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Plant Biotechnology29, 495499 (2012)

Copyright 2012 The Japanese Society for Plant Cell and Molecular Biology

Evaluation of anti-herbivory genes using anAgrobacterium-mediated transient expression system

Kei Kawazu1,a,, Naoya Wasano1,b, Kotaro Konno2, Yuko Ohashi2,

Atsushi Mochizuki 1,, Ichiro Mitsuhara2,*1 National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki 305-8604, Japan; 2 Plant-microbe InteractionsResearch Unit, National Institute of Agrobiological Science, Tsukuba, Ibaraki 305-8602, Japan*E-mail: [email protected] Fax:+81-29-838-7044

Received May 15, 2012; accepted July 11, 2012 (Edited by Y. Nagano)

Abstract A high-throughput system or the evaluation o anti-herbivory genes in plant is proposed, in which genes aretransiently expressed in a small area o lea via Agrobacterium-inltration, and the results can be obtained within 1 week.ransient expression o the cry1Ab gene, a well-known Bt toxin gene, in the leaves o tobacco (Nicotiana tabacum cv. SamsunNN) exhibited a lethal eect on both cotton cutworm ( Spodoptera litura) and cabbage armyworm (Mamestra brassicae)

larvae. InArabidopsis leaves, this gene also provided potent inhibition o eeding o diamondback moth (Plutella xylostella)larvae. Te expression o another anti-herbivory gene, MLX56, in tobacco and tomato (Solanum lycopersium cv. Micro-om) leaves resulted in enhanced mortality oM. brassicae and S. litura larvae. Signicantly higher growth inhibition oS.litura larvae and eeding inhibition oP. xylostella larvae were also observed in tobacco and Arabidopsis leaves expressingtheMLX56gene. All results could be obtained within 57 days, indicating that thisAgrobacterium-mediated transient geneexpression system enables a high throughput evaluation o anti-herbivory genes.

Key words: Anti-herbivory gene, B toxin, Lepidoptera, MLX56, transient assay

o date, many types o genetically modiied (GM)

crops have been grown commercially worldwide. All

GM soybean and canola hybrids are herbicide tolerant,

whereas some GM corn and cotton are resistant to insect

pests. Te most well-known insect resistant GM cropsare transormed with the insecticidal crystal (cry) gene

rom Bacillus thuringiensis, which encodes a protein

called Bt toxin with strong insecticidal activity against

lepidopteran insects. However, Cry proteins are very

selectively active against insects and no Bt toxins have

been ound to be available or agriculturally important

insect pests such as hemipteran and orthopteran insects,

and the emergence o pests resistant to Bt toxins has

also been reported (Janmaat and Myers 2003; abashnik

et al. 2008; Wang et al. 2003). Tereore, other novel

genes with anti-herbivory eects are being sought or

agricultural pest control (Foissac et al. 2000; Fowler et al.2009; Gatehouse 2008; Mochizuki et al. 1999; Wang et al.

2005; Wang and Constabel 2004; Yarasi et al. 2008).

Although searching o new anti-herbivory genes

is needed or the development o herbivory resistant

transgenic plants, the evaluation o such genes is labor

intensive and time consuming. For example, testing

the toxic or resistance activity o candidate anti-

herbivory proteins requires large amounts o the puried

protein. However, it is sometimes very diicult to

puriy candidate anti-herbivory proteins rom a sourceorganism or to produce candidate proteins using ectopic

expression system (Pechan et al. 2004; Mohan et al.

2006). Carrying out bioassays with the protein is more

dicult or insect species or which an articial diet has

not been established. Furthermore, the evaluation o

anti-herbivory genes in stable transgenic plants requires

substantial time, labor and cost or the introduction o

candidate anti-herbivory genes into a crop, and then the

selection and maintenance o strains exhibiting stable

expression levels, beore the bioassay or herbivory

resistance can be perormed.

Agrobacterium-iniltration methods enable thetransient and local expression o genes o interest

in the iniltrated area o a lea, in a broad range o

plants, including Nicotiana tabacum, N. benthamiana,

Arabidopsis thaliana, Linum usitattissium, Pisum

sativum, and Lactuca sativa (Kapila et al. 1997; Van

Note

DOI: 10.5511/plantbiotechnology.12.0711a

a Present address: Kyoyu Agri Co., Ltd., 173-2 Guze, omitake, Nagano 381-0006, Japan.b Present address: International Environmental and Agricultural Sciences, okyo University o Agriculture and echnology, 3-5-8 Saiwai-cho, Fuchu,

okyo 183-8509, Japan. Tese authors contributed equally to this work.

Tis article can be ound at http://www.jspcmb.jp/

Published online November 30, 2012


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496 High throughout evaluation o anti-herbivory genes


der Hoorn et al. 2000; Wroblewski et al. 2005). Tis in

planta transient expression system using Agrobacterium

has been applied to analysis o genes unction, promoter

analysis and protein production (Bendahmane et al.

2000; Sawers et al. 2006; Vaquero et al. 1999; Yang et al.

2000). A strikingly successul example o the application

o transient expression systems is the characterization

o signaling events involved in the induction o thehypersensitive reaction, which is associated with plant

resistance against pathogens (e.g. Van der Hoorn et al.

2000).

In the present study, we attempted to establish

a transient evaluation system or anti-herbivory

genes using the Agrobacterium-iniltration system

(Agrobacterium-mediated transient expression system).

We expressed two anti-herbivory genes in the leaves o

tobacco, tomato and Arabidopsis byAgrobacterium-

iniltration and then assessed the eect o transient

expression on lepidopteran larvae ed on the leaves. A

CaMV 35S promoter-driven, codon-optimized versiono a synthetic cry1Ab gene (pBIK102mcbt, Shinoyama

et al. 2003) was inltrated into leaves o N. tabacum

cv. SamsunNN using Agrobacterium (GV3101), as

described by Kobayashi et al. (2011). Native cry1 genes

are barely expressed in plant systems because o their

prokaryotic type codon usage with highly A-rich

sequences. hus, we used a synthesized cry1Ab gene

with codons optimized to the chrysanthemum codon

usage or expression in higher plants (Shinoyama et al.

2003). Te negative control consisted oAgrobacterium

transormed with pIG121-Hm, as described by Ohta et

al. (1990), which has a 35S::int-GUS construct. Liquid-cultured Agrobacterium containing the -DNA vectors

(O.D.600=0.4 to 0.8) were harvested and resuspended

in buer containing acetosyringone (10 mM MES (pH

5.6), 10 mM MgCl2, 20M acetosyringone) at O.D.=0.1.

Te suspension oAgrobacterium was le on bench at

room temperature or 3 h to induce virulence and then

inltrated into the intercellular space o leaves using

needleless syringe (Figure 1AC). Quantitative R-PCR

analysis was used to monitor transgene expression with

the use o primers 5-AAC C CC AAC GGG CC

C G-3 (Cry1Ab5) and 5-GG CAC GAA CC

GA CC GC -3 (Cry1Ab3) as described previously(Kobayashi et al. 2010) (Figure 2A). Te transcript o

the cry1Ab gene was detected at 1 day post inltration

(DPI), its level peaked at 2 DPI and remained distinct

until 5 DPI. Protein gel blot analysis using a polyclonal

antibody against Bacillus thuringinesis Cry1Ab toxin

(Abcam, Cambridge UK) was used to conirm the

accumulation o the cry1Ab gene product (Bt protein)

(Figure 2B). One day aer inltration, 10 newly hatched

larvae oMamestra brassicae (cabbage armyworm) (eggs

were kindly provided by the Japan Plant Protection

Association, Ibaraki, Japan) or Spodptera litura (cotton

cutworm) (Sumika echnoservice Co., akarazuka,

Japan) were inoculated onto each iniltrated tobacco

lea area and kept within a miniature chamber to inhibit

escaping the larvae rom the area o inoculation (Figure1DF). At 4 days ater inoculation, the numbers o

surviving M. brassicae larvae on the leaves expressing

cry1Ab gene decreased signiicantly compared with

those on the control leaves (Figure 2C). All larvae o S.

litura on the leaves expressing cry1Ab gene died within 4

days, whereas most larvae on the control leaves survived

(Figure 2D).

Plutella xylostella larvae (diamondback moth) were

also used to assess the eect o expression o cry1Ab in

Agrobacterium-inltrated leaves oArabidopsis. Rosette

leaves oArabidopsis thaliana ecotype Columbia were

Figure 1. Procedures o the Agrobacterium-mediated transient assay

or insect resistance. (A) Photograph o a tobacco (Nicotiana tabacum)

lea. (B) Inltration o anAgrobacterium suspension into a tobacco lea.

(C) Te margin o the inltrated tobacco lea area was marked. (D)

Schematic gure o the miniature chamber. Te chamber containing

a larva was positioned onto each inltrated tobacco lea area. (E)

Larvae were placed into the miniature chamber. (F) Photograph o a

tobacco lea, part o which was iniltrated with an Agrobacteriumsuspension. One day aer inltration, larvae were inoculated onto the

area and enclosed in a miniature chamber (20 mm diameter). All plants

and insects were kept at 251C, LD 16 : 8 h. (G) Photograph o an

Arabidopsis lea, part o which was inltrated with an Agrobacterium

suspension and then inoculated with one rst instar diamondback

moth (P. xylostella) larva (neonate larvae). Te larva on the lea was

enclosed in a 20-mm diameter chamber. Arabidopsis and P. xylostella

larva were kept at 251C, LD 16 : 8 h.


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K. Kawazu et al. 497


inltrated with cry1Ab transormed Agrobacterium orthe control, and one P. xylostella larva was inoculated on

the inltrated area in the same manner as M. brassicae

(Figure 1G). Because the rst instar o P. xylostella larva

is very small (approximately 1 mm in length) and they

tend to mine within the plant mesophyll tissue to eed,

larval mortality is dicult to assess. We considered that

the area o lea consumed reected the degree o larvalperormance; thereore, we measured the area consumed

byP. xylostella larva. At 3 days aer inoculation, the area

consumed byP. xylostella larvae in leaves expressing the

cry1Ab gene was signicantly less than that in the control

leaves (Figure 2E). Tese results indicated clearly that

activity o the well-known anti-herbivory gene cry1Ab

can be demonstrated with the use o our Agrobacterium-

inltration system.

o urther assess the present method, we analyzed the

eect o transient expression o a second anti-herbivory

gene, MLX56. MLX56 is a 56-kDa protein contained

in the latex o cultivated mulberry, Morus alba cv,Shin-Ichinose, and it acts a deensive protein against

lepidopteran insects (Wasano et al. 2009). When the

larvae o the cabbage armyworm,M. brassicae, and o Eri

silkworm, Samia ricimi ate an articial diet containing

MLX56 protein, larval growth was retarded. However,

MLX56 showed no activity against silkworm larvae,

Bombyx mori, the mulberry specialist, suggesting the

adaptation oB. mori to this mulberry deensive protein.

he MLX56 gene was inserted into binary vector

pEl2 (Ohtsubo et al. 1999) under the control

o a modiied 35S promoter and introduced into

Agrobacterium GV3101 and then iniltrated intotobacco, tomato or Arabidopsis leaves. Quantitative

R-PCR analysis o iniltrated tobacco leaves using

primers 5-AAC C CC AAC GGG CC C G-3

(MLX565) and 5- CCG AGG GC C CCA

CA C-3 (MLX563) identiied expression o the

MLX56 gene (Figure 3A). he numbers o survivinglarvae oM. brassicae or S. litura on the areas o tobacco

leaves iniltrated with Agrobacterium containing the

35S::MLX56 construct was signiicantly reduced

compared with those on control areas (Figure 3B, C).

he numbers o surviving S. litura larvae on tomato

(cv. micro om) lea areas expressing MLX56gene alsodecreased signicantly (Figure 3D). Tus, the transient

expression o the MLX56 gene in tobacco and tomato

leaves decreased to the survival oM. brassicae and

S. litura larvae. One 3rd instar larvae o S. litura onto

each tobacco lea area expressing the MLX56 gene in

order to assess the eect o expression oMLX56 gene

on the growth o larvae. wo days aer inoculation, the

weight gain o the larvae was signicantly suppressed

compared with those exposed to a control area (Figure

3E). o assess the eect o expression o the MLX56gene

on the rate o consumption by larvae, one 1st instar P.

Figure 2. Agrobacter ium-mediated transient expression o a

cry1Ab gene was used to induce resistance to lepidopteran larvae. (A)

Quantitative PCR was used to assess transcript levels o the cry1Ab gene

in Agrobacterium-inltrated leaves o tobacco. Te transcript levels

o cry1Ab were normalized using those o the actin gene. Values are

meansSE. Te assay was replicated three times (df=3, F=11.2562,

p=0.0030). Means with dierent letters are signicantly dierent by

ukey-Kramer HSD test (p


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498 High throughout evaluation o anti-herbivory genes


xylostella larvae was inoculated onto each Arabidopsis

lea area expressing the MLX56 gene. At 3 days aer

inoculation, the area oArabidopsis leaves expressing

the MLX56 gene consumed by P. xylostella larvae wassignicantly less than those or control areas (Figure 3F).

B toxin is known to have strong insecticidal activity

against lepidopteran insects, whereas MLX56 showed

only growth inhibition against the larvae o the M.

brassicae and S. ricimi (Wasano et al. 2009). However,

in present transient assay system, the expression o

MLX56 gene in the leaves exhibited not only a growthinhibition eect but also induced eeding inhibition and

lethality. Tis may have been achieved by a higher level

o accumulation o theMLX56gene product as a result o

Agrobacterium inltration or by a combination o MLX56

and unidentied anti-herbivory agents in the test plants.

In the present study, the inhibitory eect on

lepidopteran larvae oAgrobacterium-mediated transient

expression o the cry1Ab gene was veried in tobacco,

tomato and Arabidopsis . Furthermore, our transient

assay demonstrated that expression o the gene or the

anti-herbivory protein MLX56 enhanced plant resistance

against some lepidopteran larvae ed on tobacco, tomatoand Arabidopsis. Consequently, we are now trying to

produce stable transgenic plants with enhanced herbivore

resistance by introduction o the MLX56 gene. he

Agrobacterium iniltration method is a powerul tool

or plant molecular biology, because it enables high

throughput expression o a gene o interest. Furthermore,

our current system involving the combination o

Agrobacterium-mediated transient expression and a

specialized miniature inoculation chamber permit the

high throughput evaluation o anti-herbivory genes

within 1 week, thereby reducing the time and eort or

the selection o transgenic lines and the establishment ostable transormants. Because this system can be applied

to a variety o plant species (tobacco, tomato, Arabidopsis

etc.), it will be straight-orward to perorm bioassays

with insect pests that cannot be reared on articial diets,

and the anti-insect perormance o transgenic plants can

be measured using a variety o indices (larval weights,

mortalities, consumption rates etc.).

Acknowledgements

We thank Harue Shinoyama or providing the modied cry1Ab

gene, Kayoko Furukawa or insect rearing, Chiaki Kimoto or plant

growing, Masumi eruse or technical assistance, and akeshi

Shiraishi or valuable inormation. Tis work was supported by

the Program or the Promotion o Basic Research Activities or

Innovative Biosciences.

References

Bendahmane A, Querci M, Kanyuka K, Baulcombe DC (2000)

Agrobacter ium transient expression system as a tool or the

isolation o disease resistance genes: application to the Rx2 locus

in potato. Plant J21: 7381

Foissac X, Ti Loc N, Christou P, Gatehouse AMR, Gatehouse JA

(2000) Resistance to green leafopper (Nephotettix virescens)

Figure 3. Agrobacterium-mediated transient expression o the

MLX56 gene was used to induce resistance to lepidopteran larvae.

(A) Quantitative PCR was used to assess transcript levels o the

MLX56gene in Agrobacterium-inltrated leaves o tobacco. Te assay

was replicated three times (df=3, F=5.5330,p=0.0237). Means with

dierent letters are signicantly dierent by ukey-Kramer HSD test

(p


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K. Kawazu et al. 499


and brown planthopper (Nilaparvata lugens) in transgenic rice

expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA).

J Insect Physiol46: 573583

Fowler JH, Narvez-Vsquez J, Aromdee DN, Pautot V, Holzer FM,

Walling LL (2009) Leucine aminopeptidase regulates deense and

wound signaling in tomato downstream o jasmonic acid. Plant

Cell21: 12391251

Gatehouse JA (2008) Biotechnological prospects or engineering

insect-resistant plants. Plant Physiol146: 881887Janmaat AF, Myers J (2003) Rapid evolution and the cost o

resistance to Bacillus thuringiensis in greenhouse populations o

cabbage loopers, Trichoplusia ni. Proc Biol Sci 270: 22632270

Kapila J, De Rycke R, Van Montagu M, Angenon G (1997) An

Agrobacterium-mediated transient gene expression system or

intact leaves. Plant Sci 122: 101108

Kobayashi M, Seo S, Hirai K, Yamamoto-Katou A, Katou S, Seto

H, Meshi , Mitsuhara I, Ohashi Y (2010) Silencing o WIPK

and SIPK mitogen-activated protein kinases reduces tobacco

mosaic virus accumulation but permits systemic viral movement

in tobacco possessing the N resistance gene. Mol Plant Microbe

Interact23: 10321041

Kobayashi M, Yamamoto-Katou A, Katou S, Hirai K, Meshi ,

Ohashi Y, Mitsuhara I (2011) Identication o an amino acid

residue required or dierential recognition o a viral movement

protein by the Tomato mosaic virus resistance gene Tm-2(2). J

Plant Physiol168: 11421145

Mochizuki A, Nishizawa Y, Onodera H, abei Y, oki S, Habu Y,

Ugaki M, Ohashi Y (1999) ransgenic rice plants expressing a

trypsin inhibitor are resistant against rice stem bores, Chilo

suppressalis. Entomol Exp Appl93: 173178

Ohta S, Mita S, Hattori , Nakamura K (1990) Construction and

expression in tobacco o-glucuronidase (GUS) reporter gene

containing an intron within the coding sequence. Plant Cell

Physiol31: 805813

Ohtsubo N, Mitsuhara I, Koga M, Seo S, Ohashi Y (1999) Ethylene

promotes the necrotic lesion ormation and basic PR geneexpression in MV-inected tobacco. Plant Cell Physiol 40:

808817

Sawers RJH, Farmer PR, Moett P, Brutnell P (2006) In planta

transient expression as a system or genetic and biochemical

analyses o chlorophyll biosynthesis. Plant Methods 2: 15

Shinoyama H, Mochizuki A, Komano M, Nomura Y, Nagai

(2003) Insect resistance in transgenic Chrysanthemum

[Dendranthema grandiflorum (Ramat.) Kitamura] by the

introduction o a modiied -endotoxin gene o Bacillus

thuringiensis.Breed Sci 53: 359367

Sokal RR, Rohl FJ (1995) Biometry. Freeman, New York, pp 1937

abashnik BE, Gassmann AJ, Crowder DW, Carrire Y (2008)

Insect resistance to Bt crops: evidence versus theory. NatBiotechnol26: 199202

Vaquero C, Sack M, Chandler J, Drossard J, Schuster F, Monecke

M, Schillberg S, Fischer R (1999) ransient expression o a

tumor-specic single-chain ragment and a chimeric antibody in

tobacco leaves. Proc Natl Acad Sci USA 96: 1112811133

Van der Hoorn RA, Laurent F, Roth R, De Wit PJ (2000)

Agroinltration is a versatile tool that acilitates comparative

analyses oAvr9/Cf-9-induced and Avr4/Cf-4-induced necrosis.

Mol Plant Microbe Interact13: 439446

Wang J, Boets A, Van Rie J, Ren G (2003) Characterization o cry1,

cry2, and cry9 genes in Bacillus thuringiensis isolates rom China.

J Invertebr Pathol82: 6371

Wang J, Constabel CP (2004) Polyphenol oxidase overexpression

in transgenic Populus enhances resistance to herbivory by orest

tent caterpillar (Malacosoma disstria). Planta 220: 8796

Wang Z, Zhang K, Sun X, ang K, Zhang J (2005) Enhancement o

resistance to aphids by introducing the snowdrop lectin genegna

into maize plants.J Biosci 30: 627638

Wasano N, Konno K, Nakamura M, Hirayama C, Hattori M,

ateishi K (2009) A unique latex protein, MLX56, deends

mulberry trees rom insects. Phytochemistry70: 880888

Wroblewski , omczak A, Michelmore R (2005) Optimization o

Agrobacterium-mediated transient assays o gene expression in

lettuce, tomato andArabidopsis. Plant Biotechnol J3: 259273

Yang Y, Li R, Qi M (2000) In vivo analysis o plant promoters and

transcription actors by agroinltration o tobacco leaves. Plant

J22: 543551Yarasi B, Sadumpati V, Immanni CP, Vudem DR, Khareedu VR

(2008) ransgenic rice expressingAllium sativum lea agglutinin

(ASAL) exhibits high-level resistance against major sap-sucking

pests. BMC Plant Biol8: 102
http://dx.doi.org/10.1016/S0022-1910(99)00143-2http://dx.doi.org/10.1016/S0022-1910(99)00143-2http://dx.doi.org/10.1016/S0022-1910(99)00143-2http://dx.doi.org/10.1016/S0022-1910(99)00143-2http://dx.doi.org/10.1016/S0022-1910(99)00143-2http://dx.doi.org/10.1016/S0022-1910(99)00143-2http://dx.doi.org/10.1016/S0022-1910(99)00143-2http://dx.doi.org/10.1016/S0022-1910(99)00143-2http://dx.doi.org/10.1105/tpc.108.065029http://dx.doi.org/10.1105/tpc.108.065029http://dx.doi.org/10.1105/tpc.108.065029http://dx.doi.org/10.1105/tpc.108.065029http://dx.doi.org/10.1105/tpc.108.065029http://dx.doi.org/10.1105/tpc.108.065029http://dx.doi.org/10.1104/pp.107.111096http://dx.doi.org/10.1104/pp.107.111096http://dx.doi.org/10.1104/pp.107.111096http://dx.doi.org/10.1104/pp.107.111096http://dx.doi.org/10.1098/rspb.2003.2497http://dx.doi.org/10.1098/rspb.2003.2497http://dx.doi.org/10.1098/rspb.2003.2497http://dx.doi.org/10.1098/rspb.2003.2497http://dx.doi.org/10.1098/rspb.2003.2497http://dx.doi.org/10.1098/rspb.2003.2497http://dx.doi.org/10.1098/rspb.2003.2497http://dx.doi.org/10.1098/rspb.2003.2497http://dx.doi.org/10.1098/rspb.2003.2497http://dx.doi.org/10.1016/S0168-9452(96)04541-4http://dx.doi.org/10.1016/S0168-9452(96)04541-4http://dx.doi.org/10.1016/S0168-9452(96)04541-4http://dx.doi.org/10.1016/S0168-9452(96)04541-4http://dx.doi.org/10.1016/S0168-9452(96)04541-4http://dx.doi.org/10.1016/S0168-9452(96)04541-4http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1046/j.1570-7458.1999.00576.xhttp://dx.doi.org/10.1046/j.1570-7458.1999.00576.xhttp://dx.doi.org/10.1046/j.1570-7458.1999.00576.xhttp://dx.doi.org/10.1046/j.1570-7458.1999.00576.xhttp://dx.doi.org/10.1046/j.1570-7458.1999.00576.xhttp://dx.doi.org/10.1046/j.1570-7458.1999.00576.xhttp://dx.doi.org/10.1046/j.1570-7458.1999.00576.xhttp://dx.doi.org/10.1046/j.1570-7458.1999.00576.xhttp://dx.doi.org/10.1093/oxfordjournals.pcp.a029609http://dx.doi.org/10.1093/oxfordjournals.pcp.a029609http://dx.doi.org/10.1093/oxfordjournals.pcp.a029609http://dx.doi.org/10.1093/oxfordjournals.pcp.a029609http://dx.doi.org/10.1093/oxfordjournals.pcp.a029609http://dx.doi.org/10.1093/oxfordjournals.pcp.a029609http://dx.doi.org/10.1186/1746-4811-2-15http://dx.doi.org/10.1186/1746-4811-2-15http://dx.doi.org/10.1186/1746-4811-2-15http://dx.doi.org/10.1186/1746-4811-2-15http://dx.doi.org/10.1186/1746-4811-2-15http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1038/nbt1382http://dx.doi.org/10.1038/nbt1382http://dx.doi.org/10.1038/nbt1382http://dx.doi.org/10.1038/nbt1382http://dx.doi.org/10.1038/nbt1382http://dx.doi.org/10.1073/pnas.96.20.11128http://dx.doi.org/10.1073/pnas.96.20.11128http://dx.doi.org/10.1073/pnas.96.20.11128http://dx.doi.org/10.1073/pnas.96.20.11128http://dx.doi.org/10.1073/pnas.96.20.11128http://dx.doi.org/10.1073/pnas.96.20.11128http://dx.doi.org/10.1094/MPMI.2000.13.4.439http://dx.doi.org/10.1094/MPMI.2000.13.4.439http://dx.doi.org/10.1094/MPMI.2000.13.4.439http://dx.doi.org/10.1094/MPMI.2000.13.4.439http://dx.doi.org/10.1094/MPMI.2000.13.4.439http://dx.doi.org/10.1094/MPMI.2000.13.4.439http://dx.doi.org/10.1094/MPMI.2000.13.4.439http://dx.doi.org/10.1094/MPMI.2000.13.4.439http://dx.doi.org/10.1094/MPMI.2000.13.4.439http://dx.doi.org/10.1016/S0022-2011(02)00202-1http://dx.doi.org/10.1016/S0022-2011(02)00202-1http://dx.doi.org/10.1016/S0022-2011(02)00202-1http://dx.doi.org/10.1016/S0022-2011(02)00202-1http://dx.doi.org/10.1016/S0022-2011(02)00202-1http://dx.doi.org/10.1016/S0022-2011(02)00202-1http://dx.doi.org/10.1016/S0022-2011(02)00202-1http://dx.doi.org/10.1016/S0022-2011(02)00202-1http://dx.doi.org/10.1016/S0022-2011(02)00202-1http://dx.doi.org/10.1016/S0022-2011(02)00202-1http://dx.doi.org/10.1016/S0022-2011(02)00202-1http://dx.doi.org/10.1007/s00425-004-1327-1http://dx.doi.org/10.1007/s00425-004-1327-1http://dx.doi.org/10.1007/s00425-004-1327-1http://dx.doi.org/10.1007/s00425-004-1327-1http://dx.doi.org/10.1007/s00425-004-1327-1http://dx.doi.org/10.1007/s00425-004-1327-1http://dx.doi.org/10.1007/s00425-004-1327-1http://dx.doi.org/10.1007/s00425-004-1327-1http://dx.doi.org/10.1007/s00425-004-1327-1http://dx.doi.org/10.1007/BF02703563http://dx.doi.org/10.1007/BF02703563http://dx.doi.org/10.1007/BF02703563http://dx.doi.org/10.1007/BF02703563http://dx.doi.org/10.1007/BF02703563http://dx.doi.org/10.1007/BF02703563http://dx.doi.org/10.1007/BF02703563http://dx.doi.org/10.1016/j.phytochem.2009.04.014http://dx.doi.org/10.1016/j.phytochem.2009.04.014http://dx.doi.org/10.1016/j.phytochem.2009.04.014http://dx.doi.org/10.1016/j.phytochem.2009.04.014http://dx.doi.org/10.1016/j.phytochem.2009.04.014http://dx.doi.org/10.1111/j.1467-7652.2005.00123.xhttp://dx.doi.org/10.1111/j.1467-7652.2005.00123.xhttp://dx.doi.org/10.1111/j.1467-7652.2005.00123.xhttp://dx.doi.org/10.1111/j.1467-7652.2005.00123.xhttp://dx.doi.org/10.1111/j.1467-7652.2005.00123.xhttp://dx.doi.org/10.1111/j.1467-7652.2005.00123.xhttp://dx.doi.org/10.1111/j.1467-7652.2005.00123.xhttp://dx.doi.org/10.1111/j.1467-7652.2005.00123.xhttp://dx.doi.org/10.1046/j.1365-313x.2000.00760.xhttp://dx.doi.org/10.1046/j.1365-313x.2000.00760.xhttp://dx.doi.org/10.1046/j.1365-313x.2000.00760.xhttp://dx.doi.org/10.1046/j.1365-313x.2000.00760.xhttp://dx.doi.org/10.1046/j.1365-313x.2000.00760.xhttp://dx.doi.org/10.1046/j.1365-313x.2000.00760.xhttp://dx.doi.org/10.1046/j.1365-313x.2000.00760.xhttp://dx.doi.org/10.1186/1471-2229-8-102http://dx.doi.org/10.1186/1471-2229-8-102http://dx.doi.org/10.1186/1471-2229-8-102http://dx.doi.org/10.1186/1471-2229-8-102http://dx.doi.org/10.1186/1471-2229-8-102http://dx.doi.org/10.1186/1471-2229-8-102http://dx.doi.org/10.1186/1471-2229-8-102http://dx.doi.org/10.1186/1471-2229-8-102http://dx.doi.org/10.1186/1471-2229-8-102http://dx.doi.org/10.1186/1471-2229-8-102http://dx.doi.org/10.1186/1471-2229-8-102http://dx.doi.org/10.1186/1471-2229-8-102http://dx.doi.org/10.1046/j.1365-313x.2000.00760.xhttp://dx.doi.org/10.1046/j.1365-313x.2000.00760.xhttp://dx.doi.org/10.1046/j.1365-313x.2000.00760.xhttp://dx.doi.org/10.1111/j.1467-7652.2005.00123.xhttp://dx.doi.org/10.1111/j.1467-7652.2005.00123.xhttp://dx.doi.org/10.1111/j.1467-7652.2005.00123.xhttp://dx.doi.org/10.1016/j.phytochem.2009.04.014http://dx.doi.org/10.1016/j.phytochem.2009.04.014http://dx.doi.org/10.1016/j.phytochem.2009.04.014http://dx.doi.org/10.1007/BF02703563http://dx.doi.org/10.1007/BF02703563http://dx.doi.org/10.1007/BF02703563http://dx.doi.org/10.1007/s00425-004-1327-1http://dx.doi.org/10.1007/s00425-004-1327-1http://dx.doi.org/10.1007/s00425-004-1327-1http://dx.doi.org/10.1016/S0022-2011(02)00202-1http://dx.doi.org/10.1016/S0022-2011(02)00202-1http://dx.doi.org/10.1016/S0022-2011(02)00202-1http://dx.doi.org/10.1094/MPMI.2000.13.4.439http://dx.doi.org/10.1094/MPMI.2000.13.4.439http://dx.doi.org/10.1094/MPMI.2000.13.4.439http://dx.doi.org/10.1094/MPMI.2000.13.4.439http://dx.doi.org/10.1073/pnas.96.20.11128http://dx.doi.org/10.1073/pnas.96.20.11128http://dx.doi.org/10.1073/pnas.96.20.11128http://dx.doi.org/10.1073/pnas.96.20.11128http://dx.doi.org/10.1038/nbt1382http://dx.doi.org/10.1038/nbt1382http://dx.doi.org/10.1038/nbt1382http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1270/jsbbs.53.359http://dx.doi.org/10.1186/1746-4811-2-15http://dx.doi.org/10.1186/1746-4811-2-15http://dx.doi.org/10.1186/1746-4811-2-15http://dx.doi.org/10.1093/oxfordjournals.pcp.a029609http://dx.doi.org/10.1093/oxfordjournals.pcp.a029609http://dx.doi.org/10.1093/oxfordjournals.pcp.a029609http://dx.doi.org/10.1093/oxfordjournals.pcp.a029609http://dx.doi.org/10.1046/j.1570-7458.1999.00576.xhttp://dx.doi.org/10.1046/j.1570-7458.1999.00576.xhttp://dx.doi.org/10.1046/j.1570-7458.1999.00576.xhttp://dx.doi.org/10.1046/j.1570-7458.1999.00576.xhttp://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1016/j.jplph.2011.01.002http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1094/MPMI-23-8-1032http://dx.doi.org/10.1016/S0168-9452(96)04541-4http://dx.doi.org/10.1016/S0168-9452(96)04541-4http://dx.doi.org/10.1016/S0168-9452(96)04541-4http://dx.doi.org/10.1098/rspb.2003.2497http://dx.doi.org/10.1098/rspb.2003.2497http://dx.doi.org/10.1098/rspb.2003.2497http://dx.doi.org/10.1104/pp.107.111096http://dx.doi.org/10.1104/pp.107.111096http://dx.doi.org/10.1105/tpc.108.065029http://dx.doi.org/10.1105/tpc.108.065029http://dx.doi.org/10.1105/tpc.108.065029http://dx.doi.org/10.1105/tpc.108.065029http://dx.doi.org/10.1016/S0022-1910(99)00143-2http://dx.doi.org/10.1016/S0022-1910(99)00143-2http://dx.doi.org/10.1016/S0022-1910(99)00143-2

anti herb ivory

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