Genetic and Cytological Analysis of Host Root Responses to Cyst Nematode Infection in Wheat

Abstract

Cereal cyst nematodes (CCN, Heterodera avenae) are soil-dwelling parasites that substantially reduce yields of cereal crops including wheat (Triticum aestivum). They establish feeding sites within the root vascular tissue and divert nutrients from the host plant to serve their life cycle. Female nematodes continue to feed and mature into cysts which contain hundreds of eggs. These eggs remain in the soil and release infective juvenile nematodes the following crop season. To better understand this host-parasite relationship, it was first necessary to establish protocols for nematode-infected plant materials. Methods for growing and maintaining infected plants, sample preparation, and staining for microscopic observation were evaluated. Once appropriate methods, including methods for hydroponic growth and confocal microscopy, were established, it was possible to maintain nematode infected tissue for long periods (up to 40 d after inoculation (DAI)) and observe syncytia within the vascular tissue. Methods were then developed to obtain high-quality three-dimensional images of thick (up to 150 μm) sections of root tissue. Specialised clearing provided unprecedented views of the feeding sites and surrounding tissues. Surprisingly, segments of the central metaxylem (cMX) vessels near the feeding sites looked very different from the expected narrow hollow tubes. In the atypical cMX segments, individual elements were short and plump rather than long, narrow and cylindrical. It was determined that during a period of 15 d in which cMX vessel elements would normally elongate and then mature to form a hollow tube, cMX vessel elements near CCN infection sites do not elongate. Instead, they expand radially, becoming plump. Their outer walls undergo secondary thickening and not all walls between elements degrade. Similar to other parasites, CCN must secure nutrients from its host – without killing the host. The results presented here lead to new hypotheses about how CCN diverts water and nutrients for its own use and how wheat plants survive this attack. Mobile tracer dyes were used to trace water flow in CCN-infected roots. Results indicated that transport is hindered in infected regions. Although CCN can cause significant yield loss in wheat, much of this can be prevented through the use of resistant varieties, which limit the build-up of CCN populations in soils. In Australia, breeding for CCN resistance has been very successful, but little is known about the mechanisms of resistance. One of the resistance genes used in wheat breeding is Cre8, which maps on chromosome 6B. Previous reports on Cre8 have associated it with plant vigour and resistance. To further investigate this, differences in root and shoot development were quantified between +Cre8 and -Cre8 materials. Sister lines were genetically selected to diminish background differences observed between parental lines TMDH6 and TMDH82. Analysis of these experiments indicated no evidence of Cre8 affecting plant vigour but established that differences in CCN resistance can be detected within 21 DAI. To evaluate differences in feeding site structure between CCN-infected -Cre8 and +Cre8 plants, protocols established in this research were used to compare transverse and longitudinal sections of feeding sites. This revealed that feeding sites in -Cre8 plants developed closer to the cMX and contained more intricate cell walls within the feeding site than those in roots of +Cre8 plants. It was also observed that the structural modification of the cMX was more extreme in roots of -Cre8 plants than in +Cre8 plants. Differences in cell wall composition were also investigated. Microscopy images revealed that the roots of +Cre8 plants contained more lignified xylem vessels and more (1,3;1,4)-β-glucan deposition surrounding feeding sites than in roots of -Cre8 plants. Finally, this thesis reports a detailed genetic map of the Cre8 region of chromosome 6B. Through a repetitive process of marker development, recombinant identification and resistance testing, Cre8 was mapped to a region of 0.22 cM, corresponding to a physical region of 334 kbp on the IWGSC RefSeq 2.0 reference genome assembly. Within that region there are ten high-confidence gene models, of which only two were previously shown to be expressed in roots. One of these genes, TraesCS6B02G466600, encodes a SUS III class sucrose synthase. It was found to contain non-synonymous polymorphisms between resistant and susceptible lines resulting in five amino acid residue substitutions within the protein sequence. As sucrose synthase provides substrates for polysaccharide synthesis, it is hypothesised that differences in the enzymes localisation or activity could indirectly affect nematode development. These differences may change the synthesis of starch and/or cell wall polysaccharides and explain possible resistance mechanisms. Therefore, TraesCS6B02G466600 is a plausible candidate gene for Cre8 resistance against CCN in wheat. The research outcomes reported in this thesis have provided new insights into CCN parasitism and plant defence, and have improved the understanding of how the Cre8 locus affects the resistance of wheat against CCN.