Cherry Leaf Roll Nepovirus Research Articles

Occurrence of nepoviruses in small fruits and fruit trees in Slovakia

A survey of nepoviruses in small fruits and fruit trees in thirteen localities of Slovakia was undertaken during the last three years. The samples of spontaneously infected small fruits and fruit trees with symptoms such as leaf yellowing, mottling, chlorotic spotting, vein clearing, chlorosis, dwarfing, and in some cases even plant dying, were analysed. Virus identification in collected samples from affected crops was performed by ELISA using polyclonal antibodies. The following quarantine nepoviruses were detected: Tobacco ringspot nepovirus, Tomato ringspot nepovirus, Tomato black ring nepovirus, Raspberry ringspot nepovirus, Cherry leafroll nepovirus and Arabis mosaic nepovirus. Screening of virus vector nematodes in the rhizosphere of infected plants was also undertaken. Three Longidorus (L. elongatus, L. picenus and L. leptocephalus) and four Xiphinema phytonematode species (X. vuittenezi, X. diversicaudatum, X. taylori and X. pachtaicum) were recorded in localities tested.

Subcellular localisation of cherry leaf roll virus coat protein and genomic RNAs in tobacco leaves

The in vivo subcellular location of the coat protein and RNAs of cherry leaf roll nepovirus (CLRV) was studied in infected tobacco plants by two different approaches and it was correlated with the cytopathic structures induced by the virus. Subcellular fractions were obtained by differential centrifugation, visualised by electron microscopy and analysed for their viral RNA and coat protein content by Northern blot and Western blot analysis, respectively. Results indicate that viral RNAs accumulated preferentially at the microsomal fraction. Immunocytochemical studies revealed a clear association of the coat protein of CLRV with the virus-induced cytopathological structures. In situ hybridisation studies confirmed the cytoplasmic location of the virus and allowed one to elucidate the distribution of the CLRV genomic RNAs in the different cell types of infected tissue.

Detection of cherry leaf roll nepovirus (CLRV) in birch, beech and petunia by immuno‐capture RT‐PCR using a conserved primer pair

SummaryViruses in woody forest plants are known to be difficult to detect owing to phenolic compounds in plant extracts and an often irregular distribution of the pathogens within the plants. We describe a modified immunocapture reverse transcription polymerase chain reaction (RT‐PCR) to detect cherry leaf roll virus (CLRV) in forest trees. The application of a conserved primer pair is shown to be suitable for the detection of different isolates of CLRV from birch from distant forest stands in Germany and also in strains of CLRV from other host plants, including beech, walnut and petunia hybrids. The CLRV isolates and strains could be distinguished by direct sequencing of their PCR products. CLRV was easily detectable throughout the year in tissue of male inflorescences, leaf buds, leaves, single seeds, cortical tissues of young twigs and also in single aphids and bugs (Kleidocerys resedae) collected from a CLRV‐infected birch‐ tree.

A Subgenomic RNA Associated with Cherry Leafroll Virus Infections

Cherry leafroll nepovirus (CLRV) genomic RNA 1 (8 kb) and genomic RNA 2 (7 kb) have 3′ polyadenlyate tracts and, extending 5′ from the polyadenylate, nearly identical sequences of 1.6 kb termed the 3′ common region. We observed RNAs 1 and 2 and a third RNA of 1.5 kb in nucleic acid extracts of CLRV-infected Nicotiana tabacum suspension cell protoplasts and Chenopodium quinoa plants, using a hybridization probe complementary to 1 kb of the 3′ common region. The third RNA was partially purified by preparative gel electrophoresis and chromatography on an oligodeoxythymidylate column. Analyses of transcripts primed by a complementary oligodeoxyribonucleotide and of cDNA clones revealed that the third RNA corresponds to the 3′ 1500 nucleotide residues of RNA 1. Hence we designate the newly characterized RNA as RNA 1A. RNA 1A was not detected as encapsidated RNA in extracts of either protoplasts or C. quinoa plants. The amount of accumulated RNA 1A declined between 24 and 48 hr after inoculation of protoplasts with CLRV virions, although CLRV RNAs 1 and 2 continued to accumulate. Other results were not consistent with cleaved RNA 1 being the origin of RNA 1A. RNA 1A has the properties of a subgenomic RNA, presumably synthesized from negative-sense RNA 1 as template.

Transient Gene Expression of Antisense RNA and Coat Protein-Encoding Sequences Reduced Accumulation of Cherry Leafroll Virus in Tobacco Protoplasts

Cherry leafroll nepovirus (CLRV) is the causative agent of blackline disease, which results in a fatal necrosis of the graft union of English walnut scion on certain rootstocks. Tobacco suspension cell protoplasts were electroporated with plasmid constructions, bearing or not bearing RNA-derived sequences, and, subsequently, were electroporated with CLRV virions or virion RNA. Replication of CLRV in protoplasts was demonstrated by accumulation of both positive- and negative-sense CLRV genomic RNAs 1 and 2, capsid antigen, and virions. Three plasmids were tested for antiviral action. These have inserts that were derived from the coat protein gene, inserted in both orientations, and from the 3′ terminal sequence that is nearly identical in RNA 1 and RNA 2, oriented for expression of antisense RNA. Plasmids were introduced into protoplasts 12 hr prior to introducing the virions or virion RNA. CLRV accumulation was reduced significantly by prior electropotation of plasmids intended to express coat protein or 3′ antisense RNA, but not by electropotation of plasmid without insert or plasmid with coat protein encoding sequences in the antisense orientation. These results demonstrate the utility of transient expression in a protoplast system for comparing the efficacy of a variety of virus-derived and other sequences for their potential application in virus control strategies.

Transgenic resistance genes from nepoviruses: Efficacy and other properties

Abstract Evidence of RNA‐RNA recombination or transcapsidation was sought in transgenic tobacco (Nicotiana tabacum L. ‘Xanthi‐nc') containing the 3'‐non coding sequence (1.3 kb) from a birch isolate of cherry leaf roll nepovirus (CLRV). Following mechanical inoculation with a rhubarb isolate of the virus, no evidence of recombination was obtained using polymerase chain reactions whether the transgenic gene produced transcripts in the sense or the antisense orientation. Transgenic tobacco containing the CLRV‐derived sequence (in either orientation) did not lessen the pathological effects of the rhubarb isolate of the virus althdugh there was amelioration of the disease caused by the birch isolate. When expressed as a transgenic gene in tobacco, the capsid coding sequence of arabis mosaic nepovirus (ArMV) lessened infectibility by nematodes carrying that virus. This construct did not influence the pathogenicity of potato virus Yo(PVY0), tobacco rattle tobravirus (PRN), alfalfa mosaic ilarvirus (A1MV), or CL...

The multiplication in plants of arabis mosaic virus satellite RNA requires the encoded protein.

Oligonucleotide-directed mutagenesis was used to create two mutations at each of three positions within the open reading frame (ORF) of a cDNA clone representing a satellite RNA from a lilac isolate of arabis mosaic nepovirus (ArMV). Three of the six mutants, in which stop codons were introduced at three different sites, did not direct synthesis of a translation product. The other three mutants, in which stop codons were not introduced, directed synthesis of a translation product (39K) although, in two of these, the mutation led to a single amino acid substitution. When Chenopodium quinoa plants were inoculated with in vitro transcripts from each of the six mutants together with the genomic RNA molecules (RNA-1 and RNA-2) of ArMV, progeny RNA was detected only with two of the three mutants in which the nucleotide changes did not introduce a stop codon to the coding region. To look for complementation, two deletion mutants were made. In these, 113 or 117 nucleotides were removed from two consecutive regions within the ORF. Two insertion mutants (in which the deleted sequences were replaced with a 130 nucleotide sequence from RNA-2 of cherry leaf roll nepovirus) were also made. Transcripts from none of these mutants retained messenger activity and none was detected either in C. quinoa plants or in virions, even in the presence of wild-type satellite RNA.

The identification, cloning, and sequence analysis of the coat protein coding region of a birch isolate (I2) of cherry leaf roll nepovirus.

The coat protein gene of RNA-2 of cherry leaf roll nepovirus (CLRV) birch isolate I2 was cloned, identified, and sequenced. Transcripts derived from cDNA to the coat protein gene made a polypeptide of M(r) 51.5 k when translated in vitro. The predicted amino acid sequence of the coat protein showed little identity with nepoviruses having small RNA-2s. It did, however, have 27% sequence identity with the coat protein of tomato ringspot nepovirus which, like CLRV, has a relatively large RNA-2.

A 1.5 kb sequence homology in 3'-terminal regions of RNA-1 and RNA-2 of a birch isolate of cherry leaf roll nepovirus is also present, in part, in a rhubarb isolate.

A series of cDNA clones has been made from the birch isolate of cherry leaf roll nepovirus. Restriction enzyme analysis and sequencing showed that at the 3' end, RNA-1 and RNA-2 are identical for 1.5 kb. Also a 0.7 kb 3' end homology exists between the birch and rhubarb isolate. These sequences do not seem to code for any proteins; however, the sequence conservation points to a role in virus replication.

Synthesis and proteolytic processing of arabis mosaic nepovirus, cherry leaf roll nepovirus, and strawberry latent ringspot nepovirus proteins in reticulocyte lysate

The genomic RNA components of three nepoviruses, arabis mosaic (ArMV), cherry leaf roll (CLRV), and strawberry latent ringspot (SLRV), were translated in rabbit reticulocyte lysate. Each component (except the RNA-2 of CLRV) directed the synthesis of proteins that corresponded in size to their theoretical coding capacity. The RNA-1 components of all three viruses were translated to yield polyproteins of Mr 250k, which were autocatalytically processed to yield up to five cleavage products. The primary products of translation of the RNA-2 components of ArMV (Mr 115k and 105k), CLRV (Mr 165k) and SLRV (Mr 99k and 96k) were polyproteins that were stable on incubation, but which underwent proteolytic processing in the presence of the corresponding RNA-1 and its translation products. These polyproteins were immunoprecipitated using antisera to appropriate virions indicating that the RNA-2 sequences encode the coat protein cistrons.