gene editing I have requested funds for an industry workshop. I would develop my webpage with a section that is relevant to industry as part of the preparation for the workshop.
Milestone I4- 12 months - PI webpage as resource for public understanding of gene editing in plants.
Milestone I5- 36 months - workshop to convene key industry players in gene editing for agriculture.

3. Regulatory engagement in the development of rational, risk-based regulatory framework for gene editing.
As part of the engagement with regulatory bodies we will apply to ACRE for permission to "field-test" gene edited plants that have been generated in connection with other projects in the lab.
Milestone I6 - 24 months - decision from ACRE over field testing of gene edited tomato generated in this project.

3. Training of a postdoctoral researcher on a project that will introduce involvement in commercialization and regulatory aspects of plant biotechnology.
In this project, with the unusually prominent opportunities for engagement with the public, industry and regulators, there is a corresponding opportunity for training of the PDRA. The PI will involve this individual fully in the impact activity in the expectation that she or he will substitute for the PI on many occasions.
Milestone I7- 36 months - postdoctoral researcher with skills in molecular biology and plant pathology and familiarity with regulatory, commercialization and public engagement aspects of a research project.