Supplemental Figure S1.
A
AtDIR1
NtDIR1
NtDIR2
NtDIR3
------------------------------------------------MASKKAAMVMMA
----------------------------------------------MHMIGNKVGVLVTV
------------------------------------------------MEAKQKLVIFVA
MLDPPLPLPINPYPVIHTLLLYKSFMSCCFSTYTHRKKERKRNMDMEHYLAKKPVALALV
12
14
12
60
AtDIR1
NtDIR1
NtDIR2
NtDIR3
MIVIMAMLVDTSVAID---LCGMSQDELNECKPAVSKENPTSPSQPCCTALQHADFACLC
FLGILLLIAELTNGLS---LCNMGDDGLTACKPSVTKPNPVEPSASCCEALSGADLQCLC
LVMVAAVGFEMAAAGSGDSPCGLSIGDLMSCKPAVSGPKPLPPSEKCCAALGKADLPCLC
AILLSSFSIEVSRAQG---ICNISGEGLMSCKPSVTPPNPSAPTAKCCSALAHADWGCLC
** ** ** ***
* *
* *** *
*
GYKNSPWLGSFGVDPELASALPKQCGLANAPTC-- 102
SYRNSLLLPSLGIDPELALALPPKCNLTSPANC-- 104
TFKNSPMISAFKINATLAMDLPSKCNLNSP-NCAA 106
SYMNSHWLPSLGVDPTLAMQLPQKCKLPNPPHC-- 150
*
** ** * *
**
69
71
72
117
AtDIR1
NtDIR1
NtDIR2
NtDIR3
B
Identity (%) to AtDIR1
Similarity (%) to AtDIR1
NtDIR1
43
58
NtDIR2
40
60
NtDIR3
46
66
35S::EV
Atdir1-1
Atdir1-1
35S::NtDIR3
35S::EV
35S::NtDIR2
35S::NtDIR1
35S::EV
C
Atdir1-1
NtDIR1
NtDIR2
NtDIR3
ACT2
ACT2
ACT2
Supplemental Figure S1. The tobacco homologs of AtDIR1. A. Alignment of the
protein sequences for AtDIR1, NtDIR1, NtDIR2, and NtDIR3. Asterisks below the
sequences indicate identical amino acids. B. Percentages of identity and similarity of
NtDIR1, NtDIR2, and NtDIR3 in comparison to AtDIR1. C. Analysis of CaMV 35Sdriven over expression of NtDIR1, NtDIR2 and NtDIR3 in the T1 generation of Atdir1-1
transgenics. The 35S-driven empty vector (EV) served as the control. ACT2 was used as
an internal control. NtDIR1, NtDIR2 and NtDIR3 were PCR amplified using 25 cycles
while 20 cycles were used to amplify ACT2. The T2 generations of these transgenic
plants were used in the SAR experiments shown in Figure 2A.
Liu et al.
Supplemental Figure S2.
AtDIR1
NtDIR1
NtDIR2
NtDIR3
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ATGCTGGATCCGCCCCTGCCCCTGCCCATAAACCCATACCCAGTCATACACACATTGTTA 60
AtDIR1
NtDIR1
NtDIR2
NtDIR3
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CTATATAAATCCTTTATGTCATGTTGTTTCTCTACATACACTCACAGAAAGAAAGAAAGA 120
AtDIR1
NtDIR1
NtDIR2
NtDIR3
---------------ATGGCGAGCAAGAAAGCAGCTATGGTTATGA---TGGCGATGATC
---------ATGCATATGATAGGAAACAAAGT---GGGAGTATTGG---TTACGGTGTTT
---------ATGGAAGCAAAGCAAAAGCTTGT---AATTTTTGTGGCACTTGTGATGGTT
AAGAGAAATATGGACATGGAGCATTACTTGGCCAAAAAACCTGTGGCATTGGCTTTGGTT
*
*
**
*
** *
GTGATAATGG----CTATGTTGGTC--GATACATCAGTAGCGATAGATCTCTGCGGCATG
CTAGGAATAC-TACTACTTATAGCA--GAATTAACAAATGGGTTGAGCTTGTGTAACATG
GCAGCCGTAG-GATTTGAAATGGCG--GCGGCAGGATCCGGAGATTCACCGTGTGGTTTA
GCCATTTTGTTGAGCAGTTTTAGCATAGAAGTGTCGAGAGCACAGGGAATATGTAACATA
*
* *
*
*
**
*
AGCCAGGATGAGTTGAATGAGTGCAAACCAGCGGTTAGCAAGGAGAATCCGACGAGCCCA
GGCGATGATGGACTCACGGCGTGTAAGCCATCAGTAACAAAGCCAAATCCAGTGGAGCCA
AGCATTGGTGATCTAATGTCATGTAAGCCAGCAGTATCTGGTCCTAAACCATTGCCACCA
TCAGGAGAAGGTTTAATGTCATGTAAACCATCAGTAACACCCCCGAACCCGTCGGCGCCC
* *
* *
** ** *** * **
** **
*
**
TCACAGCCTTGCTGCACCGCTCTGCAACACGCTGATTTTGCATGTCTTTGTGGTTACAAG
TCAGCTTCTTGCTGTGAAGCCTTGTCTGGTGCAGATTTGCAGTGCTTGTGTTCTTATAGG
TCTGAAAAATGTTGCGCTGCTTTAGGCAAAGCAGATTTGCCTTGCCTTTGCACTTTCAAG
ACAGCCAAGTGCTGCAGCGCGCTGGCACATGCAGACTGGGGATGCCTTTGCTCTTATATG
*
** **
** *
** ** *
** * **
** * *
AACTCTCCATGGCTCGGTTCTTTCGGTGTTGATCCTGAACTCGCTTCTGCTCTCCCCAAA
AATTCACTCTTATTGCCTTCTCTTGGAATTGATCCAGAACTTGCTTTGGCTCTTCCTCCT
AACAGTCCAATGATATCTGCATTCAAGATTAATGCAACTCTTGCCATGGATCTCCCCTCA
AATTCTCACTGGTTGCCTTCTTTAGGAGTTGATCCAACACTTGCAATGCAACTCCCTCAG
**
*
*
* * *
** ** *
** **
** **
CAGTGTGGTCTAGCCAACGCC--CCAACTTGTTAA- 309
AAATGCAATCTCACTTCTCCTG-CTA-ATTGTTAA- 315
AAATGCAATCTTAATTCTCCAAACTGCGCCGCTTAA 321
AAATGCAAACTCCCTAATCCTCCCCA--TTGCTAA- 453
* **
**
*
*
* * *
AtDIR1
NtDIR1
NtDIR2
NtDIR3
AtDIR1
NtDIR1
NtDIR2
NtDIR3
AtDIR1
NtDIR1
NtDIR2
NtDIR3
AtDIR1
NtDIR1
NtDIR2
NtDIR3
AtDIR1
NtDIR1
NtDIR2
NtDIR3
42
45
48
180
96
102
105
240
156
162
165
300
216
222
225
360
276
282
285
420
Supplement Figure S2. Alignment of the full-length cDNAs of AtDIR1, NtDIR1,
NtDIR2, and NtDIR3. NtDIR1, NtDIR2, and NtDIR3 contain 315, 321, and 453
nucleotides, respectively. Asterisks below the sequences indicate identical nucleotides.
Liu et al.
Supplemental Materials and Methods S1
Plant Materials and Growth Conditions
The Arabidopsis dir1-1 mutant (Atdir1-1) and its control plant (wt Ws-2) were obtained
from Robin K. Cameron. Seeds for soil-grown plants were sown on autoclaved soil. After
3 days of pre-chilling at 4ºC, the seeds were germinated and grown under a 9h-light/15hdark photoperiod (140 μE m-2s-1) at 22ºC and 70% relative humidity (RH) for SAR
induction by the coronatine-deficient Pseudomonas syringae pv. maculicola carrying
AvrRpt2. For the complementation experiment, the construct for overexpression of the
full length NtDIR1 (GeneBank no. JF275846), NtDIR2 (GeneBank no. JF275847) and
NtDIR3 (GeneBank no. JF275848) genes under the CaMV 35S promoter and their empty
vector control (named 35S::NtDIR1, 35S::NtDIR2, 35S::NtDIR3, and 35S::EV) were
transformed into Atdir1-1. Antibiotic resistant T2 transgenic plants were used in the SAR
experiments. For silencing of NtDIR gene expression in tobacco, partially overlapping
segments of approximately 300 nucleotides from each of the three NtDIR genes were
sequentially amplified by PCR using 5 combinations of the primer sets: 1. NtDIR1-5’XhoI (5′-CTCGCGGTGGGAGTATTGGTTACGGTG-3′) and NtDIR1-3’+NtDIR2 (5′CAAACTGCGCCGCGCAGGAGAAGTGAGATTG-3′); 2. NtDIR1-5’-XbaI (5′TCTAGAGTGGGAGTATTGGTTACGGTG-3′) and NtDIR1-3’+NtDIR2 (5′CAAACTGCGCCGCGCAGGAGAAGTGAGATTG -3′); 3. NtDIR2-5’+NtDIR1 (5′CACTTCTCCTGCGCGGCGCAGTTTGGAGAA-3′) and NtDIR2-3’+NtDIR3 (5′CAATGTGTGTATGACGCGGCGCAGTTTGGAGAA-3′); 4.NtDIR3-5’+NtDIR2 (5′CAAACTGCGCCGCGTCATACACACATTGTTA-3′) and NtDIR3-3’-KpnI (5′GGTACCGCAATGGGGAGGATTAGGGAGTTTGC-3′); 5. NtDIR3-5’+NtDIR2 (5′CAAACTGCGCCGCGTCATACACACATTGTTA -3′) and NtDIR3-3’-ClaI (5′ATCGATGCAATGGGGAGGATTAGGGAGTTTGC-3′). The correct sequences of the
sense NtDIR1+NtDIR2+NtDIR3 fragment with XhoI/KpnI and antisense
NtDIR3+NtDIR2+NtDIR1 fragment with ClaI/XbaI were inserted into pHANNIBAL.
This NtDIRs cassette was transferred to the binary vector pART27 (Gleave 1992) using
NotI (pART27::NtDIRs) for Agrobacterium transformation. The pART27::NtDIRs
constructs were transformed into Agrobacterium tumefaciens LBA4404 (Invitrogen,
Liu et al.
Carlsbad, CA, U.S.A.) by electroporation and then transformation into the Nicotiana
tabacum (tobacco) cv. Xanthi nc (NN) plants. The T1 and T2 generation of transgenic
plants were use in this study. See “Pathogen Experiments” below for more details.
Pathogen Experiments
In Arabidopsis SAR experiments, coronatine-deficient Pseudomonas syringae pv.
maculicola carrying AvrRpt2 at a concentration of 1 × 106 CFU/ml in 10 mM MgCl2 was
infiltrated into abaxial surfaces of three leaves per plant with a 1-ml syringe to induce
SAR in 3 to 4 week old plants grown under a 9h-light/15h-dark photoperiod (140 μE m2 -1
s ) at 22°C and 70% RH. Plants infiltrated with 10 mM MgCl2 served as mock controls.
At 2 to 3 days post primary infection (dpl°i), two or three uninoculated distal systemic
leaves were challenged with virulent Pseudomonas syringae pv. tomato DC3000 (Pst) at
a density of 1 × 105 CFU/ml in 10 mM MgCl2. Pst growth was determined by shaking 4
mm leaf disks taken at 3 dp secondary (2°)i in 10 mM MgCl2 supplemented with 0.1 M
sucrose at room temperature for 4 h. The resulting bacterial suspensions were serially
diluted and spots of 20 μl per dilution were grown on Kings’ B medium with 20 μg/ml
rifampicin, 50 μg/ml kanamycin, and 1.7% agar at 28ºC for 48 h, after which time, the
number of colonies formed were counted. In tobacco SAR experiments, primary TMV
and mock inoculations were carried out on approximately 6 week old tobacco plants as
previously described (Guo et al., 2000). Five dpi with TMV or mock, three uninoculated
systemic leaves of all plants were challenged with TMV. The size of 2° TMV lesions
were measured after 5 dp2°i with a vernier caliper as previously described (Liu et al.
2010a). Percent reduction in size of the 2° lesions on plants previously receiving the 1°
TMV infection compared to 2° lesions on 1° mock-infected tobacco served as a measure
of SAR.
MeSA Quantification
MeSA contents in approximately 100 mg of tissues per sample were measured using GCMS (CP-3800/Quadrupode- 1200L system; Varian, Walnut Creek, CA, U.S.A.), as
previously described (Liu et al. 2010b). Three to four leaves per plant from
Liu et al.
approximately 20 plants grown under short-day conditions were used for MeSA
quantification for each genotype and time point.
SA and SAG Quantification
SA and SAG were extracted and quantified from approximately 250 mg of tissue per
sample using high-performance liquid chromatography analysis on an ARH-601 organic
acids column (100 mm × 6.5 mm; Transgenomic Inc., Omaha, NE, U.S.A.) run at 55°C
in 0.01 N H2SO4 with a flow rate of 0.6 ml/min, as previously described (Liu et al.
2010b). Three to four leaves per plant from approximately 40 plants grown under shortday conditions were used for each genotype and time point.
In planta MeSA assay
MeSA was obtained from Sigma. Three leaves of 3 to 4 week old Arabidopsis wt Ws-2
and Atdir1-1 plants grown under a 9h-light/15h-dark photoperiod (140 μE m–2s–1) at 22°C
and 70% RH were infiltrated, using a 1-ml syringe with concentrations of 0 μM, 3.3 μM,
and 6.6 μM of MeSA (kept at 4ºC) as previously described (Liu et al. 2010b). At 3 dpi,
two untreated distal leaves were challenged with the virulent Pst for 2 days. Growth of
virulent bacteria was determined as described in the Arabidopsis SAR assay above. The
three lower leaves of approximately 6 week-old tobacco wt and RNAi::NtDIRs #9 plants
were infiltrated with 0 μM and 5 μM of MeSA. At 5 dpi, 3 uninoculated systemic tissues
were challenged with TMV. The size of 2°TMV lesions were measured as described in
the tobacco SAR assays above.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from leaves, using Trizol reagent (Invitrogen Carlsbad, CA,
U.S.A.) according to the manufacturer’s instructions. DNase-treated total RNA (2 μg)
was used for reverse transcription with a Superscript II kit (Invitrogen). The RT product
was subjected to semi-quantitative PCR using the gene-specific forward and reverse
primers with control primers, EF1α (Elongation Factor 1α), EIF4a (Eukaryotic
Translation Initiation Factor 4a) and ACT2 (Actin 2) as previously described (Liu et al.
2005; Liu et al. 2010a; Liu et al. 2010b). The conditions for semi-quantitative PCR to
Liu et al.
amplify EF1α and ACT2 were: one cycle at 94°C (4 min), one cycle at 80°C (2 min),
touchdown cycles (94°C for 15 s, 67°C → 61°C for 15 s, and 72°C for 30 s, one cycle for
each temperature between 67 and 61°C), and 20 cycles at 94°C (15 s), 60°C (15s), and
72°C (30 s), followed by extension at 72°C (7 min). The conditions for PCR to amplify
AtBSMT1 and NtSAMT1 (Accession# FJ015052) were essentially the same as above,
except for the 25-cycle amplification. For amplifying NtDIR1, NtDIR2, NTDIR3, and
EIF4a, the PCR conditions used were essentially the same conditions as above except for
the temperatures for touchdown cycles (62°C  56°C for 15 sec) and the 20-cycle
amplification for EIF4a amplification and 25 cycles used for NtDIR1, NtDIR2, and
NTDIR3 amplification (94°C for 15 sec, 55°C for 15 sec and 72°C for 30 sec). AtBSMT1
forward (5′-TATGGAGCTCCAGGTTCCTT-3′) and AtBSMT1 reverse (5′ACGACAAGACTGACAGTCGT-3′); EIF4a forward (5′AAGAGAGCTGGCTACACAAA-3′) and EIF4a reverse 5′ATGCATTGATGAGACAGTGA-3′); NtSAMT1 forward (5′AATACCATTTTCCGCTCG-3′) and NtSAMT1 reverse (5′GGTCAAGGAGACAACAAC-3′) primers; EF1α forward (5′AAGTATGCCTGGGTGCTTG -3′) and EF1α reverse (5′AGGGACAGTACCAATTCCACC-3′); NtDIR1 forward (5′ATGCATATGATAGGAAACAAAG-3′) and NtDIR1 reverse (5′TTAACAATTAGCAGGAGAAG-3′) primers; NtDIR2 forward (5′AAGCAAAGCAAAAAGCTTG-3′) and NtDIR2 reverse (5′TTAGCAATGGGGAGGATTAG-3′) primers; NtDIR3 forward (5′CTGCCCATAAACCCATACC-3′) and NtDIR3 reverse (5′TCTGAGGGAGTTGCATTGC-3′) primers; ACT2 forward (5′GCCATCCAAGCTGTTCTCTC-3′) and ACT2 reverse (5′GAACCACCGATCCAGACACT-3′)
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