Supplemental DataMaterials and Methods

Chemicals

rac-GR24 (Supplemental Figure 1) was purchased from Chiralix (Netherlands), CN-Debranone

(Supplemental Figure 1) was kindly provided by Dr. Tadao Asami, University of Tokyo, Japan.

KAR1 (Supplemental Figure 1) was purchased from Sigma-Aldrich (Germany). N-PL was

synthesized by ReseaChem (Switzerland). All N-PL and GR24 used in this study are racemic

mixtures (Supplemental Figure S1).

Plant materials

Arabidopsis seeds: Ler-0 and Col-0 were used as wild type. Mutants max2-1, max1-1, d14-1,

max4-1 and kai2 were described before (Bennett et al., 2006; Waters and Smith, 2012). Rice

wild type Shiokari, d3 and d10 mutants were kindly provided by Dr. Junko Kyozuka. Seeds of

the S. hermonthica collected in 2012 from a sorghum field in Sudan were provided by Prof. A.

G. Babiker (University of Sudan). Seeds of P. ramosa L. collected in 2009 from sunflower

(Helianthus annuus L.) and were kindly provided by Dr. Philippe Simier, University de Nantes,

France.

N-PL synthesis

Nitro-Phenlactone ((5‘RS)-(αZ)-α-[[(2‘,5‘-dihydro-4‘-methyl-5‘-oxo-2‘-furanyl)oxy]methylene]-4-

nitrobenzeneethane) was synthesized in four steps:

Step 1: A solution of 2-(4-Nitrophenyl) propionic acid (5 g) and H2SO4 (0.28 mL) in ethanol (25

mL) was prepared and stirred under reflux for 3 hours. After cooling, the solvent was evaporated

and the residue was partitioned between water and diethylether. The diethylether was washed

twice with saturated NaHCO3 solution and water, then dried and concentrated to give 2 (5.6 g,

98%) as pale yellow oil.

Step 2: Oven dried, round-bottomed flask is charged with Ethyl 2-(4-Nitrophenyl) propionate (2

g) and dry Dichloromethane (DCM) (40 mL) under inert gas. The solution was cooled to -78°C

and DIBAL (1 M in DCM, 9.8 mL) was added dropwise over 30 min. After the addition was 1

complete, the resultant solution was stirred at -78°C for 2 hours. The reaction was quenched by

addition of MeOH (0.95 mL) and of saturated Rochelle salt (19 mL). The mixture was stirred

overnight. The suspension was prepared for extraction by adding saturated Rochelle salt (4 mL),

water (4 mL) and DCM (40 mL). The aqueous phase was separated and extracted twice with

DCM. The combined organic extracts was washed with brine, dried and concentrated.

Purification of the crude product by silica gel chromatography (eluent: hexane: ethyl acetate 5:1,

4:1, 3:1) afforded pure 3 (1.1 g, 68%).

Step 3: A solution of 3-Methyl-2(5H)-furanone (5 g) in cyclohexane (150 mL) were added N-

bromosuccinimide (9.5 g) and AIBN (50 mg). The mixture was heated under reflux for 2 hours

with exclusion of moisture. After cooling and filtration, the mother liquor was evaporated to give

5 (6.0 g, 66%) as pale orange liquid. The purity estimated using NMR was 81% and the product

was used without further purification.

Step 4: A solution of 2-(4-Nitrophenyl)propanal (2.2 g) in dry toluene (77 mL) and N-Methyl-2-

pyrrolidinone (7.7 mL) was added potassium tert-butoxide (1.51 g) and the mixture was stirred at

room temperature for 10 min. The 5-bromo-3-Methyl-2(5H)-furanone (2.8 g) was added

dropwise over 10 min. After the addition was complete, the mixture was stirred for 40 min. The

reaction was partitioned between water and ethyl acetate. The aqueous phase was separated and

extracted twice with ethyl acetate. The combined organic extracts was washed with brine, dried

and concentrated. The residue was diluted with hexane: ethyl acetate 8:2, and the precipitate

filtered off to give 6 (1g, 30%, cis/trans-Isomer 0.6/1) as yellow crystals. The mother liquor was

concentrated to give crude product that was not further purified. cis- and trans-isomers were

separated by several silica gel chromatographies (eluent: hexane : ethyl acetate 6:1, 5:1, 4:1, 3:1,

2:1, 1:1), yielding cis-Isomer (220 mg, Purity: 91%) and trans-isomer (240 mg, Purity: 84%).

Pure cis-Isomer was obtained by precipitation from hexane: ethyl acetate.

N-PL stability analysis

10 μL of N-PL or GR24 acetone solution (1 mM) were mixed with 990 μL buffer consisting of

Buffers were prepared from 5mM ammonium acetate (5 mM) adjusted to pH 6 to 8. After adding

1-Indanol (20 μL of a 2.5 mg/mL solution in acetone), as internal standard, solutions were

incubated at 21°C in amber HPLC vials. The time course of degradation was monitored by

HPLC analysis using an Zorbax Eclipse Plus C18 column (3.5 μm, 2.1 ×150 mm), eluted by a 2

gradient from 5% to 90% acetonitrile in water within 15 min. The column was operated at 35°C

with a flow rate of 0.25 mL/min. Compounds eluted from the column were detected using a

Diode Array Detector. Amounts of the compounds were determined by calculating the

corresponding peaks, compared to the internal standard.

Parasitic seed germination bioassays

Seeds were first separated from sand residue and organic debris using the sucrose gradient (40%

and 60%). Cleaned seeds were then surface-sterilized using 50 mL (2.5%) sodium hypochlorite

with 0.4% of Tween-20 for 10 minutes. Subsequently, seeds were thoroughly rinsed using sterile

MilliQ water through a glass vacuum filter holders and flask (Fisher Scientific) under a laminar

flow cabinet. The sterilized seeds were then allowed to dry under the same cabinet.

Approximately 50 to 100 seeds were evenly spread on sterilized glass fiber filter paper discs. The

discs were placed in 90 mm petri-dishes (12 discs per petri-dish) on whatman filter paper

moistened with 3 mL sterile MilliQ water. The petri dishes were sealed with parafilm, wrapped

in aluminium foil and placed in an incubator at 30/22ºC for 12/14 days for Striga/Phelipanche

seeds, respectively. The discs with preconditioned seeds were allowed to dry under laminar flow

cabinet. The discs were then placed in another petri-dish (six per petri-dish) containing a filter

paper ring, wetted with 0.9 mL sterile MilliQ water. 50 μL of each of N-PL solutions (0.0001,

0.001, 0.01, 0.1, 1 and 10 μM) were added per disc, replicated four times for each concentration.

In addition, GR24 solutions containing the same concentrations were used as a positive control.

Sterile MilliQ water was used as a negative control in each germination assays.

After application, seeds were again incubated at 30/25ºC in dark for two/seven days for

Striga/Phelipanche seeds, respectively. Germination (seeds with radicle emerging through the

seed coat) was scored using a binocular microscope. The germination rate (GR) in percentage

was calculated for each disk (replication) using the formula:

GR (%) = (Ngs / Nts;)x100

where Ngs is the number of germinated seeds per disc, Nts is the total number of seeds per disk.

Arabidopsis seed germination assays

Fresh dry ripen primary dormant Ler-0 seeds harvested from plants grown under long day

condition (16 h at 22°C/ 8h at 16°C day/night, 60% relative humidity, 4000-5000 LUX white 3

light) were stored at -80°C for germination experiments. Ler-0 seeds were sown on 1%

agar+0.1% MgCl2 supplemented with N-PL or GR24, then incubated at 22°C for 8 days.

Germinated seeds were counted daily from the third day.

Arabidopsis hypocotyl elongation assays

Sterilized Arabidopsis seeds were sown on half strength MS (with 0.5 % sucrose +1% agar,

0.5g/L MES, PH5.7) plates supplemented with N-PL or GR24. Plates were stored at 4°C in

darkness for 3 days. To encourage germination, plates were exposed to continuous white light for

24 h then transferred to continuous monochromatic red light (160 Lux, 22°C) condition for

another 4 days. For hypocotyl length measurement, at least 30 seedlings were measured using the

publicly available ImageJ software (http://rsbweb.nih.gov/ij/) after taking digital photographs.

Monochromatic red light source was applied as described previously (Wu and Yang, 2010).

Light fluence rates were measured using a Li250 quantum photometer (Li-Cor, Lincoln, NE,

USA).

RNA extraction and Quantitative Real-Time RT-PCR

Total RNA was extracted from 20 seedlings grown under the same conditions as in the hypocotyl

elongation assays, using RNeasy Kit (Qiagen). 1 µg total RNA was reverse-transcribed using

iScript™ Reverse Transcription Supermix for RT-qPCR kit (Bio Rad). Amplification was

carried out with SYBR® Green Real-Time PCR Master Mixes kit (Life technologies).

Quantitative Real-time RT PCR was performed in a StepOne™ Real-Time PCR Systems (Life

Technologies). The thermal profile for real-time PCR was 95°C for 2 min, followed by 40 cycles

of 95°C for 15s and 60°C for 30s. DLK2, STH7, IAA6 and ACTIN were amplified. Primers for

these genes are described previously (Jia et al., 2014; Scaffidi et al., 2014).

Arabidopsis lateral root density

Sterilized Arabidopsis seeds were sown on half strength MS (with 0.5 % sucrose +1% agar,

0.5g/L MES, PH5.7) plates supplemented with GR24 or N-PL. Plates were stored at 4°C in

darkness for 3 days and then vertically grown at 22°C in Percival incubator under long day

condition (16 h at 22°C/ 8h at 16°C day/night, 60% relative humidity, 4000-5000 LUX white

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light) for 8 days. Then lateral root density was calculated by dividing the total number of lateral

roots by primary root length (No./cm).

Arabidopsis shoot branching assays

Seeds were germinated for two weeks then transferred to 50 mL falcon tubes with half strength

MS liquid media solution supplemented with 1 µM GR24 or 2.5 µM N-PL (control with acetone)

under long day condition (16 h at 22°C/ 8h at 16°C day/night, 60% relative humidity, 4000-5000

LUX white light). Treatment was conducted for 4 weeks, replacing the supplemented liquid

media every 3 day. Then plants were grown for one more week without chemical treatment in ½

strength MS liquid media, before counting the secondary rosette branches.

Rice tillering and plant height assays

Seeds were surface-sterilized by washing with 70% ethanol for 1 min and then with 2.5% sodium

hypochlorite for 15 min. Seeds were then rinsed thoroughly with sterile MilliQ water and

incubated in water for 2 days at 28°C in the dark. Pre-germinated seeds were transferred to filter

papers containing half strength MS medium in 90 mm petri dishes and incubated at 28°C. Seven

days-old seedlings were transferred to 50 mL falcon tubes (one seedling per tube) containing

modified half-strength Hoagland nutrient solution and grown in the greenhouse. Plants were

treated with 2.5 M N-PL or 1 M GR24 (used as positive control) twice a week for three

weeks, changing the solution at each treatment.

Hyphal branching bioassays

Spores of Gigaspora rosea were germinated and cultivated on solid medium for six days. Stock

solutions of N-PL in acetonitrile were diluted in water to reach the appropriate concentrations

(10-6, 10-7, 10-8 and 10-9 M) of N-PL in 10% acetonitrile. Control treatments were performed with

the solvent alone (10% acetonitrile used as negative control) and with 10-7 M GR24 in 10%

acetonitrile (positive control). 5 µL samples were applied on both sides of the main hypha of a

germinated spore. The number of newly formed apices was recorded 48h after treatment.

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Supplemental Figures

Supplemental Figure 1. Structures of strigolactones, KAR1 and SL analogs.

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Supplemental Figure 2. The synthetic route of N-PL.

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Supplemental Figure 3. Effect of N-PL on Arabidopsis lateral root density. Two concentrations

(1 µM and 5 µM) of N-PL were used. Concentrations for GR24, CN-Debranone and KAR1 are 1

µM. Control was treated by acetone. The figure shows the results of one of two independent

replicates showing the same tendency. Values ± SE are from more than 30 seedlings. Values

shared the same letter indicate no significant difference (one way ANOVA, P<0.01). rac-GR24,

rac-N-PL and rac-CN-Debranone are used here.

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Supplemental Figure 4. Effect of N-PL on the transcript levels of DLK2 and IAA6. Different

genotypes of Arabidopsis seedlings were treated with 5 µM N-PL. The expression of DLK2 and

IAA6 were detected by quantitative RT-PCR. 20 seedlings for each sample were used for total

RNA extraction. ACTIN was used as reference gene, and the transcript levels in the untreated

control were normalized to 1. The data shown were obtained from one of the two biological

replicates showing the same tendency. rac-GR24 and rac-N-PL are used here.

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Supplemental Figure 5. Effect of N-PL on hypocotyl elongation is dependent on both D14 and

KAI2. (A) Hypocotyl length of different genotypes of Arabidopsis seedlings treated by 5 µM N-

PL. The expression of DLK2 (B) and IAA6 (C) were detected by quantitative RT-PCR. Values ±

SE for hypocotyl length are from 20 seedlings. All the seedlings used in (A), (B) and (C) are

treated under the same conditions. For quantitative RT-PCR, 20 seedlings for each sample were

used for total RNA extraction. ACTIN was used as reference gene, and the transcript levels in the

untreated control were normalized to 1. The data shown were obtained from one of the two

biological replicates showing the same tendency. rac-N-PL is used here.12

Supplemental Figure 6. Effect of N-PL on primary dormant arabidopsis seed germination. Two concentrations (1 µM and 10 µM) of N-PL were used. Concentrations for GR24, CN-Debranone and KAR1 are 1 µM. Data are means ± SE are from three technical replicates of more than 100 seeds for each. rac-GR24, rac-N-PL and rac-CN-Debranone are used here.

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Supplemental References

Bennett, T., Sieberer, T., Willett, B., Booker, J., Luschnig, C., and Leyser, O. (2006). The Arabidopsis MAX pathway controls shoot branching by regulating auxin transport. Curr. Biol. 16:553-563.

Jia, K.-P., Luo, Q., He, S.B., Lu, X.-D., and Yang, H.Q. (2014). Strigolactone-regulated hypocotyl elongation is dependent on cryptochrome and phytochrome signaling pathways in Arabidopsis. Mol. Plant. 7:528-540.

Scaffidi, A., Waters, M.T., Sun, Y.K., Skelton, B.W., Dixon, K.W., Ghisalberti, E.L., Flematti, G.R., and Smith, S.M. (2014). Strigolactone hormones and their stereoisomers Signal through two related receptor proteins to Induce different physiological responses in Arabidopsis. Plant Physiol. 165:1221-1232.

Waters, M.T., and Smith, S.M. (2012). KAI2-and MAX2-mediated responses to karrikins and strigolactones are largely independent of HY5 in Arabidopsis seedlings. Mol. Plant. 6:63-75

Wu, L., and Yang, H.Q. (2010). CRYPTOCHROME 1 is implicated in promoting R protein-mediated plant resistance to Pseudomonas syringae in Arabidopsis. Mol. Plant 3:539-548.

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