pTRAkc-ERH/pRIC 3.0

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	The A. tumefaciens GV3101::pMP90RK cells were electroporated using 40–400 ng of recombinant plasmid as described by Maclean, et al. [44]. Recombinant clones were selected using agar plates containing kanamycin (30 μg/mL), rifampicin (50 μg/mL) and carbenicillin (50 μg/mL) and incubated at 27° C.

Protocol tips
The A. tumefaciens GV3101::pMP90RK cells were electroporated using 40–400 ng of recombinant plasmid as described by Maclean, et al. [44]. Recombinant clones were selected using agar plates containing kanamycin (30 μg/mL), rifampicin (50 μg/mL) and carbenicillin (50 μg/mL) and incubated at 27° C.

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"The A. tumefaciens GV3101::pMP90RK cells were electroporated using 40–400 ng of recombinant plasmid as described by Maclean, et al. [44]. Recombinant clones were selected using agar plates containing kanamycin (30 μg/mL), rifampicin (50 μg/mL) and carbenicillin (50 μg/mL) and incubated at 27° C.

For infiltration, recombinant A. tumefaciens GV3101::pMP90RK were grown up overnight at 27° C with agitation in induction medium supplemented with kanamycin (30 μg/mL), rifampicin (50 μg/mL) and carbenicillin (50 μg/mL) [45]. The strain LBA4404 containing pBIN-NSs, provided by Marcel Prins from the Laboratory of Virology, Wageningen in the Netherlands, was supplemented with kanamycin (30 μg/mL), rifampicin (50 μg/mL) and 2 mM MgSO4. The addition of MgSO4 was to prevent cell clumping during incubation [44]. The NSs protein has been shown to suppress post-transcriptional gene silencing in plants, leading to an increase in transient protein expression [46]. Cells were harvested by centrifugation at 4000 g for 10 min, and resuspended in infiltration medium [45]. The suspensions were diluted to the required absorbance (OD600), for expression optimisation studies a range of OD600 values were tested, using an Ultrospec™ 10 Cell density meter (Amersham Biosciences, United Kingdom) and incubated at 22° C for 2 h. The A. tumefaciens GV3101::pMP90RK suspensions of each expression construct were co-infiltrated with strain LBA4404 containing pBIN-NSs into 6-week-old N. benthamiana plants by injecting the suspension into the abaxial spaces using a needleless 1 mL syringe. The plants were maintained in a greenhouse under a 16 h light and 8 h dark photoperiod at light intensity of 60–80 μE/m2/s and 22° C.

Vacuum infiltration of A. tumefaciens into N. benthamiana was performed as described by Maclean, et al. [44], with the following modifications. The Agrobacterium strains were combined in infiltration medium for a final OD600 of 1.00 for strain GV3101::pMP90RK and 0.25 for strain LBA4404 making a total OD600 of 1.25. The 6-week-old N. benthamiana plants were prepared for inversion into infiltration medium by sealing the base of the plant through the use of a 130 × 130 mm acrylic sheet that contained a 10 mm channel to the centre to allow for the plant stem to be inserted (Fig. 4). This prevented soil from falling into the infiltration medium while the plant was inverted and leaves and stem submerged."

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