Cell wall biosynthesis in barley powdery mildew Blumeria graminis f. sp. hordei
Date
2019
Authors
Pham, Trang Anh
Editors
Advisors
Bulone, Vincent
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Abstract
Blumeria graminis f. sp. hordei (Bgh) is the causal agent of powdery mildew in Hordeum vulgare (barley). Bgh is an obligate biotrophic pathogen, meaning that it relies on the host for survival. The Bgh asexual life cycle initiates when airborne conidia land on the host surface and germinate. An appressorium develops and penetrates the host to form an intracellular feeding structure called a haustorium. Following successful host entry, epiphytic mycelia spread along the surface and conidiophores emerge to produce vast quantities of conidia. As the fungal cell wall is essential for survival it is an obvious target for the design of novel antifungal agents for disease control. Depending on the pathogen species, the composition of the cell wall can vary significantly. In order to advance disease control practices, it is imperative to understand the composition of the cell wall and the genes involved in cell wall metabolism. This work focuses on characterising the Bgh fungal cell wall and examining the genes responsible for its synthesis during pre-penetrative events of pathogenesis. In addition, in vitro assays have been performed to investigate the biochemical activity for a number of key biosynthetic enzymes. Permethylation glycosidic linkage analysis of the Bgh conidia cell wall has revealed their cell wall composition for the first time. The conidial cell wall of Bgh is predominantly made of glucose and has a greater proportion of galactose residues compared to other well characterised fungal cell walls. Trace amounts of xylose residues were also observed. Analysis of the Carbohydrate Active enZymes (CAZy) present within the Bgh genome was conducted to identify genes involved in cell wall metabolism, as previous CAZy annotations of the genome were incomplete. A greater number of CAZy genes were identified compared to previous studies. Many of the biochemical activities of CAZy enzymes involved in cell wall synthesis are still unknown due to the technically challenging work required. In an attempt to understand cell wall synthesis in Bgh, several genes involved in chitin and b-1,3- glucan synthesiswere heterologously expressed with the aim to confirm biochemical activity and perform in vitro enzyme kinetic assays. Following successful expression of four chitin synthases, only the class I chitin synthase was able to be expressed and purified in an active state. To examine pre-penetrative events in Bgh without any contamination from the host plant it was essential to establish an in vitro system for germination of the Bgh conidia. A previously established in vitro assay using n-hexacosanal was adapted to generate in vitro fungal material for analysis. n-Hexacosanal is a known inducer of Bgh germination and appressorial development. Profiling of the transcriptome and proteome during the appressorial germ tube stage revealed that there was a notable shift towards energy and protein production during appressorial development. Linkage analysis of the appressorial cell wall showed a significant decrease in the galactose portion of the cell wall during the appressorial stage and the appearance of novel branched xylose linkages that have not been observed in fungal pathogens previously. The use of this cultivation method demonstrates that it is possible to analyse the pre-penetrative processes of Bgh development in vitro.
School/Discipline
School of Agriculture, Food and Wine
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food & Wine, 2019
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