TN HR TN - Plant Cell...(B) Hpa infection of JA signaling mutants and respective controls with and without Thr pre-treatment. 3-week-old plants of the indicated genotypes were spray-treated

Ler rar1-13 rar1-13rsp1

rar1-13rsp2

Ler

rar1-13

rar1-13rsp1

rar1-13rsp2

A

B

HR HR

TN

TN

HR

TN

Supplemental Figure 1. Phenotypes of rar1-13 rsp1 and rsp2 double mutants.(A) Disease symptom formation on rsp mutant and control plants 7 d after infection with Hpa isolate Noco2 as observed under UV illumination. HR - hypersensitive response, TN - trailing necrosis.(B) General growth phenotypes of rsp mutant and control plants. Plants were first grown under short day conditions in a growth cabinet and then shifted to long day conditions in a greenhouse. Scale = 5cm

Supplemental Data. Stuttmann et al. (2011). Plant Cell 10.1105/tpc.111.087684

Col

Col mlo2-6

Ler

Ler rar1-13 rar1-13 rsp2

rar1-13 rsp1

Supplemental Figure 2. Macroscopic disease symptom formation upon Golovinomyces orontii infection.6-week-old rsp1 and rsp2 mutant plants and controls were infected with Golovinomy-ces orontii. Fungal growth was documented 8 d after infection. The mlo2-6 mutant and the respective Col control were included as a mutant with altered Go susceptibil-ity. Fungal growth was not altered on rar1-13 rsp1, but increased on rar1-13 rsp2 double mutant plants.


100bpDHDPS1 [At3g60880]

184180

SALK_147470 LB

181 206

UTR

Actin

206/184

180/181

Col H 2O#218

#219

#220

#223

#224

gDNA

SALK_147470

0

40

80

120

Col #219 #220 #223

spor

es x

104

/ g

(FW

)

dhdps1-1 [SALK_147470]

A

BC

Supplemental Figure 3. Isolation and characterization of a Col dhdps1-1 mutant.(A) Depiction of the DHDPS1 locus with T-DNA insertion in SALK_147470 and primers used for RT-PCR indicated.(B) RT-PCR analysis of DHDPS1 transcript in different T4 families isolated from SALK_147470.(C) Conidiospore count 7 d after infection of 3-week-old plants with Hpa isolate Noco2. Biological replicates were pooled and treated as one sample, error bars indicate tech-nical error derived from five counts.


Ler

rar1-13 rar1-13 rsp2

rar1-13 rsp1

Supplemental Figure 4. Germination phenotype of rsp mutants.Photographs were taken 7 d after germination. At this time point, most seedlings of both rsp1 and rsp2 mutant plants were arrested in development. Several aborted seedlings are marked with red arrowheads. On arrested plants, cotelydons would fail to unfurl or unfurled cotelydons would become necrotic and dry out. In both cases, seedlings fail to develop any further.


147/177

LB/177

Actin

146/148

149/166

#263

#264

#265

#266

#267

#268

0

40

80

120

Col ak2#263

ak2#264

Col ak2#266 eds1-2#265

Noco2 sporulation, 7 dpi

spor

es x

104

/ g (F

W)

SAIL 258_E06

A B

Supplemental Figure 5. Characterization of a putative ak2 loss of function mutant.(A) RT-PCR analysis on families selected from SAIL_258_E06. The positions of the primers used in this analysis are indicated in Figure 4A.(B) Conidiospore count 7 d after infection of 3-week-old plants with Hpa isolate Noco2. No difference in susceptibility of putative ak2 mutant plants was detected.


35S:AK2 35S:AK2rsp1

5

17

16

15

14

13

12

11

10

9

8

7

6

18 19

20

1513 18 19

Supplemental Figure 6. Macroscopic phenotypes of T1 plants overexpressing wild type AK2 or mutant AK2rsp1.Col plants were transformed with constructs containing either 35S::AK2 or 35S::AK2rsp1 expression cassettes. Transformants were selected by BASTA resistance. Plants were then repotted and transformants were confirmed by PCR (data not shown). Four plants transgenic for 35S:AK2rsp1 showed rsp1-like phenotypes as shown enlarged in the above pictures. Numbered plants were used for amino acid measurements.


Cala2 sporulation, 6dpi

0

5

10

15

20

25

30

Ler rsp1 rsp2 rar1-13 rar1-13rsp1

rar1-13rsp2 eds1-2 Col

spor

es x

105

/ g

(FW

)

Supplemental Figure 7: Hpa resistance of rsp mutants is not rar1-13 dependent.3-week-old rsp1 and rsp2 single and rsp1 rar1-13 and rsp2 rar1-13 double mutant plants and respective controls were infected with Hpa isolate Cala2. Conidiospore formation was quantified 6 d after infection. Error bars indicate standard deviations of three biological replicates. Significantly different classes are indicated by lower case letters (One-way ANOVA, Tukey’s post-hoc test, p < 0.05 each).

aa

bb

b bb

c


rar1-13 rsp1 rsp2

7d

14d

C

0

100

200

300

400

500

600

Ler rsp1 rsp2

1/10 MS

0.5% suc

0.5% raff

0.5% inosit

0.5% sorb

B

seed

ling

grow

th [%

]; 1/

10 M

S =

100

%0

1

2

3

Ler rsp1 rsp2

1/10 MS

0.5% suc

0.5% raff

0.5% inosit

0.5% sorb

seed

ling

size

[cm

]

A

Supplemental Figure 8: Effects of sugar on growth and Hpa susceptibility of rsp mu-tants.(A) Seedling growth of rsp mutant and control plants on synthetic media containing different sugars. Seedling size (n ≥ 19) was measured after 7 d growth on the indicated media. Stan-dard deviation is shown. Significantly different classes are indicated by lower case letters (Repeated measures ANOVA , Tukey’s post-hoc test, p < 0.05 each). suc - sucrose; raff - raffinose; inosit - inositol; sorb - sorbitol. (B) Dataset as in A, but seedling growth is expressed as % of growth on media without sugar.(C) Effect of sugar on Hpa growth. Plants were grown on 1/10 MS media containing 0.5% sucrose and then transferred to soil 7 d before or just prior to infection with Hpa isolate Cala2. Leaves were stained with Trypan Blue 6 d after infection to visualize hyphae.

a

g

f,h

e

d

c

b

a,cac

c

dd,fd,f d,h


LerCol rsp1/AK2rsp1 rsp2/dhdps2-2

Supplemental Figure 9. Hpa infection structures of virulent Hpa isolate Cala2 on rsp1 and rsp2 mutant plants.Shown are additional pictures of infection structures from the experiment described in Figure 7A. Infection structures associated with cell death were observed on the resistant accession Col. Succesfully growing hyphae were visible on the compatible accession Ler. On rsp1 mutant plants hyphal growth was arrested at most infection sites. Hyphal out-growth was almost never observed on rsp2 mutant plants but oomycete structures were arrested at differ-ent developmental stages.


Indole glucosinolate content

0

20

40

60

80

100

120

I3G 4MI3G 1MI3G

nmol

/gFW

Ler

rar1-13

rar1-13 rsp1

rar1-13 rsp2

a b aa

a a bb

Supplemental Figure 10. Indole glucosinolate content of rsp mutant plant tissues.Indole-3-methylglucosinolate (I3G), 4-methoxyindol-3-ylmethylglucosinolate (4MI3G) and 1-methoxyindol-3-ylmethylglucosinolate (1MI3G) were measured by HPLC in leaf tissue extracts of 4-week-old plants. 1MI3G content was elevated in all 3 repetitions of the experiment. In 1 repetition, I3G and 4MI3G were also elevated. Significantly different classes in this experiment are indicated by lower case letters (One-way ANOVA, Tukey’s post-hoc test, p < 0.05 each).



0

100

200

300

400

500

600

Ler rsp1 rsp2

rela

tive

cont

ent [

%],

Ler =

100

%

Homoserine content

Supplemental Figure 11. Homoserine content of rsp mutant plants.Relative HS accumulation in rsp1 and rsp2 was measured in aerial tissue of 4-week-old soil-grown plants by GC-MS. Standard deviation of 5 (Ler) or 6 (rsp) independent measurements is indicated. Ler was set at 100%, and relative content is shown. As HS is almost undetectable in extracts from wild type plants, we consider that absolute accumulation of HS remains low in rsp1 and rsp2 despite the measured 2-4.5-fold accumulation of the metabolite in mutant plant tissue. Significantly different classes are indicated by lower case letters (One-way ANOVA, Tukey’s post-hoc test, p < 0.05 each).

a

b

c

0

5

10

0 0,5

serops01 x

5)

WF( g /

Thr [mM]521 10

Hpa Noco2sporulation 7dpi

Supplemental Figure 12: Dose-dependency of Hpa growth suppression by Thr.3-week-old Col eds1-2 were sprayed with solutions containing the indicated concentrations of Thr 2 d and 6 h prior to infection with Hpa isolate Noco2. Conidiospores were counted 7 dpi. Two biological replicates were pooled and error bars indicate technical error derived from three counts. Similar results were obtained in 3 independent repetitions of the experi-ment.


Ws-0 eds1-1 opr3

5mMThr

0

2

4

6

Ws-0 eds1-1 opr3sp

ores

x 1

05/g

(FW

)

no treatment5mM Threonine

Emwa 4 dpi

Colgl1aosjar1-1

gl1jin1-1

5mMThr

0

10

20

Col

jar1-1

gl1 jin

1-1

gl1 ao

s

spor

es x

105

/g (F

W)

no treatment5mM Threonine

A

B

Supplemental Figure 13. Expression of JA-regulated genes in rsp mutant plants and Thr-induced Hpa growth suppression on JA signaling mutants.(A) qRT-PCR analysis of PDF1.2 and VSP2 expression in rsp mutant leaf tissues. Total RNA was extracted from 3-week-old unchallenged plants, cDNA synthesized, expression of the indicated genes analyzed and normalized to the expression of UBQ10. Average values of three independent biological replicates, each containing two technical replicates, are shown with standard errors. No significant changes could be detected by Student’s t-test, p < 0.05.(B) Hpa infection of JA signaling mutants and respective controls with and without Thr pre-treatment. 3-week-old plants of the indicated genotypes were spray-treated with 5 mM Thr solutions or H2O 2 d and 6 h prior to infection with Hpa isolate Emwa2 (upper panel) or Noco2 (lower panel). Conidiophore formation was documented in photographs and was additionally assessed by spore counting (graphs, technical error of three counts indicated). Similar results were obtained in two repetitions of the experiment after visual inspection. Scale = 0.5 cm.

Noco 7 dpi

0

5

10

PDF1.2 VSP2

Ler

rsp1

rsp2

norm

aliz

ed fo

ld e

xpre

ssio

n



H2O 5 mM Thr

Supplemental Figure 14. Growth of Hpa on mock- or threonine-treated Col plants.Shown are pictures of leaves from plants described in Figure 9A to indicate the spectrum of pathogen growth. Scale bar = 0.2 mm.


0

40

80

120

Col-0 agd2

Threonine content

WF gm / lo

mp

*

Supplemental Figure 15: Threonine content of agd2 mutant tissues.Threonine content of Col agd2 partially defective in LL-diaminopimelate aminotransferase activity was determined from aerial tissue of 4 week-old soil grown plants by HPLC. Asterisk indicates significant difference as determined by Student’s t-test.


Supplemental Table 1: Comparative genome analysis for genes coding DAP-/ AAA-pathway enzymes in Hyaloperonospora arabidopsidis, Golovinomyces orontii and Blumeria graminis query sequences used: DHDPS: E. coli DapA; NP_416973 DAP

pathway DHDPR: E. coli DapB; ACI71848 LysA (DAP decarboxylase): E. coli LysA; NP_417315 Homocitrate synthase: S. cerevisiae LYS20 CAA98757

AAA pathway

Homoaconitase: S. cerevisiae LYS4 AAA88902 Homoisocitrate: S. cerevisiae LYS12 NP_012172 α-Aminoadipate reductase: S. cerevisiae LYS2 CAA85072 Saccharopine reductase: S. cerevisiae LYS9 CAA96331 Saccharopine dehydrogenase: S. cerevisiae LYS1 CAA86194 Hyaloperonospora arabidopsidis (Hpa)

query E-value

Score (bits)

Identities Positives reciprocal BLAST E-value present

DHDPS 2e-42 167 90/230 134/230 UNIPROT: D0N460_PHYIN; Phytophthora infestans DHDPS e-108 yes

DHDPR 8e-20 93 68/193 99/193 UNIPROT: D0NQR5_PHYIN;

Aminotransferase, putative OS=Phytophthora infestans

0.0 likely*

LysA 8e-37 150 115/392 192/392 UNIPROT:Q8TSR9_METAC; Diaminopimelate decarboxylase

Methanosarcina acetivorans8e-98 yes


Homocitrate synthase 5e-28 120 93/295 143/295

UNIPROT:D0MYD7_PHYIN; 2-isopropylmalate synthase OS=Phytophthora infestans

0.0 no

Homoaconitase

5e-52 201 181/625 280/625

UNIPROT:A3LRP7_PICST¸ 3-isopropylmalate dehydratase

(Aconitase superfamily) OS=Pichia stipitis

0.0

yes

4e-27 119 115/438 190/438 UNIPROT:D7FQN5_ECTSI;

Aconitate hydratase, OS=Ectocarpus siliculosus

0.0

Homoisocitrate DH 2e-22 99 97/340 146/340

UNIPROT:D4H8C6_DENA2; 3-isopropylmalate dehydrogenase OS=Denitrovibrio acetiphilus;

2e-37 no

α-Aminoadipate reductase 4e-31 133 82/247 132/247

UNIPROT:D8LQP2_ECTSI; Nonribosomal peptide synthetase

10 OS=Ectocarpus siliculosus 2e-93 no

Saccharopine reductase 2e-80 295 164/447 248/447

UNIPROT:D8WKY4_TRITU; Lysine ketoglutarate

reductase/saccharopine dehydrogenase OS=Triticum

turgidum

0.0 yes

Saccharopine dehydrogenase no hit N/A N/A N/A N/A N/A no

* protein contains both DHDPR N- and C-terminus pfam domains


Blumeria graminis (Bg)

query E- value

Score (bits)

identities positives reciprocal BLAST E-value present

DHDPS no hit N/A N/A N/A N/A N/A no DHDPR no hit N/A N/A N/A N/A N/A no

LysA no hit N/A N/A N/A N/A N/A no Homocitrate

synthase 5.4e-161 333 165/230 180/230 EM_FUN:FN401071; Aspergillus fumigatus

hcsA gene for homocitrate synthase 1e-152 yes

Homoaconitase 0.0 780 403/723 487/723 EM_FUN:AY955279; Aspergillus niger homoaconitate hydratase (lysA) 1e-68 yes

Homoisocitrate DH

2.19e-125 447 230/354 282/354 not conclusive N/A yes

α-Aminoadipate reductase 0.0 856 442/881 603/881

EM_FUN:AB199808; Aspergillus saitoi lys2 gene for aminoadipate reductase, partial

cds 3e-46 yes

Saccharopine reductase

3.6e-157 553 261/433 337/433

UNIPROT: A6SQH9_BOTFB; Saccharopine reductase OS=Botryotinia

fuckeliana0.0 yes

Saccharopine dehydrogenase

1.56e-96 755 154/286 186/286 not conclusive N/A yes


Golovinomyces orontii (Go)

query E-value

Score (bits)

identities positives reciprocal BLAST E-value present

DHDPS no hit N/A N/A N/A N/A N/A no DHDPR no hit N/A N/A N/A N/A N/A no

LysA no hit N/A N/A N/A N/A N/A no Homocitrate

synthase 9e-95 343 192/323 219/323 UNIPROT:A1CWS1_NEOFI; Homocitrate synthase OS=Neosartorya fischeri

1e-111 yes

Homoaconitase 3e-70 263 144/275 180/275 UNIPROT:LYS4_NEUCR; Homoaconitase, mitochondrial OS=Neurospora crassa

1e-111 yes

Homoisocitrate DH e-100 360 186/293 233/293

UNIPROT:Q7SH15_NEUCR; Homoisocitrate dehydrogenase, mitochondrial OS=Neurospora

crassa

1e-125 yes

α-Aminoadipate

reductase 0.0 679 370/717 479/717

UNIPROT:C1GLF6_PARBD¸ L-aminoadipate-semialdehyde dehydrogenase large subunit

OS=Paracoccidioides brasiliensis 0.0 yes

Saccharopine reductase e-153 539 261/396 321/396 UNIPROT:A6SQH9_BOTFB; Saccharopine

reductase OS=Botryotinia fuckeliana 0.0 yes

Saccharopine dehydrogenase 2e-57 219 111/226 139/226 UNIPROT:A6RLN2_BOTFB; Saccharopine

dehydrogenase OS=Botryotinia fuckeliana 2e-90 yes


Supplemental Table 2: Oligonucleotides used in this study

esoprup ’3>-’5 ecneuqes emanM153 ACCGTTGGAAGTTGTGGAAG VSP2 qRT-PCR M154 CCAAATCAGCCCATTGATCT R221 ACGCACCGGCAATGGTGGAA PDF1.2 qRT-PCR R222 TGCATGATCCATGTTTGGCTC GP1 AGATCCAGGACAAGGAGGTATTC Ubiquitin10 qRT-PCR GP2 CGCAGGACCAAGTGAAGAGTAG GP35 TTCTTCCCTCGAAAGCTCAA PR1 qRT-PCR GP36 AAGGCCCACCAGAGTGTATG LB-GABI ATATTGACCATCATACTCATTGC Genotyping dhdps2-3Y201 CCTAGCTTGGAATAAGCAAAACAC Genotyping dhdps2-3Y186 TTATGAATCTATGATCGATCGGTC Genotyping DHDPS2; with Y201 SALK_LB TGGTTCACGTAGTGGGCCATCG SALK_lines genotyping Y180 CCTAACTTCCATCTTCCTATGC dhdps1-1 genotyping / RT-PCR;

Supplemental Figure 3 Y181 AGTAAGGGTTAATGTGCAGTGC Y206 TGGAAATGATGATCAGTGCCATG Y184 CTAATATCGACCGATTAAGATGAAGT SAIL_LB CGTCCGCAATGTGTTATTAAG SAIL lines genotyping Y146 TTGCGGTGTCACTAACGTAG AK2 genotyping / RT-PCR; Figure 4,

Supplemental Figure 5 Y147 ACAGCTACAACGATTGGGAAAGCG Y148 GATCACAAGTCAAAACTCCATCCAC Y149 CTAACCAGCATTGTTCTGAAACG Y165 CACCATGGCTTCATTGCAGTTGTACGGA cloning AK2 Y166 TCCAGGATCGGTCTCAAAGAAGGCGGA cloning AK2 Y204 TTGAGACCGATCCTTGAAAGGGTGGGCGC introduction of STOP codon into

AK2-containing pENTR/D Y205 GCGCCCACCCTTTCAAGGATCGGTCTCAA Y222 TATCGGTTGATGTTaTGGCAACAAGTGAA introduction of rsp1 mutation into

AK2-containing plasmids Y223 TTCACTTGTTGCCAtAACATCAACCGATA Y234 CACCATGGTCGATTTGCTTCAAAGAA cloning AK2-cTP Y149 CTAACCAGCATTGTTCTGAAACG dCAPS marker (MnlI) to detect

rsp1; mutant is cut Y177 TCAATGATATACTAACTTCACTTGTTGCCT Y141 ACTGAGAATGGGGTTGTTGTGTGGACTG dCAPS marker (BsrI) for dhdps2-

2/rsp2 detection, wt is cut Y142 TGAGCCAAAGCAGTGTTGATTCC

All PCRs except qRT-PCR (see Materials and Methods section) were performed under standard conditions (annealing temperature 54°C, 1min/kb elongation, 35 cycles) with Taq polymerase. Phusion polymerase (Finnzymes) was used for cloning and site-directed mutagenesis as according to the manufacturer.

Documents

TN HR TN - Plant Cell...(B) Hpa infection of JA signaling mutants and respective controls with and without Thr pre-treatment. 3-week-old plants of the indicated genotypes were spray-treated