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Autophagy is a conserved intracellular degradation process that plays an active role in plant response to virus infections. Here we report that geminiviruses counteract activated autophagy‐mediated antiviral defense in plant cells through the C2 proteins they encode. We found that, in Nicotiana benthamiana plants, tomato leaf curl Yunnan virus (TLCYnV) infection upregulated the transcription levels of autophagy‐related genes (ATGs). Overexpression of NbATG5, NbATG7, or NbATG8a in N. benthamiana plants decreased TLCYnV accumulation and attenuated viral symptoms. Interestingly, transgenic overexpression of NbATG7 promoted the growth of N. benthamiana plants and enhanced plant resistance to TLCYnV. We further revealed that the C2 protein encoded by TLCYnV directly interacted with the ubiquitin‐activating domain of ATG7. This interaction competitively disrupted the ATG7–ATG8 binding in N. benthamiana and Solanum lycopersicum plants, thereby inhibiting autophagy activity. Furthermore, we uncovered that the C2‐mediated autophagy inhibition mechanism was conserved in three other geminiviruses. In summary, we discovered a novel counter‐defensive strategy employed by geminiviruses that enlists their C2 proteins as disrupters of ATG7–ATG8 interactions to defeat antiviral autophagy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Mitogen‐activated protein kinase (MAPK) cascades are involved in host defense against pathogens and are often activated by upstream plasma membrane leucine‐rich repeat receptor‐like kinases (LRR‐RLKs).ERECTA (ER) is an LRR‐RLK that regulates plant developmental processes through activating MAPK cascades.Tomato leaf curl Yunnan virus (TLCYnV) C4 protein interacts with BKI1, stabilizes it at the plasma membrane and impairs ER autophosphorylation through suppressing the dissociation of the BKI1/ER complex, and then inhibits the activation of downstream MAPK cascades, which ultimately creates a favorable environment for TLCYnV infection.This study provides a novel viral strategy to impair MAPK activation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Autophagy is an evolutionarily conserved degradation pathway in the cytoplasm and has emerged as a key defense mechanism against invading pathogens. However, there is no evidence showing nuclear autophagy in plants.Here, we show that a geminivirus nuclear protein, C1 of tomato leaf curl Yunnan virus (TLCYnV) induces autophagy and interacts directly with the core autophagy‐related protein ATG8h. The interaction between ATG8h and C1 leads to the translocation of the C1 protein from the nucleus to the cytoplasm and the decreased protein accumulation of C1, which is dependent on the exportin1‐mediated nuclear export pathway. The degradation of C1 is blocked by autophagy inhibitors and compromised when the autophagy‐related genes (ATGs) ATG8h, ATG5, or ATG7 are knocked down. Similarly, silencing of these ATGs also promotes TLCYnV infection in Nicotiana benthamiana and Solanum lycopersicum plants.The mutation of a potential ATG8 interacting motif (AIM) in C1 abolishes its interaction with ATG8h in the cytoplasm but favors its interaction with Fibrillarin1 in the nucleolus. TLCYnV carrying the AIM mutation displays enhanced pathogenicity in solanaceous plants.Taken together, these data suggest that a new type of nuclear autophagy‐mediated degradation of viral proteins through an exportin1‐dependent nuclear export pathway restricts virus infection in plants. [ABSTRACT FROM AUTHOR]

Summary: Tomato leaf curl Yunnan virus (TLCYnV)‐encoded C4 protein induces the upregulation of the hypersensitive induced reaction 1 (HIR1) gene but interferes with the HIR1‐mediated hypersensitive response (HR).HIR1 self‐interaction is essential for the HIR1‐induced HR.TLCYnV C4 impairs the HIR1 self‐interaction and concomitantly increases the amount of Leucine‐Rich Repeat protein 1 (LRR1), a modulator of HIR1, which binds to HIR1. LRR1 promotes the degradation of HIR1, compromising the HIR1‐mediated HR.This study provides new insights into the mechanisms employed by a viral protein to counter host resistance through the cooption of the host regulatory system. [ABSTRACT FROM AUTHOR]

Abstract Many geminivirus C4 proteins induce severe developmental abnormalities in plants. We previously demonstrated that Tomato leaf curl Yunnan virus (TLCYnV) C4 induces plant developmental abnormalities at least partically by decreasing the accumulation of NbSKη, an ortholog of Arabidopsis BIN2 kinase involved in the brassinosteroid signaling pathway, in the nucleus through directing it to the plasma membrane. However, the molecular mechanism by which the membrane-associated C4 modifies the localization of NbSKη in the host cell remains unclear. Here, we show that TLCYnV C4 is a nucleocytoplasmic shuttle protein, and that C4 shuttling is accompanied by nuclear export of NbSKη. TLCYnV C4 is phosphorylated by NbSKη in the nucleus, which promotes myristoylation of the viral protein. Myristoylation of phosphorylated C4 favors its interaction with exportin-α (XPO I), which in turn facilitates nuclear export of the C4/NbSKη complex. Supporting this model, chemical inhibition of N -myristoyltransferases or exportin-α enhanced nuclear retention of C4, and mutations of the putative phosphorylation or myristoylation sites in C4 resulted in increased nuclear retention of C4 and thus decreased severity of C4-induced developmental abnormalities. The impact of C4 on development is also lessened when a nuclear localization signal or a nuclear export signal is added to its C-terminus, restricting it to a specific cellular niche and therefore impairing nucleocytoplasmic shuttling. Taken together, our results suggest that nucleocytoplasmic shuttling of TLCYnV C4, enabled by phosphorylation by NbSKη, myristoylation, and interaction with exportin-α, is critical for its function as a pathogenicity factor. TLCYnV C4 induces severe developmental abnormalities by decreasing the accumulation of NbSKη in the nucleus. This study provides evidence that TLCYnV C4 is phosphorylated by NbSKη in the nucleus, and that phosphorylation promotes myristoylation. Myristoylation of phosphorylated C4 enables its interaction with exportin-α to mediate nuclear export of the C4/NbSKη complex and its relocalization to the plasma membrane. [ABSTRACT FROM AUTHOR]

The βC1 protein encoded by the Tomato yellow leaf curl China virus -associated betasatellite functions as a pathogenicity determinant. To better understand the molecular basis whereby βC1 functions in pathogenicity, a yeast two-hybrid screen of a tobacco cDNA library was carried out using βC1 as the bait. The screen revealed that βC1 interacts with a tobacco RING-finger protein designated NtRFP1, which was further confirmed by the bimolecular fluorescence complementation and co-immunoprecipitation assays in Nicotiana benthamiana cells. Expression of NtRFP1 was induced by βC1, and in vitro ubiquitination assays showed that NtRFP1 is a functional E3 ubiquitin ligase that mediates βC1 ubiquitination. In addition, βC1 was shown to be ubiquitinated in vivo and degraded by the plant 26S proteasome. After viral infection, plants overexpressing NtRFP1 developed attenuated symptoms, whereas plants with silenced expression of NtRFP1 showed severe symptoms. Other lines of evidence showed that NtRFP1 attenuates βC1-induced symptoms through promoting its degradation by the 26S proteasome. Taken together, our results suggest that tobacco RING E3 ligase NtRFP1 attenuates disease symptoms by interacting with βC1 to mediate its ubiquitination and degradation via the ubiquitin/26S proteasome system. [ABSTRACT FROM AUTHOR]

It is generally accepted that begomoviruses in the family Geminiviridae encode four proteins (from AC1/C1 to AC4/C4) using the complementary-sense DNA as template. Although AC5/C5 coding sequences are increasingly annotated in databases for many begomoviruses, the evolutionary relationships and functions of this putative protein in viral infection are obscure., Here, we demonstrate several important functions of the AC5 protein of a bipartite begomovirus, Mungbean yellow mosaic India virus ( MYMIV). Mutational analyses and transgenic expression showed that AC5 plays a critical role in MYMIV infection. Ectopic expression of AC5 from a Potato virus X ( PVX) vector resulted in severe mosaic symptoms followed by a hypersensitive-like response in Nicotiana benthamiana., Furthermore, MYMIV AC5 effectively suppressed post-transcriptional gene silencing induced by single-stranded but not double-stranded RNA. AC5 was also able to reverse transcriptional gene silencing of a green fluorescent protein transgene by reducing methylation of promoter sequences, probably through repressing expression of a CHH cytosine methyltransferase ( DOMAINS REARRANGED METHYLTRANSFERASE2) in N. benthamiana., Our results demonstrate that MYMIV AC5 is a pathogenicity determinant and a potent RNA silencing suppressor that employs novel mechanisms to suppress antiviral defenses, and suggest that the AC5 function may be conserved among many begomoviruses. [ABSTRACT FROM AUTHOR]

Soybean is a major legume crop that plays an important role in food production, industrial production, and animal husbandry. Here, we characterize a novel soybean-infecting monopartite geminivirus identified in China. Analysis of the contigs de novo assembled from sequenced small interfering RNAs, followed by PCR, cloning, and sequencing, the complete viral genome was determined to be 2782 nucleotides. The genome contains the conserved nonanucleotide sequence, TAATATTAC and other sequence features typical of the family Geminiviridae, and encodes two and four open reading frames in the virion-sense and the complementary-sense strands, respectively. Genome-wide pairwise identity analysis revealed that the novel virus shares less than 65.6% identity with previously characterized geminiviruses. Phylogenetic and recombination analysis indicated that this virus was placed in a unique taxon within the family Geminiviridae and potentially arose from recombination. An infectious clone of this virus was further constructed and its infectivity was tested in different species of plants. Successful infection and characteristic symptoms were observed in Glycine max, Nicotiana benthamiana, N. tabacum, N. glutinosa, and N. tabacum cv. Samsun plants. Taken together, this virus represents a member of an unclassified genus of the family Geminiviridae, for which the name soybean yellow leaf curl virus is proposed. [ABSTRACT FROM AUTHOR]

Virus-induced gene silencing (VIGS) is currently recognized as a powerful reverse genetics tool for application in functional genomics. DNA1, a satellite-like and single-stranded DNA molecule associated with begomoviruses (Family Geminiviridae), has been shown to replicate autonomously but requires the helper virus for its dissemination. We developed a VIGS vector based on the DNA1 component of tobacco curly shoot virus (TbCSV), a monopartite begomovirus, by inserting a multiple cloning site between the replication-associated protein open reading frame and the A-rich region for subsequent insertion of DNA fragments of genes targeted for silencing. When a host gene ( sulphur, Su) or transgene ( green fluorescent protein, GFP) was inserted into the modified DNA1 vector and co-agroinoculated with TbCSV, efficient silencing of the cognate gene was observed in Nicotiana benthamiana plants. More interestingly, we demonstrated that this modified DNA1 could effectively suppress GFP in transgenic N. benthamiana or endogenous Su in tobacco plants when co-agroinoculated with tomato yellow leaf curl China virus (TYLCCNV), another monopartite begomovirus that does not induce any viral symptoms. A gene-silencing system in Nicotiana spp., Solanum lycopersicum and Petunia hybrida plants was then established using TYLCCNV and the modified DNA1 vector. The system can be used to silence genes involved in meristem and flower development. The modified DNA1 vector was used to silence the AtTOM homologous genes ( NbTOM1 and NbTOM3) in N. benthamiana. Silencing of NbTOM1 or NbTOM3 can reduce tobamovirus multiplication to a lower level, and silencing of both genes simultaneously can completely inhibit tobamovirus multiplication. Previous studies have reported that DNA1 is associated with both monopartite and bipartite begomoviruses, as well as curtoviruses. This vector system can therefore be applied for the study, analysis and discovery of gene function in a variety of important crop plants. [ABSTRACT FROM AUTHOR]

A bi-directional promoter of Tomato yellow leaf curl China virus (TYLCCNV) was obtained with the total DNA from TYLCCNV isolate Y10 infected tobacco leaves as a template. Plant expression vectors were constructed by fusing the amplified DNA fragment with the gus gene and nopaline terminator in different orientations. The vectors containing promoter fragments were transferred into leaf celIs and plant stems of Nicotiana benthamiana by Agrobacterium-mediated method. Transient expression results showed that both the complementary and virion-sense promoters could drive the gus gene to express, and the GUS activity of the complementary-sense promoter was stronger than that of the virion-sense. Co-expression of the vector containing βC1 gene of TYLCCNV DNAβ with the vector containing a bi-directional promoter revealed that the βC1 protein has no impact on expression of either the virion- or the complementary-sense promoter. [ABSTRACT FROM AUTHOR]

Abstract: DNAβ is a satellite molecule associated with some monopartite begomoviruses and encodes a single gene (βC1), which is highly conserved in position and size among DNAβ molecules. A 955nt fragment of Tomato yellow leaf curl China virus (TYLCCNV) DNAβ, upstream of the translation start site of βC1 gene was tested for its promoter activity with gus as a reporter gene. Analysis of β-glucuronidase (GUS) activity following transient expression assays indicated that the 955nt fragment had promoter activity and that 3′-deletions of 399 or 173nt of the fragment resulted in complete loss of its promoter activity. The 5′-deletions of 782 or 556nt of the fragment, however, did not affect its activity. Histochemical staining revealed that this fragment can be used to express gus gene specifically in phloem tissue of stably transformed tobacco plants. Further studies have indicated that a 173nt segment from 3′-end of the 955nt fragment was responsible for basic promoter activity and phloem-specific expression. [Copyright &y& Elsevier]

Tobacco curly shoot virus, a monopartite begomovirus associated with betasatellite, causes serious leaf curl diseases on tomato and tobacco in China. Using single-particle cryo-electron microscopy, we determined the structure of tobacco curly shoot virus (TbCSV) particle at 3.57 Å resolution and confirmed the characteristic geminate architecture with single-strand DNA bound to each coat protein (CP). The CP–CP and DNA–CP interactions, arranged in a CP–DNA–CP pattern at the interface, were partially observed. This suggests the genomic DNA plays an important role in forming a stable interface during assembly of the geminate particle. [ABSTRACT FROM AUTHOR]

The occurrence of geminiviruses causes significant economic losses in many economically important crops. In this study, a novel geminivirus isolated from tobacco in Sichuan province of China, named tomato leaf curl Chuxiong virus (TLCCxV), was characterized by small RNA-based deep sequencing. The full-length of TLCCxV genome was determined to be 2744 nucleotides (nt) encoding six open reading frames. Phylogenetic and genome-wide pairwise identity analysis revealed that TLCCxV shared less than 91% identities with reported geminiviruses. A TLCCxV infectious clone was constructed and successfully infected Nicotiana benthamiana , N. tabacum , N. glutinosa , Solanum lycopersicum and Petunia hybrida plants. Furthermore, expression of the V2, C1 and C4 proteins through a potato virus X vector caused severe chlorosis or necrosis symptom in N. benthamiana. Taken together, we identified a new geminivirus in tobacco plants, and found that V2, C1 and C4 contribute to symptom development. • Geminiviruses can cause devastating diseases in many crops leading to seriously economic losses. • A novel geminivirus named tomato leaf curl Chuxiong virus (TLCCxV) were characterized. • A TLCCxV infectious clone was constructed and V2, C1 and C4 protein are virulence factors. [ABSTRACT FROM AUTHOR]

Abstract: Virus-induced gene silencing (VIGS) in tomato (Lycopersicon esculentum Mill.) is currently routinely analysed using Tobacco rattle virus (TRV)-based vector. We recently reported a new vector system modified from DNAβ (DNAmβ) of Tomato yellow leaf curl China virus (TYLCCNV) for VIGS analysis in Solanaceous species including tomato. Here, we describe DNAmβ-induced gene silencing in tomato. We found that DNAmβ-induced gene silencing was initiated from vascular tissues, and later scattered to other tissues. Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants, was expressed in a variety of tissues and organs including leaf, shoot, stem, flower and fruit, and could be achieved at any growth stage. It was insensitive to temperature as high as 32°C and no symptoms were observed in silenced plants. The DNAmβ vector worked efficiently in at least seven tomato cultivars, indicating that this system has great potential as a versatile VIGS system for routine functional analysis of genes in tomato. [Copyright &y& Elsevier]