Molecular dissection of paramutation in tomato

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


Many traits are inherited according to the well known Mendelian rules: a brown-eyed father and blue-eyed mother will have either 50% or 100% brown eyed children depending on whether the father inherited dominant brown eye genes from one or both of his parents. In turn, their brown-eyed children with a blue-eyed partner would produce 50% brown eyed offspring. Blue eyes in this situation is genetically recessive because the gene is non functional - the encoded protein lacks the ability to produce eye pigments.

Plant genetics, in most instances, is similarly straightforward with each gene having dominant and recessive versions. There are however are some exceptions that are the equivalent of a situation in which all first and subsequent progeny of a blue eyed parent have blue eyes even if the other parent has brown eyes. In this situation the underlying mechanism is referred to as paramutation. This proposal aims to understand how paramutation works so that, in future, we can assess its importance in the evolution of natural populations and the potential for its application in crops.

Much of what we know about paramutation involves plant genes that, like eye colour genes in people, encode pigment determinants. The silent genes in these systems are, however, unlike the blue-eye genes in that they are not genetically recessive. They are dominant because, in the first generation offspring (F1) the silent state of the gene from a non-pigmented parent is transferred to the equivalent gene from the pigmented parent. Both pigment genes in this offspring are silent. The situation is, however, more complicated than normal dominance and recessiveness because the previously active gene has been permanently changed. Its silent state is now inherited into subsequent generations and, like the original gene from the pigment minus parent, it can transfer its silent state in subsequent generations to an active version of the gene.

A possible explanation of paramutation is that the silent gene is somehow mutagenic and can change the sequence of the active. From earlier studies, however, in maize and tomato we know that the DNA sequence of the active and silent genes is identical. The emerging picture involves a chemical modification -methylation - of the DNA and of associated chromosomal proteins that affect gene expression. Chromatin structure and RNA has also been implicated. We refer to these factors as being epigenetic rather than genetic because there is no change to the DNA sequence.

The previous studies of paramutation do not, however, show how the silencing epigenetic mark is transferred to the active version of the gene: is there direct contact of the two genes or does the silent gene produce a diffusible factor - an RNA perhaps - that can bind to the active gene? Similarly we do not understand why some genes with the same chemical modification as those in paramutation are not paramutagenic. These other genes may be silenced and their silencing may be heritable but the silenced state is inherited in a normal Mendelian pattern.

To address these questions we are proposing to use tomato in which we have recently identified a target of paramutation. The loss of pigment in this system results in a spectacular yellow chlorosis - sulfurea. We propose to exploit gene editing technology to knock out genes that might influence sulfurea paramutation and we will delete DNA at the sulfurea locus to find out whether it affects either paramutagenicity or paramutability. Tomato has many advantages as an experimental system for these questions - it is amenable to a viral gene silencing system - VIGS - that can target DNA methylation and our recent work has identified other paramutation loci that can be compared and contrasted with sulfurea.

Technical Summary

We will use sulfurea and other loci in tomato as a model to dissect the mechanisms and biological effects of paramutation in plants. Paramutation of sulf is due to hypermethylation of the DNA spanning the transcriptional start point of a gene - SLTAB2 - required for production of photosystem I and the epigenetic effect can be triggered by virus induced gene silencing (VIGS) using that region as a target sequence. In this project we will define the features of SLTAB2 DNA/chromatin that are involved in paramutation and we will identify the proteins and RNAs that are required for establishment and maintenance of the gene silencing from one generation to the next.

To analyse the DNA/chromatin we will first generate a series of VIGS and transgene constructs to further define the minimal paramutagenic region (PGR). We will also generate a series of deletions in the genomic DNA in situ using gene editing to test the possibility that other regions in the SLTAB2 DNA are required for paramutagenicity or paramutability or both. These studies will be complemented by MNase HS to analyse chromatin structure, 3C direct assay of long-range interactions and FISH to test the potential for direct interaction of the paramutagenic and paramutable alleles during the establishment stage of paramutation.

We will test the protein and RNA requirement for paramutation with mutants and knock down lines in RdDM, RNA silencing, DNA methylation and chromatin modification. Direct analysis of RNA from the PGR will identify non coding RNAs in paramutation.

The final part of the project will integrate the findings from sulf paramutation with the analysis of other loci in the tomato genome with paramutation-like properties. We will ask whether the mechanisms at sulf and other loci are similar. These combined studies will allow for a more complete understanding of paramutation than has been possible in other systems including maize.

Planned Impact

The pathways to impact will use the findings of the project as a vehicle for enhancing understanding of evolution and epigenetics in animals and plants with an emphasis on crops.
The impact activity relevant to evolution will focus on gene drive and, in epigenetics, on the way that heritable difference between organisms need not depend on variation in the sequence of DNA. The proposed project is on plants but the underlying concepts also apply in animals (including humans) and there is scope for the impact agenda to be broader than plants. The targets of the impact activities will be:
- the general public and educators so that they can develop informed opinions about emerging technologies relevant to gene drive and epigenetics.
- regulators who will need good understanding of the basic concepts associated with gene drive and epigenetics 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 epigenetic technologies as they emerge.
- the postdoctoral researcher so that they can develop their career with experience of driving impact in science

1. Public engagement in evolution and epigenetics
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 epigenetics and gene drive.
Milestone I2 - 24 months - demonstration plot in Cambridge University Botanic Garden illustrating Mendelian and non-Mendelian inheritance due to paramutation . The "meet the scientist" sessions will be included as part of this milestone.
Milestone I3 - 36 months - presentation to popular media of the concepts in epigenetics and genetic drive.

2. Industry engagement in the importance of epigenetics in crop plant breeding.
To ensure that industry, government, policy and regulatory bodies are engaged and familiar with the potential of epigenetics 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 epigenetics in plants.
Milestone I5- 36 months - workshop to convene key industry players in epigenetics for agriculture.

3. Regulatory engagement in the development rational, risk-based regulatory framework for epigenetics.
As part of the engagement with regulatory bodies we will apply to ACRE for permission to "field-test" epigenetically modified Arabidopsis that have been generated in connection with other projects in the lab. It is not clear from the existing regulations as to whether these plants are subject to ACRE approval and this activity is intended to clarify the situation.
Milestone I6 - 24 months - decision from ACRE over field testing of Epigenetically Modified Arabidopsis and one year of trials. If successful I will apply for a second year of trials by month 36..

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 experimental and computational epigenetics and familiarity with regulatory, commercialization and public engagement aspects of a research project.


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