Comparative analysis identifies amino acids critical for citrus tristeza virus (T36CA) encoded proteins involved in suppression of RNA silencing and differential systemic infection in two plant species.

Complementary (c)DNA clones corresponding to the full-length genome of T36CA (a Californian isolate of Citrus tristeza virus with the T36 genotype), which shares 99.1% identity with that of T36FL (a T36 isolate from Florida), were made into a vector system to express the green fluorescent protein (GFP). Agroinfiltration of two prototype T36CA-based vectors (pT36CA) to Nicotiana benthamiana plants resulted in local but not systemic GFP expression/viral infection. This contrasted with agroinfiltration of the T36FL-based vector (pT36FL), which resulted in both local and systemic GFP expression/viral infection. A prototype T36CA systemically infected RNA silencing-defective N. benthamiana lines, demonstrating that a genetic basis for its defective systemic infection was RNA silencing. We evaluated the in planta bioactivity of chimeric pT36CA-pT36FL constructs and the results suggested that nucleotide variants in several open reading frames of the prototype T36CA could be responsible for its defective systemic infection. A single amino acid substitution in each of two silencing suppressors, p20 (S107G) and p25 (G36D), of prototype T36CA facilitated its systemic infectivity in N. benthamiana (albeit with reduced titre relative to that of T36FL) but not in Citrus macrophylla plants. Enhanced virus accumulation and, remarkably, robust systemic infection of T36CA in N. benthamiana and C. macrophylla plants, respectively, required two additional amino acid substitutions engineered in p65 (N118S and S158L), a putative closterovirus movement protein. The availability of pT36CA provides a unique opportunity for comparative analysis to identify viral coding and noncoding nucleotides or sequences involved in functions that are vital for in planta infection.

Completion of the mapping of transcription start sites for the five-gene block subgenomic RNAs of Beet yellows Closterovirus and identification of putative subgenomic promoters.

In the positive-sense RNA genome of Beet yellows Closterovirus (BYV), the 3'-terminal open reading frames (ORFs) 2-8 are expressed as a nested set of subgenomic (sg) RNAs. ORFs 2-6, coding for the structural and movement proteins, form a 'five-gene block' conserved in closteroviruses. We mapped the 5'-end of the ORF 4 sgRNA, which encodes the p64 protein, at adenosine-11169 in the BYV genome. This completes the mapping of the transcription start sites for the five-gene block sgRNAs of BYV. Computer-assisted analysis of the sequences upstream of BYV ORFs 2, 3, 4, 5, and 6 revealed two conserved motifs, which might constitute the subgenomic promoter elements. These motifs are conserved in the equivalent positions upstream of three orthologous genes of Citrus tristeza Closterovirus and two orthologous genes of Beet yellow stunt Closterovirus.

RNA4-encoded p31 of beet necrotic yellow vein virus is involved in efficient vector transmission, symptom severity and silencing suppression in roots.

RNA3 and RNA4 of beet necrotic yellow vein virus (BNYVV) are not essential for virus multiplication, but are associated with vector-mediated infection and disease development in sugar beet roots. Here, a unique role for RNA4 in virus transmission, virulence and RNA silencing suppression was demonstrated. Mutagenic analysis revealed that the RNA4-encoded p31 open reading frame (ORF) was involved in efficient vector transmission and slight enhancement of symptom expression in some Beta species. No effects of RNA4 on virus accumulation in infected tissue were observed. Furthermore, the p31 ORF was involved in the induction of severe symptoms by BNYVV in Nicotiana benthamiana plants without affecting viral RNA accumulation. In contrast, RNA3-encoded p25, previously identified as a major contributor to symptom induction in sugar beet, had no such effect on N. benthamiana. In two different silencing suppression assays, neither p31 nor p25 was able to suppress RNA silencing in leaves, but the presence of p31 enhanced a silencing suppressor activity in roots without alteration in viral RNA accumulation. Thus, BNYVV p31 plays a multifunctional role in efficient vector transmission, enhanced symptom expression and root-specific silencing suppression.

Detection of beet yellows virus by RT-PCR and immunocapture RT-PCR in Tetragonia expansa and Beta vulgaris.

Two sensitive methods, RT-PCR with phenol-extracted RNA or Triton X-100-released RNA and immunocapture RT-PCR (IR-RT-PCR) were used for the detection of Beet yellows virus (BYV) in young and old leaves of Tetragonia expansa and sugar beet (Beta vulgaris) and in sugar beet roots. Four oligonucleotide primer pairs proved suitable for the detection of BYV. The release of BYV RNA with Triton X-100 was shown to be a very effective and easy as compared to isolation of total RNA by phenol extraction with the same or higher sensitivity of subsequent PCR. Using the Triton X-100 release of RNA and IC-RT-PCR the sensitivity of detection was so high that pg amounts of BYV RNA occurring in dilutions up to 10(-6) of saps from young Tetragonia and sugar beet leaves could be detected.

Processing and subcellular localization of the leader papain-like proteinase of Beet yellows closterovirus.

ORF 1a of Beet yellows closterovirus (BYV) encodes the domains of the papain-like proteinase (PCP), methyltransferase (MT) and RNA helicase. BYV cDNA inserts encoding the PCP-MT region were cloned in pGEX vectors next to the glutathione S-transferase gene (GST). In a 'double tag' construct, the GST-PCP-MT cDNA was flanked by the 3'-terminal six histidine triplets. Following expression in E. coli, the fusion proteins were specifically self-cleaved into the GST-PCP and MT fragments. MT-His(6) was purified on Ni-NTA agarose and its N-terminal sequence determined by Edman degradation as GVEEEA, thus providing direct evidence for the Gly(588)/Gly(589) bond cleavage. The GST-PCP fragment purified on glutathione S-agarose was used as an immunogen to produce anti-PCP monoclonal antibodies (mAbs). On Western blots of proteins from virus-infected Tetragonia expansa, the mAbs recognized the 66 kDa protein. Immunogold labelling of BYV-infected tissue clearly indicated association of the PCP with the BYV-induced membranous vesicle aggregates, structures related to closterovirus replication.

The p23 protein of citrus tristeza virus controls asymmetrical RNA accumulation.

Citrus tristeza virus (CTV), a member of the Closteroviridae, has a 19.3-kb positive-stranded RNA genome that is organized into 12 open reading frames (ORFs) with the 10 3' genes expressed via a nested set of nine or ten 3'-coterminal subgenomic mRNAs (sgRNAs). Relatively large amounts of negative-stranded RNAs complementary to both genomic and sgRNAs accumulate in infected cells. As is characteristic of RNA viruses, wild-type CTV produced more positive than negative strands, with the plus-to-minus ratios of genomic and sgRNAs estimated at 10 to 20:1 and 40 to 50:1, respectively. However, a mutant with all of the 3' genes deleted replicated efficiently, but produced plus to minus strands at a markedly decreased ratio of 1 to 2:1. Deletion analysis of 3'-end genes revealed that the p23 ORF was involved in asymmetric RNA accumulation. A mutation which caused a frameshift after the fifth codon resulted in nearly symmetrical RNA accumulation, suggesting that the p23 protein, not a cis-acting element within the p23 ORF, controls asymmetric accumulation of CTV RNAs. Further in-frame deletion mutations in the p23 ORF suggested that amino acid residues 46 to 180, which contained RNA-binding and zinc finger domains, were indispensable for asymmetrical RNA accumulation, while the N-terminal 5 to 45 and C-terminal 181 to 209 amino acid residues were not absolutely required. Mutation of conserved cysteine residues to alanines in the zinc finger domain resulted in loss of activity of the p23 protein, suggesting involvement of the zinc finger in asymmetric RNA accumulation. The absence of p23 gene function was manifested by substantial increases in accumulation of negative-stranded RNAs and only modest decreases in positive-stranded RNAs. Moreover, the substantial decrease in the accumulation of negative-stranded coat protein (CP) sgRNA in the presence of the functional p23 gene resulted in a 12- to 15-fold increase in the expression of the CP gene. Apparently the excess negative-stranded sgRNA reduces the availability of the corresponding positive-stranded sgRNA as a messenger. Thus, the p23 protein controls asymmetric accumulation of CTV RNAs by downregulating negative-stranded RNA accumulation and indirectly increases expression of 3' genes.