A New Tymovirus Isolated From Solanum quitoense: Characterization and Prevalence in Two Solanaceous Crops in Ecuador.

Naranjilla (Solanum quitoense Lam.) and tamarillo (S. betaceum Cav.) are two important perennial solanaceous crops grown in Ecuador for the fresh market and juice production. Viruses infecting tamarillo and naranjilla are currently poorly studied, and no clean stock program exists in Ecuador. Here, we report a new virus, provisionally named as naranjilla mild mosaic virus (NarMMV) (genus Tymovirus, family Tymoviridae), isolated from naranjilla grown in an orchard in Pichincha Province, Ecuador. The complete genome of the virus consists of 6,348 nucleotides and encodes three open reading frames typical for members of the genus Tymovirus. Phylogenetically, Chiltepin yellow mosaic virus, Eggplant mosaic virus, and the recently characterized naranjilla chlorotic mosaic virus (NarCMV) were found to be the closest relatives of NarMMV. Unlike NarCMV, the new virus induced mild mosaic in naranjilla and more severe symptoms in tamarillo. Similar to NarCMV, NarMMV was unable to systemically infect potato. Virus surveys found NarMMV prevalent in naranjilla production areas of two provinces of Ecuador, especially where hybrid cultivars of naranjilla were cultivated. NarMMV was also found in field-grown tamarillo. The new virus cross-reacted with antibodies developed against NarCMV. Hence, this antibody will be useful for its field diagnosis using enzyme-linked immunosorbent assay or immunocapture reverse transcription polymerase chain reaction in future virus-free certification programs.

Live Cell Imaging Reveals the Relocation of dsRNA Binding Proteins Upon Viral Infection.

Viral infection triggers a range of plant responses such as the activation of the RNA interference (RNAi) pathway. The double-stranded RNA binding (DRB) proteins DRB3 and DRB4 are part of this pathway and aid in defending against DNA and RNA viruses, respectively. Using live cell imaging, we show that DRB2, DRB3, and DRB5 relocate from their uniform cytoplasmic distribution to concentrated accumulation in nascent viral replication complexes (VRC) that develop following cell invasion by viral RNA. Inactivation of the DRB3 gene in Arabidopsis by T-DNA insertion rendered these plants less able to repress RNA viral replication. We propose a model for the early stages of virus defense in which DRB2, DRB3, and DRB5 are invasion sensors that relocate to nascent VRC, where they bind to viral RNA and inhibit virus replication.

Multiple virus resistance using artificial trans-acting siRNAs.

Plant TAS gene encoded trans-acting siRNAs (ta-siRNAs) regulate the expression of target mRNAs by guiding their cleavage at the sequence complementary region as microRNAs. Since one TAS transcript is cleaved into multiple ta-siRNAs in a phased manner, TAS genes may be engineered to express multiple artificial ta-siRNAs (ata-siRNAs) that target multiple viruses at several distinct genomic positions. To test this hypothesis, the Arabidopsis TAS3a gene was engineered to express ata-siRNAs targeting the genome of Turnip mosaic virus (TuMV) and Cucumber mosaic virus (CMV). Transgenic Arabidopsis thaliana plants expressing these ata-siRNAs showed high level of resistance to both viruses. These results suggest that plant TAS genes can be modified to express artificial ta-siRNAs to confer multiple virus resistance and could have broad applications for future development in virus resistance strategies.

Plants Encode a General siRNA Suppressor That Is Induced and Suppressed by Viruses.

Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant-virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses.

The multifunctional protein CI of potyviruses plays interlinked and distinct roles in viral genome replication and intercellular movement.

BACKGROUND: The multifunctional cylindrical inclusion (CI) protein of potyviruses contains ATP binding and RNA helicase activities. As part of the viral replication complex, it assists viral genome replication, possibly by binding to RNA and unwinding the RNA duplex. It also functions in viral cell-to-cell movement, likely via the formation of conical structures at plasmodesmata (PD) and the interaction with coat protein (CP). METHODS: To further understand the role of CI in the viral infection process, we employed the alanine-scanning mutagenesis approach to mutate CI in the infectious full-length cDNA clone of Turnip mosaic virus (TuMV) tagged by green fluorescent protein. A total of 40 double-substitutions were made at the clustered charged residues. The effect of these mutations on viral genome amplification was determined using a protoplast inoculation assay. All the mutants were also introduced into Nicotiana benthamiana plants to assess their cell-to-cell and long-distance movement. Three cell-to-cell movement-abolished mutants were randomly selected to determine if their mutated CI protein targets PD and interacts with CP by confocal microscopy. RESULTS: Twenty CI mutants were replication-defective (5 abolished and 15 reduced), one produced an elevated level of viral genome in comparison with the parental virus, and the remaining 19 retained the same replication level as the parental virus. The replication-defective mutations were predominately located in the helicase domains and C-terminal region. All 15 replication-reduced mutants showed delayed or abolished cell-to-cell movement. Nine of 20 replication-competent mutants contained infection within single cells. Five of them distributed mutations within the N-terminal 100 amino acids. Most of replication-defective or cell-to-cell movement-abolished mutants failed to infect plants systemically. Analysis of three randomly selected replication-competent yet cell-to-cell movement-abolished mutants revealed that the mutated CI failed to form regular punctate structures at PD and/or to interact with CP. CONCLUSIONS: The helicase domain and C-terminal region of TuMV CI are essential for viral genome replication, and the N-terminal sequence modulates viral cell-to-cell movement. TuMV CI plays both interlinked and distinct roles in replication and intercellular movement. The ability of CI to target PD and interact with CP is associated with its functional role in viral cell-to-cell movement.

Mutational bias of Turnip Yellow Mosaic Virus in the context of host anti-viral gene silencing.

Plant Dicer-like (DCL) enzymes exhibit a GC-preference during anti-viral post-transcriptional gene silencing (PTGS), delivering an evolutionary selection pressure resulting in plant viruses with GC-poor genomes. However, some viruses, e.g. Turnip Yellow Mosaic Virus (TYMV, genus Tymovirus) have GC-rich genomes, raising the question as to whether or not DCL derived selection pressure affects these viruses. In this study we analyzed the virus-derived small interfering RNAs from TYMV-infected leaves of Brassica juncea showed that the TYMV population accumulated a mutational bias with AU replacing GC (GC-AU), demonstrating PTGS pressure. Interestingly, at the highly polymorphic sites the GC-AU bias was no longer observed. This suggests the presence of an unknown mechanism preventing mutational drift of the viral population and maintaining viral genome stability, despite the host PTGS pressure.

The Multiplicity of Cellular Infection Changes Depending on the Route of Cell Infection in a Plant Virus.

UNLABELLED: The multiplicity of cellular infection (MOI) is the number of virus genomes of a given virus species that infect individual cells. This parameter chiefly impacts the severity of within-host population bottlenecks as well as the intensity of genetic exchange, competition, and complementation among viral genotypes. Only a few formal estimations of the MOI currently are available, and most theoretical reports have considered this parameter as constant within the infected host. Nevertheless, the colonization of a multicellular host is a complex process during which the MOI may dramatically change in different organs and at different stages of the infection. We have used both qualitative and quantitative approaches to analyze the MOI during the colonization of turnip plants by Turnip mosaic virus. Remarkably, different MOIs were observed at two phases of the systemic infection of a leaf. The MOI was very low in primary infections from virus circulating within the vasculature, generally leading to primary foci founded by a single genome. Each lineage then moved from cell to cell at a very high MOI. Despite this elevated MOI during cell-to-cell progression, coinfection of cells by lineages originating in different primary foci is severely limited by the rapid onset of a mechanism inhibiting secondary infection. Thus, our results unveil an intriguing colonization pattern where individual viral genomes initiate distinct lineages within a leaf. Kin genomes then massively coinfect cells, but coinfection by two distinct lineages is strictly limited. IMPORTANCE: The MOI is the size of the viral population colonizing cells and defines major phenomena in virus evolution, like the intensity of genetic exchange and the size of within-host population bottlenecks. However, few studies have quantified the MOI, and most consider this parameter as constant during infection. Our results reveal that the MOI can depend largely on the route of cell infection in a systemically infected leaf. The MOI is usually one genome per cell when cells are infected from virus particles moving long distances in the vasculature, whereas it is much higher during subsequent cell-to-cell movement in mesophyll. However, a fast-acting superinfection exclusion prevents cell coinfection by merging populations originating from different primary foci within a leaf. This complex colonization pattern results in a situation where within-cell interactions are occurring almost exclusively among kin and explains the common but uncharacterized phenomenon of genotype spatial segregation in infected plants.

Genome-wide identification of turnip mosaic virus-responsive microRNAs in non-heading Chinese cabbage by high-throughput sequencing.

Turnip mosaic virus (TuMV) is the most prevalent viral pathogen infecting most cruciferous plants. MicroRNAs (miRNAs) are around 22 nucleotides long non-protein-coding RNAs that play key regulatory roles in plants. Recent research findings show that miRNAs are involved in plant-virus interaction. However we know little about plant defense and viral offense system networks throughout microRNA regulation pathway. In this study, two small RNA libraries were constructed based on non-heading Chinese cabbage (Brassica campestris ssp. chinensis L. Makino, NHCC) leaves infected by TuMV and healthy leaves, and sequenced using the Illumina-Solexa high-throughput sequencing technology. A total of 86 conserved miRNAs belonging to 25 known miRNA families and 45 novel ones were identified. Among them, twelve conserved and ten new miRNAs were validated by real-time fluorescence quantitative PCR (qPCR). Differential expression analysis showed that 42 miRNAs were down-regulated and 27 miRNAs were up-regulated in response to TuMV stress. A total of 271 target genes were predicted using a bioinformatics approach, these genes are mainly involved in growth and resistance to various stresses. We further selected 13 miRNAs and their corresponding target genes to explore their expression pattern under TuMV and/or cold (4°C) stresses, and the results indicated that some of the identified miRNAs could link TuMV response with cold response of NHCC. The characterization of these miRNAs could contribute to a better understanding of plant-virus interaction throughout microRNA regulation pathway. This can lead to finding new approach to defend virus infection using miRNA in Chinese cabbage.

Replication and packaging of Turnip yellow mosaic virus RNA containing Flock house virus RNA1 sequence.

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. In this study, RNA1 sequence of Flock house virus (FHV) was inserted into the TYMV genome to test whether TYMV can accommodate and express another viral entity. In the resulting construct, designated TY-FHV, the FHV RNA1 sequence was expressed as a TYMV subgenomic RNA. Northern analysis of the Nicotiana benthamiana leaves agroinfiltrated with the TY-FHV showed that both genomic and subgenomic FHV RNAs were abundantly produced. This indicates that the FHV RNA1 sequence was correctly expressed and translated to produce a functional FHV replicase. Although these FHV RNAs were not encapsidated, the FHV RNA having a TYMV CP sequence at the 3'-end was efficiently encapsidated. When an eGFP gene was inserted into the B2 ORF of the FHV sequence, a fusion protein of B2-eGFP was produced as expected.

Contribution of host intracellular transport machineries to intercellular movement of turnip mosaic virus.

The contribution of different host cell transport systems in the intercellular movement of turnip mosaic virus (TuMV) was investigated. To discriminate between primary infections and secondary infections associated with the virus intercellular movement, a gene cassette expressing GFP-HDEL was inserted adjacent to a TuMV infectious cassette expressing 6K2:mCherry, both within the T-DNA borders of the binary vector pCambia. In this system, both gene cassettes were delivered to the same cell by a single binary vector and primary infection foci emitted green and red fluorescence while secondarily infected cells emitted only red fluorescence. Intercellular movement was measured at 72 hours post infiltration and was estimated to proceed at an average rate of one cell being infected every three hours over an observation period of 17 hours. To determine if the secretory pathway were important for TuMV intercellular movement, chemical and protein inhibitors that blocked both early and late secretory pathways were used. Treatment with Brefeldin A or Concanamycin A or expression of ARF1 or RAB-E1d dominant negative mutants, all of which inhibit pre- or post-Golgi transport, reduced intercellular movement by the virus. These treatments, however, did not inhibit virus replication in primary infected cells. Pharmacological interference assays using Tyrphostin A23 or Wortmannin showed that endocytosis was not important for TuMV intercellular movement. Lack of co-localization by endocytosed FM4-64 and Ara7 (AtRabF2b) with TuMV-induced 6K2-tagged vesicles further supported this conclusion. Microfilament depolymerizing drugs and silencing expression of myosin XI-2 gene, but not myosin VIII genes, also inhibited TuMV intercellular movement. Expression of dominant negative myosin mutants confirmed the role played by myosin XI-2 as well as by myosin XI-K in TuMV intercellular movement. Using this dual gene cassette expression system and transport inhibitors, components of the secretory and actomyosin machinery were shown to be important for TuMV intercellular spread.

A compact viral processing proteinase/ubiquitin hydrolase from the OTU family.

Turnip yellow mosaic virus (TYMV)--a member of the alphavirus-like supergroup of viruses--serves as a model system for positive-stranded RNA virus membrane-bound replication. TYMV encodes a precursor replication polyprotein that is processed by the endoproteolytic activity of its internal cysteine proteinase domain (PRO). We recently reported that PRO is actually a multifunctional enzyme with a specific ubiquitin hydrolase (DUB) activity that contributes to viral infectivity. Here, we report the crystal structure of the 150-residue PRO. Strikingly, PRO displays no homology to other processing proteinases from positive-stranded RNA viruses, including that of alphaviruses. Instead, the closest structural homologs of PRO are DUBs from the Ovarian tumor (OTU) family. In the crystal, one molecule's C-terminus inserts into the catalytic cleft of the next, providing a view of the N-terminal product complex in replication polyprotein processing. This allows us to locate the specificity determinants of PRO for its proteinase substrates. In addition to the catalytic cleft, at the exit of which the active site is unusually pared down and solvent-exposed, a key element in molecular recognition by PRO is a lobe N-terminal to the catalytic domain. Docking models and the activities of PRO and PRO mutants in a deubiquitylating assay suggest that this N-terminal lobe is also likely involved in PRO's DUB function. Our data thus establish that DUBs can evolve to specifically hydrolyze both iso- and endopeptide bonds with different sequences. This is achieved by the use of multiple specificity determinants, as recognition of substrate patches distant from the cleavage sites allows a relaxed specificity of PRO at the sites themselves. Our results thus shed light on how such a compact protein achieves a diversity of key functions in viral genome replication and host-pathogen interaction.

Turnip yellow mosaic virus forms infectious particles without the native beta-annulus structure and flexible coat protein N-terminus.

Structural studies have implicated the TYMV N-terminal amino acids of the coat protein (CP) in both static (virion stabilization) and dynamic (RNA encapsidation and disencapsidation) roles. We have deleted residues 2-5, 2-10 and 2-26 from the N-terminus and expressed the mutant CPs in E. coli to assess assembly in the absence of genomic RNA and in plant infections to assess infectivity and virion properties. In E. coli, the deletion constructs formed virus-like particles, but in decreased yield. All mutants were infectious in Chinese cabbage, producing normal symptoms but with a slight delay and decreased viral yields. Virions were progressively less stable with increasing deletion size and also more accessible to small molecules. These results show that the N-terminal 26 amino acids are not essential for viral processes in vivo, although removal of these residues decreases stability and increases porosity, both important factors for virion integrity and survival outside the host.

The ubiquitin-proteasome system regulates the accumulation of Turnip yellow mosaic virus RNA-dependent RNA polymerase during viral infection.

Replication of positive-strand RNA viruses, the largest group of plant viruses, is initiated by viral RNA-dependent RNA polymerase (RdRp). Given its essential function in viral replication, understanding the regulation of RdRp is of great importance. Here, we show that Turnip yellow mosaic virus (TYMV) RdRp (termed 66K) is degraded by the proteasome at late time points during viral infection and that the accumulation level of 66K affects viral RNA replication in infected Arabidopsis thaliana cells. We mapped the cis-determinants responsible for 66K degradation within its N-terminal noncatalytic domain, but we conclude that 66K is not a natural N-end rule substrate. Instead, we show that a proposed PEST sequence within 66K functions as a transferable degradation motif. In addition, several Lys residues that constitute target sites for ubiquitylation were mapped; mutation of these Lys residues leads to stabilization of 66K. Altogether, these results demonstrate that TYMV RdRp is a target of the ubiquitin-proteasome system in plant cells and support the idea that proteasomal degradation may constitute yet another fundamental level of regulation of viral replication.

The Pro/Hel region is indispensable for packaging non-replicating turnip yellow mosaic virus RNA, but not replicating viral RNA.

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA. The genomic RNA has a tRNA-like structure (TLS) at the 3'-end. The 3'-TLS and hairpins in the 5'-untranslated region supposedly serve as packaging signals; however, recent studies have shown that they do not play a role in TYMV RNA packaging. In this study, we focused on packaging signals by examining a series of deletion mutants of TYMV. Analysis of encapsidated viral RNA after agroinfiltration of the deletion constructs into Nicotiana benthamiana showed that the mutant RNA lacking the protease (Pro)/helicase (Hel) region was not encapsidated by the coat proteins provided in trans, implicating that a packaging signal lies in the Pro/Hel region. Examination of two Pro(-)Hel(-) mutants showed that protein activity from the Pro/Hel domains was dispensable for the packaging of the non-replicating TYMV RNA. In contrast, the mutant TYMV RNA lacking the Pro/Hel region was efficiently encapsidated when the mutant TYMV was co-introduced with a wild-type TYMV, suggesting that packaging mechanisms might differ depending on whether the virus is replicating or not.

A tymovirus with an atypical 3'-UTR illuminates the possibilities for 3'-UTR evolution.

We report the complete genome sequence of Dulcamara mottle virus (DuMV), confirming its membership within the Tymovirus genus, which was previously based on physical and pathology evidence. The 5'-untranslated region (UTR) and coding region of DuMV RNA have the typical characteristics of tymoviral RNAs. In contrast, the 3'-UTR is the longest and most unusual yet reported for a tymovirus, possessing an internal poly(A) tract, lacking a 3'-tRNA-like structure (TLS) and terminating at the 3'-end with -UUC instead of the typical -CC(A). An expressible cDNA clone was constructed and shown to be capable of producing infectious DuMV genomic RNAs with -UUC 3'-termini. A chimeric Turnip yellow mosaic virus (TYMV) genome bearing the DuMV 3'-UTR in place of the normal TLS was constructed in order to investigate the ability of the TYMV replication proteins to amplify RNAs with -UUC instead of -CC(A) 3'-termini. The chimeric genome was shown to be capable of replication and systemic spread in plants, although amplification was very limited. These experiments suggest the way in which DuMV may have evolved from a typical tymovirus, and illuminate the ways in which viral 3'-UTRs in general can evolve.

The 5'-UTR of Turnip yellow mosaic virus does not include a critical encapsidation signal.

Turnip yellow mosaic virus (TYMV) RNA has two hairpins in the 5' untranslated region (UTR) with internal CC and CA mismatches that become protonated and are able to base pair at a pH near 5. The protonatable hairpins have previously been implicated as playing an important role in RNA encapsidation. We have examined the role of the 5'-UTR in the amplification and packaging of TYMV RNA using agroinfiltration of Chinese cabbage leaves to express various TYMV constructs with mutations affecting the 5'-UTR and the two hairpins. Mutations affecting the protonatable centers of the two hairpins, as well as deletion of one or both hairpins and deletion or mutation of the 17-nucleotide region upstream of the hairpins decreased viral amplification to varying extents (c. 10- to 1000-fold). However, in all these cases, the viral RNAs present in non-denaturing leaf extracts were predominantly ribonuclease resistant, indicative of encapsidation. These results show that, while the 5' hairpins are necessary for efficient amplification of TYMV, there appears to be no essential role for the 5'-UTR or its protonatable hairpins in the packaging of TYMV RNA. In a second set of experiments, it was demonstrated that TYMV can efficiently amplify in plants held in the dark, and that the progeny RNAs are efficiently encapsidated. Together, these observations argue for a revision of the model for TYMV encapsidation in which packaging occurs in low pH conditions that are generated by proton gradients produced by photosynthetic activity in the light and RNA packaging is dependent on the protonatable 5' hairpins.

Molecular evolution of a viral non-coding sequence under the selective pressure of amiRNA-mediated silencing.

Plant microRNAs (miRNA) guide cleavage of target mRNAs by DICER-like proteins, thereby reducing mRNA abundance. Native precursor miRNAs can be redesigned to target RNAs of interest, and one application of such artificial microRNA (amiRNA) technology is to generate plants resistant to pathogenic viruses. Transgenic Arabidopsis plants expressing amiRNAs designed to target the genome of two unrelated viruses were resistant, in a highly specific manner, to the appropriate virus. Here, we pursued two different goals. First, we confirmed that the 21-nt target site of viral RNAs is both necessary and sufficient for resistance. Second, we studied the evolutionary stability of amiRNA-mediated resistance against a genetically plastic RNA virus, TuMV. To dissociate selective pressures acting upon protein function from those acting at the RNA level, we constructed a chimeric TuMV harboring a 21-nt, amiRNA target site in a non-essential region. In the first set of experiments designed to assess the likelihood of resistance breakdown, we explored the effect of single nucleotide mutation within the target 21-nt on the ability of mutant viruses to successfully infect amiRNA-expressing plants. We found non-equivalency of the target nucleotides, which can be divided into three categories depending on their impact in virus pathogenicity. In the second set of experiments, we investigated the evolution of the virus mutants in amiRNA-expressing plants. The most common outcome was the deletion of the target. However, when the 21-nt target was retained, viruses accumulated additional substitutions on it, further reducing the binding/cleavage ability of the amiRNA. The pattern of substitutions within the viral target was largely dominated by G to A and C to U transitions.

Role of 5'-UTR hairpins of the Turnip yellow mosaic virus RNA in replication and systemic movement.

Turnip yellow mosaic virus (TYMV) RNA has two hairpins in its 5' untranslated region (5'-UTR). To investigate the role of the hairpins in replication of TYMV, mutants lacking one or both of the two hairpins were constructed. The TYMV constructs were introduced into Chinese cabbage by an Agrobacterium-mediated T-DNA transfer method, called agroinfiltration. Analysis of total RNA from agroinfiltrated leaves showed that replication of the mutant TYMV RNA lacking both hairpins was about 1/100 of wild type. This mutant was also impaired in systemic spread. Deletion analysis of each hairpin revealed that both hairpins were needed for maximal replication. The deletion analysis along with sequence modification of the hairpin structure indicates that the second hairpin plays a role in efficient long-distance systemic movement of TYMV.

Replication and encapsidation of recombinant Turnip yellow mosaic virus RNA.

Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus that infects mainly Cruciferae plants. In this study, the TYMV genome was modified by inserting an extra subgenomic RNA promoter and a multiple cloning site. This modified TYMV was introduced into Nicotiana benthamiana using a Agrobacterium-mediated T-DNA transfer system (agroinfiltration). When a gene encoding beta-glucuronidase or green fluorescent protein was expressed using this modified TYMV as a vector, replication of the recombinant viruses, especially the virus containing beta-glucuronidase gene, was severely inhibited. The suppression of replication was reduced by co-expression of viral silencing suppressor genes, such as tombusviral p19, closteroviral p21 or potyviral HC-Pro. As expected, two subgenomic RNAs were produced from the recombinant TYMV, where the larger one contained the foreign gene. An RNase protection assay revealed that the recombinant subgenomic RNA was encapsidated as efficiently as the genuine subgenomic RNA.

GCN2-dependent phosphorylation of eukaryotic translation initiation factor-2alpha in Arabidopsis.

The yeast regulatory protein kinase, general control non-derepressible-2 (GCN2) plays a key role in general amino acid control. GCN2 phosphorylates the alpha subunit of the trimeric eukaryotic translation initiation factor-2 (eIF2), bringing about a decrease in the general rate of protein synthesis but an increase in the synthesis of GCN4, a transcription factor that promotes the expression of genes encoding enzymes for amino acid biosynthesis. The present study concerned the phosphorylation of Arabidopsis eIF2alpha (AteIF2alpha) by the Arabidopsis homologue of GCN2, AtGCN2, and the role of AtGCN2 in regulating genes encoding enzymes of amino acid biosynthesis and responding to virus infection. A null mutant for AtGCN2 called GT8359 was obtained and western analysis confirmed that it lacked AtGCN2 protein. GT8359 was more sensitive than wild-type Arabidopsis to herbicides that affect amino acid biosynthesis. Phosphorylation of AteIF2alpha occurred in response to herbicide treatment but only in wild-type Arabidopsis, not GT8359, showing it to be AtGCN2-dependent. Expression analysis of genes encoding key enzymes for amino acid biosynthesis and nitrate assimilation revealed little effect of loss of AtGCN2 function in GT8359 except that expression of a nitrate reductase gene, NIA1, was decreased. Analysis of wild-type and GT8359 plants infected with Turnip yellow mosaic virus or Turnip crinkle virus showed that AteIF2alpha was not phosphorylated.