Transcriptional profile of genes involved in the production of terpenes andglyceollins in response to biotic stresses in soybean

Talitta Regina Parmezan1, Salvador Lima Brito Júnior2, Kenia de Carvalho3, Moisés de Aquino4, MichaelBirkett5, John Pickett6, Estela de Oliveira Nunes4, Ricardo Vilela Abdelnor4, Clara Beatriz HoffmannCampo4 and Francismar Corrêa Marcelino-Guimarães4

1Universidade Estadual de Londrina, Departamento de Bioquímica e Biotecnologia, Londrina, PR, Brazil.2GDM Genética do Brasil, Londrina, PR, Brazil.3Universidade Estadual de Londrina, Departamento de Genética e Biologia Molecular, Londrina, PR,

Brazil.4Empresa Brasileira de Pesquisa Agropecuária-EMBRAPA Soja, Londrina, PR, Brazil.5Rothamsted Research, Biointeractions and Crop Protection Department, Harpenden, UK.6Cardiff University, School of Chemistry, Wales, UK.

Abstract

Terpenes produced by plants comprise a diverse range of secondary metabolites, including volatile organic com-pounds (VOCs). Terpene VOC production may be altered after damage or by biological stimuli such as bacterial, fungaland insects, and subsequent triggering of plant defense responses. These VOCs originate in plants from two independ-ent pathways: the mevalonate and the methylerythritol phosphate pathways, which utilize dimethylallyl and isopentenyldiphosphates to form the terpenoidal precursors. Phakopsora pachyrhizi fungi causes Asian soybean rust, limiting soy-bean production and resulting in losses of up to 80% if no control strategies are applied. By using a transcriptomedatasets, we investigated the regulation of genes of the mevalonate pathway under different biotic stresses. We studiedthe impact of P. pachyrhizi infection in vivo expression profile of genes involved in terpenoid and glyceollin biosynthesisin genotypes harboring different resistance genes (Rpp), and across the infection cycle. In addition, we used UPLC andUPGC analysis to evaluate glyceollin and VOC production, respectively, to identify metabolites associated with soy-bean responses to pathogen infection. The regulation of soybean genes involved in terpene production was influencedby genotypes, depending on the Rpp gene, while glyceollin was induced in all genotypes. Furthermore, a sesquiterpenewas identified as a potential marker associated with rust symptoms on soybean.

Keywords: Rust, Glycine max, terpenes, glyceollin..

Received: November 27, 2019; Accepted: July 08, 2020.

Introduction

Terpenes constitute the largest and most diversifiedclass of secondary metabolites, including some volatile or-ganic compounds (VOCs) formed from the combination oftwo or more isoprenyl units, with the molecular formula(C5H8O2)n. These compounds constitute some signalingmolecules in plants and are commonly produced in responseto bacteria, fungi and insects infections (Mendgen et al.,2006; Huang et al., 2012; Pickett et al., 2012; Tamiru et al.,2012). Under infection, plants trigger an arsenal of chemicaland physical reactions, in responses to stresses and this in-duction is directly related with type of injury (Piesik et al.,2010). In addition, these plants signaling molecules mayhave negative effects on fungal infection processes (Mend-

gen et al., 2006). As signaling molecules, some terpenes canoperate by stimulating plant defense mechanisms, includingthose of its intact systemic parts, or even by stimulating rec-ognition in nearby plants (Baldwin et al., 2006; Heil andSilva Bueno, 2007), and could be useful as markers of theplant resistance or the infection process, especially if de-tected at initial steps of infection.

Biosynthesis of plant terpenes occurs in two independ-ent pathways: the methylerythritol phosphate (MEP) path-way, located in the plastid, and the mevalonate (MVA)pathway, located in the cytosol, although with some emerg-ing exceptions and crossovers. The MVA pathway is respon-sible for the production of the precursors of various terpenesand specifically the sesquiterpenes from dimethylallyl di-phosphate (DMAPP) and isopentenyl diphosphate (IPP),while the MEP pathway provides these precursors for theformation of hemiterpenes, monoterpenes, diterpenes andisoprenes, which are precursors for the prenylation step to-

Genetics and Molecular Biology 43, 4, e20190388 (2020)Copyright © 2020, Sociedade Brasileira de Genética.DOI: https://doi.org/10.1590/1678-4685-GMB-2019-0388

Send correspondence to Francismar Corrêa Marcelino-Guimarães.Rod. Carlos Strass, Postal Box 231, Warta, 86001-970, Londrina,PR, Brasil. E-mail: francismar.marcelino@embrapa.br

Research ArticlePlant Genetics

wards a variety of phytoalexins, such as glyceollins (Akashiet al., 2009).

Glyceollins are prenylated pterocarpans, regarded asthe main of soybean phytoalexins and are important in thedefense against phytopathogens (Gouvea et al., 2011). Gly-ceollins have antioxidant, antimicrobial, antinematode, andantifungal actions. The two main precursors in glyceollinproduction are daidzein and DMAPP, derived from phenyl-propanoids and the MEP pathway, respectively (Akashi et

al., 2009). According to the authors, DMAPP provides theisoprenyl unit for the enzyme prenylase 4-dimethylallyltransferase (G4DT, E.C. 2.5.1.36), which catalyzes the addi-tion of DMAPP to carbon C2 or C4 of the pterocarpan skele-ton (glycinol). The contribution of the MVA pathway toglyceollin production occurs at the step catalyzed by iso-pentenyl diphosphate �-isomerase, which converts IPP intoDMAPP, which can be transported from the cytoplasm to thechloroplast and elongate the pterocarpan skeleton formedfrom daidzein (Lygin et al., 2009).

Asian soybean rust (ASR) is a disease caused by theobligate biotrophic fungus Phakopsora pachyrhizi (Sydowand P. Syd). Damage caused by rust in Brazil varies from10% to 80% (Sinclair and Hartman, 1999; Yorinori et al.,2005), depending on the developmental stage of the plantand the degree of severity of the infection. Seven main genesconferring resistance to ASR (R genes) have been identified:Rpp1, Rpp2, Rpp3, Rpp4, Rpp5, Rpp6 and Rpp7, and geneti-cally mapped in the soybean genome (Bromfield andHartwig, 1980; Mclean and Byth, 1980; Hartwig and Brom-field, 1983; Hartwig, 1986; Hyten et al., 2007; Monteros et

al., 2007; Garcia et al., 2008; Silva et al., 2008; Chakrabortyet al., 2009; Kim et al., 2012; Li et al., 2012; Childs et al.,2018). However, the development of soybean cultivars withbroad and durable resistance against P. pachyrhizi is notachieved so far.

Recent studies have focused on identifying mecha-nisms of defense response to infection in soybean genotypes,by correlating transcriptomic, metabolomic and functionalgenomic approaches. These studies have shown that soybeanaccessions harboring different major Rpp genes can triggerresistance responses related to some metabolic pathways,and compounds formed by these pathways are involved indefense mechanisms (van de Mortel et al., 2007; Choi et al.,2008; Pandey et al., 2011; Schneider et al., 2011; Tremblayet al., 2011; Morales et al., 2013). Studies on the trans-criptomic response of soybean to P. pachyrhizi infectionidentified differentially expressed genes from the MVApathway, such as those encoding the enzymes diphospho-mevalonate decarboxylase (Choi et al., 2008), mevalonatekinase (Tremblay et al., 2010), acetyl-CoA acetyltransferaseand phosphomevalonate kinase (Tremblay et al., 2011), ge-ranyl diphosphate synthase and hydroxymethylglutaryl-CoA synthase (Schneider et al., 2011). According to Men-dgen et al. (2006), P. pachyrhizi development in soybean (cvErin) leaves was dependent on the mixture of three com-pounds (nonanal, decanal, and hexenyl acetate). This blend

was able to promote development of the pathogen 24 hoursafter inoculation (hai). However, the terpenoid farnesyl ace-tate, when separately evaluated, negatively regulated haus-torial development, by suppressing differentiation of thefungal cells, and reducing 98% of colony development.However, it is unknown whether this phenotype was causedby induction of soybean defense mechanisms by VOC orwhether the compound itself has an antifungal effect(Mendgen et al., 2006).

Studies in soybeans have demonstrated that inductionof glyceollin synthesis is a defense response of differenttypes of stress (Mazaro et al., 2008; Akashi et al., 2009;Gouvea et al., 2011; Silva, 2013; Liu et al., 2014). Akashi et

al. (2009) reported an up-regulation of glyceollin productionin soybean in response to P. pachyrhizi after one week of in-fection, associated with traditional pathway (MEP), involv-ing the precursor daidzein and dimethylallyl diphosphate(DMAPP), as well as the action of the G4DT enzyme.Naoumkina et al. (2007) and Farag et al. (2008) suggestedthat the intermediate of phytoalexins, the isoflavones, asdaidzein are stored as glycosides (daidzin), facilitating therapid synthesis of the glyceollins following a pathogenic at-tack to legume plants. Lygin et al. (2009) also reportedglyceollin accumulation when studying differences in phe-nolic metabolism during a three-week soybean-P. pachy-

rhizi interaction. Additionally, they observed that this phe-nomenon was mainly present in genotypes containingresistance genes.

In the present study, genes involved in the cytoplasmicpathway for IPP production and genes encoding differentprenylases involved in the production of mono-, di-, andsesquiterpenes and glyceollin synthesis were evaluated re-garding their response to different soybean pathogens by in

silico analysis of transcriptome databases. Using RT-qPCR,the expression levels of these genes were also evaluated inresponse to P. pachyrhizi infection, using soybean geno-types containing different Rpp genes. These genotypes werealso evaluated for glyceollin accumulation during the infec-tion cycle. We found that while glyceollin is a general me-tabolite activated in response to pathogen infection on soy-bean, including by P. pachyrhizi, and the production ofdifferent classes of terpenoids was differentially regulateddepending on the Rpp gene present in the soybean acces-sions. Additionally, VOC detection was carried out on theresistant source of Rpp2 and the susceptible genotype Em-brapa 48. Variations of the VOC (E,E)-�-farnesene were de-tected in the initial hours after P. pachyrhizi inoculation, ei-ther in compatible (genotype Embrapa 48) or incompatible(PI 230970) interactions of the soybean with rust. The levelsof this VOC were inversely proportional to the plant symp-toms; namely, the more evident the infection success was,the lower the VOC production, validating the expressionprofile observed in these soybean accessions and revealing itas a potential marker associated with of rust disease symp-toms on soybean.

2 Parmezan et al.

Material and Methods

In sílico analysis

The involvement of MVA pathway genes in soybeandisease responses was assessed by examining their expres-sion profiles during pathogen infection, which are availablein transcriptome databases. The gene models previously re-ported as differentially expressed in P. pachyrhizi studieswere used as selection criteria when different gene modelswere available for same enzyme annotation (van de Mortel et

al., 2007; Schneider et al., 2011; Morales et al., 2013).The in silico analyses were conducted using data from

nine microarray experiments performed with pathogen-in-fected soybeans, available at Genevestigator[1]. Data wereavailable from experiments with the soybean pathogens P.

pachyrhizi, and the oomycete Phytophthora sojae (Tyler,2007; Gijzen et al., 2009), the soybean aphid Aphis glycines

(Studham and Macintosh, 2013), the nematode Heterodera

glycines and the bacteria Bradyrhizobium japonicum (Zhouet al., 2009; Libault et al., 2010). The in silico expressionanalysis enabled evaluation of the transcriptional of MVApathway genes during different biotic stresses in soybean.Unexpressed gene models in at least one of the experimentalconditions were excluded from further analysis.

Seven genes encoding to the enzymes of the cytoplas-mic MVA pathway acetyl-CoA acetyltransferase (E.C.2.3.1.9), hydroxymethylglutaryl-CoA synthase (E.C.2.3.3.10), mevalonate kinase (E.C. 2.7.1.36), phosphome-valonate kinase (E.C. 2.7.4.2), diphosphomevalonate decar-boxylase (E.C. 4.1.1.33), and isopentenyl-diphosphate�-isomerase (E.C. 5.3.3.2) as well as four genes correspond-ing to the prenylases geranyl diphosphate synthase (E.C.2.5.1.1), (2E, 6E)-farnesyl-diphosphate synthetase (E.C.2.5.1.10), and geranyl-geranyl diphosphate synthase (E.C.2.5.1.29) were include in this study. Likewise, genes encod-ing the terpene synthase (E,E) -�-farnesene synthase(GmTPS) (E.C. 4.2.3.46) and glyceollin synthase (E.C.2.5.1.36) were also considered.

In vivo analysis of genes involved in terpene andglyceollin production in soybean response to P.pachyrhizi infection

Experimental design

Soybean seeds of the cv Williams 82 (susceptible –W82) and the plant introductions (PIs) containing differentRpp genes to P. pachyrhizi, were obtained from the EmbrapaSoja Germplasm Bank and soaked for 3 days in sand. Eachgenotype contains a specific previously characterized resis-tance (R) gene [PI 230970 (Rpp2), PI459025 (Rpp4) andPI200487 (Rpp5)]. The plants were grown in a greenhouseunder controlled conditions of temperature (22 � 1 °C), hu-midity (> 60%) and a photoperiod (12 h). After reaching V2stage (Fehr and Caviness, 1981), the soybean plants weresprayed with the pathogen. The experiment was carried outby a completely randomized design with three replicates,each one composed by three plants. The Williams 82 (sus-

ceptible) and resistant PI genotypes were inoculated withpure isolated LUB55 (collected in Uberlandia – MG, Brazil,2011), by manual spraying with a solution of water and0.05% (v/v) Tween-20, and a final concentration of 5 x 104

P. pachyrhizi urediniospores/ml. Samples were also mock-inoculated with the same solution lacking fungus (controlplants). The second trifoliate from each plant was collectedat 12, 24, 48, 72, 96 and 192 hours after inoculation (hai) andimmediately transferred to liquid nitrogen and stored at -80°C.

Plant RNA isolation

Each sample (100 mg per replicate) was separatelyground with a pestle, mortar and liquid nitrogen. After, thesamples were distributed into 1.5 mL microtubes and storedat -80 °C. Total RNA (~1 g) was isolated from P. pachy-

rhizi-infected or uninfected frozen leaves using an RNA ex-traction kit with TRIzol® reagent (Invitrogen, Carlsbad, CA,USA). Analysis to RNA quantifying and qualifying wereperformed with a Uniscience NanoDrop ND-1000 spectro-photometer (NanoDrop Technologies, Wilmington, DE,USA), at a wavelength of 230 nm, and via agarose gel elec-trophoresis, respectively. The RNA samples were treatedwith deoxyribonuclease I (Kit DNaseI, Invitrogen). TheSuperScriptTM III Kit (Invitrogen) was used to synthesizecDNA from treated RNA according to the manufacturer’sinstructions, and, after, samples were stored at -20 °C. Vali-dation of cDNA quality was performed using PCR withprimers designed to anneal to two different exons of the soy-bean �-actin gene (forward, 5-CCCCTCAACCCAAAGGTCAACAG-3 and reverse, 5-GGAATCTCTCTGCCCCAATTGTG-3), to avoid DNA contamination.

RT-qPCR

The expression profiles of the gene models involved inthe biosynthesis of IPP (MVA pathway), terpenes and gly-ceollins during P. pachyrhizi infection were evaluated usingRT-qPCR. The enzymes were selected using KEGG, andcomplete sequences of the gene models were obtained fromPhytozome[2] and used to design primers. Specific primersto each of the 13 gene models were designed using the soft-ware Primer3Plus[3] and Vector NTI AdvanceTM (Invi-trogen). The sequences of the primers are listed in Table S1.A schematic pathway composed of all the genes selected forthe analysis is presented in Figure 1.

The cDNA samples were amplified with specific prim-ers to each gene model at a final concentration of 0.1–0.5 �Musing 1X Kit Platinum® SYBR® Green qPCR SuperMixUDG (Invitrogen–Life Technologies), according to the man-ufacturer’s instructions. The �-actin gene was used as an en-dogenous control. The E = 10(-1/slope) formula was employedto calculate the reaction efficiency and to adjust the finalprimer concentration and the calibration curve was estab-lished based on the Ct and the log of the cDNA dilutions. Allamplification reactions were performed in triplicate usingthe 7900 Real Time System Thermocycler (Applied Bio-systems), according to the manufacturer’s instructions.

Genes involved in soybean biotic stress 3

After initial steps at 50 ºC for 2 min (UNG activity)and 95 ºC for 10 min (activation of the AmpliTaq Gold poly-merase), a two-step program of 95 ºC for 15 s and 62 ºC for 1min was run for 40 cycles. Dissociation curves were ob-tained to guarantee the absence of nonspecific amplification.The data were collected in the log phase, and the results wereanalyzed with the Sequence Detection program (Perkin El-mer, Waltham, MA, USA). The final relative quantificationof each gene compared with the control was estimated con-sidering the relative quantification (RQ) obtained in each bi-ological replicate, as represented by each independent exper-iment, with three replicates each. Significant differenceswere defined based on estimates of the Relative ExpressionSoftware Tool (REST) version 2.0.7, with a significancelevel of 5%. Additionally, expression levels were convertedto a log2-based colorimetric scale and represented as a heat-map using the software Cluster and Tree View (softwarecopyright Stanford University 1998-99 andhttp://jtreeview.sourceforge.net, respectively).

Glyceollin and (E,E) �-farnesene analysis

Aliquot portions of leaf samples from the RT-qPCRexperiments were used for the analyses of glyceollin levels.Approximately 100 mg of fresh leaves were lyophilized andextracted with 5 mL of methanol (70% MeOH) using anAP59 vortex mixer (Phoenix Luterco®) until completely ho-mogenized. After sonication (20 min) (ultrasonic cleanerLS-Logen), the samples were centrifuged (10,000 rpm for 10min) and filtered through a Millipore filter (Ø = 0.22 �m).

The ultrafiltered samples were analyzed by liquidchromatography with data photo diode array detector(UHPLC-PDA) and mass detection (Xevo Q-Tof) (both de-vices manufactured by Waters®) at the Chemical EcologyLaboratory of Embrapa Soybean, Londrina, PR. Chromato-graphic reversed-phase conditions were performed as fol-lows: a sample injection volume of 10 �L, a run time of 17min, a flow rate of 0.3 mL min-1, and a mobile phase com-posed by MeOH and ultrapure H2O. The gradient flow was:0-8 min [H2O/MeOH60:40], 8.5 min [H2O/MeOH; 56:44],14.5 min [H2O/MeOH; 46:54], 15 min of [MeOH100%],15.5-17 min, [H2O/MeOH; 60:40]. An Acquity UPLC BEHC18 column (Ø = 1.7 �m, 2.1 mm X 50 mm) was used. Datawere taken using Masslynx V4.1 software (Waters®).

Additionally, using leaf samples of Embrapa 48 andaccession PI230970 of three-week-old were analyzed foridentification of the Volatile Organic Compounds (VOC’s).These plants were maintained in glass container (60 cm highx 15 cm internal diameter), opened at the bottom and withtwo collection openings, one lateral for air entry and one atthe top (for outlet) to VOC acquisition according the stan-dard procedure of Webster et al. (2008). These experimentsand GC analyses were carried out at Rothamsted ResearchExperimental Unit, Harpenden – UK.

The samples were analyzed by gas chromatography(GC) (Agilent 6890). Conditions were as follows: The oventemperature was maintained at 30 °C for 1 min, and set at 5°C/min to 150 °C, and maintained for 0.1 min, and then 10

4 Parmezan et al.

Table 1 - List of enzymes from the mevalonate pathway identified in KEGG and the corresponding gene models obtained from Phytozome V12.0.

Enzymes ID Soybean E. C. Reference

Acetyl-CoA acetyltransferase Glyma.17g005300 2.3.1.9 Tremblay et al., 2011

Hydroxymethylglutaryl-CoA synthase Glyma.01g215500 2.3.3.10 Choi et al., 2008

Mevalonate kinase Glyma.03g239000 2.7.1.36 Tremblay et al., 2010

Phosphomevalonate kinase Glyma.06g127200 2.7.4.2 Tremblay et al., 2011

Diphosphomevalonate decarboxylase Glyma.10g279800 4.1.1.33 Choi et al., 2008

Glyma.20g109900

Isopentenyl-diphosphate �-isomerase Glyma.18g242300 5.3.3.2 -

Geranyl diphosphate synthase Glyma.17g166000 2.5.1.1 -

(2E 6E)-Farnesyl-pyrophosphate synthetase Glyma.15g121400 2.5.1.10 -

Glyma.09g015600

Geranyl-geranyl diphosphate synthase Glyma.11g063900 2.5.1.29 Choi et al., 2008

Alpha-farnesene synthase (GmTPS) Glyma.13g321100 4.2.3.46 Liu et al., 2014

Glyceollin synthase Glyma.10g295300 2.5.1.36 -

Figure 1 - Fluxogram of mevalonate pathway genes evaluated by RT-qPCR and the different terpenes formed from isopentenyl diphosphate(IPP) through the action of prenylases, as well as terpene synthases (shownin red).

°C/min to 250 °C. Four microliters of each eluted samplewere injected into the GC. Data were analyzed using HPChemStation software. The quantification of VOCs wasstandard samples (Skelton et al., 2010). The gas chromatog-raphy-mass spectrometry (GC-MS) analysis was performedusing a fused silica capillary column (50 m x 0.32 mm thick-ness, 0.52 �m thick film, DB-1), connected to a tempera-ture-controlled injector. Ionization was by electron impact(70 eV, temperature 250 °C). Helium was the carrier gas.The oven temperature was maintained at 30 °C for 5 min,and then set at 5 °C per min to 250 °C. Identifications weremade by comparing the spectra with the spectral mass data-base (NIST, 2005).

Results

In silico analysis of genes involved in thebiosynthesis of terpene and glyceollin precursorsduring pathogen infection

We conducted an in silico analysis of soybean publictranscriptome data available for 13 model genes identified inthe MVA pathway (Table S3), aiming to infer the involve-ment of genes related to terpenes production, derivated bythe MVA pathway, in soybean defense mechanisms in re-sponse to different biotic stresses, in particular by P. pachy-

rhizi. The transcriptome analysis allowed the identificationof nine studies, where at least one of 13 enzymes was differ-entially expressed, in at least one treatment in response todifferent pathogens, in at least three studies of soybean re-sponses to infection with P. pachyrhizi (Table S3).

Based on transcriptome data, the main enzymes of theMVA pathway had the same expression profile for most in-teractions. For example, up-regulation of isopentenyl-di-phosphate �-isomerase and geranyl diphosphate synthaseand down-regulation of diphosphomevalonate decarboxyl-ase (Glyma.10g426300) were observed.

It was possible to observe a differential expression ofall 13 evaluated genes in at least one of the experiments in-volving the genotypes harboring Rpp1, Rpp3 and Rpp4 ge-nes, with only a subset of the enzymes (mevalonate kinase,phosphomevalonate kinase, (2E, 6E)-farnesyl-diphosphatesynthetase and geranyl-geranyl diphosphate synthase) dis-playing down-regulation in the periods evaluated. From allsignificant expression changes, around 18% correspond tothe P. pachyrhizi and soybean interaction, independentwhether the interaction was compatible or incompatible (p <0.05, fold-change � 1.0, ratio � 0.5). The levels of expression(fold-change) ranged from -95.14 (Glyma01g42450, hydro-xymethylglutaryl-CoA synthase) to 34.66(Glyma17g05500, (E,E) -�-farnesene synthase). The differ-ential expression of the genes encoding the enzymes meva-lonate kinase, phosphomevalonate kinase, (2E, 6E) farne-syl-diphosphate synthase and geranyl diphosphate synthasewas detected in all three experiments involving soybeanbackgrounds containing the resistance genes. All significantexpression changes are summarized in Table S3.

Some of the genes studied presented a similar expres-sion profile under different pathogen infections, such as theinduction of isopentenyl diphosphate isomerase and geranyldiphosphate synthase during infection with H. glycines (Ithalet al., 2007), P. sojae (Tyler, 2007), aphids (Studham andMacintosh, 2013) and P. pachyrhizi (Choi et al., 2008); anddown-regulation of diphosphomevalonate decarboxylaseduring P. sojae infection (Zhou et al., 2009). In addition, thesame gene expression profile was observed in soybeans afterP. pachyrhizi and P. sojae infection, and differential expres-sion was shown for all 13 evaluated genes.

Expression profiles of genes involved in thebiosynthesis of terpene and glyceollin during P.pachyrhizi infection

According to the RT-qPCR analysis, in the susceptiblegenotype Williams 82 (W82), most of the genes that partici-pate in IPP production were repressed or not differentially ex-pressed (Figure 2). Only the enzymes acetyl-CoA acetyl-transferase, hydroxymethylglutaryl-CoA synthase anddiphosphomevalonate decarboxylase were induced specifi-cally at 48 hai and 192 hai. In addition, isopentenyl-diphos-phate �-isomerase was induced at 12 and 192 hai. At 72 hai,there was already intense down-regulation of the enzymesacetyl-CoA acetyltransferase, hydroxymethylglutaryl-CoAsynthase, mevalonate kinase and phosphomevalonate kinase.All prenylases were induced during at least one of thetimepoints evaluated. In this genotype, up-regulation of theenzyme (E,E) -�-farnesene synthase (GmTPS) was not ob-served; instead, a strong downregulation of this gene was ob-served.

In the Rpp2 genotype, a slight up-regulation of the en-zyme acetyl-CoA acetyltransferase was observed, while theother genes were repressed or not differentially expressed,with the exception of the later up-regulation of the enzymeisopentenyl-diphosphate �-isomerase. The prenylases en-zymes geranyl diphosphate synthase and geranyl-geranyldiphosphate synthase were induced in 12 and 24 hai, respec-tively, even after significant down-regulation of genes en-coding MVA pathway enzymes and, consequently, the IPPlevels. There was considerable down-regulation of (2E,6E)-farnesyl-diphosphate synthetase at all evaluated time-points.

In the Rpp4 genotype, slight up-regulation of hydro-xymethylglutaryl-CoA synthase, mevalonate kinase, di-phosphomevalonate decarboxylase and isopentenyl-diphos-phate �-isomerase was observed at specific timepoints.However, we observed up-regulation of prenylases involvedin the formation of FPP precursors (geranyl diphosphatesynthase and (2E, 6E)-farnesyl-diphosphate synthetase), fol-lowed by up-regulation of GmTPS (E,E) -�-farnesene syn-thase - E.C. 4.2.3.46). Significant down-regulation ofgeranyl-geranyl diphosphate synthase was observed. It wasalso observed up-regulation of glyceollin synthase involvedin production of glyceollin at all timepoints studied.

In the Rpp5 harboring genotype, differently from theother Rpp sources, the pathway was strongly induced, except

Genes involved in soybean biotic stress 5

for the enzyme hydroxymethylglutaryl-CoA synthase. Thestrongest up-regulation was observed in the enzymes phos-phomevalonate kinase and diphosphomevalonate decarbox-ylase, which reached expression levels between 79.8 and137 times more induced in inoculated samples compared tothe false-inoculated samples at 48 hai. At the same time-points, strong down-regulation of isopentenyl-diphosphate�-isomerase was observed, coinciding with the up-regu-lation of prenylases enzymes geranyl diphosphate synthaseand (2E, 6E)-farnesyl-diphosphate synthetase. At 12 hai, theenzyme geranyl diphosphate synthase was upregulated in allgenotypes, except in the Rpp5 genotype.

The enzyme glyceollin synthase was intensely inducedat almost all timepoints and in all genotypes evaluated afterinoculation with the fungus. However, GmTPS involved inthe production of sesquiterpene (E,E) �-farnesene (E.C.4.2.3.46) was generally repressed after P. pachyrhizi infec-tion, with the exception of the expression pattern observed inthe Rpp4 background. All results can be found in Figure 2and Table S2.

Glyceollin and (E,E) �-farnesene levels after P.pachyrhizi infection

Inoculation of soybean with the fungus P. pachyrhizi

showed a glyceollin peak in all genotypes tested, starting at24 hai, but at this time, the susceptible genotype presentedthe lowest glyceollin level. In general, the accumulation ofphytoalexin was increasing across the infection cycle. At192 hai, all genotypes presented nearly near the same levelsof glyceollins, as revealed by the peak area (Figure 3).

Volatiles collection in P. pachyrhizi inoculated soy-bean was started 18 hai and passed over 48 hours, totalizing66 hai of collecting process. It was observed that after soy-bean infection with P. pachyrhizi fungus, one of the VOCsemitted by the plant had its production reduced, compared tofalse inoculated plants, in both resistant and susceptible ge-notypes (Figure 4).

Comparing the spectra obtained with the spectral massdatabase, the reduced compound was identified (over 70%probability) as the metabolite (E,E) �-farnesene, that waslater confirmed by a co-injection of the VOC solution andthe external standard (E, E) �-farnesene. The peak obtainedon the chromatogram was unique at the retention time corre-sponding to (E,E) �-farnesene.

The plants were also evaluated daily for rust symp-toms. Disease symptoms appeared around seventh day afterinoculation. After comparing the chromatographic profiles,no other compounds were associated with the presence of P.

pachyrhizi in the infected leaves. Phenotypic analysisshowed that high level of disease symptoms was related tohigher (E,E) �-farnesene reduction among infected plants atthe initial times of infection evaluated (Table 2).

6 Parmezan et al.

Figure 2 - Expression profiles of the genes involved in terpenoid andglyceollin biosynthesis in soybean after infection with P. pachyrhizi. Ex-pression values are presented as the log2 of the RQ (relative quantifica-tion) values determined by the Software REST (Relative Expression Soft-ware Tool) (Pfaffl et al., 2002). These values were converted into acolorimetric scale by the Cluster and Tree View software. Legend: W82refers to the susceptible cultivar Williams 82, Rpp2 to the resistant PI230970, Rpp4 to PI459025 and Rpp5 to PI200487. Enzymes: 2.3.1.9,acetyl-CoA acetyltransferase; 2.3.3.10, hydroxymethylglutaryl-CoA syn-thase; 2.7.1.36, mevalonate kinase; 2.7.4.2-phosphomevalonate kinase;4.1.1.33, diphosphomevalonate decarboxylase; 5.3.3.2, isopentenyldiphosphate isomerase; 2.5.1.1, geranyl diphosphate synthase; 2.5.1.10,(2E,6E)-farnesyl diphosphate synthase; 2.5.1.29, geranyl-geranyl diphos-phate synthase; 4.2.3.46, �-farnesene synthase (GmTPs); 2.5.1.36, gly-ceollin synthase. The color scale can be observed below the figure corre-sponds to the interval between fold-change values.

Figure 3 - Glyceollin concentrations in soybean leaves of different geno-types [Williams 82, PI 230970 (Rpp2), PI 459025 (Rpp4), PI 200487(Rpp5)] inoculated with Phakopsora pachyrhizi, evaluated at six time pe-riods (12, 24, 38, 72, 96, and 192 hai). Data represent the mean of threereplicates of representative inoculated samples.

Tests performed with detached soybean leaves inocu-lated with P. pachyrhizi did not allow us to infer any directeffect of volatile (E,E) �-farnesene in the infectious process.Leaves developed disease symptoms characterized as TANlesions (Embrapa 48) and RB-type lesions (PI 230970), bothin which solutions were added (E,E) �-farnesene as in usedas control. However, further testing needs to be performed,especially regarding an efficient dilution of the compound(results not shown).

Discussion

Genes involved in terpene and glyceollinbiosynthesis are induced by different soybeanpathogens

The MVA pathway consists of six enzymatic reactionsinitiated by the condensation of three acetyl-CoA units,

forming 3-hydroxy-3-methylglutaryl-CoA, which is re-duced to mevalonic acid, followed by two additional steps ofphosphorylation and decarboxylation, culminating with theformation of IPP, the main precursor for the formation ofterpenoids in the cytoplasm. Six genes encoding enzymes ofthe MVA pathway, culminate with the production of IPPprecursor. The transcript levels of the enzyme isopentenyldiphosphate isomerase (IDI), which is related to the conver-sion of IPP to DMAPP, were also analyzed here, onceDMAPP is an important precursor of glyceollin, by the ac-tion of the glyceollin synthase (Figure 1).

From IPP, terpene production in plants is initially de-pendent of the prenylases (geranyl diphosphate synthase,geranyl-geranyl diphosphate synthase and (2E,6E)-farnesyldiphosphate synthase), responsible to convert IPP into GPP(geranyl diphosphate), FPP (farnesyl diphosphate) andGGPP (genranyl-geranyl diphosphate); and later by differ-ent terpene synthases, that use these precursors as substratesfor monoterpenes (10 carbons), sesquiterpenes (15 carbons)and diterpenes (20 carbons) production. The enzymeGmTPS (E,E)-�-farnesene synthase acts in the final step of(E, E) -�-farnesene (a sesquiterpene) production, convertingFPP or GGPP into (E,E)-�-farnesene (Figure 1).

The activation of genes of the MVA pathway,prenylases, and glyceollin and terpene synthases in soybean(GmTPS) was observed in different soybean-pathogen inter-actions on the transcriptome studies, corroborating that thesepathways are broadly activated by biotic stress in soybean,as part of the basal resistance response. Production of these

Genes involved in soybean biotic stress 7

Figure 4 - Chromatographic profiles of volatile solutions obtained from soybean plants, Embrapa 48 (a, c) and PI 230970 (b, d) genotypes. The arrow in-dicates the volatile compound that had its emission reduced after inoculation with the fungus. (a, b) False-inoculated soybean plants. (c, d) Soybean plantsthat were Phakoposra pachyrhizi inoculated.

Table 2 - Percentage of infected leaf area in P. pachyrhizi inoculated soy-bean plants and the production of (E, E) alpha-farnesene over 48 hours.

Embrapa 48 PI 230970

Rep % AFI ng/�L (E ,E)

�-farnesene

Rep % AFI pA (E ,E)�-farnesene

1 7.05 5.13 1 4.24 3.25

2 5.55 1.95 2 1.20 7.03

3 0.34 12.50 3 0.18 24.63

4 8.61 3.64 4 1.70 8.71

compounds may not require specific recognition by thepathogen.

Based on the transcriptome data available, the prenyla-ses involved in the synthesis of mono-, di- and sesquiter-penes and glyceollin were activated by P. pachyrhizi

infection, but was not specific for this pathogen. The dataalso did not reveal a pattern of activation or down-regulationof a specific pathway in a specific pathosystem. We ob-served only five genes differentially expressed in response toa specific pathogen, including the enzymes acetyl-CoAacetyltransferase, diphosphomevalonate decarboxylase,geranyl diphosphate synthase, (2E,6E)-farnesyl diphosphatesynthase and geranyl-geranyl diphosphate synthase in re-sponse to aphids. Similarly, acetyl-CoA synthase was spe-cifically upregulated in experiments where soybeans wereinfected with P. sojae (Tyler, 2007; Studham and Macin-tosh, 2013) and aphids (Zhou et al., 2009).

Terpene biosynthesis is part of P. pachyrhizidefense response in soybean and is dependent ofthe Rpp background in soybean genotypes

In general, we observed a slight activation of the MVApathway in the susceptible (W82) and in the Rpp4 genotypesin response to P. pachyrhizi until IPP production. In theRpp2 background, all genes evaluated in MVA pathwaywere strongly repressed, while in Rpp5 genotype, a com-pletely opposite pattern was observed, with a strong induc-tion of the pathway. Thus, considering the expression dataobtained in this study, terpene production may also contrib-utes to resistance response to P. pachyrhizi, but being tempo-ral regulated depending on the Rpp background,

Most genes in the W82 genotype leading to the pro-duction of IPP were either repressed, and it was possible toobserve a discrete up-regulation during haustorial develop-ment (48 hai) of three genes (acetyl-CoA acetyltransferase,hydroxymethylglutaryl-CoA synthase and diphosphome-valonate decarboxylase). At 192 hai, which corresponds tothe uredia formation, four genes were induced (acetyl-CoAacetyltransferase, hydroxymethylglutaryl-CoA synthase, di-phosphomevalonate decarboxylase and isopentenyl diphos-phate isomerase). However, strong down-regulation was ob-served at 72 hai, where four genes (acetyl-CoAacetyltransferase, hydroxymethylglutaryl-CoA synthase,mevalonate kinase and phosphomevalonate kinase) weresignificantly repressed, and the other genes were not differ-entially expressed, which is characteristic of a biphasic re-sponse, with two up-regulation peaks interspersed by astrong down-regulation of gene expression (Figure 2, TableS2).

A type of biphasic expression pattern in response torust had already been reported in the Embrapa 48 genotype,and in this genotype, two peaks of up-regulation were ob-served at 12 and 96 hai. In the Rpp2 genotype, expressionpeaks occurred at 12 and 72 hai. The second phase of up-regulation of resistance response was faster in resistant ge-notype, coinciding with the stages of haustorium formationand consequent secretion of proteins involved in virulence of

fungi and was indicated to be crucial to the resistancephenotype (van de Mortel et al., 2007). It should be noted,however, that the biphasic response presented in that studywas mainly related to activation of type MYB and WRKYtranscription factors, potentially regulating the production ofphenylpropanoids and lignin precursors. These pathwayswere also activated in the Rpp3 and Rpp4 genotypes (Meyeret al., 2009; Schneider et al., 2011), and a biphasic responseto fungus was also described. Subsequently, transient silenc-ing in soybean (Pandey et al., 2011) functionally validatedthe involvement of such pathways in the resistance responseto rust in the Rpp2 genotype. Thus, the production of phe-nylpropanoid secondary metabolites may be one of the mainresponses to P. pachyrhizi infection in soybean, as it is ob-served in different Rpp resistance genes sources. The terpeneproduction may also contribute to resistance response to P.

pachyrhizi, however a similar pattern of activation was notobserved among the genotypes harboring different Rpp

genes.

Interestingly, even though we could not observe a sim-ilar pattern in the MVA pathway among the different Rpps

sources, at least the prenylase geranyl diphosphate synthasewas up-regulated in all genotypes assessed at initial time-point after infection (12 hai), returning to basal levels until192 hai, when it was significantly induced again, coincidingwith the penetration and sporulation stages of the P. pachy-

rhizi infection cycle, demonstrating that at least the forma-tion of monoterpens precursors is common among thegenotypes and its production might be associated with theinitial and late steps of infection. The Rpp5 genotype was theonly one where this biphasic pattern was not observed (Fig-ure 2, Table S2).

The Rpp4 genotype likewise W82, displayed a slightup-regulation of four MVA pathway genes evaluated(hydroxymethylglutaryl CoA synthase, mevalonate kinase,diphosphomevalonate decarboxylase and isopentenyl di-phosphate isomerase) at specific timepoints. A biphasic re-sponse was not observed (Figure 2, Table S2). It should benoted that, as in W82 genotype, the enzyme isopentenyldiphosphate isomerase, responsible for reversible reaction ofIPP to DMAPP, was induced in Rpp4 genotype at the initialtimepoints. This shows that even with slight production ofIPP by the MVA pathway in these genotypes, the IPP maystill be converted into DMAPP and, consequently, may beinvolved in other terpenoids production, such as mono- andditerpenes, as demonstrated by expression profile of differ-ent prenylases observed in this study in this genotype.

In susceptible W82 genotype, all genes encodingprenylases were induced at least at one evaluated time point,indicating that mono-, di- and sesquiterpenes may be rele-vant to development of basal defense mechanisms of hostsASR infected. However, in this genotype, there was noup-regulation of enzyme (E,E) -�-farnesene synthase(GmTPS); instead, a strong down-regulation of this genewas observed (Figure 2, Table S2), suggesting that there isno (E,E) -�-farnesene production in compatible reactions, orin lower levels, corroborating with results of VOCs detec-

8 Parmezan et al.

tion in susceptible genotype Embrapa 48 after rust infection(Figure 4, Table 2).

In Rpp4 genotype, the up-regulation of prenylases as-sociated with FPP precursor formation (geranyl diphosphatesynthase and (2E, 6E)-farnesyl diphosphate synthase) estab-lishment was observed, followed by up-regulation ofGmTPS (E, E) -�-farnesene synthase), indicating the possi-ble production of (E, E), �-farnesene, at least during haus-torial development and secretion of effector proteins (48hai). Additionally, significant down-regulation of geranyl-geranyl diphosphate synthase was observed, indicating“capture” of the FPP precursor, favoring production ofsesquiterpenes over diterpenes (Figure 2, Table S2). Choi et

al. (2008) also observed the up-regulation of (E,E) -�-farne-sene synthase in a genotype containing the Rpp1 (PI200492)resistant-gene, after infection with two different isolates(HW94-1 and TW72-1) at the same timepoint of 48 hai (Ta-ble S2).

Different from the Rpp4 and W82 genotypes, Rpp2 ge-notype displayed strong down-regulation of all MVA path-way genes evaluated in response to P. pachyrhizi. A slightup-regulation was only observed in first enzyme of the path-way (acetyl-CoA acetyltransferase) at 24 hai and at finaltimepoint evaluated (192 hai). Tremblay et al. (2011) alsoverified the expression profile of this enzyme in W82 andRpp2 genotypes, with a strong up-regulation at 48 hai, a cru-cial period for fungal development, when the pathogen hasestablished many haustoria and is secreting effector proteinsto interrupt the defense mechanisms plant. At all other time-points evaluated in this study, all genes were significantlydown-regulated or were not differentially expressed (Figure2, Table S2). Repression of MVA pathway in Rpp2 genotypemay compromise the production of IPP, the main precursorfor terpenoid production in cytoplasm. This result was dem-onstrated by evaluation of the VOC sesquiterpene (E,E)�-farnesene production. This sesquiterpene was repressedafter P. pachyrhizi infection in both, susceptible Embrapa 48genotype and a resistant genotype containing Rpp2 gene(Figure 4, Table 2).

Alternatively, IPP can also be produced by plastidialMEP pathway (Akashi et al., 2009). Thus, the cytoplasmicMVA pathway appears to be out of the synthesis of this im-portant terpenoid precursor in the Rpp2 background, indicat-ing that down-regulation of cytoplasmic pathway may be thecause of reduction in VOC levels in the PI230970 genotype(Figure 4 and Table 2). Although a strong down-regulationof MVA pathway was observed, the up-regulation of iso-pentenyl diphosphate isomerase, which connects the twopathways involved in IPP production, may be an indicativeof IPP supply by plastidial MEP pathway, ensuring the pres-ence of prenyl precursors for terpenoid formation in cyto-plasm.

The prenylases enzymes geranyl diphosphate synthaseand geranyl-geranyl diphosphate synthase, involved in theformation of mono and diterpenes, were induced at 12 and24 hai, respectively, in initial timepoints of fungal develop-ment, even after significant down-regulation of MVA path-

way in this genotype, suggesting again that IPP precursorsare potentially derived from the MEP pathway. The consid-erable down-regulation of (2E, 6E)-farnesyl diphosphatesynthase at all timepoints evaluated in this genotype sug-gests that mono- and diterpenes are being preferentially gen-erated in relation to the development of (E,E)-�-farnesene(Figure 2).

Distinct from other genotypes evaluated in this study,the genotype containing the Rpp5 resistance gene displayedstrong up-regulation of MVA pathway throughout the infec-tion cycle of P. pachyrhizi. With exception of the gene en-coding hydroxymethylglutaryl-CoA synthase, all genesinvolved in IPP production were strongly upregulated,mainly at the timepoints corresponding to haustorial devel-opment (24 and 48 hai). Interestingly, at 48 hai, a strongdown-regulation of isopentenyl-diphosphate isomerase wasalso observed, suggesting a possible accumulation of IPP. Atthis time, the pathogen was able to establish plant tissue col-onization with haustorium formation and, consequently, di-rect contact with effector secretion in plant cytoplasm. Thestrong up-regulation of MVA pathway suggested immediatedetection of fungus by this genotype and the potential impor-tance of terpenoid synthesis in resistance response mediatedby Rpp5. The repression of the prenylase isopentenyl di-phosphate isomerase at intermediate timepoints, coincidingwith up-regulation of the enzymes geranyl diphosphate syn-thase and (2E, 6E)-farnesyl diphosphate synthase, indicatedthat mono- and diterpene synthesis is preferentially occur-ring in this genotype.

Based on in silico and in vivo analyses, it is possible toconclude that there is a group of terpenes produced by plantdepending on the type of biotic stress. In the case of soybeanresponse to P. pachyrhizi, we observed a differential induc-tion of genes involved in terpenes production dependent onRpp genotype. However, we could not observe a significantdifference between susceptible and resistant genotypes stud-ied, indicating that (E,E)-�-farnesene and other terpenes areinvolved in plants basal defenses, in general.

GmTPS involved in the production of thesesquiterpene (E,E)-�-farnesene is generallyrepressed after P. pachyrhizi infection

The sesquiterpene (E,E)-�-farnesene production inplants is derived from FPP or GGPP precursors. The enzymeGmTPS (E,E)-�-farnesene synthase acts in the final step ofvolatile �-farnesene, converting the precursors into (E,E)-�-farnesene.

A strong down-regulation of prenylase (2E,6E)-far-nesyl diphosphate synthase was verified, followed byGmTPS down-regulation in Rpp2 genotype, confirming thatin this genotype, the precursors generated were potentiallyused in diterpene synthesis, and subsequent (E,E) -�-far-nesene reduction observed by VOC levels.

In the susceptible genotype (W82), although FPP hadbeen synthesized, significant GmTPS down-regulation was

Genes involved in soybean biotic stress 9

observed, suggesting that in this genotype, FPP was used as asubstrate for mono- and diterpene production.

In the Rpp2 (PI230970) accession and in the suscepti-ble genotype Embrapa 48, we could demonstrate the reduc-tion of levels of the VOC (E,E) -�-farnesene across theinfection cycle, corroborating with the expression profile ofthe genes in MVA pathway, the prenylases and GmTPS ob-served. Interestingly, the (E,E) -�-farnesene production wasnegatively associated with the P. pachyrhizi symptoms atleast in these two genotypes. The reduction of the VOCs lev-els could be detected before the symptoms appearance.

GmTPS up-regulation was only observed in Rpp4background and was coordinated with prenylases activationinvolved in FPP formation, indicating that the VOC (E,E)�-farnesene may be produced at least at specific time of 48hai in this genotype. Additionally, the down-regulation ofthe enzyme geranyl-geranyl diphosphate synthase may be astrategy to save the prenyl precursor FPP, repressing diter-pene production and contributing to sesquiterpene produc-tion.

Finally, the strong up-regulation of the MVA pathwayin Rpp5 followed by up-regulation of the enzymes geranyldiphosphate synthase and (2E, 6E)-farnesyl diphosphatesynthase and no activation of GmTPS, indicated that mono-and diterpene synthesis might be preferentially occurring inthis genotype.

Further studies to determine the levels of the VOC(E,E) �-farnesene in these genotypes (Rpp4 and Rpp5) arenecessary to confirm the expression profile results.

Glyceollin levels are strongly induced by P.pachyrhizi infection

The enzyme glyceollin synthase, also known as (-)-glycinol 4-dimethylallyltransferase (G4DT), was dramati-cally induced after fungal inoculation at almost all timepoints and in all genotypes evaluated (Figure 2, Table S2),independent of which prenylases associated with differentclasses of terpenes were preferentially induced. Induction ofglyceollin production has been reported in soybean in re-sponse to infection by P. pachyrhizi (Choi et al., 2008;Pandey et al., 2011; Schneider et al., 2011).

The induction of the glyceollin synthase expressionwas consistent with the gliceollin levels. The inoculation ofsoybean leaves with P. pachyrhizi fungus resulted in an in-crease in the peak area for glyceollin in all genotypes testedin comparison to the control (mock-inoculated). The gly-ceollin level was detected from 24 hai, culminating with thepeak area at 192 hai (Figure 3). There was no significant dif-ference between genotypes containing resistance genes andsusceptible genotypes; the peak area of phytoalexin was sim-ilar in the W82, Rpp2 and Rpp5 genotypes. These results arein agreement with the observations of Lygin et al. (2009),also studying the same pathosystem interaction (soybeanand Asian soybean rust), observed accumulation three weeksafter inoculation, especially in resistant genotypes Rpp1, 2and 3.

The higher levels of glyceollin detected only in latertimepoint suggested a possible posttranscriptional controlmechanism, such as stability, transport or maturation. How-ever, little is known regarding posttranscriptional factors,and the relationship between glyceollin synthase gene ex-pression and effective glyceollin production remains elu-sive.

There are also reports of soybean phytoalexin produc-tion in other soybean interactions and microorganisms, suchas with the fungi P. sojae, P. citricola (Gijzen et al., 2009;Zhou et al., 2009), and B. japonicum (Gijzen et al., 2009;Zhou et al., 2009; Libault et al., 2010; Silva, 2013) (TableS3). These results confirm the importance the glyceollins inbasal plant defense mechanisms (Akashi et al., 2009).

Therefore, considering the slight up-regulation of theMVA pathway after infection in genotypes evaluated, withthe exception of the Rpp2 genotype, it is possible to infer anunlikely involvement of cytoplasmic pathway in generationof precursors for glyceollin biosynthesis. Only Rpp5 geno-type showed a dramatic up-regulation of MVA pathway,thus being able to generate precursors for glyceollin produc-tion by different prenylases action, including G4DT. How-ever, it should be noted that, in this genotype, a strongdown-regulation of isopentenyl diphosphate isomerase wasalso observed, indicating no conversion of IPP to DMAPP.Since high levels of G4DT expression were also observed inRpp5 genotype, it is possible infer that the supply of DMAPPto glyceollin production came from MEP pathway, corrobo-rating the results of Akashi et al. (2009).

The down-regulation of some genes encoding MVApathway enzymes observed in the W82, Rpp2 and Rpp4 ge-notypes is consistent with the expression profile observed inthe in silico analysis, as most of these genes were down-regulated in different plant-stress interactions (Table S3). Itis possible that the gene down-regulation observed in thesegenotypes is a plant mechanism to concentrate energy ex-penditure to ensure glyceollin production, an important phy-toalexin in soybean with great influence in host defensereactions. Similar observations in this study, the in silico

analysis found that the enzyme isopentenyl diphosphate iso-merase was induced at all timepoints and stresses studied, in-dicating the importance of this enzyme in plant defensemechanisms and connecting IPP production between the cy-toplasm and plastids, and providing the cytoplasm with al-ternative source of IPP.

Based on the observed transcript expression profileand glyceollin levels peak, it is possible to suggest that thisphytoalexin has an important role in basal resistance re-sponse to P. pachyrhizi infection in soybean. Even though,when compared to mRNA levels, protein elevation displaysa slower response after detection.

Acknowledgments

We acknowledge the Empresa Brasileira de PesquisaAgropecuária– EMBRAPA Soja, who provided supplies, in-frastructure grant aids towards the costs of this work. Wealso thank the Universidade Estadual de Londrina. This

10 Parmezan et al.

study was financed in part by the Coordenação de Aper-feiçoamento de Pessoal de Nível Superior - Brasil (CAPES)- Finance Code 001. Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sci-ences Research Council (BBSRC) of the United Kingdom.A BBSRC International Partnering Award to RothamstedResearch and Embrapa (BB/J02029X/1) also supported thisproject.

Conflict of Interest

The authors declare that there is no conflict of interestthat could be perceived as prejudicial to the impartiality ofthe reported research.

Author Contributions

FCMG, CBHC, RVA, EON, MB and JP conceivedand designed the study; TRP, SLBJ, KC, MA conducted theexperiments; TRP, SLBJ, and MA analyzed the data; TRPand SLBJ wrote the manuscript and FCMG critically revisedthe manuscript; all authors read and approved the final ver-sion.

ReferencesAkashi T, Sasaki K, Aoki T, Ayabe S and Yazaki K (2009) Molecu-

lar cloning and characterization of a cDNA for pterocarpan4-dimethylallyltransferase catalyzing the key prenylation stepin the biosynthesis of glyceollin, a soybean phytoalexin. PlantPhysiol 149:683-693.

Baldwin IT, Halitschke R, Paschold A, von Dahl CC and PrestonCA (2006) Volatile signaling in plant-plant interactions:“Talking trees” in the genomics era. Science 311:812-815.

Bromfield KR and Hartwig EE (1980) Resistance to soybean rustand mode of inheritance. Crop Sci 20:254-255.

Chakraborty N, Curley J, Frederick RD, Hyten DL, Nelson RL,Hartman GL and Diers BW (2009) Mapping and confirma-tion of a new allele at Rpp1 from soybean PI 594538A confer-ring RB lesion type resistance to soybean rust. Crop Sci49:783-790.

Childs SP, King ZR, Walker DR, Harris DK, Pedley KF, Buck JWand Li Z (2018) Discovery of a seventh Rpp soybean rust re-sistance locus in soybean accession PI 605823. Theor ApplGenet 131:27-41.

Choi JJ, Alkarouf NW, Schneider KT, Matthews BF and FrederickRD (2008) Expression patterns in soybean resistant toPhakopsora pachyrhizi reveal the importance of peroxidasesand lipoxygenases. Funct Integr Genomics 8:341-359.

Farag MA, Huhman DV, Dixon RA and Sumner LW (2008) Meta-bolomics reveals novel pathways and differential mechanisticand elicitor specific responses in phenylpropanoid and iso-flavonoid biosynthesis in Medicago truncatula cell cultures.Plant Physiol 146:387-402.

Fehr WR and Caviness CE (1981) Stage of soybean development.Special Report 80. Iowa State University, Ames.

Garcia A, Calvo ES, Souza-Kiihl R, Harada A, Hiromoto DM andVieira LG (2008) Molecular mapping of soybean rust(Phakopsora pachyrhizi) resistance genes: Discovery of anovel locus and alleles. Theor Appl Genet 117:545-553.

Gijzen, Dong S, Qutob D, Tedman-Jones J and Kuflu K (2009) ThePhytophthora sojae avirulence locus Avr3c encodes a multi-

copy RXLR effector with sequence polymorphisms amongpathogen strains. PLoS One 4:5556.

Gouvea A, Zanotti J, Luckmann D, Pizzatto M, Mazaro SM andPossenti JC (2011) Effect plant extracts on soybeans underlaboratory and field conditions. Rev Bras Agroecol 6:70-78.

Hartwig EE (1986) Identification of a fourth major gene conferringresistance to soybean rust. Crop Sci 26:1135-1136.

Hartwig EE and Bromfield KR (1983) Relationships among 3genes conferring specific resistance to rust in soybeans. CropSci 23:237-239.

Heil M and Silva-Bueno JC (2007) Within-plant signaling by vola-tiles leads to induction and priming of an indirect plant de-fense in nature. Proc Natl Acad Sci U S A 104:5467-5472.

Huang M, Sanchez-Moreiras AM, Abel C, Sohrabi R, Lee S,Gershenzon J and Tholl D (2012) The major volatile organiccompound emitted from Arabidopsis thaliana flowers, thesesquiterpene -beta-caryophyllene, is a defense against a bac-terial pathogen. New Phytol 193:997-1008.

Hyten DL, Hartman GL, Nelson RL, Frederick RD, Concibido VC,Narvel JM and Cregan PB (2007) Map location of the Rpp1

locus that confers resistance to soybean sust in soybean. CropSci 47:837-840.

Ithal N, Recknor J, Nettleton D, Hearne L, Maier T, Baum TJ andMitchum MG (2007). Parallel genome-wide expression pro-filing of host and pathogen during soybean cyst nematode in-fection of soybean. Mol Plant Microbe Interact 20:293-305.

Kim KS, Unfried JR, Hyten DL, Frederick RD, Hartman GL, Nel-son RL, Song Q and Diers BW (2012) Molecular mapping ofsoybean rust resistance in soybean accession PI561356 andSNP haplotype analysis of the Rpp1region in diversegermplasm. Theor Appl Genet 125:1339-1352.

Li S, Smith JR, Ray JD and Frederick RD (2012) Identification of anew soybean rust resistance gene in PI 567102B. Theor ApplGenet 125:133-142.

Libault M, Farmer A, Brechenmacher L and Drnevich J (2010)Complete transcriptome of the soybean root hair cell, a sin-gle-cell model, and its alteration in response to Bradyrhi-

zobium japonicum infection. Plant Physiol 152:541-52.Liu JZ, Braun E, Qiu W, Shi YF, Marcelino-Guimarães FC, Navar-

re D, Hill JH and Whitham SA (2014) Positive and negativeroles for soybean mpk6 in regulating defense responses. MolPlant Microbe Interact 8:824-834.

Lygin VA, Li S, Vittal R, Widholm JM, Hartman GL and LozovayaVV (2009) The importance of phenolic metabolism to limitthe growth of Phakopsora pachyrhizi. Phytopathology99:1412-1420.

Mazaro SM, Citadin I, Gouvêa A, Luckmann D and Guimarães SS(2008) Phytoalexins induction in soybean cotyledons in re-sponse to pitangueira leaf derivatives. Cienc Rural 7:1824-1829.

Mclean RJ and Byth D (1980) Inheritance of resistance to rust(Phakopsora pachyrhizi) in soybean. Aust J Agric Res31:951-956.

Mendgen K, Wirsel SG, Jux A, Hoffmann J and Boland W (2006)Volatiles modulate the development of plant pathogenic rustfungi. Plant 6:1353-1361.

Meyer JDF, Silva DCG, Yang C, Pedley KF, Zhang C, van deMortel M, Hill JH, Shoemaker RC, Abdelnoor RV andWhitham SA (2009) Identification and analyses of candidategenes for Rpp4-mediated resistance to Asian soybean rust insoybean. Plant Physiol 150:295-307.

Monteros MJ, Missaoui AM, Phillips DV, Walker DR and BoermaHR (2007) Mapping and confirmation of the ‘hyuuga’ red-

Genes involved in soybean biotic stress 11

brown lesion resistance gene for Asian soybean rust. Crop Sci47:829-834.

Morales AMAP, O’ Rourke JA, van de Mortel, Scheider KT,Bancroft TJ, Borém A, Nelson RT, Nettleton D, Baum TJ,Shoemaker RC et al. (2013) Transcriptome analyses and virusinduced gene silencing identify genes in the Rpp4-mediatedAsian soybean rust resistance pathway. Funct Plant Biol40:1029-1047.

Naoumkina M, Farag MA, Sumner LW, Tang Y, Liu CJ and DixonRA (2007) Different mechanisms for phytoalexin inductionby pathogen and wound signals in Medicago truncatula. ProcNatl Acad Sci U S A 104:17909-17915.

NIST (2005) Mass Spectral Search for the NIST/EPA/NIH MassSpectral Library Version 2.0. National Institute of Standardsand Technology, Gaithersburg.

Pandey AK, Yang C, Zhang C, Graham MA, Horstman HD, Lee Y,Zabotina OA, Hill JH, Pedley KF and Whitham SA (2011)Functional analysis of the Asian soybean rust resistance path-way mediated by Rpp2. Mol Plant Microbe Interact 24:194-206.

Pfaffl MW, Horgan GW and Dempfle L (2002) Relative expressionsoftware tool (REST©) for group-wise compassion and statis-tical analysis of relative expression results in real-time PCR.Nucleic Acids Res 30:1-10.

Pickett JA, Aradotti GI, Birkett MA, Bruce TJA, Chamberlain K,Khan ZR, Midega CAO, SmarT LE and Woodcock CM(2012) Aspects of insect chemical ecology: Exploitation ofreception and detection as tools for deception of pests andbeneficial insects. Physiol Entomol 1:2-9.

Piesik D, Wenda-Piesik A, LamparskI R, Tabaka P, Ligor T andBuszewski B (2010) Effects of mechanical injury and insectfeeding on volatiles emitted by wheat plant. Entomol Fennica21:117-128.

Schneider KT, van de Mortel M, Bancroft TJ, Braun E, Nettleton D,Nelson RT, Frederick RD, Baum TJ, Graham MA and Whi-tham SA (2011) Biphasic gene expression changes elicited byPhakopsora pachyrhizi in soybean correlates with fungalpenetration and haustoria formation. Plant Physiol157:355-371.

Silva PJT (2013) Indução de fitoalexina gliceolina em cotilédonesde soja por filtrados de Phytophthora spp. B. Sc. Thesis,Universidade Federal do Paraná, Palotina, 20 p.

Silva DC, Yamanaka N, Brogin RL, Arias CA, Nepomuceno AL,Di Mauro AO, Pereira SS, Nogueira LM, Passianotto AL andAbdelnoor RV (2008) Molecular mapping of two loci thatconfer resistance to Asian rust in soybean. Theor Appl Genet117:57-63.

Sinclair JB and Hartman GL (1999) Soybean rust. In: Hartman GL,Sinclair JB, Rupe JC (eds). Compendium of soybean diseases.APS Press, Saint Paul, pp 25-26.

Skelton A, Cameron M, Pickett J and Birkett M (2010) Identificationof neryl formate as the airborne aggregation pheromone for theAmerican house dust mite and the European house dust mite(Acari: Epidermoptidae). J Med Entomol 47:798-804.

Studham ME and Macintosh GC (2013) Multiple phytohormonesignals control the transcriptional response to soybean aphidinfestation in susceptible and resistant soybean plants. MolPlant Microbe Interact 26:116-129.

Tamiru A, Bruce TJA, Midega CAO, Woodcock CM, Birkett MA,Pickett JA and Khan ZR (2012) Oviposition induced volatileemissions from African smallholder farmers’ maize varieties.J Chem Ecol 38:231-234.

Tremblay A, Hosseini P, Alkharouf NW, Li S and Matthewsb F(2010) Transcriptome analysis of a compatible response byGlycine max to Phakopsora pachyrhizi infection. Plant Sci179:183-193.

Tremblay A, HosseinI P, Alkharouf NW, Li S and Matthews BF(2011) Gene expression in leaves of susceptible glycine maxduring infection with Phakopsora pachyrhizi using next gen-eration sequencing. Sequencing 2011:1-14.

Tyler BM (2007) Phytophthora sojae: Root rot pathogen of soy-bean and model oomycete. Mol Plant Pathol 8:1-8.

van de Mortel M, Recknor JC, Graham MA, Nettleton D, DittmanJD, Nelson RT, Godoy CV, Abdelnoor RV, Almeida ÁMR,Baum TJ et al. (2007) Distinct biphasic mRNA changes in re-sponse to Asian soybean rust infection. Mol Plant Microbe In-teract 20:887-899.

Webster B, Bruce T, Dufour S, Birkemeyer C, Birkett M, Hardie Jand Pickett J (2008) Identification of volatile compoundsused in host location by the black bean aphid, Aphis fabae. JChem Ecol 34:1153-1161.

Yorinori JT, Paiva WM, Frederick RD, Costamilan LM,Bertagnolli PF, Hartman GE, Godoy CV and Nunes-Junior J(2005) Epidemics of soybean rust (Phakopsora pachyrhizi) inBrazil and Paraguay from 2001 to 2003. Plant Dis89:675-677.

Zhou L, Mideros SX, Bao L and Hanlon R (2009) Infection and ge-notype remodel the entire soybean transcriptome. BMCGenomics 26:10-49.

Supplementary material

The following online material is available for this article:Table S1 - Primers designed and used in this study.Table S2 - Expression profile of soybean genes after P.

pachyrhizi infection.Table S3 - Transcriptional characterization of genes in re-sponse to different soybean diseases.

Associate Editor: Marcio C. Silva-Filho

License information: This is an open-access article distributed under the terms of theCreative Commons Attribution License (type CC-BY), which permits unrestricted use,distribution and reproduction in any medium, provided the original article is properly cited.

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