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A new cyanogenic metabolite in Arabidopsis required for inducible pathogen defence

Jakub Rajniak 1,Brenden Barco 2,Nicole K.Clay 2&Elizabeth S.Sattely 1

Thousands of putative biosynthetic genes in Arabidopsis thaliana have no known function,which suggests that there are numerous molecules contributing to plant fitness that have not yet been discov-ered 1,2.Prime among these uncharacterized genes are cytochromes P450upregulated in response to pathogens 3,4.Here we start with a single pathogen-induced P450(ref.5),CYP82C2,and use a combina-tion of untargeted metabolomics and coexpression analysis to uncover the complete biosynthetic pathway to 4-hydroxyindole-3-carbonyl nitrile (4-OH-ICN),a previously unknown Arabidopsis metabolite.This metabolite harbours cyanogenic functionality that is unprecedented in plants and exceedingly rare in nature 6,7;further-more,the aryl cyanohydrin intermediate in the 4-OH-ICN pathway reveals a latent capacity for cyanogenic glucoside biosynthesis 8,9in Arabidopsis .By expressing 4-OH-ICN biosynthetic enzymes in Saccharomyces cerevisiae and Nicotiana benthamiana ,we reconstit-ute the complete pathway in vitro and in vivo and validate the func-tions of its enzymes.Arabidopsis 4-OH-ICN pathway mutants show increased susceptibility to the bacterial pathogen Pseudomonas syringae ,consistent with a role in inducible pathogen defence.Arabidopsis has been the pre-eminent model system 10,11for studying the role of small molecules in plant innate immunity 12;our results uncover a new branch of indole metabolism distinct from the canon-ical camalexin pathway,and support a role for this pathway in the Arabidopsis defence response 13.These results establish a more com-plete framework for understanding how the model plant Arabidopsis uses small molecules in pathogen defence.

To identify cytochromes P450potentially involved in the biosyn-thesis of novel defence-associated small molecules,we obtained raw data sets for all transcriptomics experiments dealing with biotic stress in A.thaliana from the NASCArrays database.We examined CYP genes present in the probeset and selected a candidate,CYP82C2,that is highly expressed under a variety of pathogen treatment conditions,but whose native function in Arabidopsis is unknown (Fig.1a).

To identify small molecules whose levels change in a CYP82C2-dependent manner,we performed comparative metabolomics 14with a homozygous transfer-DNA insertion line of CYP82C2.We used the bacterial pathogen P.syringae pv.tomato DC3000harbouring the avrRpm1avirulence gene (Psta )as an elicitor since CYP82C2express-ion is strongly upregulated 24h after inoculation with this strain (Fig.1a).We analysed tissue methanolic extracts of 11-day-old seed-lings grown hydroponically in the presence of Psta by liquid chromato-graphy–mass spectrometry (LC–MS),and computationally compared mutant and wild-type (WT)Col-0metabolomes.From this analysis,we identified 11compound mass signals that reproducibly and signifi-cantly differ between WT and cyp82C2(Fig.1b);these mass ions are induced after pathogen elicitation and are not bacterially derived (Extended Data Fig.1a).

We next sought to obtain clues about the structure of these com-pounds from their tandem mass spectra (MS/MS).MS/MS analysis revealed that the 11compounds could be divided into two classes (A and B in Fig.1b),assigned as indole-3-carboxaldehyde (IAL)deri-vatives with (B )and without (A )hydroxylated indole systems.Moreover,the fact that the cyp82C2mutant lacked all the hydroxylated derivatives but accumulated excess amounts of their non-hydroxylated counterparts suggested that CYP82C2acts as an indolic hydroxylase.However,except for compound A1(Fig.2b),which was confirmed to be indole-3-carboxylic acid methyl ester,the structures of these com-pounds remained elusive.

To facilitate structural analysis,we investigated whether any of these compounds were exuded into the medium in the cyp82C2mutant seedling experiments (Fig.1d).Filtered spent medium was loaded onto a C18silica gel cartridge,and non-polar metabolites were eluted with acetonitrile and analysed by LC–MS.Surprisingly,the profile of spent medium extracted in this manner was notably different from that of tissue methanolic extracts:while small amounts of A2–A7were present,no A1could be detected;instead,a new ultraviolet-active compound with m /z 5171.0553[M 1H]1dominated the LC–MS trace (Fig.1d).NMR analysis of this compound followed by compar-ison with a synthetic standard established its identity as the novel metabolite indole-3-carbonyl nitrile (ICN)(Fig.1c and Extended Data Fig.2).

Chemically,the most striking feature of ICN is the presence of a highly reactive a -ketonitrile moiety that,to our knowledge,has not been found in any plant natural product;however,benzoyl cyanide has been previously identified in the secretions of millipedes 6,7.The a -ketonitrile is susceptible to nucleophilic attack,resulting in the dis-placement of cyanide ion:in alkaline aqueous solution,ICN degrades to indole-3-carboxylic acid (ICA)(an alternative route to ICA in Arabidopsis has been reported 15);in methanol,ICA methyl ester (A1)is formed instead,explaining the presence of A1and the absence of ICN in methanolic extracts (Fig.1c).Modifying the tissue extraction procedure by using an acidified 1:1acetonitrile/water mixture enabled direct detection of ICN by LC–MS;additionally,when deuterated methanol was used,only the deuterated form of A1was observed (Extended Data Fig.1b–e).On the basis of its molecular formula and the synthesis of an authentic standard,A6was shown to be a serine-ICN addition product (see Fig.2b).However,in the presence of cysteine and structurally related compounds,ICN can undergo a spon-taneous cycloaddition,resulting in the formation of a thiazoline ring and the net loss of ammonia.This last observation allowed us to deter-mine the structures of and synthesize standards for compounds A2–A5,which are the cycloaddition products of ICN and cysteine (A4)or Cys-Gly dipeptide (A2)and their thiazole analogues (A5and A3,respectively;see Fig.2b,Extended Data Fig.3,and Supplementary Table 1).

The absence of the hydroxylated analogues B1–B6in the cyp82C2insertion line pointed to ICN as the likely substrate for this enzyme.Incubation of ICN with yeast-expressed CYP82C2yielded only a trace amount of hydroxylated ICN,but a significant amount of 4-hydroxy-ICA (4-OH-ICA)(structure shown in Fig.3),as confirmed by NMR spectroscopy and comparison with a synthetic standard (Extended Data Fig.4a–d).Since CYP82C2shows no activity on ICA,we deduced

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Department of Chemical Engineering,Stanford University,Stanford,California 94305,USA.2Department of Molecular,Cellular,and Developmental Biology,Yale University,New Haven,Connecticut 06511,USA.

that CYP82C2converts ICN to 4-OH-ICN,competing with hydrolysis of ICN to ICA (Extended Data Fig.4e,f).Further experiments with chemically synthesized 4-OH-ICN showed that its half-life is approxi-mately 3min in aqueous solution at pH 57.5(Supplementary Table 2),rendering direct isolation of the 4-OH-ICN product infeasible.Chemical synthesis of 4-OH-ICN further enabled the synthesis of the 4-hydroxy derivatives of A1–A6,confirming that these correspond to compounds B1–B6seen in WT tissue extracts (Extended Data Fig.3and Supplementary Table 1).Therefore,all the metabolites identified in our initial metabolomics experiment with cyp82C2are ultimately derived from ICN,whether as artefacts of the extraction (A1and B1),or as in vivo addition products (A2–A7,B2–B6).

We next investigated the biosynthesis of ICN,using the CYP82C2gene as bait for coexpression analysis.For our pathogen data set,the CYP79B2gene,whose encoded enzyme converts tryptophan (Trp)into indole-3-acetaldoxime (IAOx)16,has the second highest correlation (Pearson’s r )with CYP82C2among all genes profiled (Supplementary Table 3).We performed a metabolomic analysis of the cyp79B2cyp79B3

double knockout line 17,which is deficient in IAOx production.No ICN-derived metabolites are produced in this mutant (Fig.2a),indicating that ICN is derived from IAOx.

In searching for the enzyme(s)responsible for further conversion of IAOx to ICN,we postulated a biosynthetic route paralleling that of the cyanogenic glycoside dhurrin 8:a CYP79-catalysed formation of an aldoxime,followed by a CYP71-catalysed formation of a cyanohydrin intermediate.In the dhurrin pathway,the cyanohydrin is glucosylated to yield the final product,whereas in ICN biosynthesis,a final dehy-drogenation is required to produce an a -ketonitrile (Fig.2c).

Correlation analysis implicated CYP71A12,a P450linked to cama-lexin biosynthesis 18,as the most likely candidate gene for the cyano-hydrin formation step (Supplementary Table 3).Profiling of the cyp71A12transfer-DNA insertion line,as well as transfer-DNA inser-tion lines of its two closest Arabidopsis homologues,CYP71A13and CYP71A18,demonstrated that the CYP71A12gene is in fact probably responsible:all ICN derivatives with the exception of A6are at ,10%of WT levels in the cyp71A12mutant,but unaffected in the cyp71A13

l i s

i c a e

5 10 15 20

A b s o r b a n c e (280 n m )

Retention time (min)

N H

O OMe

R R

ICN R = H 4-OH-ICN R = OH

A1 R = H B1 R = OH

MeOH extraction

I o n a b u n d a n c e (×106)

Compound

cyp82C2 + Psta

WT + Psta WT + mock

4-OH-ICN ICN

Camalexin

log 2(fold change vs control)

–4–2024

Figure 1|Transcriptomic and metabolomic analyses implicate CYP82C2in the biosynthesis of novel pathogen defence-related secondary metabolites.a ,Heat map of relative gene

expression levels for cytochrome P450genes in Arabidopsis under various pathogen stress

conditions.The enlarged map shows the top 10P450genes after sorting by mean expression level over all conditions.Cytochromes P450in grey have previously been biochemically characterized.b ,Levels of the most significantly differing metabolites identified in seedling comparative metabolomics experiments with cyp82C2.Data represent the mean 6s.d.of six biological

replicates.c ,ICA methyl ester (A1)and 4-OH-ICA methyl ester (B1)are methanolic degradation products of ICN and 4-OH-ICN.d ,High-performance liquid chromatography traces of growth medium for WT and cyp82C2seedlings,showing Psta-dependent accumulation of ICN and 4-OH-ICN.

–202

at1g26380 (fox1at1g26390 (at1g26400 (at1g26410 (at1g26420 (N H

NH 2

CO 2H N H

IAOx

N H

CYP71A12(At2g30750)

N H

FOX1N H

4-OH-ICN ICN

CYP82C2(At4g31970)

CYP79B2(At4g39950)

OH B2

GGP1 (At4g30530)

L -Trp

N H

O OCH 3

N H

N O

N H

S N

OH O

N H

S N

OH O

N H

O S

OH NH 2

N H

O O

OH NH 2

A1/B1

A3/B3

A4/B4

A5/B5

A6/B6

A R = H

B R = OH

Key:

log 2Figure 2|Targeted metabolic profiling of candidate transfer-DNA insertion lines helps uncover the entire ICN biosynthetic pathway.a ,Heat map of mean ICN-derived metabolite levels relative to WT in Psta -elicited transfer-DNA insertion lines.Mutants in bold have significantly decreased levels of ICN derivatives.Note that A6levels are not affected to the same extent as levels of other metabolites in any line except for cyp79B2/B3,hinting at an alternative biosynthetic route from IAOx for this metabolite.b ,Structures of all ICN derivatives,confirmed by comparison with synthetic standards (see Extended Data Fig.3and Supplementary Table 1).c ,Proposed

biosynthetic pathway from Trp to 4-OH-ICN and downstream metabolites.

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and cyp71A18mutants(Fig.2a).Levels of camalexin and other indolic metabolites were only slightly changed in whole-seedling tissue extracts of the cyp71A12mutant(Extended Data Fig.5c).

Further correlation analysis using CYP71A12as bait revealed a clus-ter of five tandemly arrayed homologous genes,At1g26380–At1g26420, that are highly coexpressed with CYP71A12(Supplementary Table3). At1g26380encodes a flavin-dependent oxidoreductase known as FOX1(ref.19).We profiled the corresponding homozygous transfer-DNA insertion lines for these genes and found a three-to fivefold reduction in levels of ICN metabolites in the fox1mutant,with no significant changes observed for the other mutants(Fig.2a). Additionally,we observed a build-up of IAL,the expected hydrolysis product of the indole-3-cyanohydrin intermediate(Extended Data Fig.5d).More strikingly,the fox1mutant accumulates new mass signals corresponding to indole cyanogenic glycosides(ICGs),not previously observed in plants(Extended Data Fig.6a–e,structures shown in Fig.4d).Cyanogenic glycoside compounds are widely dis-tributed in the plant kingdom,but have not yet been detected in Arabidopsis9.Disruption of the ICN pathway at the FOX1-catalysed step therefore leads to capture of some portion of the cyanohydrin intermediate by non-specific glycosyltransferases,exactly paralleling dhurrin synthesis8.

We sought to confirm the proposed biochemical transformations (Fig.2c)by reconstituting the complete pathway in vitro.A combination of yeast microsomal CYP71A12and CYP82C2and N.benthamiana-expressed FOX1was sufficient to catalyse the conversion of IAOx to ICN,as illustrated in Fig.3;the production of4-OH-ICN is inferred from the accumulation of4-OH-ICA.We also reconstituted the bio-synthesis of4-OH-ICN in the heterologous host N.benthamiana, using transient expression of the four pathway genes necessary for production of4-OH-ICN from Trp via Agrobacterium-mediated transient transformation20.We observed significant accumulation of B1(from methanol extraction of4-OH-ICN)only when all pathway genes were present;however,we also noted background levels of ICA and IAL when only early pathway genes were expressed(Extended Data Fig.7).Notably,when we expressed CYP79B2and CYP71A12 but not FOX1,we again observed the accumulation of ICG mass signals(Extended Data Fig.6f).

The Trp-derived metabolites camalexin and4-methoxy indol-3-ylmethylglucosinolate(4-methoxyglucobrassicin)have been shown to play a key role in Arabidopsis immunity(Fig.4d)10,11,18,21.To evalu-ate whether4-OH-ICN pathway products also contribute to Arabidopsis disease resistance,we challenged4-OH-ICN biosynthetic mutants with a diverse panel of https://www.wendangku.net/doc/9912611336.html,ing surface inoculation to mimic the natural infection process,we found that,compared with

ICN

4-OH-ICA IAN

IAL N

Retention time (min)

Figure3|In vitro reconstitution of4-OH-ICN biosynthesis from IAOx.

Combined extracted ion chromatograms(EICs)for IAOx substrate and

reaction products for various subsets of enzymes in the4-OH-ICN pathway;

4-OH-ICN could not be detected directly and its hydrolysis product4-OH-ICA

is shown instead.

WT cyp79B2

cyp79B3

cyp71A12 cyp82C2 cyp71A13 pad3-1 ?s2

solvent

control

ICN 4-OH-ICN camalexin

(

–

)

(

–

)

WT cyp71A12 cyp82C2 cyp71A13 pad3-1 ?s2

(

–

)

Figure4|Camalexin and CYP82C2-synthesized4-OH-ICN contribute

non-redundantly to disease resistance against the virulent bacterial

pathogen P.syringae.a,Growth analysis of the virulent P.syringae pv.tomato

DC3000(Pst)in surface-inoculated adult leaves.Data represent the

mean6s.e.m.of four biological replicates.Data points labelled with different

letters are significantly different(P,0.05,two-tailed t test);data points labelled

with the same letter are not significantly different.WT,Col-0ecotype;c.f.u.,

colony-forming units.b,Growth analysis of Pst in10-day-old seedlings pre-

treated with water or1m M bacterial MAMP flg22for6h.Data represent the

median6s.e.m.of four biological replicates of10–15seedlings each.Different

letters denote statistically significant differences(P,0.05,two-tailed t test).

c,Growth analysis of Pst in WT adult leaves pre-immunized with1m M

flg22and100m M ICN,4-OH-ICN,camalexin or solvent control

(dimethylsulfoxide(DMSO))for24h before infiltration with Pst.Data

represent the median6s.e.m.of three biological replicates.Asterisk denotes

statistical significance relative to WT(P,0.01,two-tailed t test).Experiment

was repeated three times,producing similar results.d,Summary of known

major Trp-derived secondary metabolites in Arabidopsis and

oxidative biosynthetic enzymes that have been used to reconstitute the

pathways in vitro or in planta.

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WT,the adult leaves of cyp71A12and cyp82C2are more susceptible to the virulent bacterial hemibiotroph Pst(P.syringae pv.tomato DC3000)and comparable to the immuno-deficient fls2mutant,which cannot perceive the bacterial microbe-associated molecular pattern (MAMP)flg22(refs22,23)(Fig.4a).Similarly,seedlings of the 4-OH-ICN pathway mutants are more susceptible to Pst than WT in the presence and absence of flg22(Fig.4b),indicating a role for 4-OH-ICN in basal disease resistance against a bacterial pathogen. Notably,the adult leaves and seedlings of the camalexin pathway mutants cyp71A13and pad3are also more susceptible to Pst infection than WT(Fig.4a,b),suggesting a previously unrecognized role for camalexin in the antibacterial defence response.To test for a direct role of the ICN pathway metabolites in the plant innate immune response, either as inducible antibacterial or signalling compounds,we mea-sured their protective effect against subsequent bacterial infection by infecting WT adult leaves with Pst after pre-immunizing them with pure compounds and https://www.wendangku.net/doc/9912611336.html,pared with a solvent control,pre-treatment with4-OH-ICN(but not ICN or camalexin)conferred greater bacterial resistance(Fig.4c),which supports a direct mech-anism of action for4-OH-ICN in inducible plant defence.

We also observed increased disease symptoms in adult leaves of the cyp82C2mutant upon inoculation with spores from the avirulent fungal necrotroph Alternaria brassicicola(Extended Data Fig.8e,f) and—consistent with a previous report24—the virulent necrotroph Botrytis cinerea(Extended Data Fig.8a,b),but not from the obligate fungal biotroph Golovinomyces orontii(Extended Data Fig.8c,d). Furthermore,purified ICN and4-OH-ICN have a growth inhibitory effect on B.cinerea and A.brassicicola comparable to that of cama-lexin25(Extended Data Fig.9).However,we cannot rule out the pos-sibility that the role of the4-OH-ICN pathway in fungal defence is indirect,as adult leaves of the cyp82C2mutant appear partly impaired in camalexin production after Alternaria treatment(Extended Data Fig.10).

The camalexin and4-OH-ICN pathways rely on a pair of paralo-gous genes,CYP71A12and CYP71A13,which are members of the CYP71family linked to innovations in plant metabolism26(Fig.4d). Strikingly,the4-OH-ICN pathway resembles the widespread cyano-genic glucoside pathway that has been lost in the Brassicaceae,and appears to be a metabolic re-invention leading to a novel cyanogenic metabolite type derived from Trp27,28.It is possible that4-OH-ICN acts in concert with other Trp-derived metabolites,each contributing to protection against overlapping sets of specific pathogens.Collectively, our data provide additional insight into the Arabidopsis defence res-ponse and,more generally,how plants use metabolic innovation to expand innate immunity.

Online Content Methods,along with any additional Extended Data display items and Source Data,are available in the online version of the paper;references unique to these sections appear only in the online paper.

Received14August2014;accepted14July2015.

Published online9September2015.

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Supplementary Information is available in the online version of the paper. Acknowledgements We thank F.Ausubel,M.B.Mudgett,C.Khosla,A.Saghatelian, https://www.wendangku.net/doc/9912611336.html,let,C.Danna,S.Galanie,and members of the Sattely and Clay laboratories for advice on experiments and comments on the manuscript.We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis transfer-DNA insertion mutants.We thank G.Lomonossoff(John Innes Centre)for providing plasmid pEAQ.This work was supported by R00GM089985and DP2

AT008321(to E.S.S.),T32GM008412-20(to J.R.),and T32GM007499-38(to B.B.). The early stages of this work were supported by National Science Foundation grant MCB-0519898and National Institutes of Health grant R37GM48707(awarded to Fred Ausubel,Massachusetts General Hospital,Boston,Massachusetts,USA). Author Contributions J.R.,B.B.,N.K.C.,and E.S.S.designed experiments.J.R.and B.B. performed experiments.J.R.,B.B.,N.K.C.,and E.S.S.analysed data and wrote the paper. Author Information Reprints and permissions information is available at

https://www.wendangku.net/doc/9912611336.html,/reprints.The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper.Correspondence and requests for materials should be addressed to E.S.S.(sattely@https://www.wendangku.net/doc/9912611336.html,)or N.K.C.(nicole.clay@https://www.wendangku.net/doc/9912611336.html,).

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Extended Data Figure1|Elicitation of compounds identified in metabolomics screen by flg22peptide and origin of ICA methyl ester as artefact of the methanol extraction method.a,Levels of compounds in Flg22-elicited Arabidopsis Col-0seedling tissue,quantified as mean[M1H]1 ion(m/z610ppm)abundances extracted from raw data;error bars,s.d.based on three biological replicates.Production of these compounds in axenic plant culture demonstrates that they are plant-derived.b,c,Structure and mass peaks of ICA methyl ester(compound A1)seen in LC–MS analysis(b),and EICs for the expected m/z using a standard extraction with80:20CH3OH/H2O or with80:20CD3OD/D2O(c).d,e,Structure and mass spectrum peaks seen for the triply deuterated A1analogue(d),and EICs for the expected m/z using extraction with80:20CH3OH/H2O,or with80:20CD3OD/ D2O(all EICs are to scale)(e).The presence of the deuterated analogue of ICA methyl ester and the complete absence of the non-deuterated compound in plant extracts when CD3OD is substituted for CH3OH show that the methyl ester is not a product of Arabidopsis metabolism,but arises because of the extraction method as a degradation product of ICN.

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Extended Data Figure2|Comparison of spectra for plant-extracted and synthetic compound establishes identity of ICN as new indolic metabolite produced by A.thaliana.a,Full-range(d510.5to20.5)and,b,downfield region partial(d58.5–7.0)1H NMR spectra in CD3CN.Upfield contaminants in the full-range spectra are presumed to be residual solvent.c,Ultraviolet–visible absorbance spectra obtained via a diode array detector during LC analysis.Note that the prominent peak at230nm is due to acetonitrile in the LC mobile phase.d,Targeted MS/MS spectra for the parent ICN[M1H]1 ion(m/z5171.0550)at a collision energy of20V.See Supplementary Table1 for relative peak intensities at other collision energies.e,Aligned EICs for the ICN[M1H]1ion for a Col-01Psta tissue sample extracted with DMSO and synthetic compound,showing identical retention times.

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Extended Data Figure3|Comparison of plant-extracted ICN derivatives, 4-OH-ICN derivatives,and synthetic standards shows identical column elution times for all compounds.Col-01Psta combined EICs were extracted for the relevant compound[M1H]1m/z values for a DMSO-extracted medium sample(4-OH-ICN trace),or a MeOH-extracted seedling tissue sample(all other traces),while synthetic EICs were extracted for a mixed standard in DMSO.Note that chromatograms are not to scale,and the synthetic standard is not equimolar with respect to all compounds because of partial degradation.

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Extended Data Figure4|CYP82C2is an ICN4-hydroxylase.a,b,1H NMR spectra in CD3OD of synthetic ICA(a)and4-OH-ICA(b).c,Spectrum for large-scale enzymatic reaction extract of ICN incubated with CYP82C2.In addition to ICA,resulting from hydrolysis of ICN,peaks for a singly hydroxylated analogue of ICA are seen;these are qualitatively consistent with, but shifted slightly upfield(,30–60Hz)from the4-OH-ICA spectrum, possibly because of impurities or a pH effect in the enzymatic reaction sample. d,To confirm the identity conclusively,80m g of4-OH-ICA dissolved in

CD3OD was added to the enzymatic reaction NMR sample before acquiring another spectrum:no new peaks are seen,while the prior hydroxylated ICA peaks grow in intensity,establishing the product of the enzymatic reaction as 4-OH-ICA.e,EICs for enzymatic reactions of CYP82C2on ICN or ICA,or empty vector control incubation with ICN.Only trace amounts of the expected 4-OH-ICN product but significant amounts of4-OH-ICA are seen for the CYP82C2/ICN reaction.No hydroxylated products are seen for the CYP82C2/ ICA or empty vector/ICN reactions,indicating that CYP82C2catalyses

only the hydroxylation of ICN to4-OH-ICN,but4-OH-ICA is seen as

the predominant end product due to rapid hydrolysis of4-OH-ICN(f). Chromatograms in this figure were obtained using the20min LC–MS gradient (see Supplementary Information,Methods section1.9LC–MS analysis).

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Extended Data Figure5|Levels of numerous Arabidopsis indolic metabolites are altered in ICN pathway gene insertion lines compared with WT plants.a–e,Relative compound levels for mock treatment condition and indicated pathway insertion line mutants,and,f,absolute levels in Psta-treated WT(Col-0)seedlings.For a–e,data bars represent a logarithmically scaled ratio of mean metabolite levels in the indicated line or treatment condition,quantified as[M1H]1ion abundances by LC–MS analysis with XCMS processing,to levels in Psta-treated WT Arabidopsis seedlings.In

f,absolute levels for all compounds except RA were quantified by measuring [M1H]1ion abundances and comparing to standard curves.Error bars,s.d., based on six biological replicates.Cam,camalexin;RA,raphanusamic acid; other abbreviations as detailed previously.

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Extended Data Figure6|Putative ICGs observed in Arabidopsis and in N.benthamiana expressing ICN pathway enzymes.a,EICs for putative ICGs in WT Arabidopsis and fox mutant elicited with Psta.The m/z values shown are median values calculated by XCMS.b,Hypothesized structures and theoretical m/z values for the two ICGs identified.c,MS/MS spectrum for ICG1;m/z values and relative abundances are shown above each peak.The ion analysed here(m/z5691.2210)represents a[2M1Na]1dimer that is significantly more abundant than the[M1Na]1ion.Direct analysis of the [M1Na]1ion(m/z5357.1057)yielded low abundance spectra that could not be easily analysed.At lower collision energies,the[2M1Na]1ion fragments to[M1Na]1,but yields a rich spectrum at40V,which is shown.Predicted peak assignments for the ICG1MS/MS spectrum are shown in the accompanying table.For peaks in bold,exact counterparts could be identified in the dhurrin[M1Na]120V MS/MS spectrum in the METLIN metabolite database.d,MS/MS spectrum obtained for the ICG2[M1Na]1ion and predicted peak assignments.While the[2M1Na]1peak(m/z5864.2225)is also seen for this compound(not shown),[M1Na]1is more abundant in this case,and was analysed directly.e,f,Levels of ICG1and ICG2in ICN pathway mutants(e)and in WT plants elicited with Psta and N.benthamiana expressing ICN pathway enzymes(f).For e and f,levels are quantified as mean [M1Na]1ion(m/z610ppm)abundances extracted from raw data;error bars,s.d.,based on six biological replicates.

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Extended Data Figure7|ICN pathway metabolites are produced in

N.benthamiana transiently expressing pathway genes.Levels of ICN and 4-OH-ICN derivatives(left axis)and other relevant indolic compounds (right axis),quantified as mean[M1H]1ion(m/z610ppm)abundances extracted from raw data;error bars,s.d.,based on six biological replicates.The set of transiently expressed genes is indicated for each panel.Background levels of ICA and IAL detected when only the early pathway genes CYP71A12and/or CYP79B2are expressed indicate potential involvement of endogenous N. benthamiana enzymes.

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Extended Data Figure8|ICN pathway metabolites contribute to disease resistance towards B.cinerea but not towards G.orontii.a,Top:typical lactophenol trypan blue staining of leaves drop-inoculated with spores from the virulent fungal necrotroph B.cinerea to visualize the extent of host cell death (darkly stained areas within and beyond the fungal spore droplet region). Middle:microscopic analysis of stained leaves to visualize the extent of fungal colonization(stained filamentous fungal hyphae within and beyond the fungal spore droplet region).Images were taken at the same magnification (325)and are representative of five biological replicates.Bottom:close-up images of the fungal hyphae beyond the fungal spore droplet region for cyp82C2 and cyp71A13mutants.Images were taken at the same magnification(3100). b,Measurement of the disease lesion diameters in infected leaves.Data represent the median6s.e.m.for five biological replicates.Asterisks denote statistical significance relative to WT(P,0.05,two-tailed t test).c,Typical lactophenol trypan blue staining of fungal conidiophores(spore-bearing structures)formed in leaves infected with the adapted powdery mildew G.orontii.The pad4-1mutant is more susceptible to fungal growth by G.orontii and thus produces significantly more conidiophores.Images were taken at the same magnification(3100)and are representative of three biological replicates. d,Measurement of the number of conidiophores in infected leaves.Data represent the mean6s.d.for three biological replicates.e,Top:typical disease symptoms3days after drop inoculation of leaves with spores from the avirulent fungal necrotroph A.brassicicola.Bottom:microscopic analysis of infected leaves after lactophenol trypan blue staining confirming that disease symptoms are consistent with extent of fungal colonization(lightly stained fungal hyphae extending from the fungal spore droplet region)and host cell death(darkly stained areas along and beyond the border of the spore droplet region).Images were taken at the same magnification(325)and are representative of ten biological replicates.f,Measurement of the disease lesion diameters in infected leaves.Data represent the median6s.e.m.of eight(top graph)or ten biological replicates(bottom graph).Different letters denote statistically significant differences(P,0.05,two-tailed t test).

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Extended Data Figure9|ICN and4-OH-ICN but not their degradation products inhibit fungal growth in vitro.a,b,Fungal growth inhibition assays on B.cinerea SF1(a)or A.brassicicola FSU218(b)with the tested compound (or compound combination)indicated.For compound combinations,the concentration indicated is for each compound;the given combinations approximate the hydrolysis products of ICN or4-OH-ICN.Growth of fungi in potato dextrose broth on a microplate was quantified by measuring absorbance at600nm(OD600nm)72h after spore inoculation and subtracting the absorbance at0h;see Methods for further details.Error bars,s.d.based on three biological replicates.Note that the half-maximum inhibitory concentrations (IC50)for both camalexin and ICN are approximately25m M against B.cinerea and50m M against A.brassicicola.For4-OH-ICN,the inhibitory effect is not as pronounced,possibly because of rapid degradation of4-OH-ICN in potato dextrose broth(see Supplementary Table2).

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Extended Data Figure10|Levels of indolic compounds in leaves of mature plants after mock treatment or fungal infection.Tissue extracts were analysed by LC–MS7days post-infection for A.brassicicola FSU218and5days post-infection for B.cinerea SF1.a–e,Levels of indicated compound,quantified as EIC integral for the[M1H]1ion(m/z610ppm)and converted to absolute amounts by comparison with a standard curve.f,Ion count integrals for indole glucosinolates([M-H]2ion,m/z610ppm).Error bars in all panels,s.d. based on six biological replicates.

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