Supplementary information
Results
We note that PR-1 gene expression is delayed in inoculated leaves in this particular
experiment (Fig.1-Supplementary Information), but that in 6 of 8 experiments, PR-1
expression was similar in Ws and dir1-1. Therefore we can sustain the conclusion that
there is little or no delay in defense gene activation in dir1-1. More importantly, avirulent
and virulent Pst grow to similar levels in both naïve dir1-1 and wild type Ws plants,
indicating that dir1-1 is affected in just the SAR pathway.
Table 1
Genotype
Ws
Free Salicylic Acid (ng gfw-1) in Ws and dir1-1
Control
(M,M)1
5
8213
Induction
(M,A-inoc)2
1633243
Establishment
(M,A-uninoc)3
12718
Manifestation
(A,V)4
1685384
dir1-1
8212
1469528
13256
1478529
1
˚
(M,M) – M, primary mock inoculation, M, 2 mock-inoculation in other leaves.
2,3
(M,A) – M, primary mock-inoculation, A, 2˚ inoculation with avirulent Pst (106 cfu ml-1)
in other leaves, SA levels determined 2 days post inoculation (dpi) in 2˚ inoculated leaves
(inoc) or remaining uninoculated leaves (uninoc).
4
(A,V) – A, primary inoculation with avirulent Pst (106 cfu ml-1), V, 2˚ challenge
inoculation with virulent Pst (106 cfu ml-1) in other leaves. SA levels determined 2 dpi in
2˚ inoculated leaves in MM and AV.
5
Average and standard deviation of three replicates, significant differences were not
observed (t-test, p>0.5). This experiment was repeated twice with similar results.
SAR is abolished to P.parasitica in dir1-1
Ws plants that were mock-inoculated and dipped in P. parasitica displayed visible
sporulation on 9 of 10 leaves, while Ws plants that were induced by local inoculation with
PstavrRpt2 displayed sporulation on only 2 of 12 challenged distant leaves. In contrast,
dir1-1 plants that had been inoculated with PstavrRpt2 allowed extensive sporulation on
susbequent challenge with P. peronospora (12 of 13 leaves), comparable to that
observed in uninduced controls (8 of 9 leaves). Thus Pst-induced SAR to P. parasitica
was also abolished in the dir1-1 mutant.
Methods
Salicylic Acid Determination
Extraction of salicylic acid from 0.5g batches of Arabidopsis leaf tissue (20-30 leaves)
that had been ground in liquid N2 and analysed by HLPC was as described previously1.
P. parasitica infections and INA applications
Arabidopsis ecotype Ws leaves with sporulating P. parasitica (Emco5 711198) were
provided by John McDowell (U. of North Carolina) and used to make spore suspensions
of 2-5 x 105 spores ml-1. Detached Arabidopsis leaves from plants inoculated 2-3 days
previously with MgCl2 or PstavrRpt2 were dipped in spore suspensions containing 0.02%
silwet L-77 (OSI Specialities, Danbury CT., USA) for five minutes, then floated on water
in sterile petri dishes for 10 d. Sporulation was assessed by eye and using a dissecting
microscope (repeated once with similar results). In INA rescue experiments2 plants were
sprayed with either sterile water or INA (100 g ml-1), then challenge-inoculated 5 d later
with virulent Pst (106cfu ml-1) (repeated twice with similar results each time). Symptoms
were visually scored 3 d later. A plant was considered SAR competent if at least three of
four leaves were symptomless.
Amplification of T-DNA flanking sequences
Genomic sequences flanking the T-DNA insertion were amplified using a thermal
asymmetric interlaced (TAIL)-PCR. The reaction conditions and the thermal cycling
settings were as described by Liu et al.7. Taq polymerase (Perkin Elmer) was used for all
PCR reactions. TAIL-PCR from the right border (RB) of the T-DNA was performed with
specific primers designed to anneal at the end of the RB. The T-DNA specific primers
were:
R1:
5’-TGCTAGCTGATAGTGACCTTAGG-3’,
R2:
5’-
CTGACGTATGTGCTTAGCTC-3’, R3: 5’-GGTTCTGTCAGTTCCAAACG-3’
TAIL-PCR from the left border (LB) of the T-DNA was performed using specific primers
designed to anneal at the end of the LB: (L1:5’-ATCTCGACGATATAGAGCAAGATGG3’), (L2:5’GAATCCTGTATATCAGACATTAGGAAGCA-3’),
(L3:5’ CTGTAATGACTCCGCGCAATATTTACAC-3’).
Eight different degenerate arbitrary primers were used. Their sequences are:
(AD1:
5’-NTCGA(G/C)T(A/T)T(G/C)G(A/T)GTT-3’),
(AD2:
5’-
NGTCGA(G/C)(A/T)GANA(A/T)GAA-3’), (AD3: 5’-(A/T)GTGNAG(A/T)ANCANAGA-3’),
(AD4:
5’-TG(A/T)GNAG(A/T)ANCA(G/C)AGA-3’),
(AD5:
5’-
AG(A/T)GNAG(A/T)ANCA(A/T)AGG-3’), (AD6: 5’-CA(A/T)CGICNGALA(C/G)GAA-3’, I
indicates inosine), (AD7: 5’-TCTTICGNATCTTNGGA-3’).
DNA sequence analysis
Tertiary TAIL-PCR products were separated by electrophoresis on a 1.5% agarose gel.
PCR products were purified from agarose gel slices with QiaquickGel extraction Kit
(Qiagen). The purified products were cloned in pGEM-T Easy Vector System (Promega)
and sequenced.
DNA blot hybridization
Southern blots, hybridization screening of the Arabidopsis CD4-7 library in Zip Lox8,
clone purification, subcloning and nucleotide sequencing were performed using standard
techniques.
Construction of transgenic plants
A 353 bp fragment containing the DIR1 protein coding region was amplified by PCR
using as template the DIR1 cDNA. The primers used for the amplification contained the
appropriate endonuclease restriction sites for posterior cloning into the binary vector
pCHF23: BamHI
primers P45: 5’-GGCGGATCCAGAGAGGAGGATTAATAATATG-3’,
and P44: 5’-GGCGGATCCAGAGAGTTCTTTTAACAAGTTGG-3’ were used for the
sense
and
anti-sense
construct
respectively)
GGCCTGCAGAGAGTCTTTTAACAAGTTGG-3’,and
and
PstI
primers
P43,
P46,
5’5’-
GGCCTGCAGAGAGGAGGATAATAATATGGC-3’ were used for the sense and antisense construct respectively. The PCR products were subcloned into the pGEM-T Easy
Vector System (Promega), sequenced and cloned into the BamHI-PstI sites of the
pCHF2 binary vector between the 35S promoter and the tm 3’ terminator, to give
pCHF2.LTP and pCHF2.anti-DIR1.
Arabidopsis transformation
Agrobacterium tumefaciens strain ASE and the floral dipping method were used in all
transformations4. The plasmid pCHF2.LTP was introduced into Ws and dir1-1 plants and
pCHF2.anti-LTP was introduced into Ws. Seeds resulting from self-pollination of the
transformants (T2) were scored for antibiotic resistance on MS medium(Gibco BRL)
containing
100 g ml-1 gentamycin or both kanamycin (50 µg ml-1) and 100 µg ml-1
gentamycin. Those progeny giving a 3:1 (resistant : susceptible) segregation of the
gentamycin marker were checked for expression of the transgene. Homozygous lines
were identified by screening 10 gentamycin resistant T2 progeny from each independent
transformant (T3 generation) for non-segregation of the marker.
DIR1 antibody production
A 249 bp fragment containing the DIR1 mature protein without the signal peptide was
amplified by PCR using as template the DIR1/LTP cDNA. The primers used for the
amplification and the appropriate endonuclease restriction sites for posterior cloning into
the
expression
vectors
were:
ACAGAATTCAGCGATAGATCTCTGCGGCA-3’)
EcoRI
and
(primer
XhoI
(primer
P47:5’P48:5’-
AGTCTCGAGACAAGTTGGGCGTTGGC-3’). The PCR products were subcloned in the
pGEM-T Easy Vector System (Promega) and sequenced. These were then cloned into
the EcoRI-XhoI sites of the pET-29b expression vector (Novagen) and transformed into
the E.coli strain BL21 (DE3). The protein expression and purification was performed
according to the pET System Manual (Novagen). The purified protein was used to
immunize rabbits at 3-week intervals.
References
1. Cameron, R.K, Paiva, N.L., Lamb C.J., Dixon, RA. Accumulation of salicylic acid and
PR-1 gene transcripts in relation to the Systemic Acquired Resistance (SAR)
response induced by Pseudomonas syringae pv. tomato in Arabidopsis. Physiol.
Mol. Plant Pathol. 55,121-130 (1999).
2. Uknes, S. et al. Acquired resistance in Arabidopsis. Plant Cell 4, 645-656 (1992).
3. Hajdukiewicz, P. Svab, Z., Maliga, P. The small, versatile pPZP family of
Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25, 989-994
(1994).
4. Bent, A.F. Arabidopsis in planta transformation. Uses, mechanisms and prospects
for transformation of other species. Plant Physiol. 124,1540-1547 (2000).
Figure Legend
Figure 1 – Defense gene expression in Ws and dir1-1 plants
Plants were inoculated with avirulent Psm-avrRpm1(107 cfu ml-1) to induce SAR, leaves
were collected at 0, 12, 24, 36, 48 hours post inoculation (hpi) from both the inoculated
leaves representing a), the induction stage of SAR and from uninoculated or systemic
leaves b), representing the establishment stage of SAR. RNA gel blots were prepared
and hybridized with gene probes for PR-1, PR-5, GST and rDNA.