protein gene (PVX/ΔCP) or (PVX/GFPΔCP) resulted in immobile PVX. Insertion of key FT or StBEL5 sequences into the RNA
genome of PVX/ΔCP or PVX/GFPΔCP, however, led to recovery
of movement-deficiency of PVX/GFP-FTΔCP (Fig. 1) or PVX/
BEL5ΔCP (Fig. 2).
Here, we describe the PVX-based RNA movement assay to
identify RNA sequences that facilitate movement, using the FT
(Fig. 1) or StBEL5 (Fig. 2) model systems as examples. This protocol outlines easy steps for cloning the target RNA as cDNA into the
PVX/GFP-mFTΔCP
a
b
c
PVX/GFP-FTΔCP
PVX/GFPΔCP
FT
GFP
PVX
18S
530bp
420bp
430bp
185bp
gRNA
sgRNA1
gfp-ft sgRNA
Viral inoculated leaf
Young leaf
D
N
A
m
a r k e r
m
o c k G
F P G
F P - F T G
F P - m
F T
D
N
A
m
a r k e r
m
o c k G
F P G
F P - F T G
F P - m
F T
RDRP
25K 8K
12K
GFP
mFT
*
FT
Fig. 1 PVX-based FT RNA mobility assay (RMA). (a) PVX-based PVX 7.0 kb
genomic gRNA, 2.6 kb subgenomic sgRNA1, and 1.4 kb GFP-FT sgRNA are
indicated. The coat protein (CP) gene was deleted. PVX functions with the
RNA-dependent RNA polymerase (RDRP), an enzyme that catalyzes the
replication of RNA from a RNA template. The positions of a stop codon (∗)
replacing FT start codon in PVX/GFP-mFTΔCP, and three sets of primers for
detection of FT (red arrows), GFP (green arrows), and PVX (blue arrows) RNAs are
indicated. Individual epidermal cells expressing free GFP or GFP-FT fusion
proteins show green fluorescence. (b and c) RT-PCR analysis of FT, GFP, and
PVX RNA and 18S ribosomal RNA in inoculated and newly growing systemic
young leaves, including shoot apices of N. benthamiana. RNAs were extracted
from leaves of plants mock inoculated (mock) or inoculated with PVX/GFPΔCP
(GFP), PVX/GFP-FTΔCP (GFP-FT), or PVX/GFP-mFTΔCP (GFP-mFT). The 1.0 Kb
DNA ladder and sizes of RT-PCR products are indicated. This experiment
demonstrates that wild-type or mutated FT mRNA (∗ panel a) can promote
the long-distance spread of PVX/GFP-FTΔCP (GFP-FT), or PVX/GFP-mFTΔCP
(GFP-mFT), respectively, from virus-inoculated source leaves to systemic new
leaves. This figure is taken from Li et al. [24] with permission of the publisher
PVX-Based RNA Movement Assay
183
genome of PVX/ΔCP or PVX/GFPΔCP, however, led to recovery
of movement-deficiency of PVX/GFP-FTΔCP (Fig. 1) or PVX/
BEL5ΔCP (Fig. 2).
Here, we describe the PVX-based RNA movement assay to
identify RNA sequences that facilitate movement, using the FT
(Fig. 1) or StBEL5 (Fig. 2) model systems as examples. This protocol outlines easy steps for cloning the target RNA as cDNA into the
PVX/GFP-mFTΔCP
a
b
c
PVX/GFP-FTΔCP
PVX/GFPΔCP
FT
GFP
PVX
18S
530bp
420bp
430bp
185bp
gRNA
sgRNA1
gfp-ft sgRNA
Viral inoculated leaf
Young leaf
D
N
A
m
a r k e r
m
o c k G
F P G
F P - F T G
F P - m
F T
D
N
A
m
a r k e r
m
o c k G
F P G
F P - F T G
F P - m
F T
RDRP
25K 8K
12K
GFP
mFT
*
FT
Fig. 1 PVX-based FT RNA mobility assay (RMA). (a) PVX-based PVX 7.0 kb
genomic gRNA, 2.6 kb subgenomic sgRNA1, and 1.4 kb GFP-FT sgRNA are
indicated. The coat protein (CP) gene was deleted. PVX functions with the
RNA-dependent RNA polymerase (RDRP), an enzyme that catalyzes the
replication of RNA from a RNA template. The positions of a stop codon (∗)
replacing FT start codon in PVX/GFP-mFTΔCP, and three sets of primers for
detection of FT (red arrows), GFP (green arrows), and PVX (blue arrows) RNAs are
indicated. Individual epidermal cells expressing free GFP or GFP-FT fusion
proteins show green fluorescence. (b and c) RT-PCR analysis of FT, GFP, and
PVX RNA and 18S ribosomal RNA in inoculated and newly growing systemic
young leaves, including shoot apices of N. benthamiana. RNAs were extracted
from leaves of plants mock inoculated (mock) or inoculated with PVX/GFPΔCP
(GFP), PVX/GFP-FTΔCP (GFP-FT), or PVX/GFP-mFTΔCP (GFP-mFT). The 1.0 Kb
DNA ladder and sizes of RT-PCR products are indicated. This experiment
demonstrates that wild-type or mutated FT mRNA (∗ panel a) can promote
the long-distance spread of PVX/GFP-FTΔCP (GFP-FT), or PVX/GFP-mFTΔCP
(GFP-mFT), respectively, from virus-inoculated source leaves to systemic new
leaves. This figure is taken from Li et al. [24] with permission of the publisher
PVX-Based RNA Movement Assay
183