Retracted: An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus

Abstract

SummaryTransient gene expression is a fast, flexible and reproducible approach to high-level expression of useful proteins. In plants, recombinant strains of Agrobacterium tumefaciens can be used for transient expression of genes that have been inserted into the T-DNA region of the bacterial Ti plasmid. A bacterial culture is vacuum-infiltrated into leaves, and upon T-DNA transfer, there is ectopic expression of the gene of interest in the plant cells. However, the utility of the system is limited because the ectopic protein expression ceases after 2-3 days. Here, we show that post-transcriptional gene silencing (PTGS) is a major cause for this lack of efficiency. We describe a system based on co-expression of a viral-encoded suppressor of gene silencing, the p19 protein of tomato bushy stunt virus (TBSV), that prevents the onset of PTGS in the infiltrated tissues and allows high level of transient expression. Expression of a range of proteins was enhanced 50-folds or more in the presence of p19 so that protein purification could be achieved from as little as 100 mg of infiltrated leaf material. The effect of p19 was not saturated in cells that had received up to four individual T-DNAs and persisted until leaf senescence. Because of its simplicity and rapidity, we anticipate that the p19-enhanced expression system will have value in industrial production as well as a research tool for isolation and biochemical characterisation of a broad range of proteins without the need for the timeconsuming regeneration of stably transformed plants.

The ROXY1 C-Terminal L**LL Motif Is Essential for the Interaction with TGA Transcription Factors

“…[See online article for color version of this figure. ] terminus of YFP (YC) was fused N terminally to PAN or TGA3. By performing Agrobacterium tumefaciens coinfiltration, constructs were pairwise transiently expressed in abaxial epidermal cells of tobacco (Nicotiana benthamiana) leaves (Voinnet et al, 2003). Reconstitution of YFP fluorescence was examined by confocal microscopy 3 to 5 d after transient coexpression of protein pairs.…”

“…Constructs were transformed into A. tumefaciens strain GV3101::pMP90 (RK). To avoid cosuppression, A. tumefaciens strain GV3101::pMP90 (RG) carrying the p19 viral silencing suppressor gene was always included in the infiltration mixture (Voinnet et al, 2003). Infiltrated tobacco leaves stayed attached to the plants, were examined after 3 to 5 d for the reconstitution of YFP fluorescence, and images were captured with a Leica TCS SP2 AOBS confocal laser scanning microscope.…”

Manufacturing antibodies in the plant cell

“…Using complementary vectors derived from these viruses, H and L chains were successfully co-expressed in N. benthamiana leaves, reaching commercially viable levels of up to 10% TSP [82]. It is noteworthy that the cell secretion machinery is apparently able to cope with this vast amount of newly Deleted: [75,76] Deleted: [77] Deleted: MAb Deleted: [78] Page 23 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 …”

Characterization of a cDNA from Beta maritima that confers nickel tolerance in yeast

“…Transient expression of the fluorescence construct was carried out in intact plant leaves of 4-week-old Nicotania benthamiana by co-infiltration with Agrobacterium tumefaciens strain GV3101 containing the Venus-NIC6 construct in combination with the C58C1 strain carrying the pCH32 helper plasmid which contains the RNA silencing suppressor protein p19 of tomato bushy stunt virus (Voinnet et al, 2003). Fluorescence was observed 3 days after infiltration by confocal laser scanning microscopy (CLSM) with a Leica DMIRE2 microscope (Leica Microsystems) using the excitation/ emission wavelengths of 514/540-580 nm.…”