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Editorial
. 2020 May;18(5):1115-1117.
doi: 10.1111/pbi.13274. Epub 2019 Nov 12.

Development of an autonomously replicating viral expression system tailored for Catharanthus roseus

Cara Mortimer  1 , Benjamin Dugdale  1 , Peter Waterhouse  1
Affiliations
Editorial

Development of an autonomously replicating viral expression system tailored for Catharanthus roseus

Cara Mortimer et al. Plant Biotechnol J. 2020 May.
No abstract available

Keywords: Catharanthus roseus; MIA; gene expression; monoterpinoid indole alkaloid; transient gene expression; viblastine; vincristine; viral vector.

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Conflict of interest statement

The authors have no competing interests.

Figures

Figure 1
Figure 1
Development of an autonomously replicating viral expression system tailored for Catharanthus roseus . An autonomously replicating viral expression system for C. roseus was constructed and its efficacy demonstrated using the fluorescent reporter protein Orange. (a) The Catharanthus yellow mosaic virus (CaYMV)‐based system was mobilized into binary plasmid pBIN+creating a ~4 kb vector pBIN+CaYMV. (b) An Orange expression cassette was inserted into the MCS creating a ~5.5 kb vector pBIN+CaYMV‐Orange. (c) The region between the IR repeats (the replication cassette ~4 kb including Orange) serves as a template for rolling circle replication initiated by CaYMV Rep. This results in a circular, single‐stranded DNA episome, which is converted to a double‐stranded (ds) form by host polymerases. The dsDNA episomes are transcriptionally active and serve as a template for further amplification. (d) pBIN+CaYMV‐Orange was agroinfiltrated into C. roseus and expression visualized 6 days after infiltration. Fluorescence was compared to (i) wild‐type (WT) leaves, (ii) leaves infiltrated with a non‐replicating vector (pBIN+35S35S‐ORANGE) and (iii) leaves infiltrated with a CaYMV vector in which the Rep gene was disrupted (pBIN+CaYMV‐Orange‐NOREP). (e) DNA was extracted from infiltrated leaf tissues and probed for replication by PCR: Lane 1, WT DNA; Lane 2, pBIN+CaYMV‐Orange; Lane 3, pBIN+CaYMV‐Orange‐NOREP. Intron, potato gbbs gene intron IV; 2X35S, enhanced CaMV 35S promoter; AV2, CaYMV anti‐defence protein coding sequence; nosT and nopaline synthase terminator; AMV 5′, UTR translational enhancer sequence from alfalfa mosaic virus; MCS, multiple cloning site; 35ST CaMV 35S terminator; CaYMV Rep‐Ren‐TrAP, CaYMV replicase, replication enhancer and transcriptional activator coding regions; OrE1/OrE2 Exons 1/2 of Orange.
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