control of a promoter driving abundant and ubiquitous expression.
Golden Gate or GreenGate systems provide rapid and seamless
cloning into binary vectors suitable for Agrobacterium-mediated
transformation [8, 9]. The binary plasmids should be verified by
DNA sequencing before transformation into A. tumefaciens, which
can then be used for transient expression in noninfected and virusinfected 35S:B2:GFP/N. benthamiana leaves. The virus itself can
be delivered either by A. tumefaciens-mediated transient expression
of an infectious clone or by rub inoculation with a crude extract of
infected plant tissue or purified virus. The former method will be
described here, since it is the one used in ref. 8. Also, since the virus
clones are delivered via A. tumefaciens in the same solution containing the A. tumefaciens for transient protein expression, it can be
expected that all the cells expressing the dsRNA-binding protein
candidate will also be infected.
1. Grow a culture of A. tumefaciens containing the binary plasmid
of interest in 10 mL LB medium with appropriate antibiotic
selection on a shaker in the dark for 24 h at 28
C in 50 mL
Falcon tubes. In parallel, grow the A. tumefaciens containing a
plasmid encoding the virus of interest.
2. Pellet the bacteria by centrifugation for 10 min at 3000 Â g,
discard the liquid, and resuspend in MMA buffer. Measure the
absorbance (A) of the suspension at 600 nm (OD 600 ) with a
spectrophotometer. Prepare two separate bacterial solutions in
MMA buffer, one containing only the A. tumefaciens for
expression of the protein of interest at final A of 0.2 (noninfected sample) and the other containing both A. tumefaciens
for expression of the protein of interest at final A of 0.2 and
A. tumefaciens containing the virus infection plasmid at final A
of 0.01 or less (virus-infected sample). Incubate in the dark for
1 h.
3. Use a syringe without needle to infiltrate the A. tumefaciens
solution into the abaxial side of young expanded leaves. To aid
in infiltration, punch a small hole into the abaxial side of the
leaf with a pipette tip. Place the tip of the syringe on the hole
and a finger on the other side of the leaf and gently press the
finger against the syringe tip so it is well in contact with the
hole. Gently infiltrate the bacterial solution into the leaf
through the hole. After completing the infiltration, water the
plants and return them to the growth chamber/greenhouse
(see Note 15).
4. 4 days after A. tumefaciens infiltration (see Note 16), collect
leaf disks from the infiltrated areas, place them with their lower
adaxial side oriented upwards onto a microscope slide, cover
them with a coverslip, add water, and vacuum-infiltrate to
replace the air in the intercellular spaces with the water.
324
Marco Incarbone and Christophe Ritzenthaler
Golden Gate or GreenGate systems provide rapid and seamless
cloning into binary vectors suitable for Agrobacterium-mediated
transformation [8, 9]. The binary plasmids should be verified by
DNA sequencing before transformation into A. tumefaciens, which
can then be used for transient expression in noninfected and virusinfected 35S:B2:GFP/N. benthamiana leaves. The virus itself can
be delivered either by A. tumefaciens-mediated transient expression
of an infectious clone or by rub inoculation with a crude extract of
infected plant tissue or purified virus. The former method will be
described here, since it is the one used in ref. 8. Also, since the virus
clones are delivered via A. tumefaciens in the same solution containing the A. tumefaciens for transient protein expression, it can be
expected that all the cells expressing the dsRNA-binding protein
candidate will also be infected.
1. Grow a culture of A. tumefaciens containing the binary plasmid
of interest in 10 mL LB medium with appropriate antibiotic
selection on a shaker in the dark for 24 h at 28
C in 50 mL
Falcon tubes. In parallel, grow the A. tumefaciens containing a
plasmid encoding the virus of interest.
2. Pellet the bacteria by centrifugation for 10 min at 3000 Â g,
discard the liquid, and resuspend in MMA buffer. Measure the
absorbance (A) of the suspension at 600 nm (OD 600 ) with a
spectrophotometer. Prepare two separate bacterial solutions in
MMA buffer, one containing only the A. tumefaciens for
expression of the protein of interest at final A of 0.2 (noninfected sample) and the other containing both A. tumefaciens
for expression of the protein of interest at final A of 0.2 and
A. tumefaciens containing the virus infection plasmid at final A
of 0.01 or less (virus-infected sample). Incubate in the dark for
1 h.
3. Use a syringe without needle to infiltrate the A. tumefaciens
solution into the abaxial side of young expanded leaves. To aid
in infiltration, punch a small hole into the abaxial side of the
leaf with a pipette tip. Place the tip of the syringe on the hole
and a finger on the other side of the leaf and gently press the
finger against the syringe tip so it is well in contact with the
hole. Gently infiltrate the bacterial solution into the leaf
through the hole. After completing the infiltration, water the
plants and return them to the growth chamber/greenhouse
(see Note 15).
4. 4 days after A. tumefaciens infiltration (see Note 16), collect
leaf disks from the infiltrated areas, place them with their lower
adaxial side oriented upwards onto a microscope slide, cover
them with a coverslip, add water, and vacuum-infiltrate to
replace the air in the intercellular spaces with the water.
324
Marco Incarbone and Christophe Ritzenthaler