3 Methods
Use kits according to manufacturers’ instructions unless otherwise
indicated.
3.1 Generation
of Mutant AGO
Expression Constructs
Recently, we have provided a detailed report of the cloning of the
N. benthamiana AGO proteins [6]. Here we only describe the
generation of expression constructs producing various mutant
forms of these molecules. The full-length cDNAs of AGOs are
initially assembled in pGEM-T easy plasmid vector. Due to their
simplicity and relatively small size these pGEM-AGO plasmids can
be used as templates for site-directed mutagenic PCR reactions.
The core biochemical properties of AGO proteins are evolutionarily
conserved. Thus, multiple sequence alignments (Clustal) of AGOs
from evolutionarily distant organisms can help to predict regions of
potential functional importance. Appropriately designed forward
and reverse mutagenic primers are used to introduce the desired
mutations into the AGO proteins by the Quickchange method [10]
(see Note 4). The presence of the intended mutation has to be
confirmed by sequencing of the mutagenized pGEM-AGO plasmid
clones. Finally, the altered AGO ORFs are swapped into plant
binary expression vectors, which allows their functional testing in
the in vivo reporter system.
3.1.1 Site-Directed
Mutagenesis
of N. benthamiana
AGO cDNAs
1. Set up a 50 μL PCR mixture by mixing 10 ng of pGEM-AGO
plasmid, 10 μL of 5 Â Phusion buffer, 1 μL of 10 μM mutagenic forward primer, 1 μL of 10 μM mutagenic reverse primer,
1 μL of 10 mM dNTP mix, appropriate amount of water, and
finally 0.5 μL of Phusion polymerase.
2. Place the tube into the thermocycler and apply the following
temperature cycling conditions: 98
C for 2 min (initial denaturation); 18 cycles of 98
C for 10 s (denaturation), 54–64
C
for 20 s (annealing) (see Note 5), and 72
C for 15 s/kb of
plasmid to be amplified (extension); and 72
C for 5 min
(polishing).
3. Add 2 μL (20 U) of DpnI restriction enzyme to the completed
PCR reaction. Incubate at 37
C for 2 h to eliminate original,
methylated plasmid template.
4. Transform chemically competent TOP10 E. coli cells with
10 μL of the digested PCR reaction (see Note 6). Spread
bacteria onto an LB agar plate containing 100 μg/mL of
ampicillin. Incubate the plates overnight at 37
C.
5. Purify plasmids from 4 to 6 colonies and verify the presence of
the desired mutation in the AGO cDNA by sequencing (see
Note 7).
An In Vivo Reporter System for RNA Silencing
437
Use kits according to manufacturers’ instructions unless otherwise
indicated.
3.1 Generation
of Mutant AGO
Expression Constructs
Recently, we have provided a detailed report of the cloning of the
N. benthamiana AGO proteins [6]. Here we only describe the
generation of expression constructs producing various mutant
forms of these molecules. The full-length cDNAs of AGOs are
initially assembled in pGEM-T easy plasmid vector. Due to their
simplicity and relatively small size these pGEM-AGO plasmids can
be used as templates for site-directed mutagenic PCR reactions.
The core biochemical properties of AGO proteins are evolutionarily
conserved. Thus, multiple sequence alignments (Clustal) of AGOs
from evolutionarily distant organisms can help to predict regions of
potential functional importance. Appropriately designed forward
and reverse mutagenic primers are used to introduce the desired
mutations into the AGO proteins by the Quickchange method [10]
(see Note 4). The presence of the intended mutation has to be
confirmed by sequencing of the mutagenized pGEM-AGO plasmid
clones. Finally, the altered AGO ORFs are swapped into plant
binary expression vectors, which allows their functional testing in
the in vivo reporter system.
3.1.1 Site-Directed
Mutagenesis
of N. benthamiana
AGO cDNAs
1. Set up a 50 μL PCR mixture by mixing 10 ng of pGEM-AGO
plasmid, 10 μL of 5 Â Phusion buffer, 1 μL of 10 μM mutagenic forward primer, 1 μL of 10 μM mutagenic reverse primer,
1 μL of 10 mM dNTP mix, appropriate amount of water, and
finally 0.5 μL of Phusion polymerase.
2. Place the tube into the thermocycler and apply the following
temperature cycling conditions: 98
C for 2 min (initial denaturation); 18 cycles of 98
C for 10 s (denaturation), 54–64
C
for 20 s (annealing) (see Note 5), and 72
C for 15 s/kb of
plasmid to be amplified (extension); and 72
C for 5 min
(polishing).
3. Add 2 μL (20 U) of DpnI restriction enzyme to the completed
PCR reaction. Incubate at 37
C for 2 h to eliminate original,
methylated plasmid template.
4. Transform chemically competent TOP10 E. coli cells with
10 μL of the digested PCR reaction (see Note 6). Spread
bacteria onto an LB agar plate containing 100 μg/mL of
ampicillin. Incubate the plates overnight at 37
C.
5. Purify plasmids from 4 to 6 colonies and verify the presence of
the desired mutation in the AGO cDNA by sequencing (see
Note 7).
An In Vivo Reporter System for RNA Silencing
437