ABSTRACT
Positive-strand RNA viruses induce the biogenesis of unique membranous organelles called viral replication organelles (VROs), which perform virus replication in infected cells. Tombusviruses have been shown to rewire cellular trafficking and metabolic pathways, remodel host membranes, and recruit multiple host factors to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely related carnation Italian ringspot virus (CIRV) usurp Rab7 small GTPase to facilitate building VROs in the surrogate host yeast and in plants. Depletion of Rab7 small GTPase, which is needed for late endosome and retromer biogenesis, strongly inhibits TBSV and CIRV replication in yeast and in planta. The viral p33 replication protein interacts with Rab7 small GTPase, which results in the relocalization of Rab7 into the large VROs. Similar to the depletion of Rab7, the deletion of either MON1 or CCZ1 heterodimeric GEFs (guanine nucleotide exchange factors) of Rab7 inhibited TBSV RNA replication in yeast. This suggests that the activated Rab7 has proviral functions. We show that the proviral function of Rab7 is to facilitate the recruitment of the retromer complex and the endosomal sorting nexin-BAR proteins into VROs. We demonstrate that TBSV p33-driven retargeting of Rab7 into VROs results in the delivery of several retromer cargos with proviral functions. These proteins include lipid enzymes, such as Vps34 PI3K (phosphatidylinositol 3-kinase), PI4Kα-like Stt4 phosphatidylinositol 4-kinase, and Psd2 phosphatidylserine decarboxylase. In summary, based on these and previous findings, we propose that subversion of Rab7 into VROs allows tombusviruses to reroute endocytic and recycling trafficking to support virus replication. IMPORTANCE The replication of positive-strand RNA viruses depends on the biogenesis of viral replication organelles (VROs). However, the formation of membranous VROs is not well understood yet. Using tombusviruses and the model host yeast, we discovered that the endosomal Rab7 small GTPase is critical for the formation of VROs. Interaction between Rab7 and the TBSV p33 replication protein leads to the recruitment of Rab7 into VROs. TBSV-driven usurping of Rab7 has proviral functions through facilitating the delivery of the co-opted retromer complex, sorting nexin-BAR proteins, and lipid enzymes into VROs to create an optimal milieu for virus replication. These results open up the possibility that controlling cellular Rab7 activities in infected cells could be a target for new antiviral strategies.
Subject(s)
Nicotiana/virology , Organelles/virology , Saccharomyces cerevisiae/virology , Tombusvirus/physiology , Viral Proteins/metabolism , Virus Replication , rab GTP-Binding Proteins/physiology , 1-Phosphatidylinositol 4-Kinase/metabolism , Endosomes/metabolism , Gene Knockdown Techniques , Guanine Nucleotide Exchange Factors/physiology , Host Microbial Interactions , Organelles/metabolism , Plant Diseases/virology , Protein Binding , Protein Transport , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/physiology , Sorting Nexins/metabolismABSTRACT
Biogenesis of viral replication organelles (VROs) is critical for replication of positive-strand RNA viruses. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely related carnation Italian ringspot virus (CIRV) hijack the retromer to facilitate building VROs in the surrogate host yeast and in plants. Depletion of retromer proteins, which are needed for biogenesis of endosomal tubular transport carriers, strongly inhibits the peroxisome-associated TBSV and the mitochondria-associated CIRV replication in yeast and in planta. In vitro reconstitution revealed the need for the retromer for the full activity of the viral replicase. The viral p33 replication protein interacts with the retromer complex, including Vps26, Vps29, and Vps35. We demonstrate that TBSV p33-driven retargeting of the retromer into VROs results in delivery of critical retromer cargoes, such as 1) Psd2 phosphatidylserine decarboxylase, 2) Vps34 phosphatidylinositol 3-kinase (PI3K), and 3) phosphatidylinositol 4-kinase (PI4Kα-like). The recruitment of these cellular enzymes by the co-opted retromer is critical for de novo production and enrichment of phosphatidylethanolamine phospholipid, phosphatidylinositol-3-phosphate [PI(3)P], and phosphatidylinositol-4-phosphate [PI(4)P] phosphoinositides within the VROs. Co-opting cellular enzymes required for lipid biosynthesis and lipid modifications suggest that tombusviruses could create an optimized lipid/membrane microenvironment for efficient VRO assembly and protection of the viral RNAs during virus replication. We propose that compartmentalization of these lipid enzymes within VROs helps tombusviruses replicate in an efficient milieu. In summary, tombusviruses target a major crossroad in the secretory and recycling pathways via coopting the retromer complex and the tubular endosomal network to build VROs in infected cells.
Subject(s)
Vesicular Transport Proteins/metabolism , Virus Replication/physiology , Class III Phosphatidylinositol 3-Kinases/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Host-Pathogen Interactions/genetics , Lipid Metabolism/physiology , Lipids/physiology , Peroxisomes/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphatidylinositols/metabolism , RNA, Viral/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Tombusvirus/genetics , Tombusvirus/metabolism , Viral Proteins/metabolism , Viral Replication Compartments/metabolism , Viral Replication Compartments/physiologyABSTRACT
Positive-strand RNA [(+)RNA] viruses assemble numerous membrane-bound viral replicase complexes (VRCs) with the help of viral replication proteins and co-opted host proteins within large viral replication compartments in the cytosol of infected cells. In this study, we found that deletion or depletion of Sac1 phosphatidylinositol 4-phosphate [PI(4)P] phosphatase reduced tomato bushy stunt virus (TBSV) replication in yeast (Saccharomyces cerevisiae) and plants. We demonstrate a critical role for Sac1 in TBSV replicase assembly in a cell-free replicase reconstitution assay. The effect of Sac1 seems to be direct, based on its interaction with the TBSV p33 replication protein, its copurification with the tombusvirus replicase, and its presence in the virus-induced membrane contact sites and within the TBSV replication compartment. The proviral functions of Sac1 include manipulation of lipid composition, sterol enrichment within the VRCs, and recruitment of additional host factors into VRCs. Depletion of Sac1 inhibited the recruitment of Rab5 GTPase-positive endosomes and enrichment of phosphatidylethanolamine in the viral replication compartment. We propose that Sac1 might be a component of the assembly hub for VRCs, likely in collaboration with the co-opted the syntaxin18-like Ufe1 SNARE protein within the TBSV replication compartments. This work also led to demonstration of the enrichment of PI(4)P phosphoinositide within the replication compartment. Reduction in the PI(4)P level due to chemical inhibition in plant protoplasts; depletion of two PI(4)P kinases, Stt4p and Pik1p; or sequestration of free PI(4)P via expression of a PI(4)P-binding protein in yeast strongly inhibited TBSV replication. Altogether, Sac1 and PI(4)P play important proviral roles during TBSV replication.IMPORTANCE Replication of positive-strand RNA viruses depends on recruitment of host components into viral replication compartments or organelles. Using TBSV, we uncovered the critical roles of Sac1 PI(4)P phosphatase and its substrate, PI(4)P phosphoinositide, in promoting viral replication. Both Sac1 and PI(4)P are recruited to the site of viral replication to facilitate the assembly of the viral replicase complexes, which perform viral RNA replication. We found that Sac1 affects the recruitment of other host factors and enrichment of phosphatidylethanolamine and sterol lipids within the subverted host membranes to promote optimal viral replication. In summary, this work demonstrates the novel functions of Sac1 and PI(4)P in TBSV replication in the model host yeast and in plants.
Subject(s)
Host-Pathogen Interactions/genetics , Phosphatidylinositol Phosphates/metabolism , Phosphoric Monoester Hydrolases/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Tombusvirus/genetics , Virus Replication/genetics , 1-Phosphatidylinositol 4-Kinase/genetics , 1-Phosphatidylinositol 4-Kinase/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis/virology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Endosomes/metabolism , Gene Expression Regulation , Phosphatidylethanolamines/genetics , Phosphatidylethanolamines/metabolism , Phosphoric Monoester Hydrolases/deficiency , Phosphoric Monoester Hydrolases/metabolism , Plant Cells/metabolism , Plant Cells/virology , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Leaves/virology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protoplasts/metabolism , Qa-SNARE Proteins/genetics , Qa-SNARE Proteins/metabolism , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/virology , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction , Sterols/metabolism , Nicotiana/genetics , Nicotiana/metabolism , Nicotiana/virology , Tombusvirus/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , rab5 GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/metabolismABSTRACT
Bacterial virulence factors or effectors are proteins targeted into host cells to coopt or interfere with cellular proteins and pathways. Viruses often coopt the same cellular proteins and pathways to support their replication in infected cells. Therefore, we screened the Legionella pneumophila effectors to probe virus-host interactions and identify factors that modulate tomato bushy stunt virus (TBSV) replication in yeast surrogate host. Among 302 Legionella effectors tested, 28 effectors affected TBSV replication. To unravel a coopted cellular pathway in TBSV replication, the identified DrrA effector from Legionella was further exploited. We find that expression of DrrA in yeast or plants blocks TBSV replication through inhibiting the recruitment of Rab1 small GTPase and endoplasmic reticulum-derived COPII vesicles into the viral replication compartment. TBSV hijacks Rab1 and COPII vesicles to create enlarged membrane surfaces and optimal lipid composition within the viral replication compartment. To further validate our Legionella effector screen, we used the Legionella effector LepB lipid kinase to confirm the critical proviral function of PI(3)P phosphoinositide and the early endosomal compartment in TBSV replication. We demonstrate the direct inhibitory activity of LegC8 effector on TBSV replication using a cell-free replicase reconstitution assay. LegC8 inhibits the function of eEF1A, a coopted proviral host factor. Altogether, the identified bacterial effectors with anti-TBSV activity could be powerful reagents in cell biology and virus-host interaction studies. This study provides important proof of concept that bacterial effector proteins can be a useful toolbox to identify host factors and cellular pathways coopted by (+)RNA viruses.
Subject(s)
Bacterial Proteins/metabolism , DNA-Binding Proteins/metabolism , Legionella pneumophila/metabolism , Tombusvirus/growth & development , Virulence Factors/metabolism , rab1 GTP-Binding Proteins/metabolism , Agrobacterium tumefaciens/virology , COP-Coated Vesicles/virology , Legionella pneumophila/pathogenicity , Saccharomyces cerevisiae/virology , Nicotiana/virology , Tombusvirus/metabolism , Virus Replication/physiologyABSTRACT
Similar to other (+)RNA viruses, tomato bushy stunt virus (TBSV) utilizes metabolites, lipids, membranes, and co-opted host factors during replication. The coordination of cell metabolism and growth with environmental cues is performed by the target of rapamycin (TOR) kinase in eukaryotic cells. In this paper, we find that TBSV replication partially inhibits TOR activity, likely due to recruitment of glycolytic enzymes to the viral replication compartment, which results in reduced ATP levels in the cytosol. Complete inhibition of TOR activity with rapamycin in yeast or AZD8055 inhibitor in plants reduces tombusvirus replication. We find that high glucose concentration, which stimulates TOR activity, enhanced tombusvirus replication in yeast. Depletion of yeast Sch9 or plant S6K1 kinase, a downstream effector of TOR, also inhibited tombusvirus replication in yeast and plant or the assembly of the viral replicase in vitro. Altogether, the TOR pathway is crucial for TBSV to replicate efficiently in hosts.
Subject(s)
Mechanistic Target of Rapamycin Complex 1/metabolism , Nicotiana/virology , RNA, Viral/biosynthesis , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/virology , Tombusvirus/genetics , Transcription Factors/metabolism , Virus Replication , Adenosine Triphosphate/metabolism , Glycolysis , Host-Pathogen Interactions , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Morpholines/pharmacology , RNA-Dependent RNA Polymerase/metabolism , Ribonucleotides/biosynthesis , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Sirolimus/pharmacology , Nicotiana/drug effects , Nicotiana/metabolism , Tombusvirus/physiology , Transcription Factors/antagonists & inhibitorsABSTRACT
Reconstituted antiviral defense pathway in surrogate host yeast is used as an intracellular probe to further our understanding of virus-host interactions and the role of co-opted host factors in formation of membrane-bound viral replicase complexes in protection of the viral RNA against ribonucleases. The inhibitory effect of the RNA interference (RNAi) machinery of S. castellii, which only consists of the two-component DCR1 and AGO1 genes, was measured against tomato bushy stunt virus (TBSV) in wild type and mutant yeasts. We show that deletion of the co-opted ESCRT-I (endosomal sorting complexes required for transport I) or ESCRT-III factors makes TBSV replication more sensitive to the RNAi machinery in yeast. Moreover, the lack of these pro-viral cellular factors in cell-free extracts (CFEs) used for in vitro assembly of the TBSV replicase results in destruction of dsRNA replication intermediate by a ribonuclease at the 60 min time point when the CFE from wt yeast has provided protection for dsRNA. In addition, we demonstrate that co-opted oxysterol-binding proteins and membrane contact sites, which are involved in enrichment of sterols within the tombusvirus replication compartment, are required for protection of viral dsRNA. We also show that phosphatidylethanolamine level influences the formation of RNAi-resistant replication compartment. In the absence of peroxisomes in pex3Δ yeast, TBSV subverts the ER membranes, which provide as good protection for TBSV dsRNA against RNAi or ribonucleases as the peroxisomal membranes in wt yeast. Altogether, these results demonstrate that co-opted protein factors and usurped lipids are exploited by tombusviruses to build protective subcellular environment against the RNAi machinery and possibly other cellular ribonucleases.
Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , RNA Interference , RNA, Double-Stranded/genetics , RNA, Viral/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/virology , Tombusvirus/genetics , Endosomal Sorting Complexes Required for Transport/genetics , RNA, Double-Stranded/metabolism , RNA, Viral/metabolism , Saccharomyces/genetics , Saccharomyces/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Tombusvirus/physiology , Virus ReplicationABSTRACT
Primary infection of a plant with a pathogen that causes high accumulation of salicylic acid in the plant typically via a hypersensitive response confers enhanced resistance against secondary infection with a broad spectrum of pathogens, including viruses. This phenomenon is called systemic acquired resistance (SAR), which is a plant priming for adaption to repeated biotic stress. However, the molecular mechanisms of SAR-mediated enhanced inhibition, especially of virus infection, remain unclear. Here, we show that SAR against cucumber mosaic virus (CMV) in tobacco plants (Nicotiana tabacum) involves a calmodulin-like protein, rgs-CaM. We previously reported the antiviral function of rgs-CaM, which binds to and directs degradation of viral RNA silencing suppressors (RSSs), including CMV 2b, via autophagy. We found that rgs-CaM-mediated immunity is ineffective against CMV infection in normally growing tobacco plants but is activated as a result of SAR induction via salicylic acid signaling. We then analyzed the effect of overexpression of rgs-CaM on salicylic acid signaling. Overexpressed and ectopically expressed rgs-CaM induced defense reactions, including cell death, generation of reactive oxygen species, and salicylic acid signaling. Further analysis using a combination of the salicylic acid analogue benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and the Ca2+ ionophore A23187 revealed that rgs-CaM functions as an immune receptor that induces salicylic acid signaling by simultaneously perceiving both viral RSS and Ca2+ influx as infection cues, implying its autoactivation. Thus, secondary infection of SAR-induced tobacco plants with CMV seems to be effectively inhibited through 2b recognition and degradation by rgs-CaM, leading to reinforcement of antiviral RNA silencing and other salicylic acid-mediated antiviral responses.IMPORTANCE Even without an acquired immune system like that in vertebrates, plants show enhanced whole-plant resistance against secondary infection with pathogens; this so-called systemic acquired resistance (SAR) has been known for more than half a century and continues to be extensively studied. SAR-induced plants strongly and rapidly express a number of antibiotics and pathogenesis-related proteins targeted against secondary infection, which can account for enhanced resistance against bacterial and fungal pathogens but are not thought to control viral infection. This study showed that enhanced resistance against cucumber mosaic virus is caused by a tobacco calmodulin-like protein, rgs-CaM, which detects and counteracts the major viral virulence factor (RNA silencing suppressor) after SAR induction. rgs-CaM-mediated SAR illustrates the growth versus defense trade-off in plants, as it targets the major virulence factor only under specific biotic stress conditions, thus avoiding the cost of constitutive activation while reducing the damage from virus infection.
Subject(s)
Cucumovirus/growth & development , Immunity, Innate/genetics , Nicotiana/immunology , Nicotiana/virology , Plant Diseases/immunology , Plant Proteins/immunology , Calcimycin/pharmacology , Calcium Ionophores/pharmacology , Cells, Cultured , Cucumovirus/immunology , Gene Expression Regulation, Plant , Plant Diseases/virology , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/immunology , Plants, Genetically Modified/virology , RNA Interference/immunology , Reactive Oxygen Species/metabolism , Salicylic Acid/metabolism , Signal Transduction/immunology , Thiadiazoles/pharmacology , Nicotiana/geneticsABSTRACT
Lunasin is a 44 amino acid peptide with multiple functional domains including an aspartic acid tail, an RGD domain, and a chromatin-binding helical domain. We recently showed that Lunasin induced a phenotype switch of cancer initiating cells (CIC) out of the stem compartment by inducing melanocyte-associated differentiation markers while simultaneously reducing stem-cell-associated transcription factors. In the present study, we advance the hypothesis that Lunasin can reduce pools of melanoma cells with stem cell-like properties, and demonstrate that Lunasin treatment effectively inhibits the invasive potential of CICs in vitro as well as in vivo in a mouse experimental metastasis model. Mice receiving Lunasin treatment had significantly reduced pulmonary colonization after injection of highly metastatic B16-F10 melanoma cells compared to mice in the control group. Mechanistic studies demonstrate that Lunasin reduced activating phosphorylations of the intracellular kinases FAK and AKT as well as reduced histone acetylation of lysine residues in H3 and H4 histones. Using peptides with mutated activity domains, we functionally demonstrated that the RGD domain is necessary for Lunasin uptake and its ability to inhibit oncosphere formation by CICs, thus confirming that Lunasin's ability to affect CICs is at least in part due to the suppression of integrin signaling. Our studies suggest that Lunasin represents a unique anticancer agent that could be developed to help prevent metastasis and patient relapse by reducing the activity of CICs which are known to be resistant to current chemotherapies.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Peptides/pharmacology , Soybean Proteins/pharmacology , Acetylation , Animals , Cell Line, Tumor , Extracellular Signal-Regulated MAP Kinases/metabolism , Focal Adhesion Kinase 1/metabolism , Gene Expression , Histones/metabolism , Humans , Integrins/genetics , Integrins/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/secondary , Melanoma/drug therapy , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Melanoma, Experimental , Mice , Peptides/chemistry , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins c-akt/metabolism , Retinal Dehydrogenase/metabolism , Signal Transduction/drug effects , Soybean Proteins/chemistryABSTRACT
Plant viral symptoms are rarely explained by direct molecular interaction between a viral protein and a host factor, but rather understood as a consequence of arms race between host RNA silencing and viral silencing suppressors. However, we have recently demonstrated that the 2b protein (2b) of Cucumber mosaic virus (CMV) HL strain could bind to Arabidopsis catalase that is important in scavenging cellular hydrogen peroxide, leading to the induction of distinct necrosis on Arabidopsis. Because we previously used virulent strains of subgroup I CMV in the study, we here further analyzed mild strains of subgroup II CMV, which share 70 to 80% sequence homology with subgroup I, to understand whether the necrosis induction is a general phenomenon to compromise host defense system mediated by catalase in the pathosystem of any CMV strains and Arabidopsis. Based on the results, we concluded that 2bs of subgroup II could also bind to catalase, resulting in decrease in catalase activity and weak necrosis on Arabidopsis. Because the 2b-catalase interaction did not prevent CMVs from spreading, it may eventually operate in favor of CMV.
Subject(s)
Arabidopsis/virology , Cucumovirus/physiology , Viral Proteins/physiology , Cucumovirus/pathogenicity , VirulenceABSTRACT
Nucleotide-sequence-specific interactions mediated by double-stranded RNA (dsRNA) can induce gene silencing. Gene silencing through transcriptional repression can be induced by dsRNA targeted to a gene promoter. However, until recently, no plant has been produced that harbors an endogenous gene that remains silenced in the absence of promoter-targeting dsRNA. We have reported for the first time that transcriptional gene silencing can be induced by targeting dsRNA to the endogenous gene promoters in petunia and tomato plants, using a Cucumber mosaic virus (CMV)-based vector and that the induced gene silencing is heritable. Efficient silencing depended on the function of the 2b protein encoded in the vector, which facilitates epigenetic modifications through the transport of short interfering RNA (siRNA) to the nucleus. Here we show that gene silencing that is mediated by targeting dsRNA to a gene promoter via the CMV vector can be as strong as co-suppression in terms of both the extent of mRNA decrease and phenotypic changes. We also show that the expression of genes involved in RNA-directed DNA methylation and in demethylation are upregulated and downregulated, respectively, in Arabidopsis plants infected with CMV. Thus, along with the function of the 2b protein, that transports siRNA to the nucleus, the promoter-targeted silencing system using the CMV vector has some property that facilitates heritable epigenetic changes on endogenous genes, enabling the production of a novel class of modified plants that do not have a transgene.
Subject(s)
Epigenesis, Genetic , Gene Expression Regulation, Plant , Gene Silencing , Nicotiana/genetics , Petunia/genetics , RNA, Double-Stranded/genetics , Cucumovirus/genetics , DNA Methylation , Genetic Vectors , Plants, Genetically Modified/genetics , Promoter Regions, Genetic , Transgenes , Viral Proteins/geneticsABSTRACT
Many plant host factors are known to interact with viral proteins during pathogenesis, but how a plant virus induces a specific disease symptom still needs further research. A lily strain of Cucumber mosaic virus (CMV-HL) can induce discrete necrotic spots on infected Arabidopsis (Arabidopsis thaliana) plants; other CMV strains can induce similar spots, but they are not as distinct as those induced by CMV-HL. The CMV 2b protein (2b), a known RNA-silencing suppressor, is involved in viral movement and symptom induction. Using in situ proximity ligation assay immunostaining and the protoplast assays, we report here that CMV 2b interacts directly with Catalase3 (CAT3) in infected tissues, a key enzyme in the breakdown of toxic hydrogen peroxide. Interestingly, CAT3, normally localized in the cytoplasm (glyoxysome), was recruited to the nucleus by an interaction between 2b and CAT3. Although overexpression of CAT3 in transgenic plants decreased the accumulation of CMV and delayed viral symptom development to some extent, 2b seems to neutralize the cellular catalase contributing to the host defense response, thus favoring viral infection. Our results thus provide evidence that, in addition to altering the type of symptom by disturbing microRNA pathways, 2b can directly bind to a host factor that is important in scavenging cellular hydrogen peroxide and thus interfere specifically with that host factor, leading to the induction of a specific necrosis.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Arabidopsis/virology , Catalase/metabolism , Cucumovirus/metabolism , Host-Pathogen Interactions , RNA Interference , Viral Proteins/metabolism , Arabidopsis/enzymology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Catalase/genetics , Cell Nucleus/enzymology , Disease Resistance/immunology , Gene Expression Regulation, Plant , Hydrogen Peroxide/metabolism , Necrosis , Plant Diseases/immunology , Plant Diseases/virology , Plant Leaves/cytology , Plant Leaves/metabolism , Plant Leaves/virology , Plants, Genetically Modified , Protein Binding , Protein Transport , Protoplasts/cytology , Protoplasts/metabolism , Protoplasts/virology , Two-Hybrid System TechniquesABSTRACT
Symptoms on virus-infected plants are often very specific to the given virus. The molecular mechanisms involved in viral symptom induction have been extensively studied, but are still poorly understood. Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is a non-coding subviral RNA and modifies the typical symptom induced by CMV in specific hosts; Y-sat causes a bright yellow mosaic on its natural host Nicotiana tabacum. The Y-sat-induced yellow mosaic failed to develop in the infected Arabidopsis and tomato plants suggesting a very specific interaction between Y-sat and its host. In this study, we revealed that Y-sat produces specific short interfering RNAs (siRNAs), which interfere with a host gene, thus inducing the specific symptom. We found that the mRNA of tobacco magnesium protoporphyrin chelatase subunit I (ChlI, the key gene involved in chlorophyll synthesis) had a 22-nt sequence that was complementary to the Y-sat sequence, including four G-U pairs, and that the Y-sat-derived siRNAs in the virus-infected plant downregulate the mRNA of ChlI by targeting the complementary sequence. ChlI mRNA was also downregulated in the transgenic lines that express Y-sat inverted repeats. Strikingly, modifying the Y-sat sequence in order to restore the 22-nt complementarity to Arabidopsis and tomato ChlI mRNA resulted in yellowing symptoms in Y-sat-infected Arabidopsis and tomato, respectively. In 5'-RACE experiments, the ChlI transcript was cleaved at the expected middle position of the 22-nt complementary sequence. In GFP sensor experiments using agroinfiltration, we further demonstrated that Y-sat specifically targeted the sensor mRNA containing the 22-nt complementary sequence of ChlI. Our findings provide direct evidence that the identified siRNAs derived from viral satellite RNA directly modulate the viral disease symptom by RNA silencing-based regulation of a host gene.
Subject(s)
Chlorophyll/biosynthesis , Cucumber Mosaic Virus Satellite/genetics , Nicotiana/virology , Plant Diseases/virology , RNA Interference , RNA, Viral/genetics , Arabidopsis/genetics , Arabidopsis/virology , Base Sequence , Capsicum/genetics , Capsicum/virology , Chlorophyll/genetics , Cucumber Mosaic Virus Satellite/metabolism , Cucumovirus/metabolism , Cucumovirus/pathogenicity , Down-Regulation , Gene Expression Regulation, Plant , Genes, Plant , Host-Pathogen Interactions , Lyases/genetics , Lyases/metabolism , Solanum lycopersicum/genetics , Solanum lycopersicum/virology , Molecular Sequence Data , Phenotype , Plant Diseases/genetics , Plants, Genetically Modified/enzymology , Plants, Genetically Modified/genetics , Plants, Genetically Modified/virology , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA, Viral/metabolism , Nicotiana/enzymology , Nicotiana/geneticsABSTRACT
Gene silencing through transcriptional repression can be induced by targeting double-stranded RNA (dsRNA) to a gene promoter. It has been reported that a transgene was silenced by targeting dsRNA to the promoter, and the silenced state was inherited to the progeny plant even after removal of the silencing inducer from cells. In contrast, no plant has been produced that harbors silenced endogenous gene after removal of promoter-targeting dsRNA. Here, we show that heritable gene silencing can be induced by targeting dsRNA to the endogenous gene promoters in petunia and tomato plants, using the Cucumber mosaic virus (CMV)-based vector. We found that efficient silencing of endogenous genes depends on the function of the 2b protein encoded in the vector virus, which has the ability to facilitate epigenetic modifications through the transport of short interfering RNA to nucleus. Bisulfite sequencing analyses on the targeted promoter in the virus-infected and its progeny plants revealed that cytosine methylation was found not only at CG or CNG but also at CNN sites. The observed inheritance of asymmetric DNA methylation is quite unique, suggesting that plants have a mechanism to maintain even asymmetric methylation. This CMV-based gene silencing system provides a useful tool to artificially modify DNA methylation in plant genomes and elucidate the mechanism for epigenetic controls.
Subject(s)
Cucumovirus/genetics , Gene Silencing/physiology , Genetic Vectors/genetics , Plants, Genetically Modified/metabolism , DNA Methylation , Flowers/genetics , Flowers/metabolism , Flowers/physiology , Petunia/genetics , Petunia/metabolism , Petunia/physiology , Plants, Genetically Modified/genetics , Plants, Genetically Modified/physiology , Pollen/genetics , Pollen/metabolism , Pollen/physiology , RNA, Double-Stranded/genetics , RNA, Small Interfering/geneticsABSTRACT
The RNA silencing suppressor 2b protein of Cucumber mosaic virus (CMV) is difficult to produce in Escherichia coli. We compared two CMV 2b proteins that differ in their toxicity against E. coli and found that the acidic amino acid residues in the C-terminal significantly affected the toxicity and expression level of the protein in E. coli. In addition, in a DNA-binding assay, 2b had the ability to bind to DNA, and this ability was affected by the charge on the C-terminal residues of 2b. We concluded that the C-terminal residues were important for 2b's DNA-binding ability, which may partly explain the toxicity of the protein.