G. Jonard

P0 of Beet Western Yellows Virus Is a Suppressor of Posttranscriptional Gene Silencing

ABSTRACT Higher plants employ a homology-dependent RNA-degradation system known as posttranscriptional gene silencing (PTGS) as a defense against virus infection. Several plant viruses are known to encode proteins that can suppress PTGS. Here we show that P0 of beet western yellows virus (BWYV) displays strong silencing suppressor activity in a transient expression assay based upon its ability to inhibit PTGS of green fluorescent protein (GFP) when expressed in agro-infiltrated leaves of Nicotiana benthamiana containing a GFP transgene. PTGS suppressor activity was also observed for the P0s of two other poleroviruses, cucurbit aphid-borne yellows virus and potato leafroll virus. P0 is encoded by the 5′-proximal gene in BWYV RNA but does not accumulate to detectable levels when expressed from the genome-length RNA during infection. The low accumulation of P0 and the resulting low PTGS suppressor activity are in part a consequence of the suboptimal translation initiation context of the P0 start codon in viral RNA, although other factors, probably related to the viral replication process, also play a role. A mutation to optimize the P0 translation initiation efficiency in BWYV RNA was not stable during virus multiplication in planta. Instead, the P0 initiation codon in the progeny was frequently replaced by a less efficient initiation codon such as ACG, GTG, or ATA, indicating that there is selection against overexpression of P0 from the viral genome.

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.

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.

In vitro synthesis of biologically active beet necrotic yellow vein virus RNA

Beet necrotic yellow vein virus (BNYVV) has a quadripartite plus-strand RNA genome in which the two smallest genome components, RNA 3 and 4, are not necessary for virus multiplication in leaves. Infectious transcripts of BNYVV RNA 3 and 4 have already been described (V. Ziegler-Graff, S. Bouzoubaa, I. Jupin, H. Guilley, G. Jonard, and K. Richards (1988) J. Gen. Virol. 69, 2347-2357). In this paper we describe synthesis of a full-length RNA-1 transcript by bacteriophage T7 RNA polymerase-directed run-off transcription of cloned viral cDNA. A recombinant plasmid containing a full-length cDNA insert of RNA 2 could not be maintained in Escherichia coli. Therefore full-length transcript of RNA 2 was produced by transcription of cDNA ligation products without amplification in bacteria. When inoculated together to leaves of Chenopodium quinoa or Tetragonia expansa the RNA 1 and 2 transcripts were infectious; they also supported multiplication of the BNYVV RNA 3 and 4 transcripts, providing a totally synthetic inoculum of the virus. In one recombinant clone of RNA 2 a point mutation causing an arginine to serine substitution at position 119 of the viral coat protein was discovered. The mutation was detected because the resulting coat protein had altered electrophoretic mobility. RNA 2 transcripts containing this mutation were infectious but viral RNA was not encapsidated. The mutation also interfered with long distance movement of the virus in spinach, presumably as a consequence of the packaging deficiency.

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 of DNA from an altered-virulence isolate D/H of the cauliflower mosaic virus

The double-stranded DNA from the isolate D/H with an altered virulence of the cauliflower mosaic virus (CaMV) contains 8016 bp. The DNA is circular and possesses, like the DNA of most CaMV strains, three sequence interruptions. The comparison of its sequence with the previously published sequences of two other CaMV strains (Cabb-S and CM 1841) leads to the following conclusions: (1) The genetic organization of all three CaMV strains is identical with six potential genes (open reading frames) and two intergenic regions; (2) considered pairwise, the three DNAs differ from one another by only about 5% with base substitutions accounting for most of the changes although several deletions and insertions are also observed. The sequence differences among the three strains are spread in a uniform manner upon the genome except for the two intergenic regions, which are more highly conserved. The stability of the noncoding regions is probably linked to the fact that they carry sequences important for the initiation and termination of transcription. On the other hand, the sequence variation in the open reading frames has relatively little effect on the sequence of the corresponding polypeptides as changes occur preferentially in the third position of the reading frame triplets. It is anticipated that knowledge of the DNA sequences of several CaMV strains will facilitate construction of inter-strain recombinants which, once available, can be used to correlate gene structure and function.

Immunodetection in Vivo of beet necrotic yellow vein virus-encoded proteins

Open reading frames identified on the four genomic RNAs of beet necrotic yellow vein virus were cloned into bacterial expression vectors and resulting cl-fusion proteins expressed in Escherichia coli were used to raise polyclonal antibodies. This set of antisera was used to show the presence of 7 of 9 predicted viral proteins in mechanically inoculated Chenopodium quinoa leaves by the Western blot technique. Viral coat protein (p22) and its readthrough protein p85 encoded by RNA-2 could be detected in all subcellular fractions. Two other RNA-2-encoded proteins, p42 and p13, are predominantly associated with membranous structures. Another RNA-2-encoded protein, p14, as well as the two polypeptides p25 and p31, encoded by RNA-3 and -4, respectively, are soluble proteins. The viral proteins could first be detected about the time lesions became visible and increased thereafter except for p85, in which case the amount of the soluble form decreased with time. No protein could be detected corresponding to the RNA-1-encoded p237 protein or to the p15 species encoded by open reading frame V of RNA-2.

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.

Effect of beet necrotic yellow vein virus RNA composition on transmission by Polymyxa betae.

Beet necrotic yellow vein virus (BNYVV) is naturally transmitted by the soil-borne fungus Polymyxa betae and usually remains confined to the roots of infected sugarbeets. In naturally infected sugarbeets the virion RNA always consists of four components which are uniform in size in different isolates but when BNYVV is propagated by mechanical inoculation to leaves of Chenopodium quinoa the two smallest RNA components, RNA-3 and -4, may undergo deletion or disappear from the isolate, suggesting that they are only essential for the natural mode of infection. To test this hypothesis, several C. quinoa isolates of BNYVV with different RNA-3 and -4 contents have been retransmitted to sugarbeet root via P. betae. The results show that the two isolates containing no detectable full-length RNA-3 and -4 are poorly transmitted and that cases of successful infection are associated with the reappearance of full-length RNA-3 and -4.