The Full-Size ABCG Transporters Nb-ABCG1 and Nb · PDF fileYusuke Shibata,a Makoto Ojika,a Akifumi Sugiyama,b Kazufumi Yazaki,b David A. Jones,c Kazuhito ... Pre- and Postinvasion

The Full-Size ABCG Transporters Nb-ABCG1 and Nb-ABCG2Function in Pre- and Postinvasion Defense againstPhytophthora infestans in Nicotiana benthamiana

Yusuke Shibata,a Makoto Ojika,a Akifumi Sugiyama,b Kazufumi Yazaki,b David A. Jones,c Kazuhito Kawakita,a

and Daigo Takemotoa,1

aGraduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japanb Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji 611-0011,JapancResearch School of Biology, The Australian National University, Acton ACT 2601, Australia

ORCID ID: 0000-0002-9816-7882 (D.T.)

The sesquiterpenoid capsidiol is the major phytoalexin produced by Nicotiana and Capsicum species. Capsidiol is producedin plant tissues attacked by pathogens and plays a major role in postinvasion defense by inhibiting pathogen growth. Usingvirus-induced gene silencing-based screening, we identified two Nicotiana benthamiana (wild tobacco) genes encodingfunctionally redundant full-size ABCG (PDR-type) transporters, Nb-ABCG1/PDR1 and Nb-ABCG2/PDR2, which are essentialfor resistance to the potato late blight pathogen Phytophthora infestans. Silencing of Nb-ABCG1/2 compromised secretion ofcapsidiol, revealing Nb-ABCG1/2 as probable exporters of capsidiol. Accumulation of plasma membrane-localized Nb-ABCG1 and Nb-ABCG2 was observed at the site of pathogen penetration. Silencing of EAS (encoding 5-epi-aristolochenesynthase), a gene for capsidiol biosynthesis, reduced resistance to P. infestans, but penetration by P. infestans was notaffected. By contrast, Nb-ABCG1/2-silenced plants showed reduced penetration defense, indicating that Nb-ABCG1/2 areinvolved in preinvasion defense against P. infestans. Plastidic GGPPS1 (geranylgeranyl diphosphate synthase) was also foundto be required for preinvasion defense, thereby suggesting that plastid-produced diterpene(s) are the antimicrobialcompounds active in preinvasion defense. These findings suggest that N. benthamiana ABCG1/2 are involved in theexport of both antimicrobial diterpene(s) for preinvasion defense and capsidiol for postinvasion defense against P. infestans.

INTRODUCTION

Plants have multiple mechanisms to prevent or limit infection bymicroorganisms. Constitutively produced chemicals on plantsurfaces can inhibit penetration of plant tissues or cells by po-tential pathogens, while invaded plant cells can induce rapiddefense responses including the accumulation of antimicrobialcompounds that limit host colonization by pathogens. Suchcompounds include cell wall-degrading enzymes (e.g., chitinasesand glucanases; Kombrink et al., 1988), antimicrobial proteins (e.g., thionins and defensins; Balls et al., 1942; Osborn et al., 1995),and the low molecular weight secondary metabolites phytoa-lexins (Müller and Borger, 1940; Kuc, 1995; Ahuja et al., 2012).

Plant species produce a diverse structural range of phytoa-lexins. In the Solanaceae, the major phytoalexins are sesqui-terpenes, such as capsidiol for Nicotiana and Capsicum speciesand rishitin for Solanum species (Katsui et al., 1968; Bailey et al.,1975; Kuc 1995). In contrast, rice (Oryza sativa) produces phe-nylpropanoid-derived sakuranetin and diterpene momilactones(Kato et al., 1973; Kodama et al., 1992), and the model plantArabidopsis thaliana produces the indole alkaloid camalexin

(Tsuji et al., 1992). Although the importance of phytoalexins inplant defense is well established, their mechanisms of actionagainst microorganisms and their transport to the sites ofpathogen infection are largely unknown.In the solanaceaous model plant Nicotiana benthamiana

(wild tobacco), the sesquiterpenoid phytoalexin, capsidiol, isproduced from isopentenyl pyrophosphate (IPP) (McGarveyand Croteau, 1995; Matsukawa et al., 2013). IPP and its allylicisomer dimethylallyl pyrophosphate (DMAPP) are converted tofarnesyl pyrophosphate (FPP), an intermediate in the bio-synthesis of sterols and terpenoids (Figure 1). Cyclization ofFPP to 5-epi-aristolochene, catalyzed by 5-epi-aristolochenesynthase (EAS) (Facchini and Chappell, 1992), and twohydroxylation reactions, catalyzed by 5-epi-aristolochene dihy-droxylase (EAH) (Ralston et al., 2001), are specific steps in thebiosynthesis of capsidiol.In plant cells, there are two pathways for the production of IPP

(Figure 1). In the cytosol, IPP is produced via the mevalonate (MVA)pathway using acetyl-CoA as precursor (McGarvey and Croteau,1995). The MVA pathway, conserved among all eukaryotic or-ganisms, is a primary metabolic pathway for the production ofsterols and an enormous variety of isoprenoid-derived secondarymetabolites, including sesquiterpenes and triterpenes. Anotherpathway for IPP production is the plastidic methylerythritolphosphate (MEP) pathway, which uses triose phosphates andpyruvate as precursors (Rohmer, 1999). Monoterpenes, di-terpenes, and tetraterpenes synthesized in plastids are produced

1Address correspondence to [email protected] author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Daigo Takemoto([email protected]).www.plantcell.org/cgi/doi/10.1105/tpc.15.00721

The Plant Cell, Vol. 28: 1163–1181, May 2016, www.plantcell.org ã 2016 American Society of Plant Biologists. All rights reserved.

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from IPP provided by the MEP pathway (Lichtenthaler, 2000).Although IPP-derived compounds in the cytosol and plastids aremainly synthesized from IPP produced in their respective com-partments, previous studies have provided evidence for bi-directional exchangeof isoprenoid intermediatesbetween the twocompartments (Laule et al., 2003; Flügge and Gao, 2005;Dudareva et al., 2005; Figure 1). InN.benthamiana, the expressionof genes encoding enzymes in the MVA pathway is significantlyenhanced by treatment with the elicitin INF1, an elicitor proteinsecreted by the potato late blight pathogen Phytophthora in-festans (Kamoun et al., 1997), indicating that production of IPP bythe cytosolic MVA pathway, which is necessary for subsequentcapsidiol production, is accelerated during the induction of dis-ease resistance (Ohtsu et al., 2014).

Effective transport of antimicrobial compounds to the site ofpathogen attack is an important process for plant disease re-sistance. There are several reports showing the involvement ofABC transporters in defense against pathogens. A full-size ABCtransporter ofNicotiana plumbaginifolia, Np-PDR1, is reported asthe exporter of a constitutively produced antifungal diterpene,sclareol (Bailey et al., 1974; Jasinski et al., 2001).Gene silencing ofNp-PDR1 caused increased sensitivity to sclareol and decreasedresistance to the necrotrophic pathogen Botrytis cinerea(Stukkens et al., 2005) and many other pathogens (Bultreyset al., 2009).

The Arabidopsis ABC transporter PEN3/PDR8/At-ABCG36was identified from the forward genetic screening of mutants forcompromised penetration resistance to a nonadapted powderymildewpathogen,Blumeria graminis (Stein et al., 2006). Bednareket al. (2009) reported the accumulation of 4-methoxy-indol-3-yl-methylglucosinolate (4MI3G) in the pen3mutant, suggesting thatPEN3 is a potential transporter of antifungal compounds derivedfrom 4MI3G. The wheat (Triticum aestivum) ABC transporter Lr34confers durable resistance to multiple biotrophic fungal patho-gens, including rust and powdery mildew (Krattinger et al., 2009).In its interaction with N. benthamiana, the hemibiotrophic oo-

mycete pathogen P. infestans grows in the intercellular spaces ofthe mesophyll cell layer and penetrates the cell walls of adjacentplant cells to form haustoria (Shibata et al., 2010). Therefore, to beeffective againstP. infestans, phytoalexins need to be exported tothe apoplast. Chappell and Nable (1987) reported the accumu-lation of capsidiol in the culture medium of elicitor-stimulatedtobacco (Nicotiana tabacum) cells, indicating that capsidiol isexported from plant cells. Export of capsidiol is probably alsoimportant for plant cells because sesquiterpenoid phytoalexinsare phytotoxic (Lyon, 1980; Smith, 1982; Polian et al., 1997).Phytoalexin production is regulated by diverse mechanisms

among plants (Nojiri et al., 1996; Nakazato et al., 2000; Ren et al.,2008). In N. benthamiana, capsidiol production is principallyregulated via ethylene signaling. Gene silencing of EIN2 (a gene

Figure 1. Schematic Representation of Cytosolic MVA and Plastidic MEP Pathways Leading to the Production and Secretion of Antimicrobial Terpenes.

x1, x2, and x3 indicate the number of IPP molecules required for the biosynthesis of particular metabolites.

1164 The Plant Cell

required for ethylene signaling) compromised the production ofcapsidiol induced by treatment with INF1 (Ohtsu et al., 2014).Consistent with this finding, EIN2-silenced N. benthamianashowed reduced resistance to P. infestans (Shibata et al., 2010).The expression of EAS and EAH, specialized genes for capsidiolproduction, is upregulated in N. benthamiana by treatment withethylene, and their INF1-induced expression is reduced by si-lencing EIN2 (Shibata et al., 2010).

In this study, virus-induced gene silencing (VIGS)-basedscreening was employed to identify N. benthamiana genes re-quired for the resistance against P. infestans. Several genes in-volved in ethylene production were identified, confirming theimportance of ethylene signaling for disease resistance in N.benthamiana. Five genes encoding enzymes in the MVA pathwaywere also identified by this screen, suggesting a central role forcapsidiol production in the resistance of N. benthamiana to P.infestans. Genes encoding two functionally redundant full-sizeABCG (PDR-type) transporters, Nb-ABCG1/PDR1 and Nb-ABCG2/PDR2, were identified as essential for disease resistancein N. benthamiana. Analysis of Nb-ABCG1/2-silenced N. ben-thamiana plants suggested that Nb-ABCG1/2 are involved insecretion of both capsidiol required for postinvasion resistanceand an unidentified diterpene required for preinvasion defenseagainst P. infestans in N. benthamiana.

RESULTS

Identification of N. benthamiana Genes Required forResistance against P. infestans

Mature N. benthamiana (over 35 d old) is resistant to P. infestans(Shibata et al., 2010, 2011). VIGS-basedscreeningwasperformedto identify genes required for resistance to P. infestans in N.benthamiana. pTV00 vectors with cloned random cDNA frag-ments from N. benthamiana were constructed and transformedinto Agrobacterium tumefaciens for gene silencing in N. ben-thamiana. Over 3000 Agrobacterium transformants were used forthe induction of VIGS inN. benthamiana, and VIGS-induced plantlines were inoculated with P. infestans isolate 08YD1 (Shibataet al., 2011). Approximately 15% of VIGS-induced plants showedsignificant growth defects caused by VIGS per se, and such lineswere eliminated from the inoculation assay. Eighty-two N. ben-thamiana linesshoweddiseasesymptomswithin10dofpathogeninoculation (Figure2A). Thirty-three uniquegeneswere identifiedfrom the VIGS constructs used on these 82 plant lines ascandidate genes required for resistance against P. infestans.Six of the 33 candidate genes encoded enzymes related to theproduction of ethylene, including S-adenosylmethionine syn-thetase (line A4-85), cystathionine gamma synthase (A4-174),S-adenosylhomocysteine hydrolase (A7-109), and three ami-nocyclopropanecarboxylate oxidases (A2-228, A5-45, and A9-256) (Table1, Figure2C). This result is consistentwithourpreviousresults indicating that phytoalexin production in N. benthamianais regulatedbyethylenesignaling (Shibataet al., 2010;Ohtsuet al.,2014). Six other candidate genes encoded enzymes of theMVA pathway, including acetoacetyl-CoA thiolase (A4-77), hy-droxymethylglutaryl CoA synthase (A4-61 and A8-369),

hydroxymethylglutaryl CoA reductase (A7-131 and A8-159),farnesyl diphosphate synthase (FPPS) (A1-152), and EAS (A4-31) (Table 1, Figure 2D), suggesting a central role for ses-quiterpenoid phytoalexins in resistance against P. infestans inN. benthamiana. Five silencing constructs (A1-190, A2-117,A2-158, A2-189, and A6-96) causing a significant reduction inresistance against P. infestans were found to encode full-sizeABCG (PDR-type) transporters (Figure 2B, Table 1). Furtheranalyses of other lines not listed in Table 1 have been reported(Matsukawa et al., 2013; Ohtsu et al., 2014) or are in progressand will be reported elsewhere.

The ABCG Transporter Nb-ABCG1 Is Essential for DiseaseResistance against P. infestans

The five fragments of ABCG (PDR-type) transporter cDNAs iso-lated from the VIGS vectors showed highest sequence homologywith PDR1 from N. tabacum (Sasabe et al., 2002). Minor differ-encesbetween thesequencesof thesecDNA fragments indicatedthat there are at least two highly homologous Nb-ABCG1 genesinN.benthamiana. Full-length cDNAsequenceofNb-ABCG1wasgenerated by 59- and 39-RACE ends PCR, and two highly ho-mologous copies of Nb-ABCG1, designated Nb-ABCG1a andNb-ABCG1b, were identified. Analysis of the full-length cDNAsequences showed that Nb-ABCG1 possess two sets of sixtransmembrane domains, ABC signature domains, and Walker Aand B domains, indicating that Nb-ABCG1 is a full-size ABCGtransporter (Figure 2B; Supplemental Figure 1). Following releaseof the draft genome sequence (Bombarely et al., 2012), weidentified two Nb-ABCG1 genes in the N. benthamiana genome.Given that N. benthamiana has an allopolyploid genome (Goodinet al., 2008), the two copies of Nb-ABCG1 were presumablyderived from the Nicotiana species ancestral to N. benthamiana.In fact,weoftenfind twocopiesof highly homologous genes in thegenome sequence of N. benthamiana (Table 1; Matsukawa et al.,2013; Ohtsu et al., 2014). Because the nucleic acid sequences ofthe two Nb-ABCG1 genes were highly similar, we could notsuppress expression of a single Nb-ABCG1 gene.

Expression of Nb-ABCG1 and Nb-ABCG2 Is Enhancedduring the Induction of Disease Resistance

At least 17 genes for full-size ABCG transporters can beidentified from the draft genome sequence of N. benthamiana(Supplemental Table 1 and Supplemental Figure 2). Interestingly,two of these genes, Nb-ABCG1a and 1b, are adjacent tothe Nb-ABCG2a and 2b genes in N. benthamiana genomesequence scaffolds Niben101Scf01719 and Niben101Scf06583,respectively (Supplemental Table 1 and Supplemental Figure 3).Expression analysis of N. benthamiana leaves treated with wateror 150 nM INF1 elicitor, a secretory protein produced by P. in-festans (Kamoun et al., 1997), indicated that the expression ofNb-ABCG1a/band their closesthomologsNb-ABCG2a/b, butnotother Nb-ABCG genes, is induced by the treatment of INF1(Supplemental Figure 4). The expression profile of Nb-ABCG1a/b in N. benthamiana leaves treated with 150 nM INF1 was in-vestigated by quantitative RT-PCR. Nb-ABCG1a/b transcriptlevels peaked 1 h after treatment with INF1 and decreased again

ABC Transporters for Phytoalexin Export 1165

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by 3 h. Another peak in Nb-ABCG1a/b transcript accumulationwas observed around 6 to 12 h after treatment with INF1 (Figure3A). A similar biphasic expression pattern was observed for Nb-ABCG2a/b (Figure 3A).

To identify the signaling factors involved in the elicitor-inducedexpression of Nb-ABCG1 and Nb-ABCG2, gene silencing wasemployed in N. benthamiana, including silencing of ICS1 (a genefor salicylic acid production),EIN2 (ethylene signaling), andWIPK/SIPK/NTF4 (MAP kinases) (Shibata et al., 2010; Ishihama et al.,2011;Ohtsu et al., 2014). Increased expression ofNb-ABCG1 andNb-ABCG2 at 12 h after INF1 treatment was reduced in EIN2- andWIPK/SIPK/NTF4-silenced plants (Figure 3B). Given that WIPKand SIPK/NFT4 are involved in the INF1-induced productionof ethylene (Ohtsu et al., 2014), these results suggest that ex-pression of Nb-ABCG1 and Nb-ABCG2 is at least partially

regulated by ethylene signaling. Consistent with these findings,we identified typical GCC boxes for binding of the ethylene-responsive transcription factor ERF in thepromoter regionsofNb-ABCG1a/b and Nb-ABCG2a/b (Supplemental Figure 5).

Preferential Localization of Nb-ABCG1 and Nb-ABCG2 tothe Penetration Sites of P. infestans

To determine the intracellular localization of Nb-ABCG1 and Nb-ABCG2,Nb-ABCG1a-GFP andNb-ABCG2a-GFP fusion proteinswere expressed under the control of the CaMV 35S promoter byagroinfiltration of N. benthamiana leaves. Only the peripheries ofcells were labeled by Nb-ABCG1a-GFP and Nb-ABCG2a-GFP(Figure 4A).Colocalization of ABCG1a-GFPandNbABCG2a-GFPwith the plasma membrane-localized aquaporin PIP2-RFP was

Figure 2. Screening of N. benthamiana Genes Required for the Resistance to P. infestans.

ID numbers of VIGS-induced N. benthamiana lines with reduced resistance to P. infestans (green letters) shown in (B) to (D) indicate the downregulatedgenes in these lines.(A) An example of gene-silenced N. benthamiana inoculated with P. infestans. Silenced line A2-189 showed reduced resistance to P. infestans comparedwith TRV-infected control plant. Photographs were taken 9 d after inoculation.(B) Domain structure of ABC-transporter Nb-ABCG1. TMD, transmembrane domain; NBD, nucleotide binding domain. See Supplemental Figure 1 fordetails of domains.(C) Ethylene biosynthetic pathway.(D) MVA pathway and capsidiol biosynthetic pathway.

1166 The Plant Cell



observed, indicating that Nb-ABCG1a and Nb-ABCG2a localizeto the plasma membrane in N. benthamiana cells (Figure 4A).Furthermore, plasmolysis ofN. benthamiana cells expressing Nb-ABCG1a-GFP or Nb-ABCG2a-GFP was induced by treatmentwith0.5Mmannitol.GFPfluorescencewasdetected in theplasmamembrane extensions (Hechtian strands; Oparka, 1994) con-necting the plasma membrane to the cell wall of plasmolysedcells (Figure 4B), further confirming that Nb-ABCG1a and Nb-ABCG2a localize to the plasma membrane in N. benthamianacells. N. benthamiana leaves expressing Nb-ABCG1a-GFP orNb-ABCG2a-GFP were inoculated with P. infestans, and pref-erential localization of Nb-ABCG1a-GFP and Nb-ABCG2a wasobserved at penetration sites or around infection hyphae ofP. infestans (Figure 4C). Fluorescence of plant tissues was notdetected at P. infestans penetration sites or around infectionhyphaewhenN. benthamianawas inoculatedwith Agrobacteriumcarrying a control vector (Figure 4D). In contrast to the preferential

focal accumulation of Nb-ABCG1a-GFP or NbABCG2a-GFPbeneath appressorium-like swellings of P. infestans, little accu-mulation at penetration sites of P. infestans was observed forPIP2-RFP (Figure 4E; Supplemental Figure 6).

Both Nb-ABCG1 and Nb-ABCG2 Are Involved in theResistance of N. benthamiana to P. infestans

The closest homologs of Nb-ABCG1, Nb-ABCG2a and Nb-ABCG2b, share ;85% nucleotide identity with Nb-ABCG1a/b.Because the sequences of the ABCG1 gene fragments in theVIGS vectors showed high similarity to Nb-ABCG2, the expres-sion of Nb-ABCG2 was investigated in A1-190-silenced plants.The expression of both Nb-ABCG1 and Nb-ABCG2 was signifi-cantly suppressed in A1-190 plants (Figure 5A). Further analysisusing theSGNVIGStool (Fernandez-Pozoetal.,2015) identifiedNb-ABCG3 and Nb-ABCG11 as potential silencing targets of A1-190

Table 1. Summary of cDNA Fragments Identified by VIGS-Mediated Screening for N. benthamiana Genes Required for the Resistance to P. infestans

Plant ID Gene Name Gene Product Accession No.

Length of cDNAFragment inVIGS Vector Scaffold of Nb Genome (ver. 1.0.1)a

Ethylene productionA2-228 ACO1a Aminocyclopropanecarboxylate

oxidase (ACO)LC008352 291 Niben101Scf02543 (Niben101Scf08039)

A4-85 SAMS1a S-adenosylmethionine synthetase (SAMS) LC008353 238 Niben101Scf03535 (Niben101Scf04643)A4-174 CGS1a Cystathionine gamma synthase (CGS) LC008354 276 Niben101Scf10866 (Niben101Scf00136)A5-45 ACO2a Aminocyclopropanecarboxylate oxidase

(ACO)LC008355 409 Niben101Scf09590 (Niben101Scf02433)

A7-109 SAHH1a S-adenosylhomocysteine hydrolase(SAHH)

LC008356 >540 Niben101Scf10608 (Niben101Scf09136)

A9-256 ACO1b Aminocyclopropanecarboxylate oxidase(ACO)

LC008357 221 Niben101Scf08039 (Niben101Scf02543)

ABC transporterA1-190 Nb-ABCG1a Pleiotropic drug resistance protein 1

(ABCG1/PDR1)LC015759 259 Niben101Scf01719 (Niben101Scf06583)

A2-117 Nb-ABCG1b Pleiotropic drug resistance protein 1(ABCG1/PDR1)


A2-158 Nb-ABCG1a Pleiotropic drug resistance protein 1(ABCG1/PDR1)






MVA pathway and capsidiol productionA1-152 FPPS1a Farnesyl diphosphate synthase (FPPS) LC015753 187 Niben101Scf04739 (Niben101Scf00414)A1-244 MVD1a Mevalonate 5-disphosphate

decarboxylase (MVD)LC015754 311 Niben101Scf03413 (Niben101Scf00173)

A4-31 EAS1a 5-epi-aristolochene synthase (EAS) LC015755 >330 Niben101Scf07725 (Niben101Scf07725)A4-61 HMGS1a Hydroxymethylglutaryl coenzyme A

synthase (HMGS)LC015756 >130 Niben101Scf01111 (Niben101Scf01729)

A4-77 ACAT1a Acetoacetyl-coenzyme A thiolase (ACAT) LC015757 480 Niben101Scf04727 (Niben101Scf01100)A7-131 HMGR1 Hydroxy 3 methylglutaryl coenzyme A

reductase (HMGR)LC015758 346 Niben101Scf02203

A8-159 HMGR1 Hydroxy 3 methylglutaryl coenzyme Areductase (HMGR)

LC015758 281 Niben101Scf02203

A8-369 HMGS1a Hydroxymethylglutaryl coenzyme Asynthase (HMGS)


aScaffold number containing highly homologous gene is shown in parentheses.



(Supplemental Table 2). However, expression analysis of Nb-ABCG3 and Nb-ABCG11 in control and A1-190-silenced plantsindicated that the expression of Nb-ABCG3 was not significantlyaffected in A1-190, and the expression of Nb-ABCG11 was notdetectable in leaf tissue of control or A1-190 silenced N. ben-thamiana (Supplemental Figure 7). Thus, we used A1-190 VIGSplants as representative Nb-ABCG1/2-silenced plants for furtheranalysis. To distinguish the roles of Nb-ABCG1 and Nb-ABCG2,gene fragments corresponding to 59 and 39 untranslated regionswereused for theconstructionofgene-specificsilencingvectors forNb-ABCG1 and Nb-ABCG2. Successful gene-specific silencing ofNb-ABCG1 and Nb-ABCG2 in the corresponding VIGS plants wasconfirmed by quantitative PCR (Figure 5A). Disease symptoms oneitherNb-ABCG1-orNb-ABCG2-silencedplants inoculatedwithP.infestans were less severe than those in A1-190 plants(Supplemental Figures 8A and 8B), indicating that the reduced re-sistance of A1-190 VIGS plants to P. infestans was a result of dualsilencing of Nb-ABCG1 and Nb-ABCG2.

Nb-ABCG1 and Nb-ABCG2 Are Probable Transporters forthe Secretion of Capsidiol

Np-PDR1 of N. plumbaginifolia, the closest homolog of Nb-ABCG2, is reported to be a transporter of the diterpene sclareol(Jasinski et al., 2001). Given that some ABCG (PDR-type)

transporters have broad substrate spectra (Kang et al., 2011), wehypothesized that Nb-ABCG1/2 may play a role in export of thesesquiterpenoid phytoalexin, capsidiol. Given that Np-PDR1-si-lenced N. plumbaginifolia plants showed enhanced sensitivity tothe toxic effects of sclareol (Stukkens et al., 2005), TRV-infectedand Nb-ABCG1/2-silenced N. benthamiana plants were treatedwith capsidiol to test their sensitivity to the phytotoxic effects ofcapsidiol reported previously by Polian et al. (1997). Treatmentwith 1 mg/mL capsidiol did not induce any cell death as detectedby microscopic observation of Trypan blue-stained dead cells incontrol TRV-infected N. benthamiana leaves (Figure 5B). In con-trast, induction of cell death was obvious in leaf tissues of Nb-ABCG1/2-silenced plants following treatment with 1 mg/mLcapsidiol (Figure 5B). No capsidiol-induced cell death was ob-served in plants silenced for either Nb-ABCG1 or Nb-ABCG2alone (Figure 5B). Damage to N. benthamiana cells by capsidioltreatmentwasdetectedby ion leakage fromplantcells (Figure5C).Although control, Nb-ABCG1-, Nb-ABCG2-, or Nb-ABCG1/2-silenced plants all showed increased ion leakage followingcapsidiol treatment, Nb-ABCG1/2-silenced plants were signifi-cantly more sensitive to capsidiol (Figure 5C).To verify the role of Nb-ABCG1/2 in capsidiol secretion, the

amount of secreted capsidiol was quantified in TRV-infectedcontrol, Nb-ABCG1-, Nb-ABCG2-, or Nb-ABCG1/2-silencedplants. As cyclohexane is commonly used as a solvent towash off

Figure 3. Expression Profiles of Nb-ABCG1 and Nb-ABCG2 in N. benthamiana Leaves after Treatment with INF1 Elicitor.

(A)N. benthamiana leaveswere treatedwithwater or 150 nM INF1, and transcript levels ofNb-ABCG1a/b andNb-ABCG2a/bwerequantified relative to thatof constitutively expressed EF-1a at the indicated times. Data are means 6 SE (n = 10). Data marked with asterisks are significantly different from leavestreated with water as assessed by two-tailed Student’s t tests: *P < 0.05 and **P < 0.01. Data are from three separate experiments.(B)Ethylene signal transduction is involved in the INF1-induced expressionofNb-ABCG1a/b andNb-ABCG2a/b. Leaves of control, ICS1-,EIN2-, orWIPK/SIPK/NTF4-silenced plants were treated with water or 150 nM INF1, and transcript levels of Nb-ABCG1a/b and Nb-ABCG2a/b were quantified relative tothat of constitutively expressed EF-1a 12 h after treatment. Data aremeans6 SE (n = 9). Datamarkedwith asterisks are significantly different from leaves ofcontrol plants treated with INF1 as assessed by two-tailed Student’s t tests: *P < 0.05 and **P < 0.01. Data are from three separate experiments.

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Figure 4. Subcellular Localization of Nb-ABCG1a-GFP and Nb-ABCG2a-GFP in Epidermal Cells of N. benthamiana.

(A) N. benthamiana leaves were inoculated with Agrobacterium strains for expression of Nb-ABCG1a-GFP (or Nb-ABCG2a-GFP) and PIP2-RFP.Transformed cells were monitored by confocal laser scanning microscopy 24 h after inoculation. Bars = 10 mm.(B) N. benthamiana leaves were inoculated with Agrobacterium strains for expression of Nb-ABCG1a-GFP or Nb-ABCG2a-GFP. Transformed cells weretreatedwith 0.5Mmannitol to induce plasmolysis. Dotted lines and arrowheads indicate cell wall and plasmamembrane of plasmolysed cells, respectively.hs, Hechtian strands; DIC, differential interference contrast image. Bars = 10 mm.(C)Preferential localizationofNb-ABCG1a-GFPandNb-ABCG2a-GFParound thepenetration sites ofP. infestans.N. benthamiana leaveswere inoculatedwith Agrobacterium for expression of Nb-ABCG1a-GFP or Nb-ABCG2a-GFP. Transformed leaves were inoculated with P. infestans 24 h after inoculationwith Agrobacterium, and the localization of GFP fluorescence wasmonitored by confocal laser scanningmicroscopy 24 h after P. infestans inoculation. P.infestans was visualized by calcofluor white (CW) staining. z, zoosporangium; a, appressorium-like swelling; ih, infection hyphae. Bars = 20 mm.(D)N.benthamiana leaveswere inoculatedwithAgrobacteriumcontainingexpressionvectorpNPP40.Transformed leaveswere inoculatedwithP. infestans24 h after inoculation with Agrobacterium, and fluorescence was monitored by confocal laser scanning microscopy 24 h after P. infestans inoculation. P.infestans was visualized by calcofluor white staining. Bars = 20 mm.(E) N. benthamiana leaves were inoculated with Agrobacterium strains for expression of Nb-ABCG1a-GFP (or Nb-ABCG2a-GFP) and PIP2-RFP.Transformed leaves were inoculated with P. infestans 24 h after inoculation with Agrobacterium, and the localization of GFP and RFP fluorescence wasmonitored by confocal laser scanningmicroscopy 24 h afterP. infestans inoculation.P. infestanswas visualized by calcofluor white staining. Bars = 20 mm.Images are representative of at least 20 sites observed from three separate experiments.


pollen coat layers but keep pollen viable (Doughty et al., 1993;Iwano et al., 2014) and capsidiol is soluble in cyclohexane(Matsukawa et al., 2013), we used cyclohexane to wash secretedcapsidiol from INF1-treated leaves. Leakage of chlorophyll wasnot detected bywashingwith cyclohexane for 30 s, indicating thatthere was no major damage to leaf cells (Supplemental Figure 9).Leaves treated with 150 nM INF1 were washed with cyclohexanefor 30 s at 9 h after INF1 treatment, and capsidiol in washing fluids(secreted) and washed leaf tissues (intracellular) was quantifiedbyHPLC. The ratio of secreted/intracellular capsidiolwas;1.75:1for control plants, but;0.5:1 forABCG1/2-silenced plants (Figure5D). Both Nb-ABCG1- and Nb-ABCG2-silenced plants showedpartial reduction in the ratio of secreted/intracellular capsidiol(0.8:1 and 0.78:1, respectively; Figure 5D). Moreover, Agro-bacterium-mediated expression of Nb-ABCG1a-GFP and Nb-ABCG2a-GFP showed an increase in the ratio of secreted/intracellular capsidiol compared with a control expressing GFP

alone (Supplemental Figure 10). These data indicate that bothNb-ABCG1 andNb-ABCG2 are probable transporters for secretion ofcapsidiol.

Effect of Nb-ABCG1/2 Silencing on Elicitor-InducedResistance Reactions

To examine the possible involvement of Nb-ABCG1/2 in the ac-tivation of defense responses, the induction of several resistanceresponses was compared between TRV-infected and Nb-ABCG1/2-silenced plants. Leaves of control or Nb-ABCG1/2-silenced N. benthamiana were treated with INF1, and productionof reactive oxygen species (ROS) was measured 12 h aftertreatment via chemiluminescence using L-012 (a luminol de-rivative). ROS production was similar in TRV-infected and Nb-ABCG1/2-silenced plants (Figure 6A). INF1-induced cell deathwas also not affected by silencing of Nb-ABCG1/2 (Figure 6B).

Figure 5. Nb-ABCG1 and Nb-ABCG2 Are Probable Transporters for Secretion of Capsidiol.

(A) Expression of Nb-ABCG1 andNb-ABCG2 in gene-silenced or controlN. benthamiana 12 h after treatmentwith 150 nM INF1. Quantitative RT-PCRwasperformed with gene-specific primers. Constitutively expressed EF-1a was used as an internal standard. Data are means 6 SE (n = 4). Data marked withasterisks are significantly different from control data (TRV) as assessed by two-tailed Student’s t tests: *P < 0.05 and **P < 0.01.(B)Sensitivity ofABCG-silenced plant to capsidiol. Leaves of TRV-infected or gene-silencedN. benthamianawere treatedwith 2% (v/v) DMSOor 1mg/mLcapsidiol and stained with Trypan blue 24 h after treatment to visualize dead cells. Bars = 50 mm.(C) Leaves of TRV-infected or gene-silenced N. benthamiana were treated with 2% (v/v) DMSO or 1 mg/mL capsidiol. Capsidiol-induced cell death wasquantifiedby ion leakage from four leaf disks (5mmdiameter) in 1mLwater at 12hafter INF1 treatment andgently shaken for another 3h.Data aremeans forsix leaves (consisting of two leaves from each of three plants) 6 SE (n = 6). The data shown for this experiment are representative of three separateexperiments. Data marked with asterisks are significantly different as assessed by two-tailed Student’s t tests: *P < 0.05 and **P < 0.01.(D) Leaves of TRV-infected or gene-silenced plants were treated with 150 nM INF1 and washing fluids were prepared 9 h after treatment. Capsidiol inwashing fluids (secreted) andwashed leaf tissue (intracellular) was quantifiedbyHPLC (n= 7 for TRV andTRV:ABCG1/2 and n=4 for TRV:ABCG1andTRV:ABCG2 fromsevenor four separateexperiments).Datamarkedwithasterisksaresignificantlydifferent asassessedby two-tailedStudent’s t tests: *P<0.05and **P < 0.01.

1170 The Plant Cell



Accumulation of transcripts encoding enzymes of the MVApathway (MVD and FPPS) and specific enzymes for capsidiolproduction (EAS and EAH) was also analyzed. Induction of thesegenes by INF1 treatment was not significantly affected by si-lencing of Nb-ABCG1/2 (Figure 6C). These results indicated thatNb-ABCG1/2 are not involved in INF1-induced defense re-sponses in N. benthamiana.

Effect of Nb-ABCG1/2 Silencing on the Accumulationof Phytoalexins

Accumulation of capsidiol was investigated in leaves of controland Nb-ABCG1/2-silenced plants after INF1 treatment. Elicitor-treated leaves were ground and the total amount of capsidiol wasquantified by HPLC. Capsidiol production was detected begin-ning at 9 h after INF1 treatment, and there was no significant

difference between control and Nb-ABCG1/2-silenced plantsuntil 12 h after treatment (Figure 6D), although reduced secretionof capsidiol was detected in Nb-ABCG1/2-silenced plants at thesame time point (Figure 5D). However, the total amount of cap-sidiol was significantly reduced in Nb-ABCG1/2-silenced plantscompared with that in control plants by 24 h after INF1 treatment(Figure 6D). Specific silencing of either Nb-ABCG1 or Nb-ABCG2hadnosignificant effect oncapsidiol accumulation (SupplementalFigure 8C). Accumulation of debneyol, another sesquiterpenoidphytoalexin structurally related to capsidiol (Burden et al., 1985),was also reduced in Nb-ABCG1/2-silenced plants (SupplementalFigure 11). As degradation of the sesquiterpenoid phytoalexinsrishitin and capsidiol by potato (Solanum tuberosum) and sweetpepper (Capsicum annuum) cells has been reported previously(Stöessl et al., 1977;Horikawaet al., 1976), it was conceivable thatcapsidiol anddebneyolmaybedegradedormetabolized incellsof

Figure 6. Nb-ABCG1 and Nb-ABCG2 Are Not Involved in INF1-Induced Defense Responses.

(A) Top: Leaves of TRV-infected or Nb-ABCG1/2-silencedN. benthamianawere treatedwith water or 150 nM INF1. Production of O22was detected via L-

012-mediatedchemiluminescence12hafter INF1 treatment.Circles indicate theareaof treatment.Chemiluminescence imageswereobtainedusingaCCDcamera. Bottom: Intensities of chemiluminescence were quantified with a photon image processor. Data are means 6 SE (n = 30).(B) Top: Leaves of control or Nb-ABCG1/2-silenced plants were treated with water or 150 nM INF1. Photographs were taken 5 d after treatment. Bottom:INF1-induced cell deathwas quantified by ion leakage from four leaf disks (5-mmdiameter) in 1mLwater at 24 h after INF1 treatment and gently shaken foranother 6 h. Data are means for six leaves (consisting of two leaves from each of three plants) 6 SE (n = 6). The data shown for this experiment arerepresentative of three separate experiments.(C)Expression of genes for capsidiol production inNb-ABCG1/2-silencedN. benthamiana. Leaves of control or Nb-ABCG1/2-silenced plantswere treatedwith 150nM INF1, and the transcript levelsof indicatedgeneswerequantified relative to that of constitutively expressedEF-1a12hafter treatment.Data aremeans 6 SE (n = 4).(D) Accumulation profiles of capsidiol in control and Nb-ABCG1/2-silenced plants treated with INF1. Leaves of TRV-infected or Nb-ABCG1/2-silencedplants were treatedwithwater or 150 nM INF1, and capsidiol productionwas quantified byHPLC at the indicated time after treatment. Data aremeans6 SE

(n = 6). Data marked with asterisks are significantly different from leaves of control plants as assessed by two-tailed Student’s t tests: **P < 0.01.






Nb-ABCG1/2-silenced plants. However, we could not detect theaccumulation of any unique metabolite in Nb-ABCG1/2-silencedplants by thin-layer chromatography (Supplemental Figure 11).

Nb-ABCG1/2 Are Involved in Both Pre- and PostinvasionDefense against P. infestans

Among the silenced N. benthamiana lines susceptible to P. in-festans found in the original screening, we noticed that Nb-ABCG1/2-silenced lines (A1-190, A2-117, A2-158, A2-189, andA6-96) showed more severe disease symptoms compared withthe other susceptible lines. To verify this difference in suscepti-bility, Nb-ABCG1/2- and EAS-silenced N. benthamiana were in-oculated with P. infestans, and development of visible diseasesymptoms was compared. EAS encodes 5-epi-aristolochenesynthase, required for production of capsidiol, the major antimi-crobial molecule underpinning postinvasion resistance (Shibataet al., 2010). Although both EAS- and Nb-ABCG1/2-silencedplants showed significant reduction in resistance against P. in-festans compared with control TRV-infected plants, developmentof disease symptoms on Nb-ABCG1/2-silenced plants wasconsistently faster and more severe compared with that on EAS-silenced plants (Figure 7). This result suggests that Nb-ABCG1/2play an additional role in disease resistance beside capsidiolsecretion.

In theN. benthamiana-P. infestanspathosystem, infection bythe majority of zoosporangia and zoospores ceases at the leafsurface without induction of plant cell death, indicating thatpreinvasion defense is an important resistance mechanismagainst P. infestans (Shibata et al., 2010). To investigate thepossible role of Nb-ABCG1/2 in preinvasion defense, EAS- andNb-ABCG1/2-silenced and control N. benthamiana plantswere inoculated with P. infestans and the number of penetrationsites was scored 24 h later. Accumulation of callose around thepenetration sites was visualized by staining with aniline blue, andfluorescent spots were counted as penetration sites (Figure 8A).The density of penetration sites (number per cm2) found in EAS-silenced plants was comparable to that in control plants (Figure8B), indicating that capsidiol and related sesquiterpenoids haveno role in preinvasion defense against P. infestans. In contrast,a significant increase in the density of penetration sites was ob-served in Nb-ABCG1/2-silenced plants (Figure 8B), suggestingthat Nb-ABCG1/2 may also transport compounds involved inpreinvasion defense against P. infestans. Specific silencing ofeither Nb-ABCG1 or Nb-ABCG2 also reduced preinvasion de-fense, but the density of penetration sites was significantly lowerthan that in Nb-ABCG1/2-silenced plants (Supplemental Figure8D). Therefore, Nb-ABCG1 and Nb-ABCG2 appear to haveoverlapping functions in both pre- and postinvasion defensesagainst P. infestans in N. benthamiana.

MVD, but Not FPPS, Is Required for Preinvasion Defense inN. benthamiana

As reported above, MVD and FPPS were also isolated fromVIGS-based screening of genes required for resistanceagainstP. infestans inN. benthamiana (Figure 2Dand9A, Table1). Reduced expression of MVD and FPPS in gene-silenced

Figure 7. Critical Role of Nb-ABCG1/2 inN. benthamianaResistance toP.infestans.

(A) Growth of control TRV-infected, EAS-silenced, and Nb-ABCG1/2-silencedN. benthamiana. Photographs were taken 10 d after inoculationwith Agrobacterium.(B) Disease symptoms on control, EAS-, and Nb-ABCG1/2-silencedN. benthamiana inoculated with P. infestans. Top: Appearance ofdisease symptoms on leaves. Middle: Disease symptoms on leaveswere categorized into five classes based on severity. 0, No visiblesymptom; 1, small spots on inoculated area; 2, wilting of whole in-oculated area; 3, browning of inoculated area; 4, development ofdisease symptoms over central leaf vein. Bottom: Plot showingpercentage of N. benthamiana leaves with disease symptoms se-verities represented in the five classes as shown in the middle panelsfor leaves of control, EAS-, or Nb-ABCG1/2-silenced plants in-oculated with P. infestans at 3 to 5 d postinoculation (dpi). At leastnine inoculated leaves from each silenced plant were scored. Datamarked with asterisks are significantly different as assessed by one-tailed Mann-Whitney U tests: *P < 0.05.

1172 The Plant Cell




plants was confirmed by quantitative RT-PCR using gene-specific primers (Supplemental Figure 12). These genes en-code enzymes of the MVA pathway for the production of FPP,a precursor of capsidiol (Figure 2D). As expected, productionof capsidiol induced by INF1 was reduced in both MVD- andFPPS-silencedplants as found forEAS-silencedplants (Figure9B), suggesting that MVD and FPPS, like EAS, should be in-volved in capsidiol-dependent postinvasion resistance. Thedensity of penetration sites on leaves of MVD- and FPPS-si-lenced N. benthamiana was scored, and, unexpectedly, thedensity of penetration sites was significantly increasedin MVD-silenced plants, but not in FPPS-silenced plants(Figures 9C and 9D). These results indicate that productionof compound(s) for pre-invasion defense requires MVD,but not FPPS.

GGPPS1 Is Involved in Preinvasion Defense inN. benthamiana

MVD catalyzes the production of IPP, and IPP and its isomerDMAPPareconverted toFPPbyFPPS (Figure10A;McGarveyandCroteau, 1995). Therefore, the requirement for MVD, but notFPPS, inpreinvasiondefense indicates that IPP,butnotFPP, is theprecursor of the compound(s) involved in preinvasion defense.Given that sclareol, an antifungal diterpene derived from IPP, isa substrate forNp-PDR1, a homologofNb-ABCG2 (Jasinski et al.,2001; Supplemental Figure 2), it is possible that diterpenes me-diate the preinvasion defense of N. benthamiana to P. infestans.To test this hypothesis, genes for plastidic geranylgeranyl pyro-phosphate synthase (Nb-GGPPS1a/b, homologs of ArabidopsisGGPPS1; Okada et al., 2000), required for the biosynthesis ofditerpenes from IPP or DMAPP precursors (Figure 10A), wereidentified from the N. benthamiana genome sequence.GGPPS1-silenced plants were produced, and reduced expression ofGGPPS1 in gene-silenced plants was confirmed by quantitativeRT-PCR (Supplemental Figure 12). In GGPPS1-silenced plants,penetration defense against P. infestans was significantlyreduced compared with control plants (Figures 10B and 10C).Inoculation of Nb-ABCG1/2-silenced plants with P. infestanscaused disease symptoms expanding from the inoculation site,whereas relatively small spot lesions were observed in GGPPS1-silenced plants (Figure 10D), indicating that GGPPS1-silencedplants can still stop the development of disease symptoms bypostinvasion resistance.

Expression of GGPPS1 Is Negatively Regulated during theInduction of Disease Resistance

The expression profile of GGPPS1 in INF1-treated leaves of N.benthamiana was investigated by quantitative RT-PCR. Tran-script levels for genes involved in capsidiol production, i.e.,MVD,FPPS, and EAS, increased after INF1 treatment, whereas that forGGPPS1 was reduced (Figure 10E). In GGPPS1-silenced plants,INF1 induced the accumulation of capsidiol, debneyol, andcapsidiol 3-acetate compared with control plants (Figure 10F).Therefore, downregulation of GGPPS1 appears to enhance theproduction of sesquiterpenoid phytoalexins.

DISCUSSION

Plants in natural environments are always under the threat ofinvasion by surrounding microorganisms. In general, plants havetwo layers of defense to resist potential pathogens. Constitutiveproduction of antimicrobial metabolites, together with physicalbarriers, can counter attempted infections by microbes. A widerange of metabolites, called phytoanticipins, including saponins,benzoxazinone glucosides, and glucosinolates, are known tobe involved in such constitutive defenses (Mithen et al., 1986;Niemeyer, 1988; VanEtten et al., 1994; Osbourn, 1996). A secondlayer of defense is activated by penetration of potentialpathogens. To stop the spread of attempted pathogen infections,plants activate robust defense responses, suchas the inductionofhypersensitive cell death and the production of phytoalexins.Although a diverse range of phytoalexins, including flavonoids,

Figure 8. Nb-ABCG1/2, but Not EAS, Is Required for PreinvasionDefenseagainst P. infestans in N. benthamiana.

(A) Control (left), EAS- (middle), or Nb-ABCG1/2-silenced (right) plantswere inoculated with P. infestans, and penetration sites (callose deposi-tions; arrowheads) were stained with aniline blue 24 h after inoculation. P.infestanswas visualized by calcofluor white staining. a, appressorium-likeswelling, z, zoosporangium. Bars = 30 mm.(B) Top: Penetration sites of P. infestans were detected as callose dep-ositions by aniline blue staining 24 h after inoculation. Arrowheads indicatepenetrationsitesofP. infestans.Bars=50mmformainpanelsand20mmforinsets. Bottom: Number of fluorescent spots was counted in leaf discs ofcontrol or gene-silenced N. benthamiana 24 h after inoculation. Data aremeans6 SE (n = 8). Datamarkedwith asterisks are significantly different asassessed by two-tailed Student’s t tests: **P < 0.01.





terpenoids, and alkaloids, are produced by different plant families(Hammerschmidt, 1999), it is generally considered that phytoa-lexins act by inducing changes in permeability of fungal, oomy-cete, and bacterial cellular membranes (Smith, 1982; Turelli et al.,1984; Robertson et al., 1985). Phytoalexin toxicity is not specificto microorganisms but is also destructive to plant cells (Shiraishiet al., 1975; Smith, 1982). For example, rishitin, a sesquiterpenoidphytoalexin produced by Solanum species, affects the perme-ability of plant liposomal membranes and disrupts chloroplasts(Lyon, 1980). Therefore, timely production and efficient transportof phytoalexins to the site of pathogen attack is important forplants to apply these double-edged weapons effectively.

Ethylene Production and Signaling Are Involved inTranscriptional Upregulation of Genes for the Productionand Secretion of Capsidiol

In our VIGS-based screen, six genes related to ethylene pro-duction were identified as essential genes for the resistance ofN. benthamiana to P. infestans (Table 1), indicating that ethyleneis a key plant hormone for resistance to P. infestans in N. ben-thamiana. Previously, we reported that MAP kinase (WIPK/SIPK/NTF4)-mediated upregulation of ethylene production is essentialfor the transcriptional activation of genes for phytoalexin

production (Shibata et al., 2010; Ohtsu et al., 2014). In addition,the expression of Nb-ABCG1 and Nb-ABCG2 is regulated byethylene signaling components (Figure 3), indicating that ethylenesignaling is involved in both production and secretion of capsidiolin N. benthamiana. N. tabacum leaves treated with cryptogein,an elicitin producedbyPhytophthora cryptogea, showedbiphasicproduction of ethylene (Wi et al., 2012). Biphasic induction of Nb-ABCG1 and Nb-ABCG2 expression might therefore be related toa biphasic pattern of ethylene production in response to elicitintreatment in N. benthamiana (Figure 3). Seo et al. (2012) showedthat sclareol-induced expression of At-ABCG40, an encodinghomolog of Nb-ABCG1/2 in Arabidopsis, was impaired in ein2mutants, implying that ethylene regulation of genes for this classof ABC transporters may be a feature common to many differentplant species.

Nb-ABCG1 and Nb-ABCG2 Are ABC Transporters Involvedin Both Pre- and Postinvasion Defense

Little hypersensitive cell death is observed in epidermal andmesophyll cells of N. benthamiana following inoculation withP. infestans (Shibata et al., 2010), indicating that the majorityof infection attempts by inoculated spores are inhibited beforeinvasion of plant cells. Assays for penetration success of P.

Figure 9. MVD, but Not FPPS, Is Required for Preinvasion Defense against P. infestans in N. benthamiana.

(A)Top:Growth of control,MVD-, andFPPS-silencedN.benthamiana.Photographswere taken10dafter inoculationwithAgrobacterium.Bottom:Diseasesymptoms of MVD- and FPPS-silenced N. benthamiana inoculated with P. infestans. P. infestans was inoculated on the right sides of the leaves. Pho-tographs were taken 7 d after inoculation.(B) Leaves of control,MVD-, FPPS-, or EAS-silencedN. benthamianawere treated with 150 nM INF1, and phytoalexin was extracted 24 h after treatment.The amount of capsidiol was quantified by HPLC. Data are means6 SE (n = 3). Data marked with asterisks are significantly different from leaves of controlplants as assessed by two-tailed Student’s t tests: *P < 0.05.(C) Penetration sites of P. infestans on leaves of control, MVD-, or FPPS-silenced N. benthamiana were detected as callose depositions by aniline bluestaining 24 h after inoculation. Arrowheads indicate penetration sites of P. infestans. Bar = 50 mm for main panels and 20 mm for insets.(D) The numbers of penetration sites of P. infestans (fluorescent spots) were counted in leaf discs of control or gene-silenced N. benthamiana 24 h afterinoculation. Data are means 6 SE (n = 8). Data marked with asterisks are significantly different as assessed by two-tailed Student’s t tests: **P < 0.01.

1174 The Plant Cell

infestans on Nb-ABCG1- and Nb-ABCG2-silenced plants in-dicated that Nb-ABCG1 and Nb-ABCG2 are both involved inpreinvasion defense (Supplemental Figure 8D). Previous reportsindicated that Nt-PDR1 (homolog of Nb-ABCG1) and Np-PDR1(homolog of Nb-ABCG2) are involved in the transport of a di-terpene, sclareol (Jasinski et al., 2001; Crouzet et al., 2013).Sclareol was therefore a possible candidate compoundmediatingpreinvasion defense of N. benthamiana. Silencing of GGPPS1,encoding an enzyme for the production of diterpene precursors,allowed increased penetration by P. infestans, further supportinga role for sclareol in preinvasion defense. However, we were

unable to detect sclareol production in N. benthamiana by HPLC(elutionpeakat 16.7min for commercially available sclareol) underour experimental conditions. Crouzet et al. (2013) reported thatN.tabacumPDR1 is involved in the transport of several diterpenes inaddition to sclareol. Therefore, the agent required for preinvasiondefense of N. benthamiana could be an unidentified diterpene(s)secreted by Nb-ABCG1/2.Arabidopsis PEN2 (an atypical myrosinase) and PEN3/PDR8/

At-ABCG36 (a plasmamembrane ABC transporter) are thought tobe involved in the production and transport of antimicrobial glu-cosinolates responsible for preinvasiondefense (Stein et al., 2006;

Figure 10. GGPPS1 Is Involved in Preinvasion but Not Postinvasion Defense against P. infestans in N. benthamiana.

(A) Pathways for the cytosol and plastidic production of terpenoids. x2 and x3 indicate the number of IPP molecules required for biosynthesis of variousmetabolites.(B)Penetration sitesofP. infestanson leavesof control (TRV),Nb-ABCG1/2-, orGGPPS1-silencedN.benthamianaweredetectedascallosedepositionsbyaniline blue staining 24 h after inoculation. Arrowheads indicate penetration sites of P. infestans. Bars = 50 mm for main panels and 20 mm for insets.(C) The numbers of penetration sites of P. infestans (fluorescent spots) were counted in leaf discs of control or gene-silenced N. benthamiana 24 h afterinoculation. Data are means 6 SE (n = 6). Data marked with asterisks are significantly different as assessed by two-tailed Student’s t tests: **P < 0.01.(D)Right sides of leaves of control, Nb-ABCG1/2-, orGGPPS1-silencedN. benthamianawere inoculatedwithP. infestans and photographswere taken 7 dafter inoculation.(E) Expression ofMVD, FPPS, EAS, and GGPPS1 in N. benthamiana leaves treated with water or 150 nM INF1. Transcript levels of indicated genes werequantified relative to that of constitutively expressed EF-1a 12 h after treatment. Data are means6 SE (n = 4). Data marked with asterisks are significantlydifferent from water-treated controls as assessed by two-tailed Student’s t tests: **P < 0.01. Results shown are representative of at least three separateexperiments.(F) Production of capsidiol and related sesquiterpenoids in control and GGPPS1-silenced N. benthamiana. Leaves of control or GGPPS1-silenced N.benthamianawere treatedwith150nM INF1, andphytoalexinswereextracted 24hafter treatment.Amounts of capsidiol, debneyol, andcapsidiol-3 acetatewere quantifiedbyHPLC.Data aremeans6 SE (n=3). Datamarkedwith asterisks are significantly different as assessedby two-tailedStudent’s t tests: **P<0.01.



Bednarek et al., 2009). Arabidopsis pen2 and pen3 mutantsshowed reduced preinvasion defense against P. infestans anddeveloped visible necrosis as a result of increased numbers ofdead cells (Lipka et al., 2005; Stein et al., 2006; Westphal et al.,2008). Similarly, in this study, visible necrosis was observed inGGPPS1-silenced N. benthamiana plants inoculated with P. in-festans as a result of compromised preinvasion defense, butfurther development of disease symptomswas halted,most likelyby postinvasion resistance (Figure 10D).

In contrast, EAS- and FPPS-silenced plants retained pre-invasion defense comparable to control plants but showedcompromised production of capsidiol required for postinvasionresistance. Given that Nb-ABCG1/2-silenced plants showedmore severe disease symptoms compared withGGPPS1-, EAS-,or FPPS-silenced plants, both pre- and postinvasion defensewould appear to play important roles in resistance against P.infestans in N. benthamiana.

The Nb-ABCG1/2 and At-ABCG40 Class of Proteins areMultifunctional ABC Transporters

The closest homolog of Nb-ABCG1/2 in Arabidopsis is At-ABCG40/PDR12 (Supplemental Figure 2). The Arabidopsisabcg40 mutant shows enhanced sensitivity to sclareol, as is thecase for PDR1-silenced N. plumbaginifolia and N. tabacum(Campbell et al., 2003; Stukkens et al., 2005; Crouzet et al., 2013),indicating that transport of diterpenes is likely a conservedfunction of this class of PDR-type ABCG transporter. Arabidopsisabcg40 mutants also showed enhanced sensitivity to lead; thus,At-ABCG40 is presumed to be a pump that excludes lead or lead-containing compounds from the cytoplasm. Lead-induced ex-pression of At-ABCG40 was impaired in ein2mutants, indicatingthat expression of this gene is also regulated by ethylene (Caoet al., 2009). A more recent report indicated that At-ABCG40 isalso involved in the import of the sesquiterpenoid hormone ab-scisic acid (ABA) (Kang et al., 2010). Conceivably, Nb-ABCG1/2could have multiple functions like At-ABCG40. Therefore, wetested the possible effect of Nb-ABCG1/2 silencing on elicitor-induced disease resistance. INF1-induced cell death, ROS pro-duction, and the expression of genes for capsidiol productionwere not significantly affected by the silencing of Nb-ABCG1/2(Figure 6), implying that Nb-ABCG1/2 is not directly involved inINF1-induceddefense responses.Aprevious report indicated thatexogenous application of ABA downregulated capsidiol bio-synthesis in elicitor-treated N. plumbaginifolia (Mialoundamaet al., 2009). However, the accumulation of capsidiol was sig-nificantly reduced in Nb-ABCG1/2-silenced N. benthamiana(Figure 6), suggesting that ABA signaling is unlikely to be re-sponsible for the reduced accumulation of capsidiol in Nb-ABCG1/2-silenced N. benthamiana.

A Defect in Capsidiol Secretion May Activate Mechanismsto Reduce the Cellular Accumulation of Capsidiol in Nb-ABCG1/2-Silenced N. benthamiana

In general, phytoalexin toxicity is not specific to pathogens, but italso affects plant cells; therefore, secretion of phytoalexinsis important for plant cells to avoid the harmful effects of

phytoalexins. Treatment with capsidiol induced more severe celldeath in Nb-ABCG1/2-silenced plants compared with controlTRV-infected plants (Figures 5B and 5C), indicating that Nb-ABCG1/2 is able to counteract the entry of capsidiol into plantcells. Interestingly, there are several examples showing that ABCtransporters of plant pathogenic fungi are likewise responsible forthe efflux of plant-derived defense compounds. These includeatrB of B. cinerea, which transports grapevine (Vitis vinifera) re-sveratrol, ABC1 of Nectria hematococca, which transportspea (Pisum sativum) pisatin, and ABC-G1 of Grosmannia clav-igera, which transports pine (Pinus contorta) monoterpenes(Schoonbeek et al., 2001;Coleman et al., 2011;Wanget al., 2013).Metabolism to nontoxic compounds is another mechanism

plant cells could potentially use to decrease the toxicity of phy-toalexins. It is widely known that some plant pathogens have the

Figure 11. AModel for the Role of Nb-ABCG1/2 in Pre- and PostinvasionDefense of N. benthamiana against P. infestans.

Top: Preinvasion defense. Constitutively produced compound is secretedby Nb-ABCG1/2 to inhibit the invasion of P. infestans. Bottom: Post-invasion defense. Recognition of the invading pathogen leads to the ac-tivation of the MVA pathway and enzymes for capsidiol production.Produced capsidiol is transported to the site of pathogen attackby Nb-ABCG1/2. GGPP, geranylgeranyl pyrophosphate; EA; 5-epi-aristolochene.

1176 The Plant Cell


ability to detoxify phytoalexins produced by host plants to enablethem to establish infection (Pedras and Ahiahonu, 2005), e.g., B.cinerea is able to convert capsidiol to nontoxic capsenone (Wardand Stoessl, 1972). Degradation of the sesquiterpenoid phytoa-lexins rishitin and capsidiol in potato and sweet pepper cells hasalso been reported (Horikawa et al., 1976; Stöessl et al., 1977), butit is not known how they are metabolized.

Arabidopsis PEN3/PDR8/At-ABCG36 is postulated to bea multifunctional transporter involved in the efflux of indole-3-butyric acid, an auxin precursor (Strader and Bartel, 2009), cad-mium (Kim et al., 2007), and tryptophan-derived compounds forpreinvasion defense (Bednarek et al., 2009; Lu et al., 2015). In theArabidopsis pen3 mutant, 4MI3G, a precursor of the antifungalcompounds secreted by PEN3, is accumulated (Bednarek et al.,2009), indicating that the accumulation of end product is nega-tively regulated in the pen3 mutant. More recently, accumulationof 4-O-b-D-glucosyl-indol-3-yl formamide (4OGlcI3F) in the pen3mutant has been reported (Lu et al., 2015). Because of thepresence of a glycosylation residue, 4OGlcI3F is predicted to bea metabolite produced as part of a detoxification strategy. Al-though the expression of genes for capsidiol production was notsignificantly affected by silencing of Nb-ABCG1/2 in N. ben-thamiana (Figure 6C), cytosolic accumulation of capsidiol mayhave inhibited enzymes for capsidiol production or activatedbranch pathways to avoid the accumulation of capsidiol in plantcells.

Metabolic Crosstalk between Cytosolic MVA and PlastidicMEP Pathways May Affect Production of AntimicrobialCompounds for Pre- and Postinvasion Defense

Gene silencing of GGPPS1 compromised preinvasion defenseagainstP. infestans, suggesting that a plastid produced diterpenemay be involved in preinvasion defense inN. benthamiana (Figure10). MVD-silenced plant also showed reduced preinvasion de-fense (Figure 9). These results may seem paradoxical, as theMEPpathway for plastid production of IPP should be intact in MVD-silenced plants. There is some evidence for metabolic crosstalkbetween the plastidic MEP and cytosolic MVA pathways for IPPbiosynthesis (Laule et al., 2003; Flügge and Gao, 2005; Dudarevaet al., 2005). In cultured tobacco (N. tabacum) cells, inhibition ofthe MVA pathway by the HMG-CoA reductase (HMGR) inhibitor,mevinolin, was recovered by application of a precursor in theMEPpathway. Moreover, incorporation studies revealed that sterols,normally producedvia theMVApathway,were synthesized via theMEP pathway in the presence of mevinolin (Hemmerlin et al.,2003). Given that the gene silencing of MVD had only a minoreffect on thegrowthofN.benthamiana (Figure 9), IPPderived fromthe MEP pathway could be exported from the plastids to sustaincytosolic sterol biosynthesis at the expense of reduced pro-duction of diterpenes. Theproduction of sesquiterpenoids relatedto capsidiol, debneyol, and capsidiol 3-acetate was enhanced bythe silencing of GGPPS1 (Figure 10F), further indicating that IPPfrom the MEP pathway could be transported to the cytosol. In-terestingly, the expression of all genes for enzymes in the MVApathway was upregulated by INF1 treatment, while transcriptionof GGPPS1 was downregulated (Ohtsu et al., 2014; Figure 10E).These results may indicate that N. benthamiana switches

terpenoid biosynthesis pathways after pathogen penetration tothe production of phytoalexins rather than antimicrobial com-pound for preinvasion defense (Figure 11).

METHODS

Biological Materials, Growth Conditions, and Inoculation

Nicotiana benthamiana plants were inoculatedwith TRV:PDS (for silencingof the phytoene desaturase gene), and plants showing effective gene si-lencing were selected. Progenies from selected plants were tested for theefficiency of gene silencing again, and a selected line originating froma single N. benthamiana plant named SNPB-A5 was used in this study. N.benthamiana plants of line SNPB-A5were grown in a growth room at 23°Cunder a16-h-light/8-h-darkcycle.P. infestans isolate08YD1 (Shibataetal.,2011) wasmaintained on ryemedia at 20°C. Inoculation ofN. benthamianaleaves (;35 d old) with a zoospore suspension of P. infestans was per-formed as described previously (Shibata et al., 2010).

Preparation and Treatment with INF1 Elicitor

INF1 elicitor was prepared from Escherichia coli cells (DH5a) carrying anexpression vector for INF1, pFB53 (Kamoun et al., 1997), as describedpreviously (Shibata et al., 2010). Leaves of N. benthamiana were treatedwith INF1 solution (150 nM) by injecting the elicitor solution into the in-tracellular spaces of the abaxial sides of leaves using a needleless syringe.

Preparation of cDNA Library in VIGS Vector

Random cDNA fragments for gene silencing were prepared using a PCR-Select cDNA Subtraction Kit (Clontech). Leaf tissues of N. benthamianawere harvested at 1, 3, 6, and 12 h after 150 nM INF1 treatment and used tothe prepare tester cDNA. Leaves without treatment were used for thepreparation of driver cDNA. BI andHIII library primers (Supplemental Table3) were used for the second round of PCR. Amplified PCR products weredigestedwithBamHI andHindIII and cloned into theBamHI-HindIII sites ofpTV00 (Ratcliff et al., 2001). Vectors with random cDNA fragments weretransformed intoAgrobacterium tumefaciens (strainGV3101) as describedpreviously (Shibata et al., 2010). Colonies of transformed Agrobacteriumfromnine separate experiments (A1 toA9 lines)were used for the inductionof VIGS in N. benthamiana.

Construction of Vectors for VIGS and Gene Expression

Base vectors, primer sets, and templates for PCR amplification of clonedDNA fragments and restriction sites used for the construction of vectorsused in this study are listed inSupplemental Table 4. Sequences of primersare listed in Supplemental Table 3. Gene fragments in pTV00 vectors forVIGS induction were assessed using the SGN VIGS tool (Fernandez-Pozoet al., 2015) to exclude the risk of unexpected off-target effects(Supplemental Table 2). Inoculation of N. benthamiana leaves with Agro-bacterium for VIGS induction and gene expression was performed asdescribed previously (Shibata et al., 2010). Unless otherwise stated in thefigure legends, at least three separate experiments were performed for alldata, and at least three gene-silenced plants were used in each round ofexperiments. Reduced expression of target genes in gene-silenced plantwas assessed by quantitative RT-PCR using gene-specific primers(Supplemental Table 3) or as reported previously (Shibata et al., 2010).

Fluorescence Microscopy

Leaves of N. benthamiana were inoculated with Agrobacterium carryingeither pNPP40, pNPP40-ABCG1a-GFP, pNPP40-ABCG2a-GFP, or








pNPP40-PIP2-RFP (Supplemental Table 4). Transformed epidermalcells of N. benthamiana leaves were observed by confocal laserscanning microscopy 48 h after inoculation with Agrobacterium and24 h after inoculation with P. infestans. Fluorescence imageswere recorded between 425 and 475 nm (calcofluor white), between495 and 520 nm (GFP) or between 570 and 590 nm (RFP) under anFV1000-D microscope (Olympus) using 405-nm (calcofluor white),488-nm (GFP), or 559-nm (RFP) excitation. Plasmolysis of N. ben-thamiana cells was induced by infiltration of 0.5 M mannitol usinga needleless syringe.

Lactophenol Trypan Blue Staining

To visualize plant cell death, leaves of N. benthamiana were stained withlactophenol trypan blue as described previously (Takemoto et al., 2003).Stained leaves were monitored under a BX51 microscope (Olympus).

Quantitative RT-PCR

Total RNA was isolated from N. benthamiana leaves using TRIzol reagent(Life Technologies), and cDNA synthesis was performed using ReverTraAce-a- (Toyobo). qRT-PCR was performed using a Thermal Cycler DiceReal Time System Single TP800 (Takara) or LightCycler Quick System350S (Roche Applied Science) with Thunderbird SYBR qPCR Mix(Toyobo). All n values shown in the figure legends indicate the number ofbiological replicates. Expression ofN. benthamiana EF-1awas used as aninternal standard. Gene-specific primers used for expression analysis arelisted in Supplemental Table 3.

Treatment with Capsidiol

Leaves ofN. benthamianawere treated with purified capsidiol (1 mg/mL in2% DMSO) (Matsukawa et al., 2013) by injecting the solution into the in-tracellular spaces of the abaxial sides of leaves using a needleless syringe.

Quantitative Analysis of ROS Production andCapsidiol Accumulation

The generation of ROSwas determined by counting photons generatedby L-012-mediated chemiluminescence as reported previously (Urumaet al., 2009). The luminol derivative L-012was obtained fromWakoPureChemical. Capsidiol, debneyol, and capsidiol 3-acetate produced inleaves ofN. benthamianawere quantified by HPLC or detected by thin-layer chromatography as described previously (Matsukawa et al.,2013). For quantitative analysis of secreted capsidiol, washing fluidsof N. benthamiana leaves were prepared as follows. N. benthamianaleaves (1 g) treated with 150 nM INF1 were washed in 50 mL ofcyclohexane for 30 s with gentle shaking. The washing fluid wasevaporated and redissolved in 150 mL of acetonitrile/water (2:1, v/v),and a 5-mL portion was analyzed by HPLC. Intracellular capsidiol wasextracted from washed leaves as previously reported (Matsukawaet al., 2013). Leakage of chlorophyll was measured using a spectro-photometer (Multiskan GO; Thermo Fisher Scientific) and calculatedaccording to Arnon (1949).

Callose Staining and Quantification of Pathogen Penetration Sites

Callose deposition at the site of pathogen penetration was visualizedby aniline blue staining (Currier and Strugger, 1956). Leaves of N.benthamianawere inoculated withP. infestans, and leaf disks (;8mmdiameter) were excised from inoculated areas 24 h after the in-oculation. The leaf disks were then immersed in fixative solution (1%[v/v] glutaraldehyde, 5 mM citric acid, and 90 mM Na2HPO4, pH 7.4)overnight. Fixed leaf disks were incubated in water at 100°C for 3 min,

immersed in 95% (v/v) ethanol, and stained with 0.1% (w/v) water-soluble aniline blue in 67 mM phosphate buffer (pH 12.0). Thenumbers of fluorescent spots were counted in leaf discs undera BX51 fluorescence microscope (Olympus) using an excitationwavelength of 365 nm.

Bioinformatic Analyses

Statistical analyses were performed using Student’s t tests and Mann-Whitney U tests.

Phylogenetic Analysis

The encoded protein sequences of plant ABCG transporter genes werealigned using the ClustalW program (Thompson et al., 1994) with defaultsettings (Supplemental File 1). Phylogenetic analysiswas conducted usingthe neighbor-joining method (Saitou and Nei, 1987) using MEGA version6.06 (Tamura et al., 2013) with 1000 bootstrap trials.

Accession Numbers

Sequence data from this article can be found in the GenBank databaseunder the accession numbers listed in Table 1 and Supplemental Table 1.

Supplemental Data

Supplemental Figure 1. Comparison of the deduced amino acidsequences for N. benthamiana ABCG1a, ABCG1b, ABCG2a, andABCG2b with N. plumbaginifolia PDR1.

Supplemental Figure 2. Phylogenetic analysis of ABCG transportersfrom N. benthamiana and Arabidopsis.

Supplemental Figure 3. Physical maps of Nb-ABCG1 and Nb-ABCG2 in the genome of N. benthamiana.

Supplemental Figure 4. Elicitor-induced expression of genes for full-size ABCG transporters in N. benthamiana.

Supplemental Figure 5. Nucleic acid sequences of the promoterregions of N. benthamiana ABCG1a, ABCG1b, ABCG2a, andABCG2b.

Supplemental Figure 6. Preferential localization of Nb-ABCG1a-GFPand Nb-ABCG2a-GFP around the penetration sites of P. infestans.

Supplemental Figure 7. Expression of Nb-ABCG3 and Nb-ABCG11is not affected in Nb-ABCG1/2-silenced plant.

Supplemental Figure 8. Specific silencing of Nb-ABCG1 orNb-ABCG2 partially compromises resistance to P. infestans inN. benthamiana.

Supplemental Figure 9. Treatment with cyclohexane causes nodetectable leakage of chlorophyll from leaf cells of N. benthamiana.

Supplemental Figure 10. Enhanced secretion of capsidiol followingAgrobacterium-mediated expression of Nb-ABCG1a-GFP or Nb-ABCG2a-GFP.

Supplemental Figure 11. TLC analysis of phytoalexin production incontrol and Nb-ABCG1/2-silenced N. benthamiana treated with INF1.

Supplemental Figure 12. Silencing of target genes in TRV:MVD, TRV:FPPS, or TRV:GGPPS-infected N. benthamiana.

Supplemental Table 1. Predicted genes for ABCG transportersidentified from the N. benthamiana genome sequence.

Supplemental Table 2. Target genes of VIGS constructs detected bySGN VIGS tool.

1178 The Plant Cell



















Supplemental Table 3. Primers used in this study.

Supplemental Table 4. Plasmids used in this study.

Supplemental File 1. Alignment data used for phylogenetic analysis.

ACKNOWLEDGMENTS

We thank David C. Baulcombe (University of Cambridge) for providingpTV00 and pBINTRA6 vectors and Sophien Kamoun (The SainsburyLaboratory) for the pFB53 vector.We also thankGregory B.Martin (CornellUniversity) for access to the N. benthamiana genome database and KayoShirai (HokkaidoCentralAgriculturalExperimentStation, Japan)andSeishiAkino (Hokkaido University, Japan) for providing P. infestans isolate08YD1. We also thank Takashi Tsuge (Nagoya University, Japan) forvaluable suggestions,MizukiMatsukawa, AkiMizutani, MinaOhtsu, HirokiKojima, Eri Miyazaki, Yohei Kondou, Yuri Mizuno, Rin Soriya, and ErikoTakinami-Shibata for technical support, andMotoyukiMori, Shogo Tsuda,and Kenji Asano (National Agricultural Research Center for HokkaidoRegion, Japan), and Yasuki Tahara (Nagoya University, Japan) for pro-viding potato tubers. Thisworkwas supported by aGrant-in-Aid for YoungScientists (B) (23780041) to D.T., a Grant-in-Aid for Scientific Research (B)(26292024) to D.T., a Grant-in-Aid for JSPS Fellows (11J02734) to Y.S.from the Japan Society for the Promotion of Science, and by a Grant forBasic Science Research Projects to D.T. from The Sumitomo Foundation.

AUTHOR CONTRIBUTIONS

D.T. designed the research. Y.S., M.O., A.S., K.Y., D.A.J., K.K., and D.T.designed experiments. Y.S., A.S., and D.T. performed experiments. Y.S.,M.O., D.A.J., and D.T. analyzed data. D.T. and Y.S. wrote the article withcontributions from D.A.J.

Received August 24, 2015; revised April 5, 2016; accepted April 19, 2016;published April 21, 2016.

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DOI 10.1105/tpc.15.00721; originally published online April 21, 2016; 2016;28;1163-1181Plant Cell

Kawakita and Daigo TakemotoYusuke Shibata, Makoto Ojika, Akifumi Sugiyama, Kazufumi Yazaki, David A. Jones, Kazuhito

Nicotiana benthamiana in Phytophthora infestansDefense against The Full-Size ABCG Transporters Nb-ABCG1 and Nb-ABCG2 Function in Pre- and Postinvasion

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The Full-Size ABCG Transporters Nb-ABCG1 and Nb · PDF fileYusuke Shibata,a Makoto Ojika,a Akifumi Sugiyama,b Kazufumi Yazaki,b David A. Jones,c Kazuhito ... Pre- and Postinvasion