Molecular dissection of paramutation in tomato

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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.