Post on 08-Oct-2020
0.1 kb
M pen2-4
PCR
product
uncut Col-0
sid2
pen2-4 M Col-0 coi1-16
0.2 kb
PEN2
pen2-4
445 195 66
640 66
AciI AciI
AciI
(Westphal et al., 2008)
COI1 122 25
coi1-16 25 78 44
Tsp509I
Tsp509I Tsp509I
Supplemental Figure 1 - Geng, Cheng, Gangadharan and Mackey
A
B
0.2 kb
0.65 kb
0.4 kb
M coi1 Col-0 M sid2
coi1
npr1
coi1 M Col-0
sid2
npr1
coi1 pen2-4
PCR
product
uncut C
D E
Supplemental Figure 1. DNA analysis of mutant bacteria and plants. (A) At left are schematics of the Cfa6 locus (top) and the Tn5-inserted cfa6 locus (bottom). The hatched bar within cfa6 indicates the region of the cfa6 mutant locus found in plasmid pDB29 and integrated by homologous recombination. P1 through P4 indicate the position of primers. At right are the products (or lack thereof) of colony PCRs including P1, P2, and P3 (top gel) or P2, P3, and P4 (bottom gel). (B) Structure of PEN2 and pen2-4. Shown are schematics of the 706-bp PCR products, including the AciI restriction sites. AciI cuts the PCR fragments from wild-type PEN2 into 445 bp, 195 bp and 66 bp and cuts those from pen2-4 into 640 bp and 66 bp. (C) AciI restriction of the 706-bp PCR fragment of PEN2/pen2-4 from Col-0, coi1-16, sid2 coi1-16, npr1 coi1-16, sid2 npr1 coi1-16, and pen2-4 plants. The results indicate that all coi1-16 lines have been “cured” of the pen2-4 mutation. (D) Structure of COI1 and coi1-16. Shown are schematics of the 147-bp PCR products, including the Tsp509I restriction sites. Tsp509I cuts the PCR fragments from wild-type COI1 into 122 bp and 25 bp and cuts those from coi1-16 into 78 bp, 44 bp and 25 bp. (E) Tsp509I restriction of the 147-bp fragment of COI1/coi1-16 from Col-0, pen2-4, sid2 pen2-4, and coi1-16 plants. The results indicate that the pen2-4 and sid2 pen2-4 lines have been “cured” of the coi1-16 mutation.
1.65KbP1+P3P1+P2
P4+P3P4+P21.65Kb
1.65KbP1+P3P1+P2
P4+P3P4+P21.65Kb
P1+P3P1+P2
P4+P3P4+P21.65Kb
1.65 kb
1.65 kb
P1 P3 Tn5
P2
~1350 bp
~1390 bp no product
no product
P4
P1 P3
1698 bp
1738 bp
P4
Cfa6
cfa6
M Pto cor- ∆CEL
∆CEL
cor-
Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312
1
0
0.5
1
1.5
2
2.5
3
Col-0
PR
1 e
xp
res
sio
n
Pto cor-
△CEL△CEL cor-buffer
* *
**
Supplemental Figure 2 - Geng, Cheng, Gangadharan and Mackey
Pto cor- ∆CEL ∆CEL cor- buffer
48 h
72 h
Pto cor- ∆CEL ∆CEL cor- buffer
Pto cor- ∆CEL ∆CEL cor- buffer
24 h
A
B
Rubisco
Rubisco Rubisco
Supplemental Figure 2. COR inhibits PR-1 expression. (A) qRT-PCR analysis of PR1 expression 24 h after infiltration of Col-0 leaves with Pto, Ptocor-, Pto∆CEL, Pto∆CEL cor-, or buffer. Shown are the mean and standard error of combined data from of four biological repeats with the level of each transcript induced by Pto∆CEL set to 1. Significant differences were determined by two-tailed t test: * p<0.05 between cor- and DCEL cor- relative to Pto and DCEL, respectively, ** p<0.005 between buffer and all other samples. (B) Immunoblot showing the accumulation of PR-1 in Col-0 leaves 24, 48, and 72 h after infiltration with the indicated bacterial strains. The experiment was done four times with similar results. Arrows point to PR-1 and the upper band is a non-specific cross-reacting protein. Ponceau staining of RuBisCo beneath the blots demonstrates equal loading of samples.
Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312
2
Supplemental Figure 3. SA accumulation after bacterial infiltration. Accumulation of free SA was measured by HPLC after infiltrating Col-0, sid2, coi1 and sid2 coi1 plants with the indicated bacterial strains or buffer. Leaves were collected 15 h post infiltration. Shown is the combined data from three biological replicates and error bars indicate standard deviations. No SA was detected in sid2 and sid2 coi1, which, based on our SA standards, corresponds to < 0.02 mg/g fresh weight. Statistical analyses of the indicated samples were by one-way ANOVA and Fisher LSD test with significant differences (p<0.05) indicated by lowercase letters.
Supplemental Figure 3
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Col 0 sid2 coi1 sid2 coi1
Ptocor-∆CEL∆CEL cor-buffer
Col-0 sid2 coi1 sid2 coi1
ab
ab
bc
c c
c
a
c
SA μ
g/g
fre
sh w
eigh
t
Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312
3
2.5
3.5
4.5
5.5
6.5
7.5
8.5
9.5
Col-0 coi1 sid2 npr1 sid2 npr1coi1
Log
CFU
/cm
2
Ptocor-ΔCELΔCEL cor-
Col-0 coi1 sid2 npr1 sid2 npr1 coi1
A
B
BC C a a a a
A’
B’
C’
BC’ a’
c’ c’ b’
Supplemental Figure 4 - Geng, Cheng, Gangadharan and Mackey A
B
Supplemental Figure 4. COR promotes bacterial virulence in sid2 npr1 plants independent of targeting COI1. (A) Growth of the indicated strains 4 days after inoculation into Col-0, coi1, sid2 npr1 and sid2 npr1 coi1 plants. The dashed line indicates the starting inoculum of bacteria. Shown are the mean and standard error of five biological replicates. Different letter types indicate significant differences (p<0.05) by one-way ANOVA and Tukey HSD test of comparisons between the different bacterial strains on individual plant genotypes. (B) Quantification of callose deposits following infiltration of the indicated strains into Col-0, coi1, sid2 npr1 and sid2 npr1 coi1 leaves. Shown are the mean and standard error of combined data from two independent biological replicates. Statistical analyses of log transformed data of indicated samples were by one-way ANOVA and Tukey HSD test (p<0.05).
0
0.5
1
1.5
2
2.5
3
3.5
Pto cor- ΔCEL ΔCEL/cor- buffer
Log
Cal
lose
dep
osits
/1.1
mm
2 Col-0coi1
sid2 npr1
sid2 npr1 coi1
b bc c
a
Pto cor- ∆CEL ∆CEL cor- buffer
Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312
4
Supplemental Figure 5 - Geng, Cheng, Gangadharan and Mackey
Supplemental Figure 5. PEN2 is not required for flg22-induced callose deposition in soil-grown plants. (A) Callose deposition 15 hours after infiltration of 30 mM flg22 or buffer into Col-0 and pen2-1 leaves. Shown are representative fluorescence microscopy images of aniline blue-stained leaves (scale bar = 0.2 mm). The experiment was done two times with similar results. (B) Callose deposition in the cotyledons of 10-day-old liquid-grown seedlings of Col-0, sid2, pen2-1, pen2-4 and sid2 pen2-4 after 18 h of exposure to 1 mM flg22 or the control treatment. Shown are representative fluorescence microscopy images of aniline blue-stained leaves (scale bar = 0.2 mm). The experiment was done two times with similar results.
A
B
Col-0 pen2-1
water
flg22
Col-0 sid2 pen2-1 sid2 pen2-4 pen2-4
water
flg22
Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312
5
0
0.5
1
1.5
2
Col-0 sid2 pen2-1 sid2pen2-4
bufferflg22 (10 μM)ΔCELΔCEL + 3 μM CORΔCEL cor- ΔCEL cor- + 3 μM COR
Supplemental Figure 6 - Geng, Cheng, Gangadharan and Mackey A
B
Supplemental Figure 6. COR inhibits indole glucosinolate accumulation upstream of PEN2. Accumulation of 1MI3G (A) and I3G (B) after infiltration of buffer, flg22, or the indicated bacterial strains, with or without 3 mM COR, into Col-0, sid2, pen2-1 and sid2 pen2-4 leaves. Quantities of 1MI3G and I3G were calculated relative to sinigrin (spiked into each sample) and normalized with the amount elicited by Pto∆CEL in pen2-1 set to 1. Shown are the means and standard error from three biological replicates. Different letter types indicate significant differences (p<0.05) by one-way ANOVA and Tukey HSD test of comparisons between the indicated bacterial strains on individual plant genotypes.
0
0.5
1
1.5
2
2.5
3
3.5
4
Col-0 sid2 pen2-1 sid2pen2-4
I3G
acc
umul
atio
n
buffer
flg22 (10 μM)
ΔCEL
ΔCEL + 3 μM COR
ΔCEL cor-
ΔCEL cor- + 3 μM COR
A
A
A
A
a
a
a a
0
0.5
1
1.5
2bufferflg22 (10 μM)ΔCELΔCEL + 3 μM CORΔCEL/cor- ΔCEL/cor- + 3 μM COR
AA A
A
a
aa
a
Col-0 sid2 pen2-1 sid2 pen2-4
I3G
acc
umul
atio
n
A A A
A
a
a
a
a
1MI3
G a
ccum
ulat
ion
0
0.5
1
1.5
2bufferflg22 (10 μM)ΔCELΔCEL + 3 μM CORΔCEL/cor- ΔCEL/cor- + 3 μM COR
AA A
A
a
aa
a
Col-0 sid2 pen2-1 sid2 pen2-4
Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312
6
Supplemental Table 1. Phenotype of Arabidopsis types used in this study
Supplemental Tables
Plant type Phenotype
Col-0 Wild-type
sid2 Lacks isochorismate synthase required for the majority of defense-
associated production of SA
npr1 Lacks a key signaling molecule for transducing the SA-signal to a
transcriptional response
coi1 Lacks the only known receptor for active JA-conjugates and
coronatine
pen2 Lacks the myrosinase that hydrolyzes 4-methoxy-indol-
3ylmethylglucosinolate (4MI3G)
sid2 pen2 Deficient in SA-production and hydrolysis of 4MI3G
sid2 coi1 Deficient in SA-production and JA-signaling
npr1 coi1 Deficient in SA-signaling and JA-signaling
sid2 npr1 Deficient in SA-production and SA-signaling
sid2 npr1 coi1 Deficient in SA-production, SA-signaling and JA-signaling
Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312
7
Allele Forward
primer Reverse primer Comments
sid2-2 5’-
gaaagacgacct
cgagttctcta-3’
5’-cgtaagtctccctgcca
attgag-3’
Deletion mutant: SID2 product
= 470bp, sid2-2 gives no
product
npr1-1 5’-
atgtctcgaatgta
cataaggc-3’
5’-catgagtgcggttctac
cttc-3’
NlaIII digestion: NPR1
products = 200 and 100bp,
npr1-1 product = 300bp
coi1-16
5’-caaagacttggaa
accatagctagaa
attg-3’
5’-ccaatatcctcattcaag
gagccaccacaagat-
3’
Tsp509I digestion: COI1
products = 122 and 25bp, coi1-
16 products = 78, 44, and 25bp
pen2-4 5’-
aaacgttgccgtt
gatttct-3’
5’-cagcaacactagcgcc
atta-3’
AciI digestion: PEN2 products
= 445, 195, and 66bp, pen2-4
products = 640 and 66bp
Cfa6
and
cfa6::Tn5
P1 = 5’-
ctggtccggtcgg
tctaggta-3’
P4 = 5’-
cgagcaactcgtc
gagcatac-3’
P3 = 5’-
cggacctacgtcgagg
taaca-3’
P2 = 5’-
agatctgatcaagaga
cag-3’
See Supplemental Figure 1 for
details on the position of these
primers within the Cfa6 and
cfa6::Tn5 loci.
Supplemental Table 2. Primers used for screening mutant plants and bacteria
Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312
8
Supplemental Table 3. Primers used for qRT-PCR
Gene Primers
ACTIN2 (AT3G18780) 5’-CTAAGCTCTCAAGATCAAAGGCTTA-3’ 5’-TTAACATTGCAAAGAGTTTCAAGGT-3’
MYB51 (AT1G18570) 5’-ACAAATGGTCTGCTATAGCT-3’ 5’-CTTGTGTGTAACTGGATCAA-3’
CYP79B2 (AT4G39950) 5’-GTAACTTCGGAGCATTCGT-3’
5’-TCGCCGGATATCACATCC-3’
CYP79B3 (AT2G22330) 5’- AGTCACTTCCGAACACTCA-3’
5’-TCGCAGGTTACCATATTCC-3’
CYP83B1 (AT4G31500) 5’-TCACGCCATATCTACCAGC-3’
5’-TGGACGTCATGACTGGAC-3’
CYP81F2 (AT5G57220) 5’-CTCATGCTCAGTATGATGC-3’ 5’-CTCCAATCTTCTCGTCTATC-3’
PR-1 (AT2G14610) 5’-CTACGCAGAACAACTAAGAGGCAAC-3’
5’-TTGGCACATCCGAGTCTCACTG-3’
Supplemental Data. Geng et al. (2012). Plant Cell 10.1105/tpc.112.105312
9