Supplementary Material
Comparison of Expression Vectors for Transient Expression of Recombinant
Proteins in Plants
Journal: Plant Molecular Biology Reporter
Kausar Hussain Shah, Bachar Almaghrabi and Holger Bohlmann
Division of Plant Protection, Department of Crop Sciences, University of Natural
Resources and Life Sciences Vienna, Austria.
Corresponding author: Holger Bohlmann, address: Division of Plant Protection,
Department of Crop Sciences, University of Natural Resources and Life Sciences
Vienna, UFT, Konrad Lorenz Strasse 24, 3430 Tulln, Austria, Phone: +43 1 47654
3360, Fax: +43 1 47654 3359, E-mail: holger.bohlmann@boku.ac.at
Supplementary Figures
Figure S1
Map of pPZPTRBO created by using PlasMapper web server (Dong, 2004).
Figure S2
GFP fluorescence and GUS expression with pEAQ-HT in LBA4404.
Previously the Agrobacterium strain LBA4404 was used for agroinfiltration of the
vector pEAQ-HT rather than the strain GV3101 used in our case (Sainsbury et al.
2009). Therefore, we have validated our results by using LBA4404. N. benthamiana
leaves were infiltrated with the Agrobacterium strain LBA4404 (OD600 = 1.0)
containing the vector pEAQ-HT-GFP (A) and
pEAQ-HT-GUS (B, C). (A), GFP
fluorescence at 5 dpi; (B), the GUS expression shown with GUS staining done at 5
dpi.; (C), black and white image of (B) to clearly see the difference of GUS
expression with and without P19. The leaf area marked with P19 shows co-infiltration
with RNA silencing inhibitor P19.
Figure S3
Comparison of GFP expression by the vector pEAQ-HT in the Agrobacterium strains
GV3101 and LBA4404.
The OD600 of the Agrobacteria suspension was 0.4. The extraction of TSP from
infiltrated leaves was done at 5 dpi. The Data were analysed by one way ANOVA (p
<0.05) and LSD test by using SPSS software (n = 6). The stars show significant
difference between samples with and without RNA silencing inhibitor P19 whereas
different letters indicate statistically significant difference between different strains.
‘ns’, not significant.
Figure S4
The timeline of GFP expression.
Representative
pictures
showing
N.
benthamiana
leaves
infiltrated
with
Agrobacterium suspensions (OD600 0.4) containing the GFP expression vectors. The
GFP fluorescence was monitored under UV light at 3, 4, 5, and 6 dpi (rows A, B, C,
and D). The columns 1-4 show the GFP expression with vectors pPZP3425,
pPZPTRBO, pPJLTRBO, and pBY030-2R, respectively. The left side of the leaf
image was co-infiltrated with the P19 RNA silencing inhibitor while the right side is
without P19.
Figure S5
GFP fluorescence at OD600 0.4
GFP relative fluorescence produced by different vectors with and without P19 was
measured. The OD600 of the Agrobacteria suspension was 0.4 in this experiment and
the samples were taken at 5 dpi to extract TSP. The RFU (relative fluorescence unit)
shown here are average ± SE of three individual replicates. The data were analysed
by one way ANOVA (p <0.05) and LSD test by using SPSS software (n = 6). The
stars show significant difference between samples with and without RNA silencing
inhibitor P19 while different letters indicate statistically significant difference between
different vectors.
Figure S6
SDS-PAGE gel of TSP from N. benthamiana leaves infiltrated with GFP vectors.
TSP samples extracted from N. benthamiana leaves with GFP expression were
resolved on a 16% Tricine SDS-PAGE under reducing condition and stained with
Coomassie-brilliant blue. The red arrow in the image shows ~27 kDa GFP band.
P19, co-infiltrated with P19 RNA silencing inhibitor; Agrobacteria, infiltrated only with
GV3101 without any expression vector; Emp Vector, infiltrated with vector
pPZPTRBO without any reporter gene; Negative, infiltrated with infiltration buffer.
Figure S7
SDS-PAGE gel of TSP from N. benthamiana leaves infiltrated with GUS vectors.
TSP samples extracted from N. benthamiana leaves with GUS expression were
resolved on a 16% Tricine SDS-PAGE under reducing condition and stained with
Coomassie-brilliant blue. The red arrow in the image shows ~68 kDa GFP band.
P19, co-infiltrated with P19 RNA silencing inhibitor.
Additional Reference
Dong X, Stothard P, Forsythe IJ,Wishart DS (2004) PlasMapper: a web server for
drawing and auto-annotating plasmid maps. Nucleic Acids Res 32 (Web
Server issue):W660-664