Integrated transcriptome and genetic analysis of early events determining tissue susceptibility in the Claviceps purpurea - wheat interaction

The need for safe and nutritious wheat is as important as ever. The aim of this project is to gain an understanding of the interaction between the alkaloid producing plant pathogen Claviceps purpurea and its host hexaploid wheat. Claviceps forms a fungal body called an ergot in the place of a wheat grain following infection of the flower. Ergots contain high levels of extremely toxic alkaloids and pose a risk to humans and animals when ingested hence a zero tolerance level for wheat entering the human food chain and strict levels allowed for sale of grain for feed. Levels of ergots found in seed lots have been increasing over recent years which is driving the need for this research. In order to understand the fundamental biology of the interaction during the infection process, we will extract and measure relative abundance of the expressed genes from a set of very specific tissues of the infected wheat flower over a time course that identifies the key stages of Claviceps infection along with an uninfected control. Claviceps hyphae grow through the same tissues of the wheat flower as the growing pollen tube during pollination so we propose to include the pollen-flower interactions in the analysis. We will identify set of genes that are up and down regulated in response to either pollen and or hyphal growth and will identify common signals involved in perception and response. There is no chemical that can be deployed to defend against infection by Claviceps, so natural resistance to this pathogen must be exploited in any long-term sustainable control strategy. Partial resistance has been identified for a UK wheat variety and there is also source of near complete resistance identified in durum wheat, and we intend to locate the genes conferring these traits and to compare their location with those of genes whose expression appears to correlate in time and space with the expression of resistance. Additional evidence for or against the putative role of genes of interest in determining resistance will be gained by using genetic modification to increase or decrease expression of specific candidates.

Claviceps purpurea infects the floral tissue of wheat via the same route as a germinated pollen tube and replaces the resulting grain with a highly toxic fungal sclerotium called an ergot. In previous work, two sources of resistance to ergot infection were identified and segregating populations generated. However, understanding more about the molecular and cellular biology of the interaction is a pre-requisite to selection of appropriate candidate genes to map in the forward genetics approach, and equally in developing hypotheses for rational targeting (using transgenesis) of particular infection stages. Our first goal is to make a unique set of cDNA libraries from microdissected stigma and ovary tissues representing a timecourse series throughout the first four days of Claviceps infection or pollination, using deep coverage next generation sequencing techniques to quantify transcript abundance. Here, the aim is to identify those genes and pathways which are specific to different infection stages, and those which are differentially regulated between resistant and susceptible lines. Our second goal is to locate Quantitative Trait Loci (QTL) for naturally occurring resistance mechanisms in two distinct backgrounds, and we will integrate the two approaches by mapping the expression QTL and structural gene locations of interesting transcripts. Finally, germline transformation will be used to test hypotheses about the role of particular genes in determining susceptibility to ergot.