Salah Bouzoubaa

Nucleotide Sequence of Beet Necrotic Yellow Vein Virus RNA-1

Summary The complete nucleotide sequence of beet necrotic yellow vein virus (BNYVV) RNA-2 has been determined from a study of cloned cDNA. The RNA sequence is 4612 nucleotides in length, excluding the poly(A) tail. There are six long open reading frames (ORFs) in the sequence. The viral coat protein cistron is the ORF nearest to the 5′ terminus and the coat protein amber termination codon is followed by a long in-phase ORF. A corresponding readthrough polypeptide with the coat protein sequence at its N terminus has been detected in previous in vitro translation studies. Four additional ORFs encoding potential polypeptides of mol. wt. 42000 (42K), 12.5K, 14.8K and 14.4K are present in the sequence and evidence is presented that the 42K polypeptide is expressed, probably from a subgenomic messenger RNA. There is extensive homology between sequences near the 3′ termini of RNA-2, RNA-3 and RNA-4 of BNYVV.

Synthesis of full-length transcripts of beet western yellows virus RNA: Messenger properties and biological activity in protoplasts

Full-length cDNA of beet western yellows virus genomic RNA has been cloned behind the bacteriophage T7 RNA polymerase promoter of the transcription vector BS(-). The in vitro run-off transcription product obtained in the presence of T7 RNA polymerase and m7GpppG cap has the same messenger properties as natural viral RNA in in vitro translation systems. The full-length transcript was also able to infect Chenopodium quinoa protoplasts inoculated by electroporation. Infection could be followed by the appearance of viral coat protein in the inoculated protoplasts and the de novo synthesis of viral RNA. Site-directed mutagenesis experiments revealed that expression of beet western yellows virus open reading frame 1 and the C-terminal portion of open reading frame 6 were not required for infection of protoplasts. Additional experiments with these mutants and mutants in the other viral open reading frames should provide information concerning the requirements for beet western yellows virus replication and, ultimately, the role of virus genes in other important steps in the virus infection cycle, such as aphid transmission.

Nucleotide Sequence Analysis of RNA-3 and RNA-4 of Beet Necrotic Yellow Vein Virus, Isolates F2 and G1

Summary The nucleotide sequences of cDNA clones corresponding to RNA-3 and RNA-4 of beet necrotic yellow vein virus isolates F2 and G1 have been determined. The cDNA of RNA-3 of isolate F2 is 1775 residues in length and contains a coding region of 219 codons. In isolate G1 this coding region has undergone an internal deletion of 354 nucleotides in such a way as to conserve a shortened reading frame. Otherwise, the RNA-3 sequences of the two isolates were closely similar. RNA-4 of isolate F2 has an extrapolated length of 1431 residues and contains an open reading frame of 282 codons. This open reading frame has undergone an internal deletion of 324 nucleotides in one cDNA clone of RNA-4(G1) with conservation of a shortened reading frame. Sequence analysis of other RNA-4(G1) cDNA clones revealed, however, that the exact boundaries of the deletion are not always the same. RNA-3 and RNA-4 of each isolate are more than 90% homologous for the 3′-terminal 200 nucleotides. Short homologous sequences are also present in RNA-3 and RNA-4 of isolate F2 flanking the regions deleted in each of these RNAs in the G1 isolate. These homologous sequences probably play a role in the deletion process.

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.

Biologically Active Transcripts of Beet Necrotic Yellow Vein Virus RNA-3 and RNA-4

Summary Synthetic transcripts of beet necrotic yellow vein virus (BNYVV) RNA-3 and RNA-4 were prepared from cloned cDNA in a bacteriophage T7 in vitro run-off transcription system. The RNA-3 transcripts tested had 5′ non-viral extensions of 1, 23 or 64 nucleotides and identical 3′ non-viral extensions of 12 nucleotides. An RNA-4 transcript with a 12 nucleotide 5′ non-viral extension and a 28 nucleotide 3′ non-viral extension was also synthesized. All of the transcripts were biologically active when coinoculated to Chenopodium quinoa with BNYVV isolates deficient in RNA-3 and/or RNA-4 but the presence of a long 5′ non-viral sequence on the RNA-3 transcripts was found to diminish their specific activities considerably.

Beet necrotic yellow vein virus coat protein-mediated protection in sugarbeet (Beta vulgaris L.) protoplasts.

Transformed Beta vulgaris L. suspension cultures were obtained after cocultivation of sugarbeet cells with Agrobacterium tumefaciens harbouring a binary vector containing the coat protein gene of beet necrotic yellow vein virus inserted between the kanamycin resistance gene and a ß-glucuronidase reporter gene. Protoplasts were isolated both from untransformed cells, and from transformed cells expressing the viral coat protein, and both were then infected with beet necrotic yellow vein virus. Comparison of the levels of infectivity shows that the expression of the coat protein gene in sugarbeet protoplasts mediates high levels of protection against infection by beet necrotic yellow vein virus.

The Benyvirus RNA Silencing Suppressor Is Essential for Long-Distance Movement, Requires Both Zinc-Finger and NoLS Basic Residues but Not a Nucleolar Localization for Its Silencing-Suppression Activity

The RNA silencing-suppression properties of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) cysteine-rich p14 proteins have been investigated. Suppression of RNA silencing activities were made evident using viral infection of silenced Nicotiana benthamiana 16C, N. benthamiana agroinfiltrated with green fluorescent protein (GFP), and GF-FG hairpin triggers supplemented with viral suppressor of RNA silencing (VSR) constructs or using complementation of a silencing-suppressor-defective BNYVV virus in Chenopodium quinoa. Northern blot analyses of small-interfering RNAs (siRNAs) in agroinfiltration tests revealed reduced amounts of siRNA, especially secondary siRNA, suggesting that benyvirus VSR act downstream of the siRNA production. Using confocal laser-scanning microscopy imaging of infected protoplasts expressing functional p14 protein fused to an enhanced GFP reporter, we showed that benyvirus p14 accumulated in the nucleolus and the cytoplasm independently of other viral factors. Site-directed mutagenesis showed the importance of the nucleolar localization signal embedded in a C4 zinc-finger domain in the VSR function and intrinsic stability of the p14 protein. Conversely, RNA silencing suppression appeared independent of the nucleolar localization of the protein, and a correlation between BNYVV VSR expression and long-distance movement was established.

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.

Molecular Basis for Mitochondrial Localization of Viral Particles during Beet Necrotic Yellow Vein Virus Infection

ABSTRACT During infection, Beet necrotic yellow vein virus (BNYVV) particles localize transiently to the cytosolic surfaces of mitochondria. To understand the molecular basis and significance of this localization, we analyzed the targeting and membrane insertion properties of the viral proteins. ORF1 of BNYVV RNA-2 encodes the 21-kDa major coat protein, while ORF2 codes for a 75-kDa minor coat protein (P75) by readthrough of the ORF1 stop codon. Bioinformatic analysis highlighted a putative mitochondrial targeting sequence (MTS) as well as a major (TM1) and two minor (TM3 and TM4) transmembrane regions in the N-terminal part of the P75 readthrough domain. Deletion and gain-of-function analyses based on the localization of green fluorescent protein (GFP) fusions showed that the MTS was able to direct a reporter protein to mitochondria but that the protein was not persistently anchored to the organelles. GFP fused either to MTS and TM1 or to MTS and TM3-TM4 efficiently and specifically associated with mitochondria in vivo. The actual role of the individual domains in the interaction with the mitochondria seemed to be determined by the folding of P75. Anchoring assays to the outer membranes of isolated mitochondria, together with in vivo data, suggest that the TM3-TM4 domain is the membrane anchor in the context of full-length P75. All of the domains involved in mitochondrial targeting and anchoring were also indispensable for encapsidation, suggesting that the assembly of BNYVV particles occurs on mitochondria. Further data show that virions are subsequently released from mitochondria and accumulate in the cytosol.

Visualisation of transgene expression at the single protoplast level

Protoplasts are currently used to study the expression of genes following transformation. Expression is followed on a population of protoplasts after total protein extraction by conventional western blotting or measure of the enzymatic activity of the transgenic protein. We describe here a new method, called protoplast printing, allowing easy detection of the fraction of cells expressing a certain protein within a population of protoplasts. It consists of immobilization of the protoplast proteins on a nitrocellulose filter, so as to retain the outlines of the cell, followed by immunological detection of the protein of interest. The only special requirement is an antibody specific for the protein. We have studied the expression of the BNYVV coat protein after electroporation of Chenopodium quinoa protoplasts with viral RNAs, and the expression of the NPT II gene in protoplasts isolated from transgenic tobacco plants as well as after direct transfer of plasmid DNA into tobacco protoplasts. In both cases — infection with viral RNAs and transformation with plasmid DNA — expressing and non-expressing cells can be distinguished as early as 12h after transfer of the transgenes.