Viral genome methylation as an epigenetic defense against geminiviruses

Geminiviruses encapsidate single-stranded DNA genomes that replicate in plant cell nuclei through double-stranded DNA intermediates that associate with cellular histone proteins to form minichromosomes. Like most plant viruses, geminiviruses are targeted by RNA silencing and encode suppressor proteins such as AL2 and L2 to counter this defense. These related proteins can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus, we hypothesized that the viral genome is targeted by small-RNA-directed methylation. Here, we show that Arabidopsis plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed methylation pathway components, or ADK are hypersensitive to geminivirus infection. We also demonstrate that viral DNA and associated histone H3 are methylated in infected plants and that cytosine methylation levels are significantly reduced in viral DNA isolated from methylation-deficient mutants. Finally, we demonstrate that Beet curly top virus L2- mutant DNA present in tissues that have recovered from infection is hypermethylated and that host recovery requires AGO4, a component of the RNA-directed methylation pathway. We propose that plants use chromatin methylation as a defense against DNA viruses, which geminiviruses counter by inhibiting global methylation. In addition, our results establish that geminiviruses can be useful models for genome methylation in plants and suggest that there are redundant pathways leading to cytosine methylation.     

Geminivirus AL2 and L2 proteins interact with and inactivate SNF1 kinase

Geminivirus AL2 and L2 proteins cause enhanced susceptibility, characterized primarily by an increase in viral infectivity, when expressed in transgenic plants. Here, we present genetic and biochemical evidence that enhanced susceptibility is attributable to the interaction of AL2 and L2 with SNF1 kinase, a global regulator of metabolism. Specifically, we show that AL2 and L2 inactivate SNF1 in vitro and in vivo. We further demonstrate that expression of an antisense SNF1 transgene in Nicotiana benthamiana plants causes enhanced susceptibility similar to that conditioned by the AL2 and L2 transgenes, whereas SNF1 overexpression leads to enhanced resistance. Transgenic plants expressing an AL2 protein that lacks a significant portion of the SNF1 interaction domain do not display enhanced susceptibility. Together, these observations suggest that the metabolic alterations mediated by SNF1 are a component of innate antiviral defenses and that SNF1 inactivation by AL2 and L2 is a counterdefensive measure. They also indicate that geminiviruses are able to modify host metabolism to their own advantage, and they provide a molecular link between metabolic status and inherent susceptibility to viral pathogens.     

A Complex Containing SNF1-Related Kinase (SnRK1) and Adenosine Kinase in Arabidopsis

SNF1-related kinase (SnRK1) in plants belongs to a conserved family that includes sucrose non-fermenting 1 kinase (SNF1) in yeast and AMP-activated protein kinase (AMPK) in animals. These kinases play important roles in the regulation of cellular energy homeostasis and in response to stresses that deplete ATP, they inhibit energy consuming anabolic pathways and promote catabolism. Energy stress is sensed by increased AMP:ATP ratios and in plants, 5′-AMP inhibits inactivation of phosphorylated SnRK1 by phosphatase. In previous studies, we showed that geminivirus pathogenicity proteins interact with both SnRK1 and adenosine kinase (ADK), which phosphorylates adenosine to generate 5′-AMP. This suggested a relationship between SnRK1 and ADK, which we investigate in the studies described here. We demonstrate that SnRK1 and ADK physically associate in the cytoplasm, and that SnRK1 stimulates ADK in vitro by an unknown, non-enzymatic mechanism. Further, altering SnRK1 or ADK activity in transgenic plants altered the activity of the other kinase, providing evidence for in vivo linkage but also revealing that in vivo regulation of these activities is complex. This study establishes the existence of SnRK1-ADK complexes that may play important roles in energy homeostasis and cellular responses to biotic and abiotic stress.     

A plant kinase plays roles in defense response against geminivirus by phosphorylation of a viral pathogenesis protein

The plant SNF1-related kinase (SnRK1) is the α-subunit of the SnRK1 heterotrimeric compleses. Although SnRK1 is widely known as a key regulator of plant response to various physiological processes including nutrient- and energy-sensing, regulation of global metabolism, and control of cell cycle, development, as well as abiotics stress, less is known about the function of SnRK1 during pathogen infection. Our previous work has demonstrated that a tomato SNF1-related kinase (SlSnRK1) can interact with and phosphorylate βC1, a pathogenesis protein encoded by tomato yellow leaf curl China betasatellite. Our results also showed that the plant SnRK1 can affect genimivirus infection in plant and reduce viral DNA accumulation. Phosphorylation of βC1 protein negatively impacts its function as a pathogenicity determinant. Here we provide more information on interaction between βC1 and SlSnRK1 and propose a mechanistic model for the SlSnRK1-mediated defense responses against geminiviruses and the potential role of SnRK1 in plant resistance to geminivirus.     

Host GRXC6 restricts Tomato yellow leaf curl virus infection by inhibiting the nuclear export of the V2 protein

Geminiviruses cause serious symptoms and devastating losses in crop plants. With a circular, single-stranded DNA genome, geminiviruses multiply their genomic DNA in the nucleus, requiring the nuclear shuttling of viral proteins and viral genomic DNAs. Many host factors, acting as proviral or antiviral factors, play key roles in geminivirus infections. Here, we report the roles of a tomato glutaredoxin (GRX), SlGRXC6, in the infection of Tomato yellow leaf curl virus (TYLCV), a single-component geminivirus. The V2 protein of TYLCV specifically and preferentially interacts with SlGRXC6 among the 55-member tomato GRX family that are broadly involved in oxidative stress responses, plant development, and pathogen responses. We show that overexpressed SlGRXC6 increases the nuclear accumulation of V2 by inhibiting its nuclear export and, in turn, inhibits trafficking of the V1 protein and viral genomic DNA. Conversely, the silenced expression of SlGRXC6 leads to an enhanced susceptibility to TYLCV. SlGRXC6 is also involved in symptom development as we observed a positive correlation where overexpression of SlGRXC6 promotes while knockdown of SlGRXC6 expression inhibits plant growth. We further showed that SlGRXC6 works with SlNTRC80, a tomato NADPH-dependent thioredoxin reductase, to regulate plant growth. V2 didn’t interact with SlNTRC80 but competed with SlNTR80 for binding to SlGRXC6, suggesting that the V2-disrupted SlGRXC6-SlNTRC80 interaction is partially responsible for the virus-caused symptoms. These results suggest that SlGRXC6 functions as a host restriction factor that inhibits the nuclear trafficking of viral components and point out a new way to control TYLCV infection by targeting the V2-SlGRXC6 interaction.     

Suppression of RNA Silencing by a Plant DNA Virus Satellite Requires a Host Calmodulin-Like Protein to Repress RDR6 Expression

In plants, RNA silencing plays a key role in antiviral defense. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different effector molecules in the RNA silencing pathway. Evidence has shown that plants also encode endogenous suppressors of RNA silencing (ESRs) that function in proper regulation of RNA silencing. The possibility that these cellular proteins can be subverted by viruses to thwart host defense is intriguing but has not been fully explored. Here we report that the Nicotiana benthamiana calmodulin-like protein Nbrgs-CaM is required for the functions of the VSR βC1, the sole protein encoded by the DNA satellite associated with the geminivirus Tomato yellow leaf curl China virus (TYLCCNV). Nbrgs-CaM expression is up-regulated by the βC1. Transgenic plants over-expressing Nbrgs-CaM displayed developmental abnormities reminiscent of βC1-associated morphological alterations. Nbrgs-CaM suppressed RNA silencing in an Agrobacterium infiltration assay and, when over-expressed, blocked TYLCCNV-induced gene silencing. Genetic evidence showed that Nbrgs-CaM mediated the βC1 functions in silencing suppression and symptom modulation, and was required for efficient virus infection. Moreover, the tobacco and tomato orthologs of Nbrgs-CaM also possessed ESR activity, and were induced by betasatellite to promote virus infection in these Solanaceae hosts. We further demonstrated that βC1-induced Nbrgs-CaM suppressed the production of secondary siRNAs, likely through repressing RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) expression. RDR6-deficient N. benthamiana plants were defective in antiviral response and were hypersensitive to TYLCCNV infection. More significantly, TYLCCNV could overcome host range restrictions to infect Arabidopsis thaliana when the plants carried a RDR6 mutation. These findings demonstrate a distinct mechanism of VSR for suppressing PTGS through usurpation of a host ESR, and highlight an essential role for RDR6 in RNA silencing defense response against geminivirus infection.     

烟粉虱传播双生病毒的特性及分子机制研究进展

烟粉虱以持久性、可循回的方式传播双生病毒。烟粉虱传毒历经获毒、持毒和传毒3个阶段,烟粉虱体内的病毒受体、病毒蛋白以及寄主植物因子都参与了这个过程。本文综述了影响烟粉虱特异性传播双生病毒的因素以及二者的直接和间接互作。烟粉虱传播双生病毒的特异性不仅与烟粉虱隐种和病毒种类有关,还与烟粉虱体内特定的器官或细胞、烟粉虱和病毒的蛋白以及烟粉虱体内的共生细菌有关。在烟粉虱和双生病毒的长期共进化中,病毒可以通过调控烟粉虱和寄主植物的特性而促进其自身的传播。     

Interactions between geminivirus replication proteins.

Geminiviruses are small DNA viruses that replicate in the nuclei of infected plant cells. The closely related geminiviruses tomato golden mosaic virus and bean golden mosaic virus each encode a protein, AL1, that catalyzes the initiation of rolling-circle replication. Both viruses also specify a second replication protein, AL3, that greatly enhances the level of viral DNA accumulation. Using recombinant proteins produced in a baculovirus expression system, we showed that AL1 copurifies with a protein fusion of glutathione S-transferase (GST) and AL1, independent of the GST domain. Similarly, authentic AL3 cofractionates with a GST-AL3 fusion protein. These results demonstrated that both AL1 and AL3 form oligomers. Immunoprecipitation of protein extracts from insect cells expressing both AL1 and AL3 showed that the two proteins also complex with each other. None of the protein interactions displayed virus specificity; the tomato and bean golden mosaic virus proteins complexed with each other. The addition of heterologous replication proteins had no effect on the efficiency of geminivirus replication in transient-replication assays, suggesting that heteroprotein complexes might be functional. The significance of these protein interactions is discussed with respect to geminivirus replication in plant cells.     

Geminivirus C3 protein: replication enhancement and protein interactions

Most dicot-infecting geminiviruses encode a replication enhancer protein (C3, AL3, or REn) that is required for optimal replication of their small, single-stranded DNA genomes. C3 interacts with C1, the essential viral replication protein that initiates rolling circle replication. C3 also homo-oligomerizes and interacts with at least two host-encoded proteins, proliferating cell nuclear antigen (PCNA) and the retinoblastoma-related protein (pRBR). It has been proposed that protein interactions contribute to C3 function. Using the C3 protein of Tomato yellow leaf curl virus, we examined the impact of mutations to amino acids that are conserved across the C3 protein family on replication enhancement and protein interactions. Surprisingly, many of the mutations did not affect replication enhancement activity of C3 in tobacco protoplasts. Other mutations either enhanced or were detrimental to C3 replication activity. Analysis of mutated proteins in yeast two-hybrid assays indicated that mutations that inactivate C3 replication enhancement activity also reduce or inactivate C3 oligomerization and interaction with C1 and PCNA. In contrast, mutated C3 proteins impaired for pRBR binding are fully functional in replication assays. Hydrophobic residues in the middle of the C3 protein were implicated in C3 interaction with itself, C1, and PCNA, while polar resides at both the N and C termini of the protein are important for C3-pRBR interaction. These experiments established the importance of C3-C3, C3-C1, and C3-PCNA interactions in geminivirus replication. While C3-pRBR interaction is not required for viral replication in cycling cells, it may play a role during infection of differentiated cells in intact plants.     

A geminivirus replication protein is a sequence-specific DNA binding protein.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. The A component encodes the only viral protein, AL1, that is required for viral replication. We showed that AL1 interacts specifically with TGMV A and B DNA by using an immunoprecipitation assay for AL1:DNA complex formation. In this assay, a monoclonal antibody against AL1 precipitated AL1:TGMV DNA complexes, whereas an unrelated antibody failed to precipitate the complexes. Competition assays with homologous and heterologous DNAs established the specificity of AL1:DNA binding. AL1 produced by transgenic tobacco plants and by baculovirus-infected insect cells exhibited similar DNA binding activity. The AL1 binding site maps to 52 bp on the left side of the common region, a 235-bp region that is highly conserved between the two TGMV genome components. The AL1:DNA binding site does not include the putative hairpin structure that is conserved in the common regions or the equivalent 5' intergenic regions of all geminiviruses. These studies demonstrate that a geminivirus replication protein is a sequence-specific DNA binding protein, and the studies have important implications for the role of this protein in virus replication.     

High-frequency reversion of geminivirus replication protein mutants during infection

The geminivirus replication protein AL1 interacts with retinoblastoma-related protein (RBR), a key regulator of the plant division cell cycle, to induce conditions permissive for viral DNA replication. Previous studies of tomato golden mosaic virus (TGMV) AL1 showed that amino acid L148 in the conserved helix 4 motif is critical for RBR binding. In this work, we examined the effect of an L148V mutation on TGMV replication in tobacco cells and during infection of Nicotiana benthamiana plants. The L148V mutant replicated 100 times less efficiently than wild-type TGMV in protoplasts but produced severe symptoms that were delayed compared to those of wild-type infection in plants. Analysis of progeny viruses revealed that the L148V mutation reverted at 100% frequency in planta to methionine, leucine, isoleucine, or a second-site mutation depending on the valine codon in the initial DNA sequence. Similar results were seen with another geminivirus, cabbage leaf curl virus (CaLCuV), carrying an L145A mutation in the equivalent residue. Valine was the predominant amino acid recovered from N. benthamiana plants inoculated with the CaLCuV L145A mutant, while threonine was the major residue in Arabidopsis thaliana plants. Together, these data demonstrated that there is strong selection for reversion of the TGMV L148V and CaLCuV L145A mutations but that the nature of the selected revertants is influenced by both the viral background and host components. These data also suggested that high mutation rates contribute to the rapid evolution of geminivirus genomes in plants.