A plant intron enhances the performance of an infectious clone in planta

• Published in: Journal of virological methods Vol. 265; pp. 26 - 34
• Main Authors: Tran, Phu-Tri, Fang, Miao, Widyasari, Kristin, Kim, Kook-Hyung
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2019
• Subjects: Agrobacterium; Agrobacterium - genetics; bacteria; clones; DNA Replication; enzymes; Escherichia coli; Escherichia coli - genetics; Genetic Vectors; Genomic Instability; Infectious clone; Intron; Introns; leaves; Nicotiana - virology; pathogenicity; PepMoV; Plant Diseases - virology; Plant Leaves - virology; plant pathology; Plasmid instability; Plasmids; Potyvirus - genetics; Potyvirus - pathogenicity; Recombination, Genetic; virology; Infectious clone; Intron; Plasmid instability; PepMoV
• ISSN: 0166-0934; 1879-0984; 1879-0984
• DOI: 10.1016/j.jviromet.2018.12.012

•An infectious clone of PepMoV in a bacterial binary vector was constructed.•The pPepMoV infectious clone was instable and lost infectivity during manipulation.•During its propagation in E. coli, the pPepMoV infectious clone spontaneously acquired a bacterial transposon.•A plant intron inserted in the pPepMoV restored infectivity and maintained plasmid stability.•We have demonstrated the in vivo enhancement of an infectious clone by a plant intron. Although infectious clones are fundamental tools in virology and plant pathology, their efficacy is often reduced by the instability of viral sequences in Escherichia coli. In this study, we constructed an infectious clone of PepMoV (pPepMoV) in a bacterial binary vector (pSNU1); the clone induces symptoms of PepMoV in agroinfiltrated plants. During its modification and maintenance in E. coli, however, the pPepMoV infectious clone was instable in the bacteria. Manipulation of this unstable clone in the bacterial strain DH10B led to the spontaneous formation of a recombined clone with high stability in the bacteria but with reduced infectivity due to an unwanted insertion of an E. coli sequence in the NIa-protease coding region. Replacement of this sequence with a plant intron restored infectivity and maintained plasmid stability. In addition to restoring plasmid growth in both E. coli and Agrobacterium, the presence of the intron in the PepMoV sequence enhanced the accumulation of PepMoV in agroinfiltrated leaves and resulted in symptom induction in upper systemic leaves that was nearly as strong as with PepMoV sap-inoculation. Plant introns have been previously used to stabilize plasmids in E. coli without any effect or with an unexpected lag in symptom development. In contrast, the current results demonstrated the in vivo enhancement of an infectious clone by a plant intron.