Molecular characterization and In Vitro synthesis of infectious RNA of a Turnip vein-clearing virus isolated from Alliaria petiolata in Hungary

Abstract

A tobamovirus was isolated from leaves of Alliaria petiolata plants, showing vein-clearing, interveinal chlorosis, and moderate deformation. Host range experiments revealed a high similarity of isolate ApH both to ribgrass mosaic viruses and turnip vein-clearing viruses. The complete nucleotide sequence of the viral genome was determined. The genomic RNA is composed of 6312 nucleotides and contains four open reading frames (ORF). ORF1 is 3324 nt-long and encodes a polypeptide of about 125.3 kDa. The ORF1 encoded putative replication protein contains an Alphavirus-like methyltransferase domain. ORF2 is 4806 nt-long and encodes a polypeptide of about 182 kDa. The ORF2 encoded putative replication protein contains an RNA-dependent RNA polymerase, catalytic domain. ORF3 encodes the putative cell-to-cell movement protein with a molecular weight of 30.1 kDa. ORF4 overlaps with ORF3 and encodes the coat protein with a size of 17.5 kDa. Sequence comparisons revealed that the ApH isolate has the highest similarity to turnip vein-clearing viruses and should be considered an isolate of Turnip vein-clearing virus (TVCV). This is the first report on the occurrence of TVCV in Hungary. In vitro transcripts prepared from the full-length cDNA clone of TVCV-ApH were highly infectious and induced typical symptoms characteristic to the original isolate of the virus. Since infectious clones of TVCV-ApH and crTMV (another isolate of TVCV) markedly differed in respect to recovery phenotype in Arabidopsis thaliana, it is feasible to carry out gene exchange or mutational studies to determine viral factors responsible for the symptom recovery phenotype.

Fig 1. Symptoms observed in different plants infected by TVCV-ApH.

(a) Mild vein clearing and mosaic in the leaf of garlic mustard (Alliaria petiolata) the source of TVCV-ApH. (b) Systemic symptoms on Solanum betaceum. (c) Nicotiana tabacum cv. Samsun, systemic symptoms. (d) Local lesions on Nicotiana tabacum cv. Xanthi-nc.

Fig 2. Phylogenetic relationship of the TVCV-ApH and some representative members of tobamoviruses.

The evolutionary history was inferred using the Neighbor-Joining method [25]. The optimal tree with the sum of branch length = 8.01862305 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches [27]. All positions containing gaps and missing data were eliminated. There were a total of 5052 positions in the final dataset. Tobacco rattle virus was used as an outgroup.

Fig 3. Phylogenetic relationship of the TVCV-ApH and the TVCV isolates with available full genome sequences.

The evolutionary history was inferred using the Neighbor-Joining method [25]. The optimal tree with the sum of branch length = 0.16283470 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [27]. All positions containing gaps and missing data were eliminated. There were a total of 5965 positions in the final dataset. Ribgrass mosaic virus was used as an outgroup.

Fig 4. Schematic representation of the genome organization of TVCV-ApH.

Similarly to other tobamoviruses, the TVCV-ApH genome contains four ORFs. The genomic RNA is a template for expression of the 125 and 182 kDa replication proteins. The 3’ distal movement protein (MP) and capsid protein (CP) ORFs are expressed from separate 3’ co-terminal sgRNAs. The tRNA structure motif at the 3’-end of the RNA is represented by a cloverleaf motif.

Fig 5. Infection of Nicotiana benthamiana plants with the in vitro transcribed TVCV-ApH.

(a) Agarose gel electrophoresis of in vitro synthesized infectious transcripts with or without the m7G cap analog. (b) Denaturing agarose gel electrophoresis of total RNAs from mock-treated, uncapped-, and capped-transcript-treated Nicotiana benthamiana plants after 5 dpi. The gel was stained with ethidium bromide. (c) The same gel was blotted to a membrane and subjected to northern blot analysis using a virus-specific probe. The genomic (gRNA) and the subgenomic RNAs (sgRNA1 and sgRNA2) are marked. (d) Total proteins were purified from the inoculated and the systemic leaves of the same mock- and capped-transcript-treated plants as in the case of northern analysis and run on 12% TGX Stain-Free^™ FastCast^™ Acrylamide Gels (Bio-Rad). The 18 kDa Coat Protein is marked.

Fig 6. Infection of Arabidopsis thaliana Bur-0 plants with TVCV-ApH- and crTMV.

(a) Symptom development and recovery in the Bur-0 ecotype of Arabidopsis thaliana plants infected either with TVCV-ApH or crTMV. (b) The viral genomic (gRNA) and subgenomic RNA (sgRNA1 and sgRNA2) levels were detected in a northern blot assay using a probe that recognizes the 3’ end of the viruses which is highly conserved at the nucleotide level. The loading was checked by running the total RNAs on an agarose gel that was stained with ethidium bromide. For quantitative analysis, the density of the gRNA northern signal was measured in every sample and was normalized to the 28S ribosomal RNA (28S rRNA) levels in the corresponding sample. The values were further normalized to the first sample. The resulting relative values were placed under the samples.