8. High-Fidelity PCR Master Mix.
9. SpeI enzyme (10 units/μL).
10. SpeI 10Â reaction buffer: 500 mM Potassium acetate, 200 mM
Tris-acetate, 100 mM magnesium acetate, 100 μg/mL BSA
(pH 7.9).
11. RNasin (40 units/μL).
12. NTP mix: 20 mM each of ATP, CTP, UTP, and 2.0 mM GTP.
13. 20 mM GTP.
14. 5.0 mM Cap analog (m7GpppG).
15. T7 RNA polymerase (50 units/μL).
16. TBE: 90 mM Tris-borate/2.0 mM EDTA, pH 8.0.
17. Appropriate gene-specific primers for selected mobile RNAs.
2.3 Plant Inoculation
and Molecular
Analysis of RNA LongDistance Trafficking
1. Nicotiana benthamiana seeds.
2. 6-in. plastic pots.
3. Carborundum dust.
4. Agarose.
2.4 Equipment
1. Bench-top microcentrifuge.
2. Real-time PCR thermocycler.
3. 37
C Incubator.
4. Growth chamber.
5. NanoDrop spectrophotometer.
3 Methods
3.1 Cloning into the
PVX Vector
The coat protein (CP) of PVX is required for PVX movement and
without it, the PVX RNA is immobile. PVX vectors with and
without the coat protein have been previously designed and tested
[23, 24]. Without its CP, PVX cannot move systemically in infected
plants and it has been shown that replacing the CP with phloemmobile RNA sequences reinstates systemic mobility [24, 26]. The
purpose of this section is to prepare RNA sequences of interest
(ROI) and to clone derived cDNA into the CP-deficient PVX
vector to determine if the ROI contains sequences for systemic
transport and thus for mobilizing PVX for systemic infection. The
PVX plasmid vectors (PVX/CP with CP and PVX/ΔCP without
CP) can be obtained from the co-author, Yiguo Hong. The protocol involves the cloning of derived cDNA (from the ROI) into the
multiple cloning site (MCS) just downstream of the three viral
movement proteins (Fig. 1). This cDNA represents ROI from the
gene sequence of interest (GOI). It is the GOI that is cloned into
186
Zhiming Yu et al.
9. SpeI enzyme (10 units/μL).
10. SpeI 10Â reaction buffer: 500 mM Potassium acetate, 200 mM
Tris-acetate, 100 mM magnesium acetate, 100 μg/mL BSA
(pH 7.9).
11. RNasin (40 units/μL).
12. NTP mix: 20 mM each of ATP, CTP, UTP, and 2.0 mM GTP.
13. 20 mM GTP.
14. 5.0 mM Cap analog (m7GpppG).
15. T7 RNA polymerase (50 units/μL).
16. TBE: 90 mM Tris-borate/2.0 mM EDTA, pH 8.0.
17. Appropriate gene-specific primers for selected mobile RNAs.
2.3 Plant Inoculation
and Molecular
Analysis of RNA LongDistance Trafficking
1. Nicotiana benthamiana seeds.
2. 6-in. plastic pots.
3. Carborundum dust.
4. Agarose.
2.4 Equipment
1. Bench-top microcentrifuge.
2. Real-time PCR thermocycler.
3. 37
C Incubator.
4. Growth chamber.
5. NanoDrop spectrophotometer.
3 Methods
3.1 Cloning into the
PVX Vector
The coat protein (CP) of PVX is required for PVX movement and
without it, the PVX RNA is immobile. PVX vectors with and
without the coat protein have been previously designed and tested
[23, 24]. Without its CP, PVX cannot move systemically in infected
plants and it has been shown that replacing the CP with phloemmobile RNA sequences reinstates systemic mobility [24, 26]. The
purpose of this section is to prepare RNA sequences of interest
(ROI) and to clone derived cDNA into the CP-deficient PVX
vector to determine if the ROI contains sequences for systemic
transport and thus for mobilizing PVX for systemic infection. The
PVX plasmid vectors (PVX/CP with CP and PVX/ΔCP without
CP) can be obtained from the co-author, Yiguo Hong. The protocol involves the cloning of derived cDNA (from the ROI) into the
multiple cloning site (MCS) just downstream of the three viral
movement proteins (Fig. 1). This cDNA represents ROI from the
gene sequence of interest (GOI). It is the GOI that is cloned into
186
Zhiming Yu et al.