Transcript WT 68L-19 - Springer Static Content Server

Supplementary table 1: Primers used for PCR amplification
Primer name
Sequence 5`-3`
Accession Number
ChiF
ACCAAGCTACTCGCAAGAGG
CD485880
ChiR
CGGAAGCGCAGTAAGATGA
GluF
CATTGATATGACCTTGATCG
GluR
GTGAGATATCCCTTGGATTG
PR1F
TGCCCAAGACTCACAACAAG
PR1R
GGCCTTCTCATTAACCCACA
Hist3F
GAAGCCTCATCGATACCGTC
Hist3R
CTACCACTACCATCATGGC
OsmF
AACTGCCCTTACACCGTTTG
OsmR
TATCCCTTGCATTCGAGGAC
CD486342
CK988133.1
(Patil et al. 2005)
AF024716
CF932065
(Patil et al. 2005)
Patil MA, Pierce ML, Phillips AL, Venters BJ, Essenberg M (2005) Identification of genes upregulated in bacterial-blight-resistant upland cotton in response to inoculation with Xanthomonas
campestris pv. malvacearum. Physiol Mol Plant Pathol 67: 319-335.
Supplementary Fig 1
Semi-quantitative Reverse Transcriptase-PCR analysis showing AtNPR1 transcript accumulation
in T2 and T3 generations of transgenic cotton.
a
b
Supplementary Fig 2
(a) Southern blot analysis on genomic DNA from transgenic cotton lines expressing AtNPR1. DNA
was digested with EcoR1 and probed with P32 labeled fragment of the AtNPR1 gene. (b) Map of
AtNPR1 gene construct that was used to generate transgenic cotton.
pmol 4MU/h/mg TSP
1.6
**
1.2
0.8
0.4
0
12
24
48
Hours post-treatment
Glucose equivalents/h/mg protein
a
WT (M)
WT (SA)
68L-19 (M)
68L-19 (SA)
*
30
25
20
15
*
10
5
0
12
24
48
Hours post-treatment
b
WT (M)
WT (SA)
68L-19 (M)
68L-19 (SA)
Supplementary Fig 3
Salicylic acid (SA)-induced (a) Chitinase and (b) Glucanase activities in the cotyledons of
transgenic cotton line, 68L-19 following treatment of the roots with 3 mM SA. Roots of two-day
old pre-germinated seedlings were treated with 3 mM SA and the cotyledons were harvested at
different time points for enzyme activity analyses. Data represent mean±SE (n=10); the induced
enzyme activity value for the transgenic line is significantly higher than that of the WT value at
*P<0.05; **P<0.01.
pmole 4MU/h/  g protein
1.6
***
1.2
0.8
0.4
0
WT (M)
WT (SA)
68L-20 (M)
68L-20 (SA)
Glucose equivalents mg/h/mg protein
a
6
***
5
4
3
2
1
0
WT (M)
WT (SA)
68L-20 (M)
68L-20 (SA)
b
Supplementary Fig 4
Salicylic acid (SA)-induced (a) Chitinase and (b) Glucanase activities in the cotyledons of
transgenic cotton line, 68L-20, following treatment of the roots with 3 mM SA for 24 h. Data
represent mean ±SE (n=10); the induced enzyme activity values for the transgenic line is
significantly higher than that of the WT value at ***P<0.001.
90
8
80
70
6
*
4
2
0
Leaf disease index
Shoot weight (g)
10
60
50
40
*
30
20
10
0
WT
DP444 68L-19
Uninfected
WT
DP444
Infected
68L-19
WT
DP444
68L-19
b
a
68L-19
WT
DP444
c
Supplementary Fig 5
Resistance to Fusarium oxysporum f. sp. vasinfectum (Isolate Fov11) in transgenic cotton
line, 68L-19 expressing AtNPR1. Non-transgenic Coker 312 plants (WT) were used as
control and a Fusarium wilt-resistant, commercial cotton variety (DP 444) was used for
comparison. Two parameters were used to score the effects of infection, three weeks
following the inoculation of roots; (a) Shoot weight, and (b) Leaf disease index. Data
represent mean ±SE, *P<0.05, n=12. (c) Photographs of various sets of plants showing their
relative state before scoring for disease severity.
68L-19
WT
Supplementary Fig 6
Longitudinal section showing vascular browning in the stem (5th internode) of cotton plant at
three weeks following inoculation with Verticillium dahliae (isolate TS2).
1000
2.5
800
No. of bolls/plant
Reniform nematodes/g infested soil
1200
600
400
**
**
200
2
*
68L-19
68L-20
1.5
1
0.5
0
0
WT
68L-19
68L-20
WT
68L-19
68L-20
WT
Uninfected
Infected
b
a
40
16
30
*
20
*
10
0
No. of bolls at given nodes
Boll weight/plant (g; FW)
*
12
8
4
0
WT
68L-19
68L-20
Uninfected
c
WT
68L-19
68L-20
WT
Infected
68L-19
Node 5-7
68L-20
Node 8-10
d
Supplementary Fig 7
Resistance to reniform nematode, Rotylenchulus reniformis, in transgenic cotton lines expressing
AtNPR1. (a) Nematode count in soil after 10 weeks of inoculation, (b) Number of bolls per plant,
(c) Boll weight per plant, (d) Boll position (Number of bolls at given nodes in 10 plants). Data
represent mean ±SE, *P<0.05; **P<0.01, n=10. Note that on the 5th day after inoculation, due to
the equipment break down, the temperature in the growth chamber rose to 42oC for several hours
and the plants were transferred to and maintained in a greenhouse for four days before returning to
the chamber for growth under controlled conditions until completion of the experiment.
Also, note that the final nematode count in this experiment is substantially higher compared to what
is presented for the experiment described in the main text. These high numbers are not unusual and
have been reported previously for reniform nematodes (Agudelo et al. 2005). The large differences
between two experiments can easily result from small variability in the number of reproductive
cycles completed.
Agudelo P, Robbins RT, Kim KS, Stewart MJ. 2005. Histological changes in Gossypium hirsutum
associated with reduced reproduction of Rotylenchulus reniformis. J Nematol 37:185 - 189.
WT
68L-19
Supplementary Fig 8
Resistance to reniform nematodes (Rotylenchulus reniformis). Transgenic and WT plants at 10
weeks following the inoculation of soil with reniform nematodes. The photograph was taken
before scoring the results that are presented in Supplementary Fig. 7a-d.