ty-5 Confers Broad-Spectrum Resistance to Geminiviruses.

The selection of resistant crops is an effective method for controlling geminivirus diseases. ty-5 encodes a messenger RNA surveillance factor Pelota with a single amino acid mutation (PelotaV16G), which confers effective resistance to tomato yellow leaf curl virus (TYLCV). No studies have investigated whether ty-5 confers resistance to other geminiviruses. Here, we demonstrate that the tomato ty-5 line exhibits effective resistance to various geminiviruses. It confers resistance to two representative begomoviruses, tomato yellow leaf curl China virus/tomato yellow leaf curl China betasatellite complex and tomato leaf curl Yunnan virus. The ty-5 line also exhibits partial resistance to a curtovirus beet curly top virus. Importantly, ty-5 confers resistance to TYLCV with a betasatellite. Southern blotting and quantitative polymerase chain reaction analyses showed that significantly less DNA of these geminiviruses accumulated in the ty-5 line than in the susceptible line. Moreover, knockdown of Pelota expression converted a Nicotiana benthamiana plant from a geminivirus-susceptible host to a geminivirus-resistant host. Overall, our findings suggest that ty-5 is an important resistance gene resource for crop breeding to control geminiviruses.

The novel C5 protein from tomato yellow leaf curl virus is a virulence factor and suppressor of gene silencing.

Tomato yellow leaf curl virus (TYLCV) is known to encode 6 canonical viral proteins. Our recent study revealed that TYLCV also encodes some additional small proteins with potential virulence functions. The fifth ORF of TYLCV in the complementary sense, which we name C5, is evolutionarily conserved, but little is known about its expression and function during viral infection. Here, we confirmed the expression of the TYLCV C5 by analyzing the promoter activity of its upstream sequences and by detecting the C5 protein in infected cells by using a specific custom-made antibody. Ectopic expression of C5 using a potato virus X (PVX) vector resulted in severe mosaic symptoms and higher virus accumulation levels followed by a burst of reactive oxygen species (ROS) in Nicotiana benthamiana plants. C5 was able to effectively suppress local and systemic post-transcriptional gene silencing (PTGS) induced by single-stranded GFP but not double-stranded GFP, and reversed the transcriptional gene silencing (TGS) of GFP. Furthermore, the mutation of C5 in TYLCV inhibited viral replication and the development of disease symptoms in infected plants. Transgenic overexpression of C5 could complement the virulence of a TYLCV infectious clone encoding a dysfunctional C5. Collectively, this study reveals that TYLCV C5 is a pathogenicity determinant and RNA silencing suppressor, hence expanding our knowledge of the functional repertoire of the TYLCV proteome.

A group I WRKY transcription factor regulates mulberry mosaic dwarf-associated virus-triggered cell death in Nicotiana benthamiana.

Geminiviruses constitute the largest group of known plant viruses and cause devastating losses to a wide range of crops and woody plants globally. Mulberry mosaic dwarf-associated virus (MMDaV), identified from Chinese mulberry trees via small RNA-based deep sequencing, is a divergent monopartite geminivirus belonging to the genus Mulcrilevirus of the family Geminiviridae. Previous studies have shown that plants employ multiple layers of defence to protect themselves from geminivirus infection. The interplay between plant and MMDaV is nevertheless less studied. This study presents evidence that MMDaV triggers hypersensitive response (HR)-mediated antiviral defence in Nicotiana benthamiana plants. We show that the RepA protein of MMDaV is engaged in HR-type cell death induction. We find that the RepA mutants with compromised nuclear localization ability impair their capabilities of cell death induction. Virus-induced gene silencing of the key components of the R protein-mediated signalling pathway reveals that down-regulation of the nucleus-targeting NbWRKY1 alleviates the cell death induction activity of RepA. We further demonstrate that RepA up-regulates the transcript level of NbWRKY1. Furthermore, expression of RepA in N. benthamiana confers plant resistance against two begomoviruses. We propose that plant resistance against RepA can be potentially used to improve plant defence against geminiviruses in crops.

RepA Promotes the Nucleolar Exclusion of the V2 Protein of Mulberry Mosaic Dwarf-Associated Virus.

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.

Identification of the Potential Virulence Factors and RNA Silencing Suppressors of Mulberry Mosaic Dwarf-Associated Geminivirus.

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.