Investigations into the molecular interactions between Claviceps purpurea, the causal agent of ergot, and cereal hosts

The fungus Claviceps purpurea infects female flowers of cereals and grasses, producing an ergot sclerotia (the overwintering structure of the pathogen) in place of a grain. C. purpurea can infect wheat, barley, rye, oats, triticale and millet, detrimentally affecting grain production and flour quality. Ergot also impacts human health directly through the ingestion of toxic alkaloids (found in very high levels in sclerotia), resulting in a condition known as ergotism. Currently, the content of ergot sclerotia in unprocessed cereal grains is set by the European Commission Regulation (EC) No. 1881/2006. In grain for human consumption the amount of sclerotia is restricted to a maximum of 0.05%. However, these limits are set to be reduced, and new regulations regarding the levels of alkaloids found in processed foods from cereals is about to be introduced through new EC regulations. The aim of this thesis was to obtain a better understanding of the genetic and molecular changes that occur in wheat ovaries following inoculation with C. purpurea, as well as to understand the routes by which cereal grains can become contaminated with ergot alkaloids. The data from an extensive transcriptomics study indicated a substantial reprogramming of wheat hormonal pathways, with a high proportion of genes involved in GA, auxin, ethylene and cytokinin metabolism showing differential expression relative to ovary tissue-type and time after inoculation. The results suggest that C. purpurea is able to co-opt the host’s hormonal pathways in order to facilitate infection. In addition, the host is able to activate several defence mechanisms during the early stages of infection, while the persistent up-regulation of certain categories of host defence-related genes in the later stages of infection is indicative for an ongoing basal defence response against C. purpurea. Of particular interest is evidence of a mobile signal sent from the inoculated stigma to the ovary base, differential expression of host genes being observed at the base of the ovary long before the arrival of C. purpurea hyphal tissue. Currently, no complete resistance against the fungal pathogen C. purpurea is known. Partial resistance to ergot has been previously shown to co-locate with the wheat Reduced height (Rht) dwarfing gene alleles Rht-B1b and Rht-D1b. The wheat Rht loci encode DELLA proteins, Rht-B1b and Rht-D1b representing GA-insensitive mutations in DELLA. A linkage between Rht-B1b and Rht-D1b and ergot resistance could therefore indicate a role of GA in C. purpurea infection. Reduced honeydew production, along with a reduction in the size and weight of sclerotia were found in lines carrying the dwarf and semi-dwarf mutant alleles Rht-D1b, Rht-D1c, Rht-B1c. Furthermore, the levels of GA4, auxin and dihydrozeatin-type (DHZ) cytokinins were found to be elevated during infection. Taken together, these results indicate in wild type wheat GA acts to increase susceptibility to C. purpurea. In addition, the profiles and levels of a range of other endogenous hormones were also are significantly altered in response to C. purpurea infection. Re-emerging concerns over the potential health risks presented by the ergot alkaloids, produced during C. purpurea infection, has led to new legislation on ergot alkaloids being proposed by the EC. In this thesis we show that a significant risk of grain contamination by ergot alkaloids exists both pre-harvest, due to contamination of grain while in the ear, as well as post-harvest, as a result of physical contact between sclerotia and grain. Very low levels of ergot alkaloids were found in honeydew with all C. purpurea isolates tested. Levels increased in sphacelia tissues and reached levels as high as 3 million parts per billion (ppb; 3 million μg of ergot alkaloids per kg of sclerotia) in mature ergot sclerotia. In wheat and barley, ergot alkaloids were found to transfer to healthy grain that developed above and below flowers infected with the C. purpurea isolates 04-97.1, EI4 and Rye 20.1. In addition, significant levels of ergot alkaloids were found to be transferred to clean grain of wheat and barley as a result of direct physical contact with broken pieces of sclerotia, compared to intact sclerotia, with significant differences also seen between wheat and barley

Reprogramming of the wheat transcriptome in response to infection with Claviceps purpurea, the causal agent of ergot.

BACKGROUND: Ergot, caused by the fungal pathogen Claviceps purpurea, infects the female flowers of a range of cereal crops, including wheat. To understand the interaction between C. purpurea and hexaploid wheat we undertook an extensive examination of the reprogramming of the wheat transcriptome in response to C. purpurea infection through floral tissues (i.e. the stigma, transmitting and base ovule tissues of the ovary) and over time. RESULTS: C. purpurea hyphae were observed to have grown into and down the stigma at 24 h (H) after inoculation. By 48H hyphae had grown through the transmitting tissue into the base, while by 72H hyphae had surrounded the ovule. By 5 days (D) the ovule had been replaced by fungal tissue. Differential gene expression was first observed at 1H in the stigma tissue. Many of the wheat genes differentially transcribed in response to C. purpurea infection were associated with plant hormones and included the ethylene (ET), auxin, cytokinin, gibberellic acid (GA), salicylic acid and jasmonic acid (JA) biosynthetic and signaling pathways. Hormone-associated genes were first detected in the stigma and base tissues at 24H, but not in the transmitting tissue. Genes associated with GA and JA pathways were seen in the stigma at 24H, while JA and ET-associated genes were identified in the base at 24H. In addition, several defence-related genes were differential expressed in response to C. purpurea infection, including antifungal proteins, endocytosis/exocytosis-related proteins, NBS-LRR class proteins, genes involved in programmed cell death, receptor protein kinases and transcription factors. Of particular interest was the identification of differential expression of wheat genes in the base tissue well before the appearance of fungal hyphae, suggesting that a mobile signal, either pathogen or plant-derived, is delivered to the base prior to colonisation. CONCLUSIONS: Multiple host hormone biosynthesis and signalling pathways were significantly perturbed from an early stage in the wheat - C. purpurea interaction. Differential gene expression at the base of the ovary, ahead of arrival of the pathogen, indicated the potential presence of a long-distance signal modifying host gene expression