Molecular identification and characterization of viral pathogens infecting sweet cherry
Abstract
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.