Rapid screening of RNA silencing suppressors by using a recombinant virus derived from beet necrotic yellow vein virus.

To counteract plant defence mechanisms, plant viruses have evolved to encode RNA silencing suppressor (RSS) proteins. These proteins can be identified by a range of silencing suppressor assays. Here, we describe a simple method using beet necrotic yellow vein virus (BNYVV) that allows a rapid screening of RSS activity. The viral inoculum consisted of BNYVV RNA1, which encodes proteins involved in viral replication, and two BNYVV-derived replicons: rep3-P30, which expresses the movement protein P30 of tobacco mosaic virus, and rep5-X, which allows the expression of a putative RSS (X). This approach has been validated through the use of several known RSSs. Two potential candidates have been tested and we show that, in our system, the P13 protein of burdock mottle virus displays RSS activity while the P0 protein of cereal yellow dwarf virus-RPV does not.

Subcellular localization of the Triple Gene Block movement proteins of Beet necrotic yellow vein virus by electron microscopy.

The Triple Gene Block proteins TGBp1, TGBp2, and TGBp3 of Beet necrotic yellow vein virus (BNYVV) are required for efficient cell-to-cell spread of the infection. The TGB proteins can drive cell-to-cell movement of BNYVV in trans when expressed from a co-inoculated BNYVV RNA 3-based 'replicon'. TGBp2 and TGBp3 expressed from the replicon were nonfunctional in this assay if they were fused to the green fluorescent protein (GFP), but addition of a hemagglutinin (HA) tag to their C-termini did not incapacitate movement. Immunogold labeling of ultrathin sections treated with HA-specific antibodies localized TGBp2-HA and TGBp3-HA to what are probably structurally modified plasmodesmata (Pd) in infected cells. A similar subcellular localization was observed for TGBp1. Large gold-decorated membrane-rich bodies containing what appear to be short fragments of endoplasmic reticulum were observed near the cell periphery. The modified gold-decorated Pd and the membrane-rich bodies were not observed when the TGB proteins were produced individually in infections using the Tobacco mosaic virus P30 protein to drive cell-to-cell movement, indicating that these modifications are specific for TGB-mediated movement.

Nonregulated expression of TGBp3 of hordei-like viruses but not of potex-like viruses inhibits beet necrotic yellow vein virus cell-to-cell movement.

Plant viruses containing a Triple Gene Block (TGB) movement protein gene cassette fall into two classes. We have shown previously that the third TGB protein (TGBp3) of beet necrotic yellow vein virus (BNYVV; Class 1) and peanut clump virus (Class 1) inhibit BNYVV intercellular movement when expressed from a co-inoculated BNYVV RNA 3-based replicon. Here we show that autonomous expression of TGBp3's of four other Class 1 viruses of various genera also inhibits BNYVV movement. No such effect was observed for four Class 2 virus TGBp3's, suggesting that the roles of Class 1 and 2 TGBp3's in movement differ significantly.

Rapid screening for dominant negative mutations in the beet necrotic yellow vein virus triple gene block proteins P13 and P15 using a viral replicon.

Point mutations were introduced into the genes encoding the triple gene bock movement proteins P13 and P15 of beet necrotic yellow vein virus (BNYVV). Mutations which disabled viral cell-to-cell movement in Chenopodium quinoa were then tested for their ability to act as dominant negative inhibiters of movement of wild-type BNYVV when expressed from a co-inoculated BNYVV RNA 3-based replicon. For P13, three types of mutation inhibited the movement function: non-synomynous mutations in the N- and C-terminal hydrophobic domains, a mutation at the boundary between the N-terminal hydrophobic domain and the central hydrophilic domain (mutant P13-A12), and mutations in the conserved sequence motif in the central hydrophilic domain. However, only the 'boundary' mutant P13-A12 strongly inhibited movement of wild-type virus when expressed from the co-inoculated replicon. Similar experiments with P15 detected four movement-defective mutants which strongly inhibited cell-to-cell movement of wild-type BNYVV when the mutants were expressed from a co-inoculated replicon. Beta vulgaris transformed with two of these P15 mutants were highly resistant to fungus-mediated infection with BNYVV.

Beet necrotic yellow vein virus particles localize to mitochondria during infection.

Fluorescent beet necrotic yellow vein virus (BNYVV) particles were produced by replacing part of the readthrough domain of the minor coat protein P75 with the green fluorescent protein (GFP). The recombinant virus was functional in plants and P75-GFP was incorporated at one end of the rod-shaped virions. Laser scanning confocal microscopy and transmission electron microscopy showed that virus-like particles, almost certainly authentic BNYVV virions, localized to the cytoplasmic surface of mitochondria at early times postinfection but relocated at later times to semiordered clusters in the cytoplasm. This is the first report of specific targeting of plant virus particles to the mitochondria in vivo.

P42 movement protein of Beet necrotic yellow vein virus is targeted by the movement proteins P13 and P15 to punctate bodies associated with plasmodesmata.

Cell-to-cell movement of Beet necrotic yellow vein virus (BNYVV) is driven by a set of three movement proteins--P42, P13, and P15--organized into a triple gene block (TGB) on viral RNA 2. The first TGB protein, P42, has been fused to the green fluorescent protein (GFP) and fusion proteins between P42 and GFP were expressed from a BNYVV RNA 3-based replicon during virus infection. GFP-P42, in which the GFP was fused to the P42 N terminus, could drive viral cell-to-cell movement when the copy of the P42 gene on RNA 2 was disabled but the C-terminal fusion P42-GFP could not. Confocal microscopy of epidermal cells of Chenopodium quinoa near the leading edge of the infection revealed that GFP-P42 localized to punctate bodies apposed to the cell wall whereas free GFP, expressed from the replicon, was distributed uniformly throughout the cytoplasm. The punctate bodies sometimes appeared to traverse the cell wall or to form pairs of disconnected bodies on each side. The punctate bodies co-localized with callose, indicating that they are associated with plasmodesmata-rich regions such as pit fields. Point mutations in P42 that inhibited its ability to drive cell-to-cell movement also inhibited GFP-P42 punctate body formation. GFP-P42 punctate body formation was dependent on expression of P13 and P15 during the infection, indicating that these proteins act together or sequentially to localize P42 to the plasmodesmata.

The first triple gene block protein of peanut clump virus localizes to the plasmodesmata during virus infection.

The subcellular localization of the first triple gene block protein (TGBp1) of peanut clump pecluvirus (PCV) was studied by subcellular fractionation and immunogold cytochemistry using TGBp1-specific antibodies raised against a fusion protein expressed in and purified from bacteria. In the inoculated and apical leaves of virus-infected Nicotiana benthamiana, TGBp1 localized to the cell wall and P30 fractions. Electron microscopy of immunogold-decorated ultrathin sections of the infected leaf tissue revealed TGBp1-specific labeling of the plasmodesmata joining mesophyll cells. In longitudinal sections of the plasmodesmata, the TGBp1-specific labeling was most commonly associated with the plasmodesmal collar region. In transgenic N. benthamiana, which constitutively expressed TGBp1, no TGBp1-specific immunogold labeling of plasmodesmata was observed, but plasmodesmata were gold decorated when the transgenic plants were infected with a TGBp1-defective PCV mutant, indicating that factors induced by the virus infection target and/or anchor the transgene TGBp1 to the plasmodesmata.

Effects of structural modifications upon the accumulation in planta of replicons derived from beet necrotic yellow vein virus RNA 3.

Beet necrotic yellow vein virus (BNYVV) RNA 3 from which all but the 3' and 5' 'core' replication origins (promoters) have been deleted replicates when coinoculated to Chenopodium quinoa with viral RNAs 1 and 2. The resulting 'replicon' can be used to express inserted heterologous sequences in planta. The effects of alterations of replicon structure on its efficiency of accumulation in planta were examined. Inclusion of up to approximately 240 nucleotides of sequence from the region immediately upstream of the core 3'-promoter sequence increased replicon accumulation, suggesting that this region contains specific replication enhancer elements. Insertion of non-viral 'spacer' sequences between the core promoters also increased replicon accumulation, provided that no strong secondary structure was present. The highly homologous 3'-terminal core promoters of BNYVV RNAs 1, 2 and 4 could substitute for the RNA 3 core promoter but were generally somewhat less effective. Co-inoculation of full-length RNA 3 but not RNA 4 interfered with accumulation of the RNA 3-based replicons.

Cell-to-cell movement of beet necrotic yellow vein virus: I. Heterologous complementation experiments provide evidence for specific interactions among the triple gene block proteins.

Cell-to-cell movement of beet necrotic yellow vein virus (BNYVV) requires three proteins encoded by a triple gene block (TGB) on viral RNA 2. A BNYVV RNA 3-derived replicon was used to express movement proteins to functionally substitute for the BNYVV TGB proteins was tested by coinoculation of TGB-defective BNYVV with the various replicons to Chenopodium quinoa. Trans-heterocomplementation was successful with the movement protein (P30) of tobacco mosaic virus but not with the tubule-forming movement proteins of alfalfa mosaic virus and grapevine fanleaf virus. Trans-complementation of BNYVV movement was also observed when all three TGB proteins of the distantly related peanut clump virus were supplied together but not when they were substituted for their BNYVV counterparts one by one. When P30 was used to drive BNYVV movement in trans, accumulation of the first TGB protein of BNYVV was adversely affected by null mutations in the second and third TGB proteins. Taken together, these results suggest that highly specific interactions among cognate TGB proteins are important for their function and/or stability in planta.

Vascular movement of beet necrotic yellow vein virus in Beta macrocarpa is probably dependent on an RNA 3 sequence domain rather than a gene product.

RNAs 1 and 2 of beet necrotic yellow vein virus (BNYVV) carry the functions enabling viral RNA replication, cell-to-cell movement, virus assembly and vascular movement of the virus in the systemic host Spinacea oleracea. In Beta macrocarpa, on the other hand, BNYVV RNA 3 is required for vascular movement. Replication-competent RNA 3 transcripts carrying various point mutations and deletions were coinnoculated with RNAs 1 and 2 to young leaves of B. macrocarpa and the ability of the virus to multiply on the inoculated leaves and to invade the plant systemically was examined. None of the RNA 3 mutants tested interfered with virus multiplication in the inoculated leaves. Point mutations designed to specifically block or truncate translation of the ORFs of the two known RNA 3 gene products, P25 and N, did not interfere with vascular movement. Vascular movement was not inhibited by deletions eliminating the short 5'-proximal ORF on RNA 3 (ORF A) or by point mutations blocking putative translation of the short 5'-proximal ORF (ORF S) on RNA 3sub, a subgenomic RNA derived from RNA 3. On the other hand, deletions in a 'core region' encompassing nucleotides 1033-1257 of RNA 3 completely blocked vascular movement of the virus while removal of sequences flanking the core region lowered its efficiency. The observations suggest that some feature of the RNA 3 sequence rather than an RNA-3 coded protein is important for vascular movement of BNYVV in B. macrocarpa.

Effects of mutations in the beet western yellows virus readthrough protein on its expression and packaging and on virus accumulation, symptoms, and aphid transmission.

Virions of beet western yellows luteovirus contain a major capsid protein (P22.5) and a minor readthrough protein (P74), produced by translational readthrough of the major capsid protein sequence into the neighboring open reading frame, which encodes the readthrough domain (RTD). The RTD contains determinants required for efficient virus accumulation in agroinfected plants and for aphid transmission. The C-terminal halves of the RTD are not well conserved among luteoviruses but the N-terminal halves contain many conserved sequence motifs, including a proline-rich sequence separating the rest of the RTD from the sequence corresponding to the major coat protein. To map different biological functions to these regions, short in-frame deletions were introduced at different sites in the RTD and the mutant genomes were transmitted to protoplasts as transcripts and to Nicotiana clevelandii by agroinfection. Deletions in the nonconserved portion of the RTD did not block aphid transmission but had a moderate inhibitory effect on virus accumulation in plants and abolished symptoms. Deletion of the proline tract and the junction between the conserved and nonconserved regions inhibited readthrough protein accumulation in protoplasts by at least 10-fold. The mutants accumulated small amounts of virus in plants, did not induce symptoms, and were nontransmissible by aphids using agroinfected plants, extracts of infected protoplasts, or purified virus as a source of inoculum. Other deletions in the conserved portion of the RTD did not markedly diminish readthrough protein accumulation but abolished its incorporation into virions. These mutants accumulated to low levels in agroinfected plants and elicited symptoms, but could not be aphid-transmitted. A preliminary map has been produced mapping these functions to different parts of the RTD.

Evidence for in vitro and in vivo autocatalytic processing of the primary translation product of beet necrotic yellow vein virus RNA 1 by a papain-like proteinase.

Beet necrotic yellow vein virus RNA 1 contains a single long ORF corresponding to the theoretical translation product of 237 kDa which contains the information necessary for replication of the viral genome. This ORF contains a putative papain-like proteinase domain which has been localized, on the basis of sequence alignments, between the helicase and polymerase domains. Here we show that the RNA 1 primary translation product can be cleaved autocatalytically in vitro into two species of 150 kDa and 66 kDa, the latter of which probably contains the entire polymerase domain. A 66 kDa protein was detected immunologically in infected C. quinoa protoplasts using an antiserum specific for the C-terminal region of the RNA 1 primary translation product, confirming that processing also occurs in vivo.

Conformation of the 3'-end of beet necrotic yellow vein benevirus RNA 3 analysed by chemical and enzymatic probing and mutagenesis.

Secondary structure-sensitive chemical and enzymatic probes have been used to produce a model for the folding of the last 68 residues of the 3'-non-coding region of beet necrotic yellow vein benevirus RNA 3. The structure consists of two stem-loops separated by a single-stranded region. RNA 3-derived transcripts were produced containing mutations which either disrupted base pairing in the helices or maintained the helices but with alterations in the base pairing scheme. Other mutants contained substitutions in single-stranded regions (loops or bulged sequences). With a few exceptions all three types of mutation abolished RNA 3 replication in vivo, suggesting that both secondary structure and specific sequences are required for efficient recognition of the 3'-terminal region of RNA 3 by viral RNA-dependent RNA polymerase.

High resolution analysis of the readthrough domain of beet necrotic yellow vein virus readthrough protein: a KTER motif is important for efficient transmission of the virus by Polymyxa betae.

The 5'-terminal cistron of beet necrotic yellow vein furovirus RNA 2 encodes the 21 kDa major viral coat protein and terminates with an amber stop codon which can undergo suppression to give rise to a 75 kDa readthrough (RT) protein referred to as P75. P75 is a minor component of virions and the 54 kDa RT domain following the coat protein sequence is important both for virus assembly and transmission by the fungal vector Polymyxa betae. To better define the regions of the RT domain involved in these two steps, RNA 2 transcripts encoding different in-frame RT domain deletion mutants were tested for their ability to form virions when inoculated to plants with the other viral RNAs and to be fungus-transmitted. All deletions in the N-terminal half of the RT domain interfered with virus assembly and partially or completely inhibited fungus transmission. A 4 1 1 nucleotide deletion within the C-terminal half of the RT domain did not inhibit assembly but blocked fungus transmission of the virus. Alanine scanning mutagenesis within the aforesaid 4 1 1 nucleotide subdomain identified a peptide motif (KTER) which is important for the fungus transmission process.

Beet necrotic yellow vein virus 42 kDa triple gene block protein binds nucleic acid in vitro.

The triple gene block (TGB) of beet necrotic yellow vein virus RNA 2 is required for cell-to-cell movement of the virus RNA. The protein P42 encoded by the 5'-proximal gene of the TGB has consensus sequence motifs characteristic of an ATP/GTP-dependent helicase. P42 was over-expressed in Escherichia coli and shown to bind both single- and double-stranded RNA and DNA by Northwestern blotting. Site-directed mutagenesis located the nucleic acid-binding domain to the N-terminal 24 amino acids of the protein and a point mutation or deletions in the region of P42 containing the helicase consensus sequences did not affect nucleic acid-binding activity of the immobilized protein. Electrophoretic mobility-shift assays revealed that P42 also binds nucleic acids in solution and that deletion of the N-terminal region inhibits this binding. Mutations in both the N-terminal nucleic acid-binding domain and the helicase domain blocked infection of leaves, indicating that both regions of P42 are important for its activity in vivo.

Synthesis of a full-length infectious cDNA clone of cucurbit aphid-borne yellows virus and its use in gene exchange experiments with structural proteins from other luteoviruses.

A full-length cDNA of cucurbit aphid-borne yellows virus (CABYV) has been constructed and expressed either as an in vitro transcript, under control of a bacteriophage T7 RNA polymerase promoter, or in vivo, under control of the cauliflower mosaic virus 35S promoter in an agroinfection vector. The biological activity of the cloned cDNA was demonstrated by the ability of its in vitro transcript to replicate in protoplasts and of the agroinfection vector to infect agroinoculated plants. Virus in the agroinfected plants cold be transmitted by the aphid vectors Myzus persicae and Aphis gossypii. The specificity of luteovirus RNA packaging was investigated by replacing (1) the CABYV coat protein gene (and the overlapping ORF5) by the corresponding region of potato leafroll luteovirus or (2) the CABYV readthrough domain by the readthrough domain of beet western yellows luteovirus. The resulting chimeric transcripts replicated in protoplasts and produced virions.

The small cysteine-rich protein P14 of beet necrotic yellow vein virus regulates accumulation of RNA 2 in cis and coat protein in trans.

The effect of null mutations of the small cysteine-rich protein P14 encoded by RNA 2 of beet necrotic yellow vein virus has been investigated using in vitro transcripts of viral RNA to infect Chenopodium quinoa protoplasts. The P14 mutations down-regulated RNA 2 accumulation by approximately 10- to 50-fold. Accumulation of minus-strand RNA 2 was also diminished but RNA 1 accumulation was much less affected. The inhibition of RNA 2 accumulation could not be complemented in trans by providing P14 from another source (either a second molecule of RNA 2 or an RNA 3-based replicon) containing and expressing the P14 gene. The P14 null mutations dramatically inhibited accumulation of viral coat protein, which is encoded by the 5'-proximal gene on RNA 2, but this effect could be complemented in trans, indicating that it occurs by a mechanism distinct from that affecting RNA 2 accumulation. Transient expression experiments were also carried out in which a plasmid expressing P14 and plasmids expressing a reporter gene placed downstream of potential translational control sequences (the 5'-noncoding sequences of RNAs 2, 3, or 4) were introduced into C. quinoa or Nicotiana tabacum leaves by microprojectile bombardment. Coexpression of P14 produced a 3- to 4-fold stimulation of reporter gene expression levels for all the constructs. The lack of sequence specificity suggests that this phenomenon is not directly related to the RNA 2-specific stimulation of coat protein accumulation observed in a viral infection.

Translation of the second gene of peanut clump virus RNA 2 occurs by leaky scanning in vitro.

The two 5'-proximal open reading frames of peanut clump virus RNA 2, which encode the coat protein of 23 kDa and a protein of 39 kDa (P39), are both translated in vitro from genomic RNA 2. We have studied the translational strategy involved in the initiation at the second AUG of RNA 2, which is the initiation codon of P39. Mutation experiments with synthetic transcripts corresponding to the 5'-half of RNA 2 ruled out mechanisms of P39 translation initiation involving termination-reinitiation and internal ribosomes entry. The results were consistent, however, with a leaky scanning mechanism for P39 initiation, in which about one-third of the ribosomes fail to initiate translation of coat protein and scan along the template to initiate translation at the AUG of the P39 gene, more than 1000 residues in from the 5'-terminus of the RNA 2.

Aphid transmission of beet western yellows luteovirus requires the minor capsid read-through protein P74.

Beet western yellows luteovirus is obligately transmitted by the aphid Myzus persicae in a circulative, non-propagative fashion. Virus movement across the epithelial cells of the digestive tube into the hemocoel and from the hemocoel into the accessory salivary glands is believed to occur by receptor-mediated endocytosis and exocytosis. Virions contain two types of protein; the major 22 kDa capsid protein and the minor read-through protein, P74, which is composed of the major capsid protein fused by translational read-through to a long C-terminal extension called the read-through domain. Beet western yellows virus carrying various mutations in the read-through domain was tested for its ability to be transmitted to test plants by aphids fed on agro-infected plants and semi-purified or purified virus preparations. The results establish that the read-through domain carries determinants that are essential for aphid transmission. The findings also reveal that the read-through domain is important for accumulation of the virus in agro-infected plants.

Nucleotide sequence of beet mild yellowing virus RNA.

The complete nucleotide sequence of the genomic RNA of beet mild yellowing virus, isolate 2ITB, is reported. The RNA consists of 5722 nucleotides and contains six long open reading frames which conform to the arrangement characteristic of Subgroup 2 luteoviruses. The three 3'-proximal open reading frames, which encode the viral coat protein, a putative movement protein and the Readthrough Domain, are highly homologous to the corresponding genes of beet western yellows luteovirus while the three 5'-proximal open reading frames are more closely related to the corresponding genes of cucurbit aphid borne yellows luteovirus. The sequence data thus indicate that beet mild yellowing virus should be considered a distinct virus rather than a strain of beet western yellows virus.