Tomato (
) plants from a commercial glasshouse were identified with symptoms compatible with a tomato brown rugose fruit virus (ToBRFV) infection. Reverse transcription-PCR and quantitative PCR ...confirmed the presence of ToBRFV. Subsequently, the same RNA sample and a second from tomato plants infected with a similar tobamovirus, tomato mottle mosaic virus (ToMMV), were extracted and processed for high-throughput sequencing with the Oxford Nanopore Technology (ONT). For the targeted detection of ToBRFV, the two libraries were synthesized by using six ToBRFV sequence-specific primers in the reverse transcription step. This innovative target enrichment technology enabled deep coverage sequencing of ToBRFV, with 30% of the total reads mapping to the target virus genome and 57% mapping to the host genome. The same set of primers applied to the ToMMV library generated 5% of the total reads mapping to the latter virus, indicating that sequencing of similar, non-target viral sequences was also allowed. Further, the complete genome of pepino mosaic virus (PepMV) was also sequenced from the ToBRFV library, thus suggesting that, even using multiple sequence-specific primers, a low rate of off-target sequencing can usefully provide additional information on unexpected viral species coinfecting the same samples in an individual assay. These results demonstrate that targeted nanopore sequencing can specifically identify viral agents and has sufficient sensitivity towards non-target organisms to provide evidence of mixed virus infections.
Macromolecule and cytosolic signal distribution throughout the plant employs a unique cellular and intracellular mechanism called plasmodesmata (PD). Plant viruses spread throughout plants via PD ...using their movement proteins (MPs). Viral MPs induce changes in plasmodesmata’s structure and alter their ability to move macromolecule and cytosolic signals. The developmental distribution of a family member of proteins termed plasmodesmata located proteins number 5 (PDLP5) conjugated to GFP (PDLP5-GFP) is described here. The GFP enables the visual localization of PDLP5 in the cell via confocal microscopy. We observed that PDLP5-GFP protein is present in seed protein bodies and immediately after seed imbibition in the plasma membrane. The effect of three different plant viruses, the tobacco mosaic virus (TMV), tomato brown rugose fruit virus (ToBRFV, tobamoviruses), and tomato yellow leaf curl virus (TYLCV, begomoviruses), on PDLP5-GFP accumulation at the plasmodesmata was tested. In tobacco leaf, TMV and ToBRFV increased PDLP5-GFP amount at the plasmodesmata of cell types compared to control. However, there was no statistically significant difference in tomato leaf. On the other hand, TYLCV decreased PDLP5-GFP quantity in plasmodesmata in all tomato leaf cells compared to control, without any significant effect on plasmodesmata in tobacco leaf cells.
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