CHARACTERIZATION OF TRAFFICKING ROUTES OF THE MOVEMENT PROTEIN (MP) OF OURMIA MELON VIRUS (OUMV) AND IDENTIFICATION OF OUMV MP-INTERACTING PROTEINS IN PLANTS

Open Access
- Author:
- Ozber, Natali
- Graduate Program:
- Plant Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 19, 2018
- Committee Members:
- Cristina Rosa, Dissertation Advisor/Co-Advisor
Cristina Rosa, Committee Chair/Co-Chair
Charles T Anderson, Committee Member
Timothy W Mcnellis, Committee Member
Gabriele Brigitte Monshausen, Outside Member - Keywords:
- plant virus
trafficking pathways
plant–pathogen interaction
virus movement
host factors - Abstract:
- This dissertation aims to identify and characterize trafficking routes of the movement protein (MP) of Ourmia melon virus (OuMV) to better understand viral movement mechanisms in plant cells and to study host trafficking pathways using OuMV MP as a tool. In the first part of the dissertation, the subcellular localization of MP was characterized. Contrary to some known viral MPs, OuMV MP was associated with endoplasmic reticulum (ER), but not with actin and microtubules. Arabidopsis synaptotagmin A labeled ER-plasma membrane contact sites were also not involved in the transport of OuMV MP. Morphological changes in ER and changes in ER diffusion were evident in OuMV-infected cells. The observations from the first part of the dissertation revealed that OuMV MP resides in vesicle-like structures, as the infection progresses. In the second part of the dissertation, the nature of these structures was further characterized, and the involvement of post-Golgi trafficking pathways was shown in the context of two endosomal sorting motifs, a tyrosine motif and a dileucine motif. Chemical inhibitor experiments revealed that OuMV MP localizes to the trans-Golgi network (TGN), but not to the multivesicular body/prevacuolar compartment or Golgi. This is the first study that suggests the TGN localization of a viral MP. Mutational analysis of the sorting motifs of OuMV MP showed that these motifs are important for transport of MP to plasmodesmata and systemic infection of OuMV. They, however, have different functions: the tyrosine motif seems not to be a functional internalization motif, but rather a structural motif, whereas the dileucine motif may be involved in endosomal pathways. In the final part of the dissertation, 55 detergent-soluble and 146 soluble OuMV MP-interacting host proteins were identified by a maltose-binding protein pull-down assay coupled with liquid chromatography-mass spectrometry analysis. These candidate host proteins can be classified in the following categories: membrane trafficking, lipid binding, protein phosphorylation/dephosphorylation, RNA binding, cell wall biosynthesis, blue light response, calcium signaling, transport across the plasma membrane, protein folding and homeostasis, proteolysis, and metabolism, suggesting that OuMV MP targets diverse pathways. Furthermore, potential phosphorylation sites that may be involved in the regulation of OuMV MP were also identified. Results from these studies provide valuable information on the molecular mechanism of virus movement and enhance our knowledge on plant-virus interactions. Ultimately, insights gained by this dissertation could lead to the development of strategies to prevent crop losses caused by virus-associated diseases.