Abstract
Plant viruses have limited coding capacities so that they rely heavily on the expression of multifunctional viral proteins to achieve a successful infection. The functional specification of viral proteins is often related to their differential interaction with plant and viral components and somewhat depends on their localization to various subcellular compartments. In this study, we analyzed the intracellular localization of the V2 protein of Mulberry mosaic dwarf-associated virus (MMDaV), an unsigned species of the family Geminiviridae. We show that the V2 protein colocalizes with the nucleolar protein fibrillarin (NbFib2) in the nucleolus upon transient expression in the epidermal cells of Nicotiana benthamiana. A yeast-two hybrid assay, followed by bimolecular fluorescence complementation assays, demonstrated the specific interaction between V2 and NbFib2. Intriguingly, we find that the presence of MMDaV excludes the V2 protein from the nucleolus to nucleoplasm. We present evidence that the replication-associated protein A (RepA) protein of MMDaV interacts with V2 and enables the nucleolar exclusion of V2. We also show that, while V2 interacts with itself primarily in the nucleolus, the presence of RepA redirects the site of V2–V2 interaction from the nucleolus to the nucleoplasm. We further reveal that RepA promotes V2 out of the nucleolus presumably by directing the NbFib2-V2 complex from the nucleolus to the nucleoplasm. Considering the critical role of the nucleolus in plant virus infection, this RepA-dependent modulation of V2 nucleolar localization would be crucial for understanding the involvement of this subcellular compartment in plant–virus interactions.
Highlights
Plant viruses are obligate intracellular parasites that have long caused significant yield losses to crops and are continuously threatening crop cultivation worldwide
We show that the Mulberry mosaic dwarf-associated virus (MMDaV) V2 protein colocalizes with the nucleolar protein fibrillarin in the nucleolus upon transient expression of V2 in the epidermal cells of N. benthamiana
The replication-associated protein A (RepA), V2, and V3 genes from MMDaV (GenBank accession number KP303687) (Ma et al, 2015), the V2dm61−77aa from pGEM-T-V2dm61−77aa (Yang et al, 2018) and the coding sequences of the fibrillarin 2 gene of N. benthamiana Complementary DNA (cDNA) (GenBank accession number AM269909, NbFib2) were cloned into pENTR-D/TOPO entry vector without stop codon by standard protocols (Invitrogen, Beijing, China) to yield pENTR-RepA, pENTR-V2, pENTR-V3, pENTR-V2dm61−77aa, and pENTR-NbFib2, respectively
Summary
Plant viruses are obligate intracellular parasites that have long caused significant yield losses to crops and are continuously threatening crop cultivation worldwide. Plant viruses usually encode multifunctional viral proteins that play important roles in several steps of a virus life cycle. Besides its well-known role in ribosome RNA synthesis, the nucleolus plays critical roles in cell cycle regulation and responses to biotic and abiotic stresses (Kalinina et al, 2018). Many plant RNA viruses replicate in the cytoplasm of host cells, an increasing number of viral proteins have been shown to localize, at least at some stage during infection, in the nucleolus (Hiscox, 2007). Viral proteins may subsequently utilize nucleolar functions for assembly of viral ribonucleoprotein (RNP) particles, virus replication, and movement, and to counteract plant antiviral defense (Kalinina et al, 2018)
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