0.0
1.0
2.0
3.0
4.0
0.0
1.0
2.0
3.0
4.0
roots
stolons
StBEL5
StBEL14
GFP
PVX
rRNA
Mock P/G P/GΔCP B5H B14H
Mock P/G P/GΔCP B5H B14H
Virus-inoculated leaf
Root (non-inoculated)
a
b
10-4
10-1
9-12
9-5
WT
10-4
10-1
9-12
9-5
WT
RelaƟve
StBEL5 RNA levels
*
*
*
**
**
**
*
*
Fig. 2 Analysis of RNA movement using a PVX-vector system in WT potato cultivar De ´ sire ´ e (a). PVX-based
StBEL5 RNA (full length plus both UTRs) movement into target roots and stolons of StPTB transgenic overexpression lines, 8d post-viral inoculation of leaves (b). StBEL5 RNA movement is enhanced in correlation with
StPTB1 and À6 over-expression [26]. StPTB1 and À6 are RNA-binding proteins that bind to specific motifs
present in the 3
0 untranslated region of StBEL5 RNA [26]. No movement is observed for the nonmobile control
RNA, StBEL14 (a). RNA was extracted and quantitative real-time RT-PCR with gene-specific primers was used
to calculate the relative amounts of StBEL5 RNA (b). For RT-qPCR, the qScript One-Step SYBR Green qRT-PCR
kit (Quanta Biosciences) with the Eco Real-Time PCR system (Illumina) was used. Each sample was measured
and normalized against StActin8 RNA. RNA values were calculated as the 2
ÀΔΔCt value relative to the mean
values obtained from WT samples [27]. StPTB1 and À6 OE lines are designated #9 and #10, respectively.
Standard deviations of the means of two biological replicates with two technical replicates are shown with one
and two asterisks indicating significant differences ( p < 0.05, p < 0.01, respectively) using a Student’s t-test.
For panel a, mock, inoculation with water; P/G, PVX vector with coat protein and GFP (mobile); P/GΔCP, PVX
vector with GFP but no coat protein (nonmobile); B5H, PVX vector with full-length StBEL5 plus histidine tag but
no GFP or coat protein; B14H, PVX vector with full-length StBEL14 plus histidine tag but no GFP or coat protein.
Ribosomal RNA (rRNA) was used as a loading control in panel a. This figure was used in accordance with a
copyright agreement with Oxford University Press [26]
184
Zhiming Yu et al.
1.0
2.0
3.0
4.0
0.0
1.0
2.0
3.0
4.0
roots
stolons
StBEL5
StBEL14
GFP
PVX
rRNA
Mock P/G P/GΔCP B5H B14H
Mock P/G P/GΔCP B5H B14H
Virus-inoculated leaf
Root (non-inoculated)
a
b
10-4
10-1
9-12
9-5
WT
10-4
10-1
9-12
9-5
WT
RelaƟve
StBEL5 RNA levels
*
*
*
**
**
**
*
*
Fig. 2 Analysis of RNA movement using a PVX-vector system in WT potato cultivar De ´ sire ´ e (a). PVX-based
StBEL5 RNA (full length plus both UTRs) movement into target roots and stolons of StPTB transgenic overexpression lines, 8d post-viral inoculation of leaves (b). StBEL5 RNA movement is enhanced in correlation with
StPTB1 and À6 over-expression [26]. StPTB1 and À6 are RNA-binding proteins that bind to specific motifs
present in the 3
0 untranslated region of StBEL5 RNA [26]. No movement is observed for the nonmobile control
RNA, StBEL14 (a). RNA was extracted and quantitative real-time RT-PCR with gene-specific primers was used
to calculate the relative amounts of StBEL5 RNA (b). For RT-qPCR, the qScript One-Step SYBR Green qRT-PCR
kit (Quanta Biosciences) with the Eco Real-Time PCR system (Illumina) was used. Each sample was measured
and normalized against StActin8 RNA. RNA values were calculated as the 2
ÀΔΔCt value relative to the mean
values obtained from WT samples [27]. StPTB1 and À6 OE lines are designated #9 and #10, respectively.
Standard deviations of the means of two biological replicates with two technical replicates are shown with one
and two asterisks indicating significant differences ( p < 0.05, p < 0.01, respectively) using a Student’s t-test.
For panel a, mock, inoculation with water; P/G, PVX vector with coat protein and GFP (mobile); P/GΔCP, PVX
vector with GFP but no coat protein (nonmobile); B5H, PVX vector with full-length StBEL5 plus histidine tag but
no GFP or coat protein; B14H, PVX vector with full-length StBEL14 plus histidine tag but no GFP or coat protein.
Ribosomal RNA (rRNA) was used as a loading control in panel a. This figure was used in accordance with a
copyright agreement with Oxford University Press [26]
184
Zhiming Yu et al.