[EMBARGOED] Analysis of cereal cyst nematode resistance mechanisms in barley

Abstract

Barley (Hordeum vulgare) is a widely grown, valuable cereal crop that is affected by various pests including nematodes. The cereal cyst nematode (CCN) Heterodera avenae is the most widely distributed and damaging species of nematodes to cereal crops grown in temperate regions, including Australia, and is estimated to cause global annual losses of around $160 billion. The use of cultivars resistant to H. avenae is the preferred approach for nematode management and four resistance loci, Rha1, Rha2, Rha3 and Rha4, have been mapped. The Rha4 locus was mapped in the Galleon/Haruna Nijo population to chromosome 5H and since Rha2 and Rha4 provide the most effective resistance against the Australian H. avenae pathotype Ha13 they have been widely used in barley breeding. Despite CCN resistance loci having been mapped in barley and other cereals, no resistance genes have been isolated and characterized. Recently both Rha2 and Rha4 have been fine-mapped and near-diagnostic markers have been developed to provide simple tools for selection. Fine-mapping indicates that approximately 105 genes are linked to the Rha4 locus, including cell wall-related genes predicted to encode (1-4)-β-xylan endohydrolases, also known as xylanases. This thesis reports on experiments that were undertaken to better understand the resistance conferred by Rha4 and to investigate the functions of the xylanases as prime candidate genes. The xylanase genes at the Rha4 locus were cloned and analysed for allelic differences between sequences from the cultivars Sloop (susceptible) and Flagship (Rha4-resistant). Although genes X1 and X2 have been characterised, the X3 gene has not been well described previously. The genomic sequences were used in reciprocal transformation experiments where, under the control of the 35S promoter, the genes from Sloop were transformed into a Flagship background, and vice versa. Tube tests were used to investigate any changes in nematode infection responses, and therefore resistance status, but no significant alterations were detected. However, overexpression of the xylanase genes proved to be detrimental to the overall health of the plant. The xylanases were concomitantly heterologously expressed in Escherichia coli and the X2 protein was characterized in terms of substrate preference and catalytic rate. In more general approaches not directly linked to Rha4 genotype, the natural variation in root polysaccharide amount and distribution, with a focus on (1,3;1,4)-β-glucan and arabinoxylan, was surveyed in a selection of susceptible and resistant barley cultivars. The effect of changes in amount of (1,3;1,4)-β-glucan was also examined through infection of the betaglucanless mutant and transgenic lines carrying either the overexpressed or silenced (1,3;1,4)-β-glucan synthase CslF6 gene. Variable amounts of (1,3;1,4)-β-glucan did not correlate to rates of nematode infection and no clear patterns of polysaccharide profiles could be linked to susceptible or resistant cultivar status. Finally, RNA sequencing was used to profile transcript changes in nematode infected and control Sloop and Flagship roots up to 24 days post inoculation. The patterns of the 105 genes linked to the Rha4 locus were extracted and a set of 27 genes which showed significant fold changes across the time course were profiled. Of these, three strong candidate genes were selected which are differentially expressed in the two cultivars and are involved in biochemical pathways that are feasibly linked to resistance mechanisms. Their putative roles in conferring resistance and how this might be tested were discussed.