N2-Acetylornithine deacetylase functions as a Cys-Gly dipeptidase in the cytosolic glutathione degradation pathway in Arabidopsis thaliana.

Glutathione (GSH) is required for various physiological processes in plants, including redox regulation and detoxification of harmful compounds. GSH also functions as a repository for assimilated sulfur and is actively catabolized in plants. In Arabidopsis, GSH is mainly degraded initially by cytosolic enzymes, γ-glutamyl cyclotransferase, and γ-glutamyl peptidase, which release cysteinylglycine (Cys-Gly). However, the subsequent enzyme responsible for catabolizing this dipeptide has not been identified to date. In the present study, we identified At4g17830 as a Cys-Gly dipeptidase, namely cysteinylglycine peptidase 1 (CGP1). CGP1 complemented the phenotype of the yeast mutant that cannot degrade Cys-Gly. The Arabidopsis cgp1 mutant had lower Cys-Gly degradation activity than the wild type and showed perturbed concentrations of thiol compounds. Recombinant CGP1 showed reasonable Cys-Gly degradation activity in vitro. Metabolomic analysis revealed that cgp1 exhibited signs of severe sulfur deficiency, such as elevated accumulation of O-acetylserine (OAS) and the decrease in sulfur-containing metabolites. Morphological changes observed in cgp1, including longer primary roots of germinating seeds, were also likely associated with sulfur starvation. Notably, At4g17830 has previously been reported to encode an N2-acetylornithine deacetylase (NAOD) that functions in the ornithine biosynthesis. The cgp1 mutant did not show a decrease in ornithine content, whereas the analysis of CGP1 structure did not rule out the possibility that CGP1 has Cys-Gly dipeptidase and NAOD activities. Therefore, we propose that CGP1 is a Cys-Gly dipeptidase that functions in the cytosolic GSH degradation pathway and may play dual roles in GSH and ornithine metabolism.

Glutathione degradation activity of γ-glutamyl peptidase 1 manifests its dual roles in primary and secondary sulfur metabolism in Arabidopsis.

Glutathione (GSH) functions as a major sulfur repository and hence occupies an important position in primary sulfur metabolism. GSH degradation results in sulfur reallocation and is believed to be carried out mainly by γ-glutamyl cyclotransferases (GGCT2;1, GGCT2;2, and GGCT2;3), which, however, do not fully explain the rapid GSH turnover. Here, we discovered that γ-glutamyl peptidase 1 (GGP1) contributes to GSH degradation through a yeast complementation assay. Recombinant proteins of GGP1, as well as GGP3, showed high degradation activity of GSH, but not of oxidized glutathione (GSSG), in vitro. Notably, the GGP1 transcripts were highly abundant in rosette leaves, in agreement with the ggp1 mutants constantly accumulating more GSH regardless of nutritional conditions. Given the lower energy requirements of the GGP- than the GGCT-mediated pathway, the GGP-mediated pathway could be a more efficient route for GSH degradation than the GGCT-mediated pathway. Therefore, we propose a model wherein cytosolic GSH is degraded chiefly by GGP1 and likely also by GGP3. Another noteworthy fact is that GGPs are known to process GSH conjugates in glucosinolate and camalexin synthesis; indeed, we confirmed that the ggp1 mutant contained higher levels of O-acetyl-l-Ser, a signaling molecule for sulfur starvation, and lower levels of glucosinolates and their degradation products. The predicted structure of GGP1 further provided a rationale for this hypothesis. In conclusion, we suggest that GGP1 and possibly GGP3 play vital roles in both primary and secondary sulfur metabolism.

Tombusviruses Target a Major Crossroad in the Endocytic and Recycling Pathways via Co-opting Rab7 Small GTPase.

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.

Excessive ammonium assimilation by plastidic glutamine synthetase causes ammonium toxicity in Arabidopsis thaliana.

Plants use nitrate, ammonium, and organic nitrogen in the soil as nitrogen sources. Since the elevated CO2 environment predicted for the near future will reduce nitrate utilization by C3 species, ammonium is attracting great interest. However, abundant ammonium nutrition impairs growth, i.e., ammonium toxicity, the primary cause of which remains to be determined. Here, we show that ammonium assimilation by GLUTAMINE SYNTHETASE 2 (GLN2) localized in the plastid rather than ammonium accumulation is a primary cause for toxicity, which challenges the textbook knowledge. With exposure to toxic levels of ammonium, the shoot GLN2 reaction produced an abundance of protons within cells, thereby elevating shoot acidity and stimulating expression of acidic stress-responsive genes. Application of an alkaline ammonia solution to the ammonium medium efficiently alleviated the ammonium toxicity with a concomitant reduction in shoot acidity. Consequently, we conclude that a primary cause of ammonium toxicity is acidic stress.

The retromer is co-opted to deliver lipid enzymes for the biogenesis of lipid-enriched tombusviral replication organelles.

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.

Identification of a Unique Type of Isoflavone O-Methyltransferase, GmIOMT1, Based on Multi-Omics Analysis of Soybean under Biotic Stress.

Isoflavonoids are commonly found in leguminous plants. Glycitein is one of the isoflavones produced by soybean. The genes encoding the enzymes in the isoflavone biosynthetic pathway have mostly been identified and characterized. However, the gene(s) for isoflavone O-methyltransferase (IOMT), which catalyzes the last step of glycitein biosynthesis, has not yet been identified. In this study, we conducted multi-omics analyses of fungal-inoculated soybean and indicated that glycitein biosynthesis was induced in response to biotic stress. Moreover, we identified a unique type of IOMT, which participates in glycitein biosynthesis. Soybean seedlings were inoculated with Aspergillus oryzae or Rhizopus oligosporus and sampled daily for 8 d. Multi-omics analyses were conducted using liquid chromatography-tandem mass spectrometry and RNA sequencing. Metabolome analysis revealed that glycitein derivatives increased following fungal inoculation. Transcriptome co-expression analysis identified two candidate IOMTs that were co-expressed with the gene encoding flavonoid 6-hydroxylase (F6H), the key enzyme in glycitein biosynthesis. The enzymatic assay of the two IOMTs using respective recombinant proteins showed that one IOMT, named as GmIOMT1, produced glycitein. Unlike other IOMTs, GmIOMT1 belongs to the cation-dependent OMT family and exhibited the highest activity with Zn2+ among cations tested. Moreover, we demonstrated that GmIOMT1 overexpression increased the levels of glycitein derivatives in soybean hairy roots when F6H was co-expressed. These results strongly suggest that GmIOMT1 participates in inducing glycitein biosynthesis in response to biotic stress.

Co-opted Cellular Sac1 Lipid Phosphatase and PI(4)P Phosphoinositide Are Key Host Factors during the Biogenesis of the Tombusvirus Replication Compartment.

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.

Screening Legionella effectors for antiviral effects reveals Rab1 GTPase as a proviral factor coopted for tombusvirus replication.

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.

Tombusvirus RNA replication depends on the TOR pathway in yeast and plants.

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.

Repression of Nitrogen Starvation Responses by Members of the Arabidopsis GARP-Type Transcription Factor NIGT1/HRS1 Subfamily.

Nitrogen (N) is often a limiting nutrient whose availability determines plant growth and productivity. Because its availability is often low and/or not uniform over time and space in nature, plants respond to variations in N availability by altering uptake and recycling mechanisms, but the molecular mechanisms underlying how these responses are regulated are poorly understood. Here, we show that a group of GARP G2-like transcription factors, Arabidopsis thaliana NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR1/HYPERSENSITIVE TO LOW Pi-ELICITED PRIMARY ROOT SHORTENING1 proteins (NIGT1/HRS1s), are factors that bind to the promoter of the N starvation marker NRT2.4 and repress an array of N starvation-responsive genes under conditions of high N availability. Transient assays and expression analysis demonstrated that NIGT1/HRS1s are transcriptional repressors whose expression is regulated by N availability. We identified target genes of the NIGT1/HRS1s by genome-wide transcriptome analyses and found that they are significantly enriched in N starvation response-related genes, including N acquisition, recycling, remobilization, and signaling genes. Loss of NIGT1/HRS1s resulted in deregulation of N acquisition and accumulation. We propose that NIGT1/HRS1s are major regulators of N starvation responses that play an important role in optimizing N acquisition and utilization under fluctuating N conditions.

The role of co-opted ESCRT proteins and lipid factors in protection of tombusviral double-stranded RNA replication intermediate against reconstituted RNAi in yeast.

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.

rgs-CaM Detects and Counteracts Viral RNA Silencing Suppressors in Plant Immune Priming.

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.

The soy-derived peptide Lunasin inhibits invasive potential of melanoma initiating cells.

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.

The 2b proteins of Cucumber mosaic virus generally have the potential to differentially induce necrosis on Arabidopsis.

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.

RNA-mediated epigenetic modifications of an endogenous gene targeted by a viral vector: a potent gene silencing system to produce a plant that does not carry a transgene but has altered traits.

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.

Virus-induced necrosis is a consequence of direct protein-protein interaction between a viral RNA-silencing suppressor and a host catalase.

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.

A viral satellite RNA induces yellow symptoms on tobacco by targeting a gene involved in chlorophyll biosynthesis using the RNA silencing machinery.

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.

Virus-mediated efficient induction of epigenetic modifications of endogenous genes with phenotypic changes in plants.

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.

The C-terminal residues of the 2b protein of Cucumber mosaic virus are important for efficient expression in Escherichia coli and DNA-binding.

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.