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.