Doctor of Philosophy
Agriculture and Agri-Food Canada
Kohalmi, Susanne E
Stone fruits are a valuable crop grown worldwide, however pathogens such as viruses threaten fruit production by reducing tree health and fruit yield. In an orchard within the Niagara region of Ontario, symptoms typical of viral infection such as chlorosis and leaf deformation were seen on sweet cherry (Prunus avium L.) trees. Next generation sequencing was performed on symptomatic and asymptomatic leaves and four viruses were identified. On the tree displaying the most severe symptoms, Prune dwarf virus (PDV), was the only virus detected. A survey conducted during this work showed 42% of cherry trees on a single orchard plot are infected by PDV. The first infectious clone of PDV was developed for molecular characterization of this virus. Introduction of the infectious clone into cherry revealed PDV caused dwarfing symptoms but did not induce the foliar symptoms found on orchard grown trees. A mass spectrometry (MS)-based label-free quantitative proteomic analysis was performed to identify host proteins affected by PDV infection. The results show in PDV infected cherry many defense related proteins are upregulated, and many photosynthesis-related proteins are downregulated. In the model plant cucumber (Cucumis sativus L.) infected by PDV, significant accumulation changes of proteins related to translation and photosynthesis were identified using proteomics, suggesting a possible role of these proteins in the viral infection cycle of PDV. Two proteins identified through proteomic analysis of cucumber were further studied. These proteins are predicted to be important in the infection cycle of PDV as both co-localized with the viral coat protein (CP) of PDV when visualized using confocal microscopy. Finally, to further understand the intra-host spread of PDV, the movement protein (MP) of PDV was characterized. In plant cells, MP expressed alone formed tubules, a typical structure for virus movement. Additionally, domains of MP crucial for tubule formation and subcellular localization were identified. Taken together, this work advances knowledge in the molecular biology of PDV and host impact caused by PDV infection. In the long run, these findings will assist the development of novel strategies against PDV for the sustainable production of cherry and related Prunus fruits.
Summary for Lay Audience
Stone fruits such as cherries are a valuable crop grown worldwide, when they are infected by viruses, both fruit yield and quality are reduced. In severe cases, infected trees die off. Many sweet cherry trees grown on an orchard in the Niagara region show typical viral symptoms on their leaves. Sequencing of the genetic material isolated from leaves of these trees identified four viruses as possible causes of these symptoms. The strongest symptoms were associated with a virus called prune dwarf virus (PDV). All the trees of a cherry plot were analyzed and almost half were infected by PDV. To study PDV in the laboratory, a copy of this virus was made, and young cherry trees were infected to see if the same leaf symptoms occurred. PDV infection caused the cherry seedlings to grow slower and smaller than healthy seedlings, but PDV didn’t cause any leaf symptoms. Another technique was used to study proteins in orchard grown cherries to see if the levels of proteins were different between sick and healthy leaves. The results suggest many proteins and their associated biological pathways are altered, that may contribute to the development of symptoms. In the laboratory, protein changes were studied in PDV infected cucumber plants which are easier to study compared to cherry plants. Some proteins were identified which are likely important in PDV infection. For example, two proteins were identified that might interact with proteins made by the virus. To understand how PDV moves in plants, the movement protein (MP) was studied. It was found that MP can make hollow tubules acting as tunnels for virus movement. Moreover, the beginning of the protein sequence was shown to be important for making tubules whereas the middle of the protein sequence was found to be essential for the MP to find its target in plant cells. We now have a better understanding of how PDV moves inside plants and what plant proteins are important for PDV infection. Protein studies also provided a list of proteins for researchers to study and in the future might be useful to breed PDV resistant plants.
Simkovich, Aaron J., "Molecular identification and characterization of viral pathogens infecting sweet cherry" (2020). Electronic Thesis and Dissertation Repository. 7454.
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Available for download on Friday, December 31, 2021
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