Hayes, Ashley (2022) The influence of cyst nematodes on the plant secretory pathway. PhD thesis, University of Leeds.
Abstract
Cyst nematodes are economically important plant pathogens that induce vast subcellular changes in host root cells to form a specialised feeding site, the syncytium. Previously characterised changes to the plant secretory pathway during syncytial formation include the proliferation of the endoplasmic reticulum (ER) and Golgi apparatus, and the replacement of the large central vacuole with numerous smaller vacuoles. To further characterise the plant secretory pathway in plant-cyst nematode interactions, novel dual fluorescence marker constructs were developed. Each construct contained a Golgi marker fused to YFP and an additional plant secretory pathway marker fused to RFP. Stable Arabidopsis marker lines expressing these constructs were infected with the beet cyst nematode Heterodera schachtii to provide a model host-cyst nematode system. Fluorescence microscopy evidenced small vacuoles throughout the syncytium as expected. However, all other plant secretory markers within the dual fluorescence lines were undetectable in syncytia, suggesting disruption to the plant secretory pathway. To support this, gene expression analysis of a subset of plant secretory pathway genes was conducted using published RNA-seq data. Results from this suggest the altered regulation of genes involved in the early secretory pathway and post-Golgi trafficking, validating the fluorescence microscopy observations. Another aim of this work was to identify novel cyst nematode effectors containing a single C-terminal transmembrane domain (TMD), that are predicted to localise to the ER. For this, a multi-step bioinformatics pipeline was created, using the proteomes of Heterodera schachtii and the potato cyst nematode Globodera pallida. Eight screened nematode cDNAs were cloned, with tobacco leaf epidermal cells transformed to analyse subcellular localisation, and in-situ hybridisations conducted to validate gland cell expression indicative of effector activity. From this, five novel putative effector genes were identified, localising to varied subcellular compartments, including the ER, nucleus, and peroxisomes. This effector screen has contributed to growing evidence that plant pathogen effectors can have transmembrane domains, and if studied further, these genes could provide cyst nematode target genes for RNA interference.
Metadata
Supervisors: | Urwin, Peter and Denecke, Jurgen |
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Awarding institution: | University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biology (Leeds) |
Identification Number/EthosID: | uk.bl.ethos.883386 |
Depositing User: | Miss Ashley Hayes |
Date Deposited: | 25 May 2023 14:21 |
Last Modified: | 11 Jul 2023 09:53 |
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