Year of Publication

2014

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department

Plant Pathology

First Advisor

Dr. Michael M. Goodin

Abstract

Potato yellow dwarf virus (PYDV) is the type species of the genus Nucleorhabdovirus and, like all members of this genus, replication and morphogenesis occurs inside the nuclei of infected cells. Protein localization prediction algorithms failed to identify a nuclear localization signal (NLS) in PYDV nucleocapsid (N) protein, although PYDV-N has been shown to localize exclusively to the nucleus when expressed as a green fluorescent protein (GFP):N fusion in plant cells. Deletion analysis and alanine-scanning mutagenesis identified two amino acid motifs, 419QKR421 and 432KR433, that were shown to be essential for nuclear import and interaction with importin-α. Additional bimolecular fluorescence complementation showed that the PYDV-N-NLS mutants cannot be ferried into the nucleus via interaction with PYDV-P or-M. In contrast, interaction with N-NLS mutants appeared to retard the nuclear import of PYDV-P. Taken together, it was determined that PYDV-N contains the bipartite NLS 419QKRANEEAPPAAQKR433. Similarly, alanine-scanning mutagenesis was performed to determine the regions responsible for the nuclear import of PYDV-M and -P. A non-canonical NLS was identified in PYDV-P, consisting of three regions in the N-terminus of the protein required for nuclear import. PYDV-P does not interact with any Nicotiana benthamiana importins, but was found to interact with importin-α7 and -α9 of the non-host plant Arabidopsis thaliana. Two amino acids of PYDV-M, 225KR226, were found to be critical for nuclear import and interaction with importin-α. In addition, site-directed mutagenesis identified that amino acids 223LL224 of PYDV-M, which are adjacent to the two amino acids identified as responsible for nuclear import, are critical for inducing invaginations of the inner nuclear membrane. Bimolecular fluorescence complementation (BiFC) was then used to identify any differences in localization and interaction caused by the mutations introduced to PYDV-P and -M. The PYDV-P and -M proteins were still able to interact with other PYDV proteins, although the localization of the interaction differs between mutants.

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