Supplementary material
Figure S1: Lr34res-specific expression levels in transgenic wheat lines produced in the genetic
background of BW26AUS.
Levels of Lr34res transcript levels were assessed in cold treated seedlings35 dpi with leaf rust. As
expected Lr34res expression was detected in the 5 transgenic lines and in Th+Lr34, but not in the
two negative controls (Th and BW26AUS). Biological replicates (n) = 3, error bars = 1 standard error
(S.E.), normalized to Tubb4. Results are relative to Th+Lr34 = 1.
Figure S2: Confidence intervals for trend lines depicted in Figure 5.
Trend lines are depicted as solid lines with 95% confidence intervals shown as dashed lines in the
corresponding colour. This data indicates that the deposition of callose in lines containing Lr34res is
not different from those lines without.
Table S1: Non-parametric Mann-Whitney statistical testing of variance in lesion area between two
Lr34res transgenic lines and controls from 3-4 flag leaves, 7 dpi with leaf rust, pathotype: Mackeller.
n = number of lesions measured, P-values (two-tailed) were calculated using the Mann-Whitney Test
calculator at http://faculty.vassar.edu/lowry/VassarStats.html
Wheat line
n
Th
190
190 sib
Th+Lr34
171
94
85
76
75
75
Median area
(m2)
3380
1260
3770
2120
1410
171
Th+Lr34
190 sib
190
P<0.01
not significant
P<0.01
not significant
N/A
P<0.05
P<0.01
P<0.05
N/A
not significant
P<0.01
N/A
P<0.01
N/A
Table S2: qPCR primer information
Accession
Gene name
Protein
family
Primer sequences (5’-3’)
U76895
Tubb4
β-tubulin 4
F GCCATGTTCAGGAGGAAGG
R TCGGTGAACTCCATCTCGT
AJ007348
PR1.1
PR1 (Basic)
F CTGGAGCACGAAGCTGCAG
R CGAGTGCTGGAGCTTGCAGT
Y18212
PR2
PR2
F CTCGACATCGGTAACGACCAG
R GCGGCGATGTACTTGATGTTC
AB029934
Chitinase 1
PR3
F AGAGATAAGCAAGGCCACGTC
R GGTTGCTCACCAGGTCCTTC
FJ436983
Lr34res
LR34res
F GGGAGCATTATTTTTTTCCATCATG
R ACTGGCAGAAGAACCTTGAAACA
AF251217
GAPDH
GAPDH
F TTAGACTTGCGAAGCCAGCA
R AAATGCCCTTGAGGTTTCCC
PCR efficiency (E)
r2 of calibration curve
Slope
E= 102%
r2 = 0.999
Slope = -3.29
E= 105.6%
r2 = 0.995
Slope = -3.21
E= 96.3%
r2 = 0.996
Slope = -3.43
E= 102.1%
r2 = 0.992
Slope = -3.29
E= 92.6%
r2 = 0.985
Slope = -3.517
E= 104.6%
r2 = 0.996
Slope = -3.185
Amplicon
length
Reference
70 bp
(Desmond et
al., 2006)
75 bp
(Molina et al.,
1999)
118 bp
(Ray et al.,
2003)
115 bp
(Desmond et
al., 2006)
241 bp
(this work)
81 bp
(Travella et
al., 2006)
Table S3: Significance levels of qPCR results shown in Figure 2. Significance was assessed through
pairwise comparison using REST 2009 software. * indicates variation in reference genes, refer to
supplementary experimental procedures.
Mock vs infected
Difference
Th+Lr34 20°C
No change
Th+Lr34 10°C
Up 5.7 fold
Th+Lr34 Flag
No change
171 20°C
Up 2.8 fold
171 10°C
No change
171 Flag
No change
190 20°C
No change
190 10°C
No change
190 Flag
No change
Seedlings (20°C and 10°C) vs Flag leaves
Difference
Th+Lr34, 171 and 190 mock
Up 6.0 fold
Th+Lr34, 171 and 190 infected
Up 4.8 fold
Mock Th+Lr34
*Up 9.3 fold
Infected Th+Lr34
*Up 6.4 fold
Mock 171
*Up 7.3 fold
Infected 171
*Up 5.7 fold
Mock 190
*No change
Infected 190
*Up 3.1 fold
Th+Lr34 vs transgenic lines
Difference
Mock Th+Lr34 vs 171 & 190 20°C
No change
Mock Th+Lr34 vs 171 & 190 10°C
Up 7.2 fold
Infected Th+Lr34 vs 171 20°C
Up 8.5 fold
Infected Th+Lr34 vs 190 20°C
Up 3.9 fold
Infected Th+Lr34 vs 171 10°C
No change
Infected Th+Lr34 vs 190 10°C
No change
Mock Th+Lr34 vs 171 Flag
Up 4.8 fold
Mock Th+Lr34 vs 190 Flag
No change
Infected Th+Lr34 vs 171 Flag
No change
Infected Th+Lr34 vs 190 Flag
No change
P(H1)
0.000
0.032
P(H1)
0.000
0.000
0.012
0.005
0.004
0.045
0.021
P(H1)
0.026
0.000
0.013
0.018
-
Table S4: Significance levels of qPCR results shown in figure 4a. Significance was assessed through
pairwise comparison using REST 2009 software.
Th PR1
Th+Lr34 PR1
190 sib PR1
171 PR1
190 PR1
Th PR2
Th+Lr34 PR2
190 sib PR2
171 PR2
190 PR2
Th PR3
Th+Lr34 PR3
190 sib PR3
171 PR3
190 PR3
Th vs Th+Lr34 mock
Th vs Th+Lr34 infected
190 sib vs 171 mock
190 sib vs 171 infected
190 sib vs 190 mock
190 sib vs 190 infected
Mock vs infected
Difference
Up 5.4 fold
Up 4.2 fold
No change
No change
No change
No change
Up 3.9 fold
No change
No change
No change
Up 4.8 fold
Up 3.6 fold
No change
No change
No change
Control vs Lr34res containing line
Difference
No change
No change
No change
No change
No change
No change
P(H1)
0.030
0.001
0.002
0.042
0.000
P(H1)
-
Table S5: Primer information
Primer name
L34DINT9F
L34MINUSR
L34SPF
L34DINT13R2
TauABC_f1
Lr34RT_r1
ABC_r9_2
ABCTEX1F
ABCTR3
ABCTEX1314F
ABCR9
L343UTRF
L34PDRPROMR
ABCTCODR1
T7 Promoter
SP6 Promoter
Primer sequences (5’-3’)
TTGATGAAACCAGTTTTTTTTCTA
TATGCCATTTAACATAATCATGAA
GGGAGCATTATTTTTTTCCATCATG
ACTTTCCTGAAAATAATACAAGCA
GAGTACGGCTAGGCATATGC
GAAGCCTAGCAACTTCACGAGGC
GGCAAGTAGCTATATCTGTAAC
ATGGAGGGCCTCGCAAGAGAGA
ATAACCATCGTGTACTCGCTG
CAGAACACCTACAGAAGAATATC
GGCAAGTAGCTATATCTGTAAC
GATGCAAGTGATTGCCATTAG
ACTTCACGAGACCTCTGCTTC
TATGCCATTTAACATAATCATGAA
ATTATGCTGAGTGATATCCC
AAGATATCACAGTGGATTTA
Reference
(Lagudah et al., 2009)
(Lagudah et al., 2009)
(Lagudah et al., 2009)
(Lagudah et al., 2009)
this work
this work
this work
this work
this work
this work
this work
this work
this work
this work
Promega (Cat.#Q5011)
Promega (Cat.#Q5021)
Supplementary experimental procedures
Genotypic characterisation of transgenic Lr34res lines
In Switzerland, genomic DNA was isolated from leaves as described in Stein et al., (2001). The PCRbased Lr34res marker cssfr2; primers L34DINT9F andL34MINUSR (Lagudah et al., 2009), was used to
determine the presence of the transgene in the primary transformants. Copy number was
determined by genomic DNA analysis on cssfr2 positive T0 plants using 20 µg genomic DNA digested
with EcoRI and probed with a 32P-labelled 727 bp PCR amplicon (TauABC_f1/ Lr34RT_r1) from the 5’
end of Lr34res (see Figure S2). Integration of the complete transgene was determined by reverse
transcriptase (RT) PCR amplification of the full-length Lr34res transcript using TauABC_f1/ABC_r9_2.
The amplicon was then sub-cloned and sequenced to distinguish the transgene transcript from the
endogenous BW26 Lr34sus-7D transcript. Segregation analysis of the Lr34res transgene in the
following generations was performed as afore mentioned using the cssfr2 marker and genomic DNA
analysis.
Genomic DNA analysis was carried out using 10 µg of genomic wheat DNA digested with NotI,
probed with a
32
P-labelled 1.1 kbp PCR amplicon (ABCTEX1F/ABCTR3), and then a 2.1kbp PCR
amplicon (ABCTEX1314F/ABCR9). These probes annealed to the 5’ and 3’ ends of the Lr34res gene
respectively, thus identifying plants with the full-length construct inserted. Identification of
homozygous lines was carried out using a combination of genomic DNA blot and PCR screening using
3 primer sets; L343UTRF/T7, SP6/L34PDRPROMR and a multiplex amplification using
L34SPF/L34DINT13R2/L34DINT9F/ABCTCODR1. Copy number was determined using genomic DNA
blot analysis with separate EcoRI and DraI genomic digests. The former was probed with the 727 bp
amplicon (TauABC_f1/ Lr34RT_r1) which hybridizes to the 5’ region of Lr34 and the latter probed
with the 3’ hybridizing 2.1kbp amplicon (ABCTEX1314F/ABCR9). Primer sequences can be found in
Table S5.
Quantitative PCR-based expression analysis
RNA from leaf tissue was harvested, frozen immediately in liquid nitrogen and stored for no more
than 3 months at -80°C. Frozen leaf tissue was ground and processed using a Qiagen RNeasy® Plant
purification kit with on-column RNase-Free DNase treatment (Qiagen, Germany) at CSIRO or a
Promega SV Total RNA isolation kit with on-column RNase-Free DNase treatment (Promega, USA) at
University of Zürich, both according to Manufacturer’s instructions. RNA was quantified using a
NanoDrop ND-1000 spectrophotometer (Thermo Scientific, USA). RNA quality was assessed
(A260/A230 ratio - optimally over 2.00) and integrity visualized by resolution of 100-250 ng of RNA on a
1.2% SB agarose gel. RNA samples of sufficient quality were then used for cDNA synthesis. cDNA was
synthesized from 1 or 2µg (Australia and Switzerland respectively) RNA using (dT)20 oligomers,
Invitrogen SuperScript™III RT and RNaseOUT™ Recombinant RNase Inhibitor according to
Manufacturer’s instructions using. Resulting cDNA was then diluted 1 in 10 and stored at -20°C. No
reverse transcriptase (NRT) samples were made by excluding the reverse transcriptase and
RNaseOUT™, diluted and stored as with the cDNA samples.
Qualitative PCR (qPCR) target and primer sequence information is summarised in Table S2. The
Lr34res primers were designed using a modified Lr34res specific forward primer sequence – L34SPF
(Lagudah
et
al.,
2009).
Primer
sequences
were
designed
using
Primer-blast
(http://www.ncbi.nlm.nih.gov/tools/primer-blast/) with the following restrictions; PCR product size
restrictions 180 - 250 bp, TM variation between primer pairs of <1 and a specificity check within
Triticum aestivum (taxid:4565), minimum of 3 mismatches to non-target sequence. In addition the
reverse primer spans an 86bp intron between exons 11 and 12 preventing amplification of
contaminating genomic DNA.
All qPCR analysis was carried out using a Bio-Rad CFX96 Real-Time System C1000™ Thermal cycler
(CSIRO, Australia) or Applied BiosystemsTm 7500 Fast Real-Time PCR System (University of Zürich,
Switzerland). The samples for qPCR analysis were prepared manually to a total volume of 10 µL using
5 µL Bio-Rad iQ™ SYBR® Green Supermix, 3 µL or 4 µL diluted cDNA (Australia and Switzerland
respectively) and Forward and Reverse primers at a final concentration of 500 nM. All analysis was
carried out in a 96 well format with interplate calibration standards included. Thermocycling
conditions for Tubb4 and Lr34res were 95°C for 3 min, followed by 40 cycles of 95°C for 10 sec then
60°C for 30 sec + plate read. Thermocycling conditions for GAPDH were as above or 95°C for 20 sec,
followed by 40 cycles of 95°C for 3 sec then 60°C for 30 sec + plate read. Following amplification,
melt curves were produced using a 60°C to 95°C temperature gradient of 0.5°C for 5 sec + plate read.
All primer sets were analysed for specificity by analysis of the melt curves and resolution of the
amplicon on a 2% TAE agarose gel. Validation and analysis of results in Australia was carried out
using proprietary software (Bio-Rad CFX Manager 2.0). This software also calculates both primer
efficiencies and normalization of results ((C)q) based on formulae from Vandesompele et al.,
(2002) and Pfaffl (2001). In Switzerland results validation and analysis was with proprietry software
(Applied Biosystems 7500 Software (2.0.2.). Both primer efficiencies and CNRQ (Calibrated
normalized relative quantities) were calculated with Biogazelle qbasePLUS 2.1 software (Hellemans
et al., 2007). Efficiencies for each primer set were established through three independent standard
curves using serial dilutions of cDNA (see Table S2). All results used in analysis fell within the
dynamic standard range established for each primer set. NRT controls were analysed and
corresponding cDNA samples used only if there was no (or low - C(q) of 10< cycles later)
contaminating genomic DNA. Analysis was carried out with at least 3 biological replicates (see figure
legends) in technical triplicate. Any individual aberrant technical replicate was deleted from analysis.
GAPDH and Tubb4 (β-tubulin 4 encoding gene) were used as reference genes and all data normalized
to these. Validation of the reference genes was carried out using REST 2009 software (Pfaffl et al.,
2002; Vandesompele et al., 2002). No significant variation between infected and mock infected
samples was found. Variation between different age and temperature treatments was observed but
the reference genes were either up regulated or down regulated to a similar degree (see Table S3 for
complete analysis). All stated significance across the experimental data has been normalized to the
reference genes using the REST 2009 software and was carried out using the technical replicate
mean C(q) values after being corrected for interplate variability.
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