Cell-free construction of disarmed Abutilon mosaic virus-based gene silencing vectors.
The bipartite Abutilon mosaic virus (AbMV) was engineered as a versatile silencing vector in which the coat protein gene of DNA A was deleted and replaced by sequences of interest. Plants transgenic for the dimeric AbMV DNA B component were used as test hosts to minimize the risk of unintended release of the recombinant DNA. The vector construct was stable genetically upon systemic infection and, in common with the parental virus, the vector remained phloem-limited. For virus-induced gene silencing (VIGS), a phytoene desaturase gene fragment was isolated from Nicotiana benthamiana (NbPDS) and inserted into the vector. After agroinfection, phytoene desaturase silencing was triggered efficiently in all leaf tissues without interference by viral symptoms. In order to facilitate further the use of the system, a technique for cell-free construction of recombinants was established using rolling circle amplification and biolistic inoculation of DNA B-transgenic plants. This novel procedure provides a convenient and safe way for delivering VIGS constructs for functional genomics.
Krenz
Björn
B
University of Stuttgart, Department of Molecular Biology and Plant Virology, Pfaffenwaldring 57, D-70550 Stuttgart, Germany.
Wege
Christina
C
Jeske
Holger
H
D053839:Begomovirus / Q000235:genetics*; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques / Q000379:methods*; D020868:Gene Silencing; D005822:Genetic Vectors; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D010088:Oxidoreductases / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D011995:Recombination, Genetic; D014026:Nicotiana / Q000235:genetics*
D004279:DNA, Viral; D010940:Plant Proteins; D010088:Oxidoreductases; C022398:phytoene dehydrogenase