Nicotiana tabacum overexpressing γ-ECS exhibits biotic stress tolerance likely through NPR1-dependent salicylic acid mediated pathway
Srijani Ghanta, Dipto Bhattacharyya, Ragini Sinha, Anindita Banerjee, Sharmila Chattopadhyay*
Plant Biotechnology Laboratory, Drug Development/Diagnostics & Biotechnology Division, Indian Institute of Chemical Biology (A unit of Council of Scientific & Industrial Research), 4, Raja S.C.Mullick Road, Kolkata 700 032, India
*Corresponding author: e-mail- sharmila@iicb.res.in
Tel: 033-2499 5868
Fax: 033-2473 5197
Supplementary Fig. S1 Analysis of NtGB lines. a Southern blot indicating the insertion of T-DNA containing LeECS introduced into tobacco genome of different NtGB lines. Genomic DNA from NtGB lines were digested with EcoRI (E) and HindIII (H) separately and blot hybridized to LeECS radiolabeled probe
GSH and SA status in NtGB lines as compared to wild-type plants. b The amount of total glutathione. c GSH: GSSG ratio. d SA content. Data are the mean ± SD for three individual experiments (n = 3) using plants grown independently
Supplementary Fig. S2 Time-dependent expression analysis of NtPR1, NtPR2, NtPR4 and NtPR5 in NtGB lines and wild-type plants infected with P. syringae pv. tabaci (OD 0.05). Samples were collected at indicated time intervals after pathogen infection for quantitative RT-PCR analysis. Actin was used as an internal control. Data are the mean ± SD for three individual experiments (n = 3) using plants grown independently
Supplementary Fig. S3 Time-dependent expression analysis of NtPR1, NtPR2, NtPR4 and NtPR5 in NtGB lines and wild-type plants infected with P. syringae pv. tabaci grown upto an OD of 0.7. Samples were collected at indicated time intervals after pathogen infection for quantitative RT-PCR analysis. Actin was used as an internal control. Data are the mean ± SD for three individual experiments (n = 3) using plants grown independently
Supplementary Table S1. List of primers used in semiquantitative and quantitative RT-PCR
Gene name
|
Forward primer (5' to 3')
|
Reverse primer (5' to 3')
|
Accession no.
|
NptII
|
GAGGCTATTCGGCTATGACTG
|
ATCGGGAGGGGCGATACCGTA
|
AF485783
|
ECS700bp
|
AATATTTCTTCACGGAATTCCTC
|
AAGAGAGCTGTAGCAATAGGC
|
AF017983
|
PR1a
|
ATGGGATTTGTTCTCTTTTCACA
|
TTAGTATGGACTTTCGCCTCT
|
X12737
|
GLS
|
ATGGCTCATCTTTCAGATCTC
|
ATGAGACCACAACTCCAGCA
|
X95932
|
MAPKK
|
ATGAAGAAAGGATCTTTAGCTCCT
|
ATAGTTGTAAGTGCCGACAAAGG
|
AF165186
|
NPR1
|
ATGGATAATAGTAGGACTGCGTT
|
AATGTCATGAGGCAAGGCTTTATC
|
AF480488
|
TRXh
|
ATGGCTGCTAACGATGCTACTTC
|
AGCAGTGACAGTAGCAGGAGC
|
X58527
|
GSNOR1
|
TGCTGTGGCATGGGAGGCTG
|
ATTCACAAACTCTGTCACACCAAA
|
EU652335
|
ICS1
|
CAGGTTGAGTTTGATGAGCT
|
ACTCATCATCTTCAGTCTGGA
|
AY740529
|
PR2
|
ATGACTTTATGCATTAAAAATGG
|
CTGCAGGATTTGCTTCTTGTT
|
M60460
|
PR5
|
ATGAGAAAGACCCACGTC
|
ATGCCTTCTTTGCAGCAG
|
AF154636
|
SDRLP
|
ATGGCCAGCTATTCTCTTCCA
|
CAGATTCATACCACTCACATATT
|
AJ223177
|
AOS
|
ATGGCAGTAGCAACAGCAACA
|
CTGGGTTCGCTCCGTACAAT
|
AJ295274
|
ACCOx
|
ATGGAGTTGCTTAACACTGAACAA
|
TCTTTCTCCAACCATCAGGCTGA
|
X83229
|
PR4
|
TGCTTTCTGCGCTACTTGGG
|
GCCCCTTCTTATTCTAAACGGC
|
X60281
|
PR10
|
GTTCGTATAGGGGGCAAGTT
|
TTAAGCATAGGCTTCAGGATT
|
AY055111
|
Hsp70
|
ATGGCCGGAAAAGGAGAAGG
|
AGTAAGGAGGGACACATCGAAAGT
|
AY253326
|
CA
|
ATGTCAACTGCTTCCATTAACAG
|
AATGACAACAATGTTCTCTACCTT
|
M94135
|
ADH
|
ATAGAAGAAGTGGAGGTTGCACC
|
ATTGAACCTGCTGGGATTCAAATC
|
X81853
|
ADC
|
ATGCCGGCCCTAGGTTGTTG
|
GAAATGTACTCAGCGTCCTTG
|
AF321137
|
CBP
|
ATGGAAGAAACTTGCAGCTTCAG
|
CCTAAGGAAGTCTTCAACAGTAAA
|
U58971
|
Actin
|
ATGGCAGACGGTGAGGATATTCA
|
TGGCGCAACACGAAGTTCGTT
|
EU938079
| |