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MP. RdRP. CP. SP6. 5‘UTR. 3‘UTR. (A). (C). 3 dpi. (D). 15 dpi. (B). Supplementary Figure 1 Infectivity analysis of Nicotiana tabacum . - PowerPoint PPT Presentation
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pT2SB9934 bp
Kan
coat protein
movement protein
183 kDa RdRP
126 kDa RdRP
trunc. SP6 promoter
first incorporated nucleotide
transcription stop
SeqTMV5943
TMV transcript
SeqTMV8496
SeqTMV7645
SeqTMV6794
SeqTMV5092SeqTMV4241
M13F MediGX
M13R2 MediGX
Kan promoter
T7 promoter
pUC origin
TMV 3' UTR
(A)
RdRP5‘UTR 3‘UTR
CPMPSP6
(B)
(C)
3 dpi
15 dpi
(D)
Supplementary Figure 1 Infectivity analysis of Nicotiana tabacum. Schematic illustration of the TMV RNA expression vector pT2SB (a) and the infection construct of this vector (b). Leaves of Nicotiana tabacum cv. Xanthi NN (c) and Xanthi nn (d) were dusted with carborundum and rub inoculated with RNA either generated by in vitro transcription with pT2SB as template (left) or purified from wtTMV (right). After infection, plants are grown under standard conditions. dpi, days post infection.
pT2SB:SP1-1
Nicotiana tabacum cv. Xanthi NN
Nicotiana tabacum cv. Xanthi nn
Supplementary Figure 2 Infectivity analysis of Nicotiana tabacum. (a) Schematic illustration of the infection construct of pT2SB:SP1-1. (b) Nicotiana tabacum cv. Xanthi NN and Xanthi nn were infected with RNA derived from in vitro transcription of pT2SB:SP1-1. Both the resistant and the susceptible cultivar show distinct HR-lesions 6 dpi that restricted the recombinant TMV from local cell-to-cell movement in the leaf and inhibited formation of infection.
RdRP5‘UTR 3‘UTR
CP - M - AMPMPSP6
(A)
(B)
Supplementary Figure 3 pAGRO::T2SB-CP_cc_SP1-1cc as example for an agroinfiltration vector. RdRP, RNA-dependent RNA polymerase; p35S, CaMV 35S promoter; Ap R, ampicillin resistance gene; UTR, untranslated region; CNBr, cyanogen bromide; RB, right border; LB, left border; Nos term, nopaline synthase terminator.
pA G RO :T2S B -S P1-1cc12130 bp
SP1-1
coat proteinmovement protein
183 kDa RdRP
126 kDa RdRPTrfa region
Ap R
LB
RB
stop
CNBr cleavage site
first incorporated nucleotide
transcription stop
charge compensation
p35S
trunc. SP6 promoter
RLK2 OriV
pBR322 Ori
Nos term
TM V 3' UTR
pAGRO:T2SB-CP_cc_SP1-1
12130 bp
55
43
34
26
17
1 2 M kDa
Supplementary Figure 4 Acetic acid extraction of T2SB_CP_cc_SP1-1. The acid extract was dialysed against water oN and proteins pI precipitated by adjusting the pH to 5.5 with NaOH. Precipitated proteins were centrifugated and resuspended in 1/5 volume of buffer corresponding to the volume of the acetic acid extract and separated on 15%-SDS-PAGE. 1, T2SB_CP_cc_SP1-1; 2, wtTMV CP as size control. Relative molecular marker standards are shown on the right. M, marker; kDa, kilodalton.