Plant viruses encode virulence factors or RNA silencing suppressors to reprogram plant cellular processes or to fine-tune host RNA silencing-mediated defense responses. In a previous study, Mulberry mosaic dwarf-associated virus (MMDaV), a novel, highly divergent geminivirus, has been identified from a Chinese mulberry tree showing mosaic and dwarfing symptoms, but the functions of its encoded proteins are unknown. In this study, all seven proteins encoded by MMDaV were screened for potential virulence and RNA silencing suppressor activities. We found that V2, RepA, and Rep affect the pathogenicity of a heterologous potato virus X. We showed that V2 could inhibit local RNA silencing and long-distance movement of the RNA silencing signal, but not short-range spread of the green fluorescent protein (GFP) silencing signal in Nicotiana benthamiana 16c plants. In addition, V2 localized to both subnuclear foci and the cytoplasm. Deletion mutagenesis of V2 showed that the basic motif from amino acids 61 to 76 was crucial for V2 to form subnuclear foci and for suppression of RNA silencing. Although the V2 protein encoded by begomoviruses or a curtovirus has been shown to have silencing suppressor activity, this is the first identification of an RNA silencing suppressor from a woody plant-infecting geminivirus.

Plant viruses are obligate intracellular pathogens that are constantly causing significant losses to crop production and are continuously threatening global food production (Jones & Naidu, 2019). With a limited coding capacity, plant viruses depend on the hosts for replication, intracellular and intercellular movement, and long-distance trafficking. Exposure to plant virus infection enables plants to develop sophisticated and efficient antiviral defence systems to respond properly at various levels (Boualem et al.,

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.