Strigolactones: host recognition signalsStrigolactones: host recognition signalsStrigolactones: host recognition signalsStrigolactones: host recognition signals

for arbuscular mycorrhizal fungifor arbuscular mycorrhizal fungifor arbuscular mycorrhizal fungifor arbuscular mycorrhizal fungi

Hideo Hayashi and Kohki Akiyama

Graduate School of Life and EnvironmentalSciences

Osaka Prefecture University

ISC 2008, Padjadjaran University, 31 October

Insecticidal compounds from Indonesian plants

O O

HO

HOH

OHO

O

O

Kalanchoe pinnata Kalanchoe daigremontiana x tubiflora

Bryophyllin C Methyl daigremonate

1) Unang Supratman, T. Fujita, K. Akiyama, H. Hayashi, Biosci. Biotechnol. Biochem., 64646464, 1310-1312 (2000).2) Unang Supratman, T. Fujita, K. Akiyama, H. Hayashi, Phytochemicstry, 58585858, 311-314 (2001).

New anti-brine shrimp compounds from Indonesian mushroom

HN

H

O

H

OCH3

OH

H

OO

OH

Microporellus subsessilis

10-phenyl-[11]-cytochalasans

Dikdik Kurnia, K. Akiyama, H. Hayashi, Phytochemistry, 68686868, 697-702 (2007).

OP053 by Dr. Dikdik

Ectomycorrhizal fungus (Matsutake)

http://www.pref.akita.jp/morigi/QA/QA_14.htm

Host recognition signalsHost recognition signalsHost recognition signalsHost recognition signals

for arbuscular mycorrhizal for arbuscular mycorrhizal for arbuscular mycorrhizal for arbuscular mycorrhizal for arbuscular mycorrhizal for arbuscular mycorrhizal for arbuscular mycorrhizal for arbuscular mycorrhizal

fungifungifungifungi

Arbuscular mycorrhizal (AM) fungi

・belong to the phylum Glomeromycota.

・ colonize the roots of 80% of terrestrial plants.

・ existed 400 million years ago (early Denovian)1)・ existed 400 million years ago (early Denovian)1)

460 million years ago (Ordovician)2)

when plants first colonized land.

・ obligate symbionts; the fungi have not been cultured in the absence of a plant host.

1) W. Remy, T.N. Taylor, H. Hass, and H. Kerp, Proc. Natl. Acad. Sci. USA 91919191, 11841-11843 (1994).2) D. Redecker, R. Kodner, and L.E. Graham, Science 289289289289, 1920-1921 (2000).

Life cycle of arbuscular mycorrhizal fungi

SugarSugarSugarSugarHyphaHyphaHyphaHypha

ArbusculeArbusculeArbusculeArbuscule

PiPiPiPi

HHHH２２２２OOOO

SugarSugarSugarSugar

SporeSporeSporeSpore

External hyphaExternal hyphaExternal hyphaExternal hyphaPromotion of growthEnhancement of resistance against pathogens

Enhancement of drought tolerance

Increase of yield

Mycorrhizal structures in Lotus japonicus roots

Mycorrhizal rootNon-mycorrhizal root

stained with trypan blue

Hyphal branching: host recognition response of AM fungi

host plant roothost plant roothost plant roothost plant root

branchingbranchingbranchingbranchingfactorfactorfactorfactor(BF)(BF)(BF)(BF)

host plant roothost plant roothost plant roothost plant root

Germ tubeGerm tubeGerm tubeGerm tube

nonnonnonnon----host plant roothost plant roothost plant roothost plant rootChenopodiaceaeChenopodiaceaeChenopodiaceaeChenopodiaceaeBrassicaceaeBrassicaceaeBrassicaceaeBrassicaceae

Plant root exudates induce hyphal branching in vitro

Microinjected test samplesMicroinjected test samplesMicroinjected test samplesMicroinjected test samples

wellwellwellwell

2% CO2% CO2% CO2% CO2222, 32 ºC, 32 ºC, 32 ºC, 32 ºC

Spore ofSpore ofSpore ofSpore ofG. giganteaG. giganteaG. giganteaG. giganteagerminated on germinated on germinated on germinated on gelled M medium gelled M medium gelled M medium gelled M medium

---- exudatesexudatesexudatesexudates + exudates+ exudates+ exudates+ exudates

“Hyphal branching” bioassay developed by Nagahashi and Douds “Hyphal branching” bioassay developed by Nagahashi and Douds “Hyphal branching” bioassay developed by Nagahashi and Douds “Hyphal branching” bioassay developed by Nagahashi and Douds

2% CO2% CO2% CO2% CO2222, 32 ºC, 32 ºC, 32 ºC, 32 ºC

24 hours24 hours24 hours24 hours

G. Nagahashi and D.D. Douds, Jr, Mycol. Res. 104104104104, 1453-1464 (2000).

0.1% NaClO0.1% NaClO0.1% NaClO0.1% NaClO10 min10 min10 min10 min

SporesSporesSporesSpores

Gigaspora margarita Gigaspora margarita Gigaspora margarita Gigaspora margarita CGC1411CGC1411CGC1411CGC1411

(Central Glass Co., Ltd. Japan)(Central Glass Co., Ltd. Japan)(Central Glass Co., Ltd. Japan)(Central Glass Co., Ltd. Japan)

32323232℃℃℃℃, 2% CO, 2% CO, 2% CO, 2% CO2 2 2 2

5555----7 days7 days7 days7 days

Paper disc bioassay for hyphal branching in Gigaspora margarita

0.2% Phytagel plate0.2% Phytagel plate0.2% Phytagel plate0.2% Phytagel plate

(containing 3 mM MgSO(containing 3 mM MgSO(containing 3 mM MgSO(containing 3 mM MgSO4444) ) ) )

32 32 32 32 ℃℃℃℃, 2% CO, 2% CO, 2% CO, 2% CO2222

24 hrs24 hrs24 hrs24 hrs

Test samples Test samples Test samples Test samples in 70% EtOHin 70% EtOHin 70% EtOHin 70% EtOH----HHHH2222OOOO

paper discpaper discpaper discpaper discf 6 mmf 6 mmf 6 mmf 6 mm

Hyphal branching

Branching factor from root exudates ofLotus japonicus

- grown in liquid M medium (pH 6.8)under low Pi condition (35 µM)(24 ℃ 16h light / 22 ℃ 8h dark)

Lotus japonicus B-129 Gifu

Hydroponic solutionHydroponic solution- adjusted to pH 2 with HCl- extracted with EtOAc

EtOAc phase Aqueous phase- partitioned with 0.2M K2HPO4 (pH 9)

EtOAc-soluble Aqueous phaseneutral extract

2222°°°°

2°°°°

3333°°°°

Hyphal branching induced by ethyl acetate extracts from root exudates of L. japonicus

Control Control Control Control (70% EtOH(70% EtOH(70% EtOH(70% EtOH----HHHH2222O)O)O)O)

1111°°°° (germ tube)(germ tube)(germ tube)(germ tube)

EtOAc EtOAc EtOAc EtOAc extract extract extract extract

15 15 15 15 µµµµg/discg/discg/discg/disc

2°°°°4444°°°°

5555°°°°

EtOAc EtOAc EtOAc EtOAc ----solublesolublesolublesolubleneutral fraction neutral fraction neutral fraction neutral fraction

16 ng/disc16 ng/disc16 ng/disc16 ng/disc(activated charcoal (activated charcoal (activated charcoal (activated charcoal

method)method)method)method)

Solid phase extraction of BF from hydroponic solution of L. japonicus

Activated charcoalActivated charcoalActivated charcoalActivated charcoalL. japonicusL. japonicusL. japonicusL. japonicus BBBB----129 Gifu129 Gifu129 Gifu129 Gifugrown hydroponicallygrown hydroponicallygrown hydroponicallygrown hydroponicallyin M medium (Pi 35 mM)in M medium (Pi 35 mM)in M medium (Pi 35 mM)in M medium (Pi 35 mM)

Isolation procedure for BFfrom root exudates of L. japonicus

Charcoal cartridge (hydroponic solution 20 L)

- placed in aquarium water pump for 3 days- eluted with acetone

Neutral EtOAc extract (4 mg)Neutral EtOAc extract (4 mg)

- chromatographed on Wakogel C-200(hexane- EtOAc)

40% EtOAc eluate

- chromatographed on Inertsil ODS-3(40% MeCN in water to 100% MeCN)

Active fraction with Rt 22.3 min (ca. 18 µg)

Purification of BF by HPLCAbsorbance at 236 nm (mV)

1000100010001000

500500500500

Rt 22.3 min

Column: Inertsil ODS-3 (φ 10 x 250 mm)Eluent: 40-100% MeCN-H2O, 30min gradientFlow rate: 3.8 ml/min

Absorbance at 236 nm (mV)

500500500500

0000

Retention time (min)

0 5 10 15 20 25 300 5 10 15 20 25 300 5 10 15 20 25 300 5 10 15 20 25 30

97979797

UVUVUVUV (MeCN) (MeCN) (MeCN) (MeCN) λλλλmaxmaxmaxmax 234 nm234 nm234 nm234 nmIRIRIRIR (KBr) (KBr) (KBr) (KBr) ννννmaxmaxmaxmax 1784, 1744, 1683, 1340, 1024 cm1784, 1744, 1683, 1340, 1024 cm1784, 1744, 1683, 1340, 1024 cm1784, 1744, 1683, 1340, 1024 cm----1111

1111HHHH----NMRNMRNMRNMR (400 MHz, CDCl(400 MHz, CDCl(400 MHz, CDCl(400 MHz, CDCl3333) ) ) ) δδδδ 1.08 (3H,s, H1.08 (3H,s, H1.08 (3H,s, H1.08 (3H,s, H----9 or H9 or H9 or H9 or H----10), 10), 10), 10), 1.10 (3H, s, H1.10 (3H, s, H1.10 (3H, s, H1.10 (3H, s, H----9 or H9 or H9 or H9 or H----10), 2.01 (3H, t, J=1.5 Hz, H10), 2.01 (3H, t, J=1.5 Hz, H10), 2.01 (3H, t, J=1.5 Hz, H10), 2.01 (3H, t, J=1.5 Hz, H----7'), 7'), 7'), 7'), 5.49 (1H, br.d. J=8.0 Hz, H5.49 (1H, br.d. J=8.0 Hz, H5.49 (1H, br.d. J=8.0 Hz, H5.49 (1H, br.d. J=8.0 Hz, H----8b), 6.12 (1H, m, H8b), 6.12 (1H, m, H8b), 6.12 (1H, m, H8b), 6.12 (1H, m, H----2'), 2'), 2'), 2'), 6.90 (1H, m, H6.90 (1H, m, H6.90 (1H, m, H6.90 (1H, m, H----3'), 7.39 (1H, d, J=2.7 Hz, H3'), 7.39 (1H, d, J=2.7 Hz, H3'), 7.39 (1H, d, J=2.7 Hz, H3'), 7.39 (1H, d, J=2.7 Hz, H----6')6')6')6')CDCDCDCD (MeCN) (MeCN) (MeCN) (MeCN) λλλλmaxmaxmaxmax ((((∆ε∆ε∆ε∆ε) 262 () 262 () 262 () 262 (----2.86), 230 (21.2) nm 2.86), 230 (21.2) nm 2.86), 230 (21.2) nm 2.86), 230 (21.2) nm

OO

O OO

H

97979797

EIEIEIEI----MSMSMSMS

Spectroscopic analysis of BF

187187187187 215215215215233233233233

330 [M]330 [M]330 [M]330 [M]++++

StrigolactonesStrigolactonesStrigolactonesStrigolactonesseed germination stimulants for the parasiticseed germination stimulants for the parasiticseed germination stimulants for the parasiticseed germination stimulants for the parasiticweeds weeds weeds weeds StrigaStrigaStrigaStriga and and and and OrobancheOrobancheOrobancheOrobanche

5555----DeoxystrigolDeoxystrigolDeoxystrigolDeoxystrigol

Synthesis of 5-deoxystrigol

O O CNCl

O

O O

COOCH3

1) NaNH2, THF

2) CH3I

1) TMSCN, ZnI2

2) POCl3, Py

1) 85% H3PO4

2) SOCl2

SnCl , CH Cl

Si(CH3)31) (CH3O)3CO, NaH, DMF

2) BrCH2COOCH3 6N HCl

O

COOCH3

COOCH3

3SnCl4, CH2Cl2 2) BrCH2COOCH3

K. Frischmuth, E. Samson, A. Kranz, P. Welzel, H. Meuer, and W.S. Sheldrick, Tetrahedron, 47474747, 9793-9806 (1991).

6N HClAcOH

NaBH4, CeCl3, MeOH

OO O

O

OHHCOOEt, KOtBuEt2O/DMF

O O

OO

O

O OBr

K2CO3

N-methylpyrrolidone

Racemic 5-deoxystirigol

Separation by HPLC

Natural (ca. 80 µµµµg)

6' 3' 2'8b 3a

9,10

4a 4b

7'

OO

O OO

H3a

8b

2'

45

9 10

7'3'

6'3 pg/disc

1H-NMR spectra of natural and synthetic 5-deoxystrigol

PPM

7 6 5 4 3 2 1

PPM

7 6 5 4 3 2 1

400 MHz, CDCl3

Synthetic (ca. 3 mg)

17 6 5 4 3 2

6' 3' 2'8b 3a

9,10

4a 4b

7'

ppm

<30 pg/disc

a: natural 5-deoxystrigol(30 pg/disc)

b: synthetic (±±±±)-5-deoxystrigol (3 ng/disc)

Branching activity of strigolactones

c: synthetic (±±±±)-sorgolactone(10 ng/disc)

d: (+)-strigol(3 ng/disc)

Branching factor : 5-Deoxystrigol

O O

O O OH

O O O

K. Akiyama, K. Matsuzaki, & H. Hayashi, Nature, 435, 824-827 (2005).

Exudated from roots of Lotus japonicus

Hyphal branching-inducing activity of known natural strigolactones and

a synthetic analogue GR24

O O O O O O

O

Strigol Sorgolactone GR24Orobanchol(natural) (synthetic, racemic) (synthetic, racemic) (4 isomers)

O O

OH

O O O O O O OOH

<1 ng/disc <30 pg/disc <300 pg/disc 1 ng/disc

Synthetic strigolactone analogues GR24 and GR7 also induce

hyphal branching in Gigaspora rosea

GR24(4 diastereomers)

1 ng/disc

GR70.1-10 ng/disc*

2222'-epi-GR71 mg/disc*

*weak induction of 3º hyphae

G. Bécard et al., WO 2005/077177 A2 (Aug. 25, 2005).

Strigolactones :

seed germination stimulants

for root parasitic weeds

Root parasitic weed

Striga sp. Orabanchae sp. http://www.kobe-u.ac.jp/shokusi/page004.html

Life cycle of Striga spp.

Germination

ConditioningDormancy

(After-ripening)

Germinationsignal

Initiation of

development Attachment andpenetration

Host

Germination

Haustoriaformation

Parasitism

ParasiteHaustorial

inducing factor

Kindly provided by Prof. Sugimoto

Natural strigolactonesO O

O O

OH

O

O O

O O

OAc

O

O O O O O O

5-Deoxystrigol Strigol Strigyl acetate

O O O O O OOH

O O OOAc

Solgolactone Orobanchol Alectrol

2’-Epiorobanchol Solanacol

O O

O O O

HO

Sorgomol

Distribution of strigolactones in the plant kingdom

Monocots

Gramineaerice plantsorghumcornmilletpearl millet

FabaceaeLotus japonicusred cloverpeanutChinese milk vetchchick peasoybean

Solanaceaeeggplanttomatotobacco

CompositaeCompositaeCompositaeCompositaeburdocksafflower

BrassicaceaeArabidopsis thaliana

Chenopodiaceaspinach

pearl millet

Dicots

Malvaceaecotton

MenispermaceaeMenispermumdauricum

Umbelliferaecarrot

soybeanalfalfacommon beangarden peawinged beancrimson clover broad beanadzuki beancowpeawhite lupin

Cucurbitaceaecucumber?

safflowerChrysanthemum coronarium

Cosmos bipinnatus

sunflowerlettusmarigold

Linaceaeflax

Gymnospermae

PinaceaJapanese red pine

Non-host plants in red letters

Branching factors = Strigolactones

O O

O O OH

AM fungi

(460 million years ago)

Obligate symbiontsRoot parasitic weeds

Obligate parasites

Pi

K. Akiyama & H. Hayashi, Annals of Botany, 97, 925-931 (2006).

Strigolactones Strigolactones

Acknowledgments

Mr. Ken-ichi Matsuzaki (Osaka Prefecture University)Professor Masayoshi Kawaguchi (Tokyo University)Professor Yukihiro Sugimoto (Kobe University)Professor Koichi Yoneyama (Utsunomiya University)

Financial support

Grand-in-Aid for Scientific Research from MEXTCore Research for Evolutional Science and

Technology (CREST) Sumitomo FoundationOsaka Prefecture University

Growth of L. japonicus under different concentrations of phosphate

Control Glomus mosseae

3 weeks after inoculation

0.1 mM 0.5 mM 1 mM 2 mM 0.1 mM 2 mM