Computational and experimental analysis of horizontally acquired, secreted enzymes for plant carbohydrate degradation - from hemibiotrophic oomycete, Phytophthora sojae
Attah, V
Date: 22 February 2021
Publisher
University of Exeter
Degree Title
PhD in Biological Sciences
Abstract
The oomycetes are heterotrophic, filamentous protists that share morphological similarities with some fungi, but according to phylogenetic evidence, are actually relatives of diatoms and brown algae, within a different eukaryotic supergroup (Stramenopiles) (Förster et al., 1990; Leclerc et al., 2000; Hudspeth et al., 2000; Hudspeth et ...
The oomycetes are heterotrophic, filamentous protists that share morphological similarities with some fungi, but according to phylogenetic evidence, are actually relatives of diatoms and brown algae, within a different eukaryotic supergroup (Stramenopiles) (Förster et al., 1990; Leclerc et al., 2000; Hudspeth et al., 2000; Hudspeth et al., 2003; Thines et al., 2007; McCarthy and Fitzpatrick., 2017). Horizontal Gene Transfer (HGT) from fungi to oomycetes has been previously suggested to have played a role in the evolution of osmotrophic and phytopathogenic traits during divergence of the oomycete lineage; such transfers include secreted proteins predicted to degrade plant cell wall-specific substrates – these are the initial barriers to pathogen invasion as well as an abundant source of fixed carbon (Torto et al., 2002; Belbahri et al., 2008; Richards et al., 2011; Savory et al., 2015). The HGTs are largely expanded in hemibiotrophic oomycetes, and their selective benefit is suggested by subsequent gene duplications following acquisition (Richards et al., 2011; Savory et al., 2015) - giving rise to paralogs hypothesised to possess functional differences. Bioinformatics and computational analysis of laterally-transferred Glycoside Hydrolase 10 (GH10) and GH12 in Phytophthora sojae (oomycete hemibiotrophic parasite of soybean) discovered unique differences amongst paralogs – P. sojae_482953 (GH12 xyloglucanase) encodes a significantly disordered, 186 amino acid ‘tail’, which improves the enzymatic activity of the protein towards xyloglucan when heterologously-expressed in Saccharomyces cerevisiae. Interestingly, knockout of the gene encoding P. sojae_482953 in vivo did not affect the ability of P. sojae to utilise xyloglucan as a sole carbon source, indicating that gene duplication events are also an important mechanism for transcriptional fidelity and maintenance of function. A second GH12 paralog, P. sojae_559651 was predicted to encode a ‘second’ carbohydrate-binding site, with the prediction being conserved for orthologs encoded by P. cactorum and P. nicotiniae; interestingly, two indels (coding for alanine and serine) were identified as being important for the prediction, and their removal abolished the second binding site prediction for all three proteins. Functional diversification of variant paralogs post-HGT was also suggested by differences in oligosaccharides released from xyloglucan breakdown by P. sojae_559651 and P. sojae_482953 proteins using mass spectrometry. Taken together, this work extends our understanding of the functional significance of gene duplication post-HGT in hemibiotrophic oomycetes. Of further interest is how N-terminal signal peptide sequences affect the transferability of secreted proteins by cross-phylum HGT – this work has optimised a functional agar plate screen (and demonstrated a promising microdroplet approach) in order to explore signal peptide evolution in future work.
Doctoral Theses
Doctoral College
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