Supplementary Information
SGT1-ORF
SGT1-5' RACE
M
1
1
1.1 kb
Lane # M- λ DNA/EcoRI+HindIII marker
1- AdSGT1 5' RACE pcr product
M
1077 bp
Lane # M- λ DNA/EcoRI+HindIII marker
1- AdSGT1 ORF pcr product
Figure S1 Cloning of AdSGT1 from Arachis diogoi. Gel picture represents 5' RACE
PCR product of AdSGT1 and its ORF.
Figure S2 Alignment of the deduced amino acid sequences of AdSGT1 with closely
related SGT1 sequences from other organisms. Ad: Arachis diogoi, Gm: Glycine
max, Nb: Nicotiana benthamiana, Sl: Solanum lycopersicum, Ca: Cicer arietinum,
Fv: Fragaria vesca.
100
67
C. arietinum SGT1 gi|502180703|
C. arietinum SGT1-2 gi|502180699|
84
G.max SGT1-2 gi|351725565|
A. diogoi SGT1 gi|301087124|
62
B. oleracea SGT1 gi|40974915|
20
A. thaliana SGT1a gi|17017308|
99
100
A. lyrata SGT1a gi|297803780|
G. sanguineum SGT1 gi|111013943|
37
M. hupehensis SGT1 gi|311692888|
S. tuberosum SGT1 gi|51511450|
91
36
25
S. lycopersicum SGT1 gi|350535094|
100
67
N. benthamiana SGT1 gi|29468339|
N. benthamiana SGT1-2 gi|22212722|
100
41
S. lycopersicum SGT1-2 gi|460376830|
66
N. attenuata SGT1 gi|315307974|
V. vinifera SGT1 gi|302143763|
100
V. pseudoreticulata SGT1 gi|390985902|
R. communis SGT1 gi|255545142|
O. sativa SGT1 gi|6581058|
H. vulgare SGT1 gi|326507506|
100
100
82
T. aestivum SGT1 gi|126544454|
A. tauschii SGT1 gi|475592110|
S. salar SGT1 gi|224587011|
0.05
Figure S3 The phylogenetic relationship of AdSGT1 with other SGT1 family
members from different plant species constructed by neighbour-joining algorithms of
MEGA 4.0 software after the multiple protein sequences alignment using the
ClustalW program. Bootstrapping was performed to obtain support values for each
branch.
p2300
AdSGT1:p2300
Figure S4 Transient constitutive expression of AdSGT1 induced cell death in
tobacco leaf. Tobacco leaf was infiltrated with the Agrobacterium strain carrying the
empty vector p2300 and the AdSGT1-p2300. Photographs were taken 96-120 hours
post infiltration. HR-like cell death phenotype was observed in cDNA of AdSGT1
under 35S promoter.
Figure S5 Subcellular localization of AdSGT1. GFP was visualized using confocal
images of representative Nicotiana benthamiana leaf epidermal cells transiently
expressed
through
agroinfiltration,
(a)
Control
vector
pCAMBIA1302,
(b)
pCAMBIA1302:AdSGT1, AdSGT1 localizing to both nucleus and cytoplasm. Bars .꞊
50 µm (c) Western blot analysis of recombinant protein from agroinfiltrated area of
leaves. Samples were collected at 48 and 72 hrs post agroinfiltration and used. GFP
was tagged with Histidine and anti-mouse Histidine used as primary antibody and
detected with ALP conjugated goat anti-rabbit secondary antibody using BCIP/NBT
as substrate. Ponceau stain confirmed equal loading of protein. M- represents
protein molecular weight marker; -ve – represents negative control; +ve- represents
leaf area infiltrated control vector containing GFP tagged with histiditine, 48 and 72
hrs- samples of plants expressing AdSGT1 fused with GFP.
M
1
2
3
4
5
6
7
8
9
M
WT 11 12 13 14
16 17 18
1077 bp
Figure S6 Semi-quantitative RT-PCR analysis of AdSGT1 expression in wild type
and T0 transgenic plants. cDNA was synthesized from total RNA of wild type and
transgenic plants and amplified with AdSGT1-F and AdSGT1-R primers. Lane M- λ
DNA/EcoRI+HindIII marker; WT- represent wild type; Lane 1, 5, 6, 7, 9 and 17
represent low expression lines; Lane 2, 3, 8, 11, 12, 16 and 18 represent high
expression lines. Actin amplification has been used as control.
Figure S7 Detached leaf assay with Alternaria alternata var. nicotianae, Transgenic
tobacco expressing AdSGT1-T2 plants of low expression line 6 and high expression
line 18 showed enhanced resistance to infection with Alternaria alternata var.
nicotianae compared to WT plant. Photograph was taken after 7 days post infection.
Assay was repeated two-three times.
M
P
N
1
2
3
4
5
7
9
11
M
N
14
15 16
17 21
22
25
27
739 bp
M
P
N
28
31 33
34 37 38
39
M
N
42
45
46
47
51
54
739 bp
Figure S8 Screening of the T1 generation peanut plants by PCR with nptII gene
specific primers. Lane M: λ DNA/EcoRI+HindIII and λ DNA HindIII marker, P: Vector
positive control, N: negative control (DNA from untransformed plants), Rest of the
lanes are genomic DNA samples of putative transformants.
M
N
4-1 4-2 4-4 16-1 16-2 16-3 16-4
P
M
N 33-1 33-2 33-3 42-142-2 42 -3 45-1 45-2 54-1 54-2
739 bp
Figure S9 Screening of the T2 generation peanut plants by PCR with nptII gene
specific primers. Lane M: λ DNA/EcoRI+HindIII marker, P- Vector positive control, N:
Negative control (DNA from untransformed plants), Rest of the lanes are genomic
DNA samples of putative transformants.
.
M
P WT 4-1 4-2 4-3 4-4 16-1 16-2 16-3 16-4 16-5
M 33-1 33-2 33-3 42-1 42-2 45-1 45-2 54-1 54-2
1256 bp
Figure S10 Screening of the T2 generation peanut plants by PCR with 35S-F and
SGT-R gene specific primers. Lane M: λ DNA/EcoRI+HindIII marker, P: Vector
positive control, WT: Wild type (DNA from untransformed plants), Rest of the lanes
are genomic DNA samples of putative transformants.
.
M
P
4 16 22 33 36 42 45 51 54
750 pb
Figure S11 Represents putative T1- SGT1 transgenic peanut plants were tested by
PCR amplification using VirD2-F & VirD2-R primers using gDNA as template. M- λ
DNA/EcoRI+HindIII marker, P-positive control (pCAMBIA2300 in EHA105), Rest of
the lanes are genomic DNA samples of putative transformants. Absence of VirD2
amplification in putative transgenic plants indicates the absence of Agrobacterium
contamination in the in planta transformed peanut plants.
.
Figure S12 Southern hybridization analysis of peanut T1 transgenic plants
expressing AdSGT1 gene. DNA samples were digested with EcoRI, which has single
site on the T-DNA and blots were probed by PCR amplified fragment of nptII. Plants
#33, #42, #45 and #54 represent single copy integration where as plants #4 and #39
represent the integration of two copies of the T-DNA.
Figure S13 Relative expression of AdSGT1 examined by real-time reverse
transcription-polymerase chain reaction (qRT-PCR) in peanut T1 transgenic
AdSGT1-overexpressing lines. Data were plotted from three technical replicates.
Figure S14 Phaeoisariopsis personata whole plant bioassay performed on T 2transgenic plants under green house conditions. 105 conidia/ml were sprayed on
peanutn plants and symptoms developed after 15 days post inoculation (dpi) and
photographs were taken after 25 dpi. Note the development of late leaf spots on
transgenic plants resisted in comparison to wild type plant.
Table S1 Primers for different amplification reactions used
Gene Name
AdSGT1
NtPR1a
NtPR1b
NtHMGR
NtHIN1
NtHSR203J
Actin
Primer
Abbreviation
AdSGT1-F
AdSGT1-R
NtPR1a-F
NtPR1a-R
NtPR1b-F
NtPR1b-R
NtHMGR-F
NtHMGR-R
NtHIN1-F
NtHIN1-R
NtHSR203J-F
NtHSR203J-R
Actin-F
Actin-R
Primer Sequence (5'- 3')
CGGGCCCATGGCTTCTGATCTGGAAG
CACTAGTCTAATATTCCCATTTCTTCAACTC
CTTCTTGTCTCTACACTTCTC
GCAAGAGACAACATATCCTC
TCTTAACCCTCACAATGCAG
AGGGTTGCTCCTCAAGATC
GAGATCTGAGAAGCCTGCAT
GATTGGCATCTCACAGCACT
CCATGTAGCTGTCTCTTTGG
GATGGCATCTGGTTTCCTCA
GATAGAGGAAGTATCCGGCT
GTTGCTCCCTACTGGTAAAG
TGGCATCACACTTTCTACAA
CAACGGAATCTCTCAGCTCC
Table S2 Primers for different amplification reactions used
TDFs
Primer
Abbreviation
LRR-RLK
AdRTLRR-F
AdRTLRR-R
CC-NB-LRR
AdRTCNLRR-F
AdRTCNLRR-R
SGT1
AdRTSGT-F
AdRTSGT-R
HSP70
AdRT70HSP-F
AdRT70HSP-R
Serine/threonine AdRTSPK-F
protein kinase
AdRTSPK-R
Protein kinase-6 AdRTPK6-F
AdRTPK6-R
Defensin
AdDefensin-F
AdDefensin-R
Alcohol deAdRTADH3-F
hydrogenase -3 AdRTADH3-R
Primer Sequence (5'- 3')
CGGAAATGATGTCAAGTGTGGTGG
TGAAGTGTCAATTCTGCCCTCCAG
GTGTGGAGACGTTGGAATGCAAG
CTGACATCGTTTGGTCAGCAAAGG
GCTTGATGGCGATGCAGCTC
GGACTTCCCTCCACCTTCTTCG
GGACCAAGGCACCATGTAAGAGC
CTGCTCCCATGGCAACTGC
GAAGAAGGGGAACAGATGCTGGT
GATTGGAGGATCGGCGTGTTC
GGACTGTGGACCTCCCTAAGC
CGAGACCAAAGCGAGACCTCTC
GGGTACCATGGAGAAGAAATCACTAGC
GGGATCCTTAACATCTTTTAGTACACCA
GACGCTTGGCGAGATCAACA
AACCGGACAACCACCACATG