biphenyl 4-hydroxylases involved in aucuparin biosynthesis ...biphenyl 4-hydroxylases involved in...

Biphenyl 4-Hydroxylases Involved in AucuparinBiosynthesis in Rowan and Apple Are CytochromeP450 736A Proteins1[OPEN]

Debabrata Sircar2,3, Mariam M. Gaid2, Cornelia Chizzali, Dennis Reckwell, David Kaufholdt, Till Beuerle,Giovanni A.L. Broggini, Henryk Flachowsky, Benye Liu, Robert Hänsch, and Ludger Beerhues*

Institute of Pharmaceutical Biology (D.S., M.M.G., D.R., T.B., B.L., L.B.) and Institute of Plant Biology (D.K.,R.H.), Technische Universität Braunschweig, 38106 Braunschweig, Germany; Plant Pathology, Institute ofIntegrative Biology, Eidgenössische Technische Hochschule Zürich, 8092 Zurich, Switzerland (C.C., G.A.L.B.);and Julius Kühn-Institute, Federal Research Centre for Cultivated Plants, Institute for Breeding Research onHorticultural and Fruit Crops, 01326 Dresden, Germany (H.F.)

ORCID IDs: 0000-0002-2788-119X (D.S.); 0000-0002-7290-0628 (M.M.G.); 0000-0001-9337-4527 (C.C.); 0000-0001-5449-0170 (D.R.); 0000-0001-5037-6653 (T.B.); 0000-0001-8527-565X (B.L.); 0000-0002-2147-2731 (L.B.).

Upon pathogen attack, fruit trees such as apple (Malus spp.) and pear (Pyrus spp.) accumulate biphenyl and dibenzofuranphytoalexins, with aucuparin as a major biphenyl compound. 4-Hydroxylation of the biphenyl scaffold, formed by biphenylsynthase (BIS), is catalyzed by a cytochrome P450 (CYP). The biphenyl 4-hydroxylase (B4H) coding sequence of rowan (Sorbusaucuparia) was isolated and functionally expressed in yeast (Saccharomyces cerevisiae). SaB4H was named CYP736A107. Nocatalytic function of CYP736 was known previously. SaB4H exhibited absolute specificity for 3-hydroxy-5-methoxybiphenyl.In rowan cell cultures treated with elicitor from the scab fungus, transient increases in the SaB4H, SaBIS, and phenylalanineammonia lyase transcript levels preceded phytoalexin accumulation. Transient expression of a carboxyl-terminal reporter geneconstruct directed SaB4H to the endoplasmic reticulum. A construct lacking the amino-terminal leader and transmembranedomain caused cytoplasmic localization. Functional B4H coding sequences were also isolated from two apple (Malus 3domestica) cultivars. The MdB4Hs were named CYP736A163. When stems of cv Golden Delicious were infected with the fireblight bacterium, highest MdB4H transcript levels were observed in the transition zone. In a phylogenetic tree, the three B4Hswere closest to coniferaldehyde 5-hydroxylases involved in lignin biosynthesis, suggesting a common ancestor. Coniferaldehydeand related compounds were not converted by SaB4H.

Rosaceous species include a number of valuable fruittrees, which are widespread in the temperate regions ofthe world. Economically important fruits are the pomefruits, such as apple (Malus spp.) and pear (Pyrus spp.),the stone fruits, including cherry, peach, plum, apricot,and almond (all Prunus spp.), and the berry fruits,such as strawberry (Fragaria spp.) and blackberry and

raspberry (both Rubus spp.). All these fruits are widelyconsumed for their tastes, nutritional values, and con-tents of health-promoting metabolites. In addition, theRosaceae include a number of widespread ornamentalspecies. A large variety of rose (Rosa spp.) cultivars werebred, and hawthorn (Crataegus spp.) and rowan (Sorbusspp.) are common as ornamental trees.

Over the centuries, cultivars with favorable proper-ties were selected; however, vegetative propagation bygrafting led to genetic uniformity and decreased re-sistance to pests and diseases (Norelli et al., 2003;Gessler and Patocchi, 2007). The defense potential ofplants consists of multiple strategies, one of which isthe accumulation of phytoalexins (Dixon, 2001). Plantshave elaborated an amazing array of more than200,000 natural products, many of which confer se-lective advantage against herbivore and pathogen at-tacks (Dixon, 2001; Hartmann, 2007). Two closelyrelated classes of phytoalexins are biphenyls and di-benzofurans, which are formed by apple, pear, andrelated fruit trees. This group of pome-bearing specieshas long been known as Rosaceae subfamily Maloi-deae but recently was reclassified as the subtribeMalinae of the subfamily Amygdaloideae (temporarily

1 This work was supported by the Deutsche Forschungsgemein-schaft (grant no. BE 1174/9–2).

2 These authors contributed equally to the article.3 Present address: Biotechnology Department, Indian Institute of

Technology Roorkee, Roorkee 247667, Uttarakhand, India.* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Ludger Beerhues ([email protected]).

D.S., M.M.G., R.H., and L.B. designed the research; D.S., M.M.G.,C.C., D.R., D.K., T.B., H.F., B.L., and R.H. performed the research;D.S., M.M.G., C.C., T.B., G.A.L.B., R.H., and L.B. analyzed the data;D.S., M.M.G., and L.B. wrote the article.

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http://orcid.org/0000-0002-2788-119X

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http://orcid.org/0000-0001-5449-0170

http://orcid.org/0000-0001-5037-6653

http://orcid.org/0000-0001-5037-6653

http://orcid.org/0000-0001-8527-565X

http://orcid.org/0000-0002-2147-2731

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Pyrinae of the Spiraeoideae; Campbell et al., 2007;Potter et al., 2007).Two decades ago, Kokubun and Harborne (1995)

identified biphenyls and dibenzofurans as the phyto-alexins of the Malinae. So far, 10 biphenyls and 17dibenzofurans have been detected, which are formedde novo in response to biotic and abiotic stress (Chizzaliand Beerhues, 2012). The occurrence of pathogen-inducedbiphenyl and dibenzofuran compounds is confined tothe Malinae (Kokubun and Harborne, 1995). However,some taxa other than the Malinae contain biphenylsand dibenzofurans as preformed constituents, whichcontribute to the constitutive defense barrier (Hüttneret al., 2010). The majority of the biphenyl and dibenzo-furan phytoalexins were found in response to fungalattack, either natural infection or artificial inoculation(Chizzali and Beerhues, 2012). A single article reports theaccumulation of biphenyls and dibenzofurans after bac-terial challenge (Chizzali et al., 2012b). The phytoalexinsinhibit spore germination, germ tube development, andmycelial growth as well as bacterial propagation, al-though the underlying mechanisms of antimicrobial ac-tion are still unknown (Chizzali and Beerhues, 2012).The most common biphenyl phytoalexin is aucu-

parin, which was detected in eight Malinae speciesafter pathogen attack (Chizzali and Beerhues, 2012).The scaffold of this compound is formed by biphenylsynthase (BIS), a type III polyketide synthase, whichcatalyzes the condensation of the unusual starter sub-strate benzoyl-CoA with three molecules of malonyl-CoA to yield 3,5-dihydroxybiphenyl (Fig. 1; Liu et al.,2004, 2007). The benzoyl moiety of the starter substratestems from L-Phe, whose carbon skeleton is channeledinto the phytoalexin biosynthetic pathway by the ac-tion of phenylalanine ammonia lyase (PAL; Hansonand Havir, 1981). Little is known about the conversionof the PAL product, trans-cinnamic acid, to benzoyl-CoA in the Malinae. Benzaldehyde dehydrogenasewas detected in chitosan-treated rowan (Sorbus aucu-paria) cell cultures, which serve as an in vitro systemfor studying biphenyl and dibenzofuran metabolism inthe Malinae (Gaid et al., 2009; Hüttner et al., 2010). Thefirst complementary DNA (cDNA) encoding BIS wasisolated from these cell cultures after elicitor treatment(Liu et al., 2007). The elicitors that efficiently inducephytoalexin formation in rowan cell cultures includepreparations from the fungus Venturia inaequalis, thecausal agent of scab, and the bacterium Erwinia amy-lovora, the causal agent of fire blight (Hüttner et al.,2010). These infections are among the most devastatingdiseases encountered in apple orchards (MacHardy,1996; Vanneste, 2000). Recently, the downstream en-zymes that metabolize the BIS product, 3,5-dihydrox-ybiphenyl, have been studied in cell-free extracts andmicrosomes from rowan cell cultures after the additionof an aqueous elicitor preparation from V. inaequalis(Khalil et al., 2013). Interestingly, the sequence of thebiosynthetic reactions leading from 3,5-dihydroxybiphenylto aucuparin was O-methylation → 4-hydroxylation →O-methylation (Fig. 1). The alkylation steps were catalyzed

by two distinct O-methyltransferases, and the hydro-xylation reaction was catalyzed by a cytochrome P450(CYP), which was named biphenyl 4-hydroxylase (B4H;Khalil et al., 2013).

Here, we report the isolation of the SaB4H codingsequence using a subtracted cDNA library of rowanand the recently published genome sequence of apple(Malus 3 domestica ‘Golden Delicious’). In addition,the MdB4H coding sequences of cv Golden Deliciousand cv Holsteiner Cox were cloned. The expression ofSaB4H and MdB4H was analyzed in rowan cell culturesafter treatment with elicitor from the scab fungus and inapple stems after infection with the fire blight bacte-rium, respectively. Besides functional and phylogeneticanalyses, the subcellular localization of SaB4H reporterfusions was studied in Nicotiana benthamiana.

RESULTS

cDNA Cloning of SaB4H Starting with a SubtractedRowan cDNA Library

Pools of mRNA were isolated from chitosan-treatedand nontreated rowan cell cultures and used to construct

Figure 1. Aucuparin biosynthesis in S. aucuparia cell cultures treatedwith V. inaequalis (Khalil et al., 2013).

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Biphenyl 4-Hydroxylases Are CYP736A Proteins



a suppression subtraction hybridization (SSH) cDNA li-brary, which consisted of differentially expressed tran-scripts. A total of 2,000 cDNA clones were sequencedand bioinformatically processed (Supplemental Fig. S1),leading to the detection of 15 CYP contigs. The individ-ual sequences were used to search the apple genomesequence, which is available via the Rosaceae GenomeDatabase (GDR). Apple sequences sharing a high degreeof similarity with the rowan contigs were filtered againstCYP sequences in the data bank related to the mod-ification of products of the shikimate pathway. Thesein silico analyses, details of which are listed inSupplemental Table S1, resulted in the selection of sixapple unigenes that shared high sequence identity(87.5%–97%) with the rowan contigs 62, 120, and 162.Gene-specific primers were derived and led to theamplification of a transcript from V. inaequalis-treatedrowan cell cultures. After 59 and 39 extensions, theresulting full-length cDNA was reamplified using aproofreading polymerase. The cDNA consisted of a1,500-bp coding sequence (cds), a 188-bp 59 untrans-lated region (UTR), a 260-bp 39 UTR, and a 32-bp poly(A) tail.

The cds encoded a 499-amino acid protein with apredicted molecular mass of 56.9 kD and a pI of 6.82.The protein was named CYP736A107, based on thestandardized CYP nomenclature system (Nelson, 2009).Its structural analysis revealed the presence of motifsthat are characteristic of plant CYPs (Fig. 2). The highlyconserved heme-binding domain FxxGxRxCxG (Chapple,1998) was present as FGSGRRRCAG. The N-terminalmembrane anchor comprising 19 amino acids wasfollowed by the Pro-rich region PPGPRGFP [consensus(P/I)PGPx(G/P)xP; Schalk et al., 1999]. In addition, theKETLR(K) helix and the PERFE motif were present (Baket al., 2011). The amino acid sequence of CYP736A107

shared 39.6% and 40.1% identities with functionallyestablished flavonoid 39-hydroxylases from Epimediumsagittatum (ADE80941) and Brassica napus (ABC58722),respectively.

cDNA Cloning of Class II CPR from V. inaequalis-TreatedRowan Cell Cultures

NADPH-cytochrome P450 reductase (CPR) is theelectron-transfer partner for CYPs (Jensen and Møller,2010). The apple unigene MDP0000167955 shared highsequence similarity with CPR genes in the data bankand the only CPR transcript fragment present in theSSH cDNA library. Using gene-specific primers and aproofreading polymerase, an SaCPR transcript wascloned from V. inaequalis-treated rowan cell cultures.The SaCPR cDNA contained a 2,139-bp cds encoding a712-amino acid polypeptide with a predicted molecu-lar mass of 78.8 kD. The protein shared 81% sequenceidentity each with Gossypium hirsutum CPR (ACN54324)and poplar (Populus trichocarpa) CPR3 (AAK15261) as wellas 75.5% and 65.5% identities with Arabidopsis (Arabi-dopsis thaliana) CPR2 (AAK17169) and CPR1 (CAA46814),respectively. The conserved binding domains for cofactorsand substrates (FMN, FAD, NADPH, and CYP) and themembrane anchor near the N terminus were present(Supplemental Fig. S2). The extended N-terminal se-quence upstream of the membrane anchor identifiedthe rowan enzyme as a class II CPR (Ro et al., 2002).

Functional Characterization of CYP736A107

The CYP736A107 cds and the CPR cds were clonedin the same expression vector and heterologouslyexpressed in yeast (Saccharomyces cerevisiae), from

Figure 2. Alignment of B4H and con-iferaldehyde 5-hydroxylase (CA5H) aminoacid sequences with common motifs high-lighted. SaB4H, B4H from S. aucuparia(CYP736A107); Md(GD)B4H, B4H fromcv Golden Delicious (CYP736A163); Md(HC)B4H, B4H from cv Holsteiner Cox(CYP736A163); CmCA5H, CA5H fromChrysanthemum3morifolium (BAM66576).Gaps introduced to maximize the alignmentare indicated by dashes.

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which microsomes containing the two proteins wereisolated. Based on the carbon monoxide differencespectrum (Supplemental Fig. S3), the CYP expressionlevel was 286 nmol g21 microsomal protein. Microsomeswere incubated with 3-hydroxy-5-methoxybiphenyl,which was recently found as a B4H substrate usingmicrosomes from elicitor-treated rowan cell cul-tures (Khalil et al., 2013). In the presence of NADPH,3-hydroxy-5-methoxybiphenyl was 4-hydroxylatedto yield noraucuparin, as demonstrated by gaschromatography-mass spectrometry (GC-MS) in com-parison with an authentic reference compound (Fig. 3).In addition, identical retention time values and UVspectra were observed in HPLC-diode array detection(DAD) analysis (Supplemental Fig. S4). No productformation was found in assays that contained heat-denatured protein or microsomes from yeast cellsharboring the empty vector. Nor was CYP736A107activity detected when the rowan CPR cds was omittedfrom the vector, indicating the lack of interactionwith yeast CPR. Substrates structurally related to3-hydroxy-5-methoxybiphenyl were converted neitherunder standard assay conditions nor at varying pHvalues (5.5–10.5), except for 3,5-dihydroxybiphenyl.However, the relative activity was less than 2%, leadingto the formation of only trace amounts of 3,4,5-trihydroxybiphenyl (Table I). Notably, 2-hydroxy-4-methoxydibenzofuran, which has the same substitutionpattern as 3-hydroxy-5-methoxybiphenyl, was not con-verted. Furthermore, stilbenes and one-ring compoundswere not substrates. Nor did the enzyme accept cinnamicacid derivatives, such as ferulic acid and coniferaldehyde.Thus, CYP736A107 was identified as SaB4H.

Enzyme activity was strictly dependent on NADPH,whose replacement with NADH reduced the activityto approximately 15%. In the presence of both NADPHand NADH, the activity was increased by approxi-mately 15%. The pH and temperature optima were 7.5and 30°C, respectively. B4H activity was linear withtime up to 45 min and with increasing CYP concen-trations in the standard assay up to 15 pmol, as de-termined by carbon monoxide difference spectroscopy.The Km values for 3-hydroxy-5-methoxybiphenyl andNADPH were 2.4 6 0.07 and 72.3 6 7.5 mM, respec-tively (Table II). Electron transfer from NADPH wasexclusively due to rowan CPR, because yeast CPRalone failed to accomplish detectable SaB4H acti-vity. The turnover number (kcat) and the catalytic ef-ficiency (kcat/Km) with 3-hydroxy-5-methoxybiphenylwere 0.342 min21 and 2,375 M

21 s21, respectively(Table II). When SaCPR was incubated with cyto-chrome c as an artificial electron acceptor (Urbanet al., 1994), the Km value for NADPH was 51 6 5.4mM. No appreciable loss of SaB4H activity was ob-served upon storage of microsomal preparations inTris-EDTA-glycerol buffer at 280°C for 6 months. At4°C, the enzyme activity decreased to about 20% to30% within 24 h.

Expression of SaPAL, SaBIS1, and SaB4H inV. inaequalis-Treated Rowan Cell Cultures

Transcript levels in rowan cell cultures after theaddition of an aqueous extract from the scab-causing fungus were analyzed by reverse transcription

Figure 3. GC-MS analysis of SaB4H assays. The enzymic product (noraucuparin) was analyzed as a trimethylsilyl derivativeresulting from N-methyl-N-(trimethylsilyl) trifluoroacetamide treatment. The control incubation contained heat-denaturedmicrosomes.







(RT)-PCR (Fig. 4). The gene-specific primer pairs usedled to the amplification of 407-, 882-, 379-, and 300-bpfragments of SaPAL, SaBIS1, SaB4H, and SaActintranscripts, respectively. Actin mRNA served as acontrol for equal template amounts. SaB4H transcriptswere detectable as early as 0.5 h after the onset of elic-itation. The mRNA level peaked at 4 h and decreased

slowly thereafter. Similar changes were found for theSaBIS1 transcript level, which, however, increased moreslowly before reaching the maximum at 4 h. A signifi-cantly faster increase was observed for the SaPALmRNA level, which already peaked after 1 to 2 h andreturned rapidly to the basal SaPAL expression levelthat was present at the onset of elicitation.

Table I. Substrate specificity of SaB4H

Substrate Formula Activity Substrate Formula Activity

% %

3-Hydroxy-5-methoxybiphenyl 100 Resorcinol 0

3,5-Dihydroxy-biphenyl ,2 Orcinol monomethyl ether 0

3,5-Dimethoxy-biphenyl 0 Orcinol 0

2’,3,5-Trihydroxy-biphenyl 0 3,5-Dihydroxy-benzoic acid 0

2,5-Dihydroxy-biphenyl 0 Benzoic acid 0

2-Hydroxy-4-methoxydibenzofuran 0 Ferulic acid

0

2,4-Dihydroxy-dibenzofuran 0 Coniferaldehyde 0

Pinosylvin 0 4-Coumaric acid 0

Resorcinol monomethyl ether 0 Cinnamic acid 0


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Subcellular Localization of SaB4H Reporter Fusions inN. benthamiana

In silico analysis of SaB4H using protein targetprediction software revealed the presence of a 19-amino acid N-terminal leader and membrane anchor(Fig. 2). To study the effect of this domain on subcel-lular localization, a modified yellow fluorescent pro-tein called Venus (Kaufholdt et al., 2013) was fusedto the 39 end of the SaB4H cds (35S::SaB4H-Venus).In addition, a construct containing the 27 N-terminalamino acids including the membrane anchor sequence(35S::SIG-Venus) and a construct lacking the mem-brane anchor and starting with amino acid 30 (35S::TRUNC-Venus) were generated. All three constructswere placed under the control of the 35S promoterfrom Cauliflower mosaic virus and were transientlyexpressed in N. benthamiana leaves using Agrobacteriumtumefaciens infiltration. The resulting fluorescence dis-tribution patterns were studied using confocal laserscanning microscopy. Both the SaB4H-Venus and theSIG-Venus fusions showed perinuclear and reticulatedistribution, indicating endoplasmic reticulum (ER)association (Fig. 5, A and C, respectively). A constructharboring an ER retention signal (HDEL-DsRed) wasexpressed as a positive control, leading to a similarfluorescence distribution pattern to the expression ofSaB4H-Venus (Fig. 5B, 1 and 2). Coexpression of the ERmarker HDEL-DsRed and the SaB4H-Venus constructsresulted in colocalization of the proteins, as indicatedby a whitish color in the case of a perfect match (Fig.5B, 3). The occasional occurrence of fluorescent spotsmay be due to either ER-derived vesicles containingprotein constructs or aggregates of Venus-DsRED fu-sions, which are caused by the high level of expression(Fig. 5B, 3). Finally, a construct that lacked the mem-brane anchor sequence (TRUNC-Venus) failed to targetthe protein to the ER; therefore, fluorescence was presentin the cytoplasm (Fig. 5D).

Detection of SaB4H Activity in Microsomes fromN. benthamiana

Microsomes from N. benthamiana leaves transientlyexpressing the 35S::SaB4H-Venus construct were incu-bated with 3-hydroxy-5-methoxybiphenyl and NADPH.Subsequent HPLC analysis demonstrated the formationof noraucuparin (Supplemental Fig. S5). The identity ofthis enzymatic product was confirmed by cochromatog-raphy with a sample of authentic reference compound

and UV spectroscopy. No noraucuparin formation wasobserved in control assays containing the microsomalpreparation from leaves of N. benthamiana after infil-tration with A. tumefaciens that contained the emptyvector.

Expression of MdB4H in Fire Blight-Infected Stems ofApple ‘Golden Delicious’

The cv Golden Delicious was selected because itsgenome sequence is available (Velasco et al., 2010).

Table II. Steady-state kinetic parameters of B4Hs

Data are means 6 SD of three replicates.

Enzyme Km Vmax kcat kcat/Km

mM nkat mg21 min21M21 s21

SaB4H 2.4 6 0.07 0.100 6 0.013 0.342 6 0.046 2,375Md(GD)B4H 1.0 6 0.23 0.054 6 0.002 0.180 6 0.007 3,000Md(HC)B4H 4.0 6 1.11 0.200 6 0.017 0.684 6 0.059 2,850

Figure 4. Changes in the SaPAL, SaBIS1, and SaB4H expression levelsin V. inaequalis-treated rowan cell cultures. A, The expression patternswere studied by RT-PCR. B to D, The transcript levels of SaPAL (B),SaBIS1 (C), and SaB4H (D) were normalized on the basis of the actintranscript amounts. SD values are indicated (n = 3).






Real-time PCR was used to quantify Md(GD)B4HmRNA amounts in cv Golden Delicious stems afterinoculation with E. amylovora. As observed previouslywith cv Holsteiner Cox, which is used as a modelsystem (Chizzali et al., 2012a), infection with the fireblight pathogen caused the typical shepherd’s crooksymptom at the shoot tip (Vanneste, 2000) and resultedin the formation of a transition zone that was framedby the necrotic and the healthy stem segments (Fig.6A). Md(GD)B4H expression in E. amylovora-infectedstems of cv Golden Delicious was measured in relationto that observed in mock-inoculated control stems6 d post inoculation, based on the previous observa-tions with cv Holsteiner Cox (Chizzali et al., 2012a). Asingle PCR product was amplified using gene-specificprimers for MdB4H, and no primer dimers were ob-served. The Md(GD)B4H transcript level in the tran-sition zone was approximately 1,350 times that in therespective stem segment of mock-inoculated controlshoots (Fig. 6B). The Md(GD)B4H mRNA amount inthe healthy stem segment, which later turns into thetransition zone, was approximately 1,150 times higherthan in the respective stem zone after mock inoculation.Isolation of mRNA from the necrotic stem segmentabove the transition zone was not feasible.

Phytoalexin Detection in Fire Blight-Infected Apple‘Golden Delicious’

E. amylovora-inoculated stems of cv Golden Deliciouswere dissected in necrotic, transition, and healthy zones(Fig. 6A), which were extracted with methanol (80%).Phytoalexins were identified by GC-MS using a librarycontaining the previously structure-elucidated biphenylsand dibenzofurans from elicitor-treated rowan cellcultures (Hüttner et al., 2010; Chizzali et al., 2012b).Three biphenyls, noraucuparin, aucuparin, and 29-hydroxyaucuparin, were detected in the transitionzone (Supplemental Fig. S6). The flanking necrotic andhealthy zones as well as segments from mock-inoculatedshoots lacked detectable amounts of phytoalexins.

MdB4H Transcript Cloning from FireBlight-Infected Apple

The transition zone of cv Golden Delicious served asan mRNA source to amplify the Md(GD)B4H codingsequence. Md(GD)B4H turned out to be identical to theapple unigenes MDP0000152900 and MDP0000149697(GDR). The transition zone of fire blight-infected stems

Figure 5. Subcellular localization of SaB4H re-porter fusions in N. benthamiana leaf epidermiscells. A, After transient expression of the SaB4H-Venus construct, fluorescence was observed inER-like reticulate structures. B, Cotransformationwith SaB4H-Venus (B1) and an ER-positive con-trol construct, HDEL-DsRED (B2), led to over-lapping fluorescences in ER structures (B3), witha perfect match being indicated by white areas.C, Fluorescent ER also resulted from a constructthat encoded only the signal sequence of SaB4H(i.e. amino acids 1–27). D, Transformation with aconstruct that contained the coding region butlacked the signal sequence (TRUNC-Venus)resulted in cytosolic protein localization. Bars =10 mm.


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of cv Holsteiner Cox was also used to isolate an Md(HC)B4H transcript. The B4H coding sequences of cvGolden Delicious and cv Holsteiner Cox shared 96.7%and 96.9% identities, respectively, with the SaB4H cds.The MdB4Hs were given the standardized nameCYP736A163 (Nelson, 2009). Upon expression of theMd(GD)B4H and Md(HC)B4H coding sequences inyeast harboring the CPR cds, both resultant enzymesconverted 3-hydroxy-5-methoxybiphenyl to noraucuparinin the presence of NADPH, as indicated by HPLC-DADanalysis. When heat denatured, both MdB4Hs failed toform the product. The kinetic properties of Md(GD)B4Hand Md(HC)B4H are indicated in Table II. The catalyticefficiencies resembled that of SaB4H.

Phylogenetic Reconstruction of B4H and Related AminoAcid Sequences

A neighbor-joining tree was constructed using CYPsthat are functionally characterized and contribute tovarious pathways of plant secondary metabolism (Fig.7). The accession numbers and the CYP nomenclatureof these proteins are indicated in Supplemental TableS2. The majority of the included CYPs catalyzed hy-droxylation reactions, besides three functionally estab-lished CYPs that accomplish C-C and C-O couplingsteps. The CYP-cam gene of Pseudomonas putida wasused to root the tree. The B4H sequences from rowanand apple ‘Golden Delicious’ and ‘Holsteiner Cox’

grouped together. They were closest to a clusterthat consisted of CA5H, also referred to as ferulate5-hydroxylases, from various angiosperm species.B4H and CA5H share around 40% amino acid se-quence identity (Fig. 2). The clade that consisted ofthe B4H and CA5H enzymes grouped together witha clade that comprised flavonoid 39-hydroxylases andflavonoid 39,59-hydroxylases.

DISCUSSION

Aucuparin is the most abundant biphenyl phyto-alexin formed in species of the economically valuableMalinae. Its substitution pattern consists of a para-hydroxy group that is framed by two methoxy func-tions. While the 3- and 5-hydroxy groups originatefrom the BIS-catalyzed biosynthesis of the scaffold, thepara-hydroxy group is introduced by B4H. SaB4H andMdB4H were named CYP736A107 and CYP736A163,respectively, based on the standardized CYP nomen-clature system (Nelson, 2009). So far, no catalyticfunctions have been demonstrated for CYP736 pro-teins, which are absent in several taxa, such as Arabi-dopsis and monocots, despite their early emergence(Nelson and Werck-Reichhart, 2011). However, recentstudies have functionally related CYP736 proteins toplant defense and cross talk with microorganisms, ei-ther pathogens or symbionts. Three CYP736A25 vari-ants are differentially regulated at the transcriptionaland posttranscriptional levels in grape (Vitis vinifera)lines, which are either susceptible or tolerant to Xylellafastidiosa (Cheng et al., 2010). In soybean (Glycine max),CYP736A34 is highly coexpressed with genes involvedin root and Rhizobium spp.-induced nodule devel-opment (Guttikonda et al., 2010). However, theseplant species do not form biphenyl and dibenzofu-ran phytoalexins.

Plant genome sequencing projects have revealed avast number of CYPs, leading to the annotation of morethan 5,000 sequences in the databases (Nelson andWerck-Reichhart, 2011). CYPs are among the largestplant gene superfamilies and constitute around 1% ofthe protein-coding genes. The angiosperm sequencediversity is encapsulated by 59 CYP families (Nelsonet al., 2008). Arabidopsis, rice (Oryza sativa), poplar, andgrape contain 245, 332, 310, and 315 CYPs, respectively(Nelson et al., 2008). Papaya (Carica papaya), with 142CYPs, is nearly minimalist in the CYP collection ofplants, whereas the human genome contains only 57CYP genes. However, the biochemical functions of themajority of CYP proteins are still unknown (Schuler andWerck-Reichhart, 2003; Mizutani, 2012). In Arabidop-sis, only 60 of the 245 CYPs are functionally clarified(Mizutani and Ohta, 2010). Even within the same sub-family, each CYP may be responsible for different roles.In this study, the SaB4H gene was identified by com-bining information from the recently published applegenome sequence (Velasco et al., 2010) and a newlyestablished rowan SSH cDNA library.

Figure 6. Md(GD)B4H expression levels in fire blight-infected stems ofcv Golden Delicious. A, Inoculation of cv Golden Delicious shootswith E. amylovora led to the formation of a transition zone betweenthe necrotic and healthy stem segments, as observed previously withcv Holsteiner Cox stems (Chizzali et al., 2012a, 2013). B, Quantitativereal-time PCR was used to determine Md(GD)B4H expression levels6 d post inoculation. Control mRNAwas obtained from stem segmentsof mock-inoculated shoots at day 6. SD values are indicated (n = 3).RNA isolation from the necrotic tissue was not feasible.







Figure 7. Neighbor-joining tree illustrating the evolutionary relationships between B4H and CYPs involved in secondarymetabolism. Numbers at the branch points are bootstrap values from 1,000 replicates and Poisson correction. The scale bar =0.2 amino acid substitutions per site. The accession numbers of the amino acid sequences included are listed in SupplementalTable S2. Pseudomonas putida CYP-cam served as an outgroup. F39H, Flavonoid 39-hydroxylase; F39,59H, flavonoid 39,59-hydroxylase; NMC39H, N-methylcoclaurine 39-hydroxylase; BS, berbamunine synthase; CS, corytuberine synthase; FS, flavonesynthase; IFS, isoflavone synthase; 2HIS, 2-hydoxyisoflavanone synthase; C4H, cinnamate 4-hydroxylase; I39H, isoflavone39-hydroxylase; I29H, isoflavone 29-hydroxylase.






SaB4H exhibits absolute specificity for 3-hydroxy-5-methoxybiphenyl. Discrimination of 3,5-dihydroxybiphenyland 3,5-dimethoxybiphenyl as substrates confirms thesequence of the aucuparin biosynthetic steps, whichwere recently detected in cell-free extracts fromelicitor-treated S. aucuparia cell cultures (Khalil et al.,2013). Furthermore, discrimination of 2-hydroxy-4-methoxydibenzofuran indicates that this compoundis not related to eriobofuran formation, which impliesa revision of the previously proposed pathway(Hüttner et al., 2010). The 4-hydroxylation reactionis framed by the two methylation steps, which arecatalyzed by distinct O-methyltransferases. OMT1and OMT2 catalyze the methylation of the 5- and 3-hydroxyl groups, respectively. SaB4H does not acceptstilbenes, which have an additional double bond, andone-ring compounds. Nor does the enzyme convertcinnamic acid derivatives, such as ferulic acid andconiferaldehyde, which are related to lignin biosyn-thesis. In a phylogenetic tree, B4Hs are closest toCA5H enzymes, which catalyze the formation of5-hydroxyconiferaldehyde. O-Methylation then yieldssinapaldehyde as a precursor of syringyl monolignols(Chiang, 2006; Bonawitz and Chapple, 2010). The5-hydroxylase of the phenylpropanoid pathway wasinitially considered a ferulate 5-hydroxylase (Grand,1984; Meyer et al., 1996); however, coniferaldehydeand coniferyl alcohol were later found to be the pre-ferred substrates, implying that the enzyme shouldbe referred to as CA5H (Humphreys et al., 1999;Osakabe et al., 1999). The CA5H gene, CYP84, is presentin angiosperms but not in moss or gymnosperms,which thus lack syringyl lignin monomers (Nelsonet al., 2008). Phylogenetic reconstruction suggests thatB4H and CA5H may arise from a common ancestor.They share around 40% amino acid sequence identity.After duplication of an ancestral gene, one copyretained the essential CA5H function, whereas thesecond gene copy has diverged. Mutations led to itsneofunctionalization and recruitment into the aucu-parin biosynthetic pathway of the Malinae. The newgene product still catalyzed a hydroxylation reaction,but on 3-hydroxy-5-methoxybiphenyl rather thanconiferaldehyde.The properties of a CYP may be affected by inter-

action with a heterologous CPR (Davin et al., 1997;Pauli and Kutchan, 1998). For this reason, a cDNAencoding rowan class II CPR was isolated and allowedinteraction of this protein with SaB4H. CPR catalyzesthe transfer of two electrons from NADPH via FADand FMN, as electron acceptor and donor flavins, re-spectively, to the prosthetic heme group of diverseCYP enzymes (Porter, 2004; Jensen and Møller, 2010).In contrast to animals, which contain a single CPR(Porter et al., 1990), plants have multiple CPR isoformsthat fall into two classes structurally distinguishable bytheir different N termini (Urban et al., 1997; Ro et al.,2002). In Arabidopsis and Centaurium erythraea, theexpression of class II CPR is induced by wounding,light treatment, and elicitation, whereas class I CPR is

constitutively regulated (Nisimoto, 1986; Meijer et al.,1993; Schwarz et al., 2009). Consistently, the transcriptfor rowan class II CPR, which contained all essentialstructural motifs, could be isolated from cell culturesafter the addition of elicitor.

Aucuparin biosynthesis is initiated by BIS (Liu et al.,2004, 2007). In apple, BIS is encoded by a gene familyconsisting of four differentially regulated subfamilies(Chizzali et al., 2012a). MdBIS3 is expressed in stemsupon fire blight infection. Inoculation of shoots withthe fire blight bacterium E. amylovora leads to the for-mation of a transition zone between the necrotic andthe healthy stem segments. Recently, an E. amylovoramutant that was deficient in producing the highlypotent cytotoxin 6-thioguanine was found to lackthe capacity of inducing disease symptoms in cvHolsteiner Cox, which is indicative of the crucial roleof the antimetabolite in the development of the fireblight disease (Coyne et al., 2013, 2014). The transitionzone gradually advances from the inoculated shoot tipdown the stem with increasing postinoculation time,as was observed here with cv Golden Delicious andpreviously with cv Holsteiner Cox (Chizzali et al.,2012b, 2013). The Md(HC)BIS3 transcript level in thetransition zone of cv Holsteiner Cox was approxi-mately 4,000 times that in the respective stem segmentof mock-inoculated control shoots and somewhatlower in the healthy stem segment. A similar expres-sion profile was found for Md(GD)B4H in cv GoldenDelicious. The maximum expression rate was observedin the transition zone, whereas the healthy tissuecontained a slightly reduced transcript level. In ac-cordance with these BIS3 and B4H expression data,phytoalexins accumulate in the transition zone, andthe flanking stem segments are devoid of detectablephytoalexin amounts. While diseased cv Golden De-licious mainly contains noraucuparin and 29-hydrox-yaucuparin, cv Holsteiner Cox primarily accumulatesnoraucuparin and aucuparin and, in addition, the di-benzofurans noreriobofuran and eriobofuran (Chizzaliet al., 2012b, 2013). Using a BLASTP analysis againstthe genome of cv Golden Delicious (GDR), Md(GD)B4H was located at the lower part of linkage group 11between the two single-nucleotide polymorphismmarkers GDsnp02800 (50.04 centimorgan [cM]) andGDsnp01705 (51.4 cM). Recently, a number of Diver-sity Arrays Technology markers correlating with re-sistance to fire blight were identified in the genome ofthe crabapple Malus fusca MAL0045 (Emeriewen et al.,2014). After DNA sequencing, two of them (970048and 969599) could be aligned to linkage group 11 of cvGolden Delicious at positions 49.11 and 49.12 cM, re-spectively. In addition, a minor quantitative trait locus(QTL) for fire blight resistance was recently describedon linkage group 11 of Malus 3 robusta 5 (Wöhneret al., 2014). This QTL is linked to marker CH04a12.Using the apple integrated map (Velasco et al., 2010),CH04a12 was located at position 34.3 cM, about 16 cMfrom marker GDsnp02800. However, whether Md(GD)B4H contributes to this minor QTL remains unknown.





Biphenyls and dibenzofurans are derivatives ofbenzoic acid, the biosynthesis of which branches fromthe general phenylpropanoid pathway, initiated byPAL. The fast increase in the SaPAL transcript level inrowan cell cultures in response to V. inaequalis treat-ment indicates a rapid redirection of carbon flow fromprimary to secondary metabolism. The basal SaPALtranscript level observed before and after the transientincrease in the expression rate is attributed to thefurther functions of the enzyme (e.g. in supplyingprecursors for phenylpropanoid derivatives such aslignin). Recently, cDNAs encoding cinnamate:CoA li-gase from Petunia hybrida and Hypericum calycinumwere cloned (Gaid et al., 2012; Klempien et al., 2012).This enzyme channels the metabolic flux from thegeneral phenylpropanoid pathway to benzenoid me-tabolism and further to aucuparin biosynthesis. Inrowan cell cultures, aucuparin accumulation starts 9 hafter the addition of V. inaequalis extract (Khalil et al.,2013). It is not only preceded by a fast increase in theSaPAL mRNA level but also by coordinated, albeitsomewhat delayed, increases in the SaBIS1 and SaB4Htranscript levels.

SaB4H was localized to the ER upon transient ex-pression of a reporter gene construct in N. benthamianaleaf epidermis cells. The majority of eukaryotic CYPsare cotranslationally inserted into the ER membraneand remain anchored by an N-terminal hydrophobichelix (Chapple, 1998). The N-terminal leader includesthe transmembrane domain (Johnson and Stout, 2013).However, CYPs are not restricted exclusively to thissubcellular location. In gibberellin biosynthesis,CYP701A, ent-kaurene oxidase, is present on the outersurface of the outer chloroplast envelope, therebylinking the plastid- and ER-located steps of this path-way (Helliwell et al., 2001). The same location wasfound for CYP86B1, which catalyzes the v-hydroxyl-ation of long-chain fatty acids in suberin biosynthesis(Watson et al., 2001; Compagnon et al., 2009). Tosupply the CYPs with electrons, a CPR isoform wasproposed to be targeted to the chloroplasts (Urbanet al., 1997). Plastidial localization was also reportedfor CYP74s, which are involved in oxylipin (e.g. jas-monic acid) metabolism (Hughes et al., 2009). Tomato(Solanum lycopersicum) CYP74A and CYP74B werelocalized to the inner chloroplast membrane facingthe stroma and the outer chloroplast membrane fac-ing the space between the two envelope membranes,respectively (Froehlich et al., 2001). Different locationswere also reported for the CYP74C and CYP74D pro-teins, such as cytosol, tonoplast, and amyloplasts andleucoplasts (Hughes et al., 2009). However, it shouldbe noted that CYP74s are atypical members of thesuperfamily, because they do not require molecularoxygen and electron donors and they exhibit unusu-ally high turnover rates (Hughes et al., 2009). In ani-mals, mitochondria contain steroid biosynthetic- andxenobiotic-metabolizing CYPs, which utilize adreno-doxin and a NADPH-dependent adrenodoxin reduc-tase for their electron transfer (Guzov et al., 1998).

CONCLUSION

B4H is an ER-anchored CYP that is involved inaucuparin biosynthesis initiated by BIS. Recently,MdBIS3 was immunochemically localized to the junc-tions between neighboring cortical parenchyma cells inthe transition zone of fire blight-infected apple stems,suggesting an association of the enzyme with plas-modesmata in primary pit fields (Chizzali et al., 2012a,2013). Thus, it is tempting to speculate that the aucu-parin biosynthetic enzymes may be assembled into ametabolon, which allows for an efficient transfer ofbiosynthetic intermediates between the active sites inclose proximity (Winkel, 2004; Jørgensen et al., 2005).Protein complexes are commonly associated with struc-tural elements within the cell, such as the ER and mi-crotubules (Zajchowski and Robbins, 2002; Chuonget al., 2004). B4H may act as an anchor inside the plas-modesmata for assembling the upstream and down-stream aucuparin biosynthetic enzymes. Which role theproducts of this metabolon might play in preventing aninfection from spreading from cell to cell via the sym-plastic connections remains unknown.

MATERIALS AND METHODS

Chemicals

3,5-Dihydroxybiphenyl, 3-hydroxy-5-methoxybiphenyl, 3,5-dimethoxybiphenyl,3,4,5-trihydroxybiphenyl, 29,3,5-trihydroxybiphenyl, noraucuparin, 2,4-dihydroxydibenzofuran, 2-hydroxy-4-methoxydibenzofuran, and 4-hydroxy-2-methoxydibenzofuran were synthesized as described previously (Liu et al.,2004; Hüttner et al., 2010; Chizzali et al., 2012b; Khalil et al., 2013). 2,5-Dihydroxybiphenyl, benzoic acid, 3,5-dihydroxybenzoic acid, cinnamic acid,pinosylvin, resorcinol, orcinol, and their monomethyl ethers were purchasedfrom Sigma-Aldrich (www.sigmaaldrich.com).

Plant Material and Treatments

Rowan (Sorbus aucuparia) cell cultures were grown and treated with Venturiainaequalis elicitor, as described previously (Liu et al., 2004; Khalil et al., 2013).Greenhouse-grown grafted shoots of apple (Malus 3 domestica ‘Golden Deli-cious’ and ‘Holsteiner Cox’) were inoculated with the Erwinia amylovora strain222 gfp (Flachowsky et al., 2008), as described previously (Chizzali et al., 2012b).

Extraction and Analysis of Phytoalexins

Freeze-dried stem segments (0.5 g) were extracted and analyzed as de-scribed previously (Hüttner et al., 2010; Chizzali et al., 2012a).

Construction of a Subtracted cDNA Library

An SSH library was constructed as described previously (Gaid et al., 2012),except that poly(A+) RNA was isolated from chitosan-treated (9 h after theonset of elicitation) and control rowan cell cultures and cDNA synthesis wasstarted with 1 mg of mRNA from each.

Identification and Cloning of Rowan cDNAs EncodingB4H and CPR

The genome sequence of cv Golden Delicious (apple genome version 0.1contigs) was searched for putative B4H sequences via the BLASTN server ofthe GDR (www.rosaceae.org), using selected CYP contigs from the SSHcDNA library as queries (Supplemental Table S1). Downloaded sequenceswere filtered against CYP data bank entries related to the modification of


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products of the shikimate pathway (http://metabolomics.jp/wiki/Index:P450). MDP0000152900 and closely related apple unigenes as the mostpromising candidates for hydroxylation in biphenyl metabolism served toderive 59 and 39 primers for the amplification of the cds of the putative rowanB4H cDNA (Supplemental Table S3). To clone a CPR transcript from rowancell cultures, the poplar (Populus trichocarpa) CPR3 sequence (AF302498.1),which encodes a class II CPR (Ro et al., 2002), was used as a query to searchthe GDR via the BLASTN server. The apple unigene MDP0000167955, whichshared high similarity with the only CPR transcript fragment in the rowanSSH cDNA library, was selected as a candidate CPR gene, from which 59 and39 primers were derived for the amplification of the CPR cds.

The coding sequences for SaB4H and SaCPR were PCR amplified from arowan cDNA pool prepared 4 h post elicitation using Phusion Hot Start IIHigh-Fidelity DNA polymerase (Thermo Scientific; www.thermoscientific.com). The SaB4H cds was obtained using forward primer 1 (with an EcoRIrestriction site) and reverse primer 2 (with a PacI restriction site; SupplementalTable S3). The PCR conditions were 98°C for 30 s, followed by 30 cycles of98°C for 10 s, 63°C for 30 s, and 72°C for 70 s, and a final extension of 10 minat 72°C.

The gene-specific forward primer 3 was used for 39 RACE to obtain the 39UTR. The 59 UTR was obtained by amplifying a fragment of genomic DNA,which was prepared using the DNeasy Mini Kit (Qiagen; www.qiagen.com)with gene-specific forward primer 4 (derived from a 300-bp upstream se-quence of MDP0000152900) and reverse primer 5 (Supplemental Table S3).The CPR cds was amplified with forward primer 6 and reverse primer 7,which introduced SmaI and HindIII restriction sites, respectively. PCR con-ditions were identical to those used for the SaB4H sequence. The PCR productof the expected size was excised from a 1% (w/v) agarose gel and purifiedusing the innuPREP DOUBLEpure Extraction Kit (Analytik Jena; www.analytik-jena.de).

The Md(GD)B4H and Md(HC)B4H coding sequences of cv Golden Deli-cious and cv Holsteiner Cox, respectively, were amplified from reverse-transcribed RNA isolated from the transition zones of fire blight-infectedstems.

Expression of B4H and CPR cDNAs in Yeast

The PCR products for apple and rowan B4Hswere digested with EcoRI andPacI and inserted into the MULTICLONING SITE1 (MCS1) of the pESC-Urayeast (Saccharomyces cerevisiae) dual expression vector (Stratagene; www.stratagene.com) behind the Gal-10 promoter in the correct orientation. ThePCR product for CPR was digested by SmaI and HindIII and inserted intoMCS2 behind the Gal-1 promoter. After verification by sequencing, the ex-pression construct was transferred to competent yeast cells (INVSc1) using theS.c. EasyComp Transformation Kit (Life Technologies; www.lifetechnologies.com). Transformants were selected on synthetic glucose inoculation medium(Pompon et al., 1996). Incubation of transformed yeast cultures and inductionof protein expression were performed using the high-density procedure(Pompon et al., 1996). After induction with Gal for 16 h, yeast cells wereharvested and immediately processed for microsome preparation, for which amechanical breaking procedure was used, as described by Pompon et al.(1996), except that ultracentrifugation at 100,000g was performed for 60 min at4°C. Microsome pellets were dissolved in 1 mL of TEG buffer (50 mM Tris-HCl,pH 7.4, 1 mM EDTA, and 20% glycerol) and stored at 280°C. The CYP con-centration in the isolated microsomes was determined from the reduced car-bon monoxide difference spectrum using a differential absorption coefficient(450 versus 490 nm) of 91 mM

21 cm21 (Omura and Sato, 1964).

Enzyme Assays

The standard B4H assay consisted of 10 mM 3-hydroxy-5-methoxybiphenyl,1 mM NADPH, and 10 pmol of recombinant CYP in a final assay volume of200 mL adjusted with 100 mM potassium phosphate buffer, pH 7.5. The re-action was started by the addition of NADPH, incubated at 30°C for 30 min,and stopped by the addition of 20 mL of ice-cold stopping mixture (acetic acid:methanol, 1:1), followed by two extractions with 250 mL of ethyl acetate. Thecombined extract was evaporated to dryness, resuspended in 100 mL ofmethanol, and analyzed by HPLC. Arrays of related compounds, as listed inTable I, were tested as potential B4H substrates at final concentrations of 10mM. For enzyme characterization, pH values ranged from 5.5 to 10.5, tem-peratures from 15°C to 60°C, incubation times from 5 to 120 min, and CYPconcentrations from 1 to 50 pmol per assay. The Km value for 3-hydroxy-5-

methoxybiphenyl was determined using concentrations from 0.2 to 30 mM

while keeping NADPH at 1 mM. To measure the Km value for NADPH,its concentration varied from 1 to 1,000 mM, keeping the concentration of3-hydroxy-5-methoxybiphenyl constant at 10 mM. Enzyme concentration(10 pmol per assay) and incubation time (30 min) were selected to get a linearreaction velocity during the assay period. All kinetic analyses were carried outin triplicate. The kinetic parameters Km and Vmax were determined by theHanes method using the Hyper 32 software (http://homepage.ntlworld.com/john.easterby/software.html).

The CPR assay (1 mL) contained 40 mM cytochrome c (equine heart; Sigma-Aldrich; www.sigmaaldrich.com), 0.1 mM EDTA, and 12 mg of microsomalfraction in 0.1 M potassium phosphate buffer, pH 7.5 (Urban et al., 1990, 1994).The reaction was started by the addition of NADPH. To determine the Kmvalue for NADPH, the concentration was varied between 1 and 500 mM whilekeeping cytochrome c at 40 mM. The rate of cytochrome c reduction wasspectrophotometrically monitored at 550 nm using a differential absorptioncoefficient of 21 mM

21 cm21 at 25°C (van Gelder and Slater, 1962).

Analysis of Enzymatic Products by HPLC-DADand GC-MS

The B4H reaction product was analyzed on an HPLC system describedpreviously (Gaid et al., 2012). The mobile phase consisted of water acidifiedwith 1 mM trifluoroacetic acid (A) and acetonitrile (B). The following gradientwas employed at a flow rate of 0.5 mL min21: 30% to 35% B in 5 min, 35% to80% B in 25 min, and 80% to 100% B in 28 min. The solvent gradient forresorcinol and orcinol monomethyl ethers, ferulic and 4-coumaric acids, andconiferaldehyde was employed at 5% to 10% B in 5 min, 10% to 40% B in 15min, and 40% to 100% B in 20 min. For the analysis of assays containing 2,4-dihydroxydibenzofuran, 2-hydroxy-4-methoxydibenzofuran, and 4-hydroxy-2-methoxydibenzofuran, the following gradient was used: 10% to 20% B in5 min, 20% to 40% B in 20 min, and 40% to 100% B in 10 min. Detectionwavelengths were at 254, 269, 280, 310, 322, and 340 nm.

For GC-MS analysis, the volume of the B4H assay was increased to 2 mL.The incubation was stopped by adding 200 mL of stopping mixture andextracted once with 1 mL of ethyl acetate, which was evaporated to dryness ina gentle stream of nitrogen gas. Trimethylsilyl derivatization of acidic protonsand further steps were done as described previously (Gaid et al., 2009), exceptthat the capillary column was coupled directly to a 5975B mass spectrometer(Agilent Technologies; www.agilent.com).

RT-PCR Expression Analysis

Total RNA was extracted from V. inaequalis-treated rowan cell cultures atdefined times (0–48 h) using the RNeasy Plant Mini Kit (Qiagen; www.qiagen.com). On-column DNase I (Qiagen) treatment was performed to eliminategenomic DNA contamination. RNA (1 mg) was reverse transcribed at 42°Cwith RevertAid H Minus Moloney murine leukemia virus reverse transcrip-tase (Fermentas; www.thermoscientificbio.com) and an oligo(dT) primer.SaPAL transcripts were analyzed using forward primer 8 and reverse primer 9(Supplemental Table S3), as described previously (Gaid et al., 2011). SaBIS1transcripts were analyzed using gene-specific primer pair 10 (forward) and 11(reverse) according to Liu et al. (2010). SaB4H transcripts were analyzed usingforward primer 12 and reverse primer 13. RT-PCR was carried out in 25-mLvolumes containing 2 mL of cDNA (1:10 diluted), 0.2 mM forward and reverseprimers, and 1 unit of Taq DNA polymerase (Peqlab; www.peqlab.de). Theactin transcript level served to normalize RT-PCR results using primer pair14 (forward) and 15 (reverse). To maintain RT-PCR amplification within theexponential phase, 28 cycles were run. The PCR conditions for the amplifi-cation of SaPAL, SaBIS1, and SaB4H transcripts were as follows: 4 min ofdenaturation at 94°C, then 28 cycles of 94°C for 30 s, 55°C for 30 s, and 72°Cfor 45 s (70 s for SaBIS1), and finally a 10-min extension at 72°C. Similar PCRconditions for 30 cycles were used for the amplification of SaActin transcripts.The PCR products were analyzed as described previously (Gaid et al., 2012).At least two biological and three technical repetitions were performed.

Quantitative Real-Time PCR

Total RNA was isolated from two combined biological replicates (100 mg).An aliquot (1 mg) was reverse transcribed at 42°C. Quantitative RT-PCR wasperformed with the 7500 Fast Real-Time PCR System (Applied Biosystems;




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www.appliedbiosystems.com) using 53 HOT FIREPol EvaGreen qPCR MixPlus (ROX; Solis BioDyne; www.sbd.ee). We followed the procedure outlinedin the manufacturer’s instructions with small modification: 45 cycles at 95°Cfor 30 s and 61°C for 1 min. Gene-specific amplification was evaluated bymelt-curve analysis. Amplification and correlation efficiencies of each PCRwere determined using six serial dilutions of cDNA from all samples. The PCRefficiency was used to transform the cycle threshold values into raw data forrelative quantification. Expression of the Md(GD)B4H gene was evaluatedusing the gene-specific primers listed in Supplemental Table S3 [primers 16forward and 17 reverse for Md(GD)B4H and primers 18 forward and 19 re-verse for Actin]. Quantitative PCR primer pairs forMd(GD)B4Hwere designedbased on the sequence of unigene MDP0000152900. All samples were nor-malized using mRNA of the reference gene, MdActin, as an internal controlsample for each line (Paris et al., 2009). Scaling of Md(GD)B4H expression wasperformed in relation to the respective mRNA expression levels in mock-inoculated stems, which were set to 1. All reactions were performed in tech-nical triplicates. Estimations of efficiency and expression level were based onthe mathematical model of Pfaffl (2001).

Phylogenetic Reconstruction

A number of functional CYP sequences, which are related to hydroxylationreactions in natural product biosynthesis, were used. The accession numbersand the CYP nomenclature are listed in Supplemental Table S2. The phylo-genetic tree was constructed by the neighbor-joining method using MEGA5.05 with 1,000 bootstrap support (Tamura et al., 2011). The evolutionarydistances were computed using the Poisson correction method (Zuckerkandland Pauling, 1965) and are in units of the number of amino acid substitutionsper site. The pairwise deletion method was used to nullify gaps and missingdata.

Agrobacterium tumefaciens-Mediated Transient Expressionand Subcellular Localization

Expression constructs were generated using the Gateway cloning tech-nology according to the manufacturer’s instructions (Life Technologies; www.lifetechnologies.com). Three pairs of Gateway-compatible attB primers(Supplemental Table S3) were used to amplify (1) the complete cds of SaB4H(SaB4H; 1,497 bp; primer pair forward 20 and reverse 21), (2) the N-terminalanchor sequence (SIG; 81 bp; primer pair forward 20 and reverse 22), and (3)the truncated sequence that lacks the 30 N-terminal amino acids including themembrane anchor sequence (TRUNC; 1,407 bp; primer pair forward 23 andreverse 21). All reverse primers lacked a stop codon to enable C-terminal fu-sions with Venus. The modified attB PCR products (SaB4H, SIG, and TRUNC)were cloned into the donor vector pDONR/Zeo (Life Technologies) using theGateway BP (Life Technologies) reaction to create entry vectors without a finalstop codon. The entry vectors were used to create expression vectors using themodified pEarly-Gate101-Venus destination vector (Kaufholdt et al., 2013),resulting in the generation of three C-terminal fusion constructs: (1) 35S::SaB4H-Venus, (2) 35S::SIG-Venus, and (3) 35S::TRUNC-Venus. The correctnessof each construct was confirmed by sequencing (MWG Biotech; www.mwg-biotech.com). An ER-positive control (35S::HDEL-DsRED; Höfer et al., 2008)was kindly provided by Dr. Boris Voigt. A construct expressing p19 proteinfrom Tomato bushy stunt virus (Voinnet et al., 2003) was used to suppress genesilencing. All vectors were transferred by electroporation in the A. tumefaciensstrain C58C1 and transiently expressed in leaves of Nicotiana benthamiana, asdescribed previously (Gaid et al., 2012). Prior to infiltration, bacterial cultureswere resuspended in activation medium (Bendahmane et al., 1999) and ad-justed to optical density at 600 nm = 1.5 (optical density at 600 nm = 0.9 forp19). Corresponding A. tumefaciens strains containing the Venus/DsREDconstructs and the p19-silencing plasmid were mixed 1:1:1 and coinfiltrated.Confocal laser-scanning microscopy was performed using the 510METAconfocal microscope connected to an Axiovert 200M device (Zeiss; www.zeiss.com). The argon laser (488 nm for Venus) together with the heliumlaser (543 nm for DsRED) and the primary beam-splitting mirrors 488 or477/543 nm were used. The emitted light was separated by a secondarybeam splitter at 545 nm. Fluorescence was detected with filter sets onchannel 3 for red fluorescent protein with BP 560 to 615 and for yellowfluorescent protein with BP 505 to 550 in the multitrack scan mode. We usedthe l mode to examine the spectral signature of fluorophores and theC-Apochromat 403/1.2 water immersion objective for all the specimen

examinations. All images were processed using the LSM Image BrowserRelease 4.2 (Zeiss).

To test the activity of transiently expressed SaB4H, an additional experimentwas carried out in N. benthamiana leaves using the A. tumefaciens strain C58C1harboring the construct 35S::SaB4H-Venus. Infiltration with A. tumefacienscontaining the empty vector served as a negative control. After infiltration (48h), a portion of the infiltrated leaf was examined with a confocal microscopeto ensure transient expression, while the remaining part of the leaf was usedto prepare microsomes, essentially as described by Karamat et al. (2014).The microsomal protein (20 mg) was incubated with 10 mM 3-hydroxy-5-methoxybiphenyl in a final B4H assay volume of 200 mL and adjusted with100 mM potassium phosphate buffer, pH 7.5. The reaction was started by theaddition of 1 mM NADPH, incubated at 30°C for 60 min, and stopped by theaddition of 20 mL of ice-cold stopping mixture (acetic acid:methanol, 1:1),followed by two extractions with 250 mL of ethyl acetate each. The combinedextract was evaporated to dryness, resuspended in 100 mL of methanol, andanalyzed by HPLC as described above.

Sequence data from this article can be found in the GenBank/EMBL datalibraries under the following accession numbers: SaB4H, KC964601; SaCPR,KC964602; Md(GD)B4H, KM987643; and Md(HC)B4H, KM987644.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Proposed functions of unigenes in a subtractedcDNA library.

Supplemental Figure S2. Alignment of CPR amino acid sequences.

Supplemental Figure S3. Carbon monoxide difference spectrum.

Supplemental Figure S4. Product analysis by HPLC-DAD.

Supplemental Figure S5. Transient SaB4H expression in N. benthamianaleaves.

Supplemental Figure S6. Detection of phytoalexins in cv Golden Delicious.

Supplemental Table S1. CYP transcript contigs in the subtracted cDNAlibrary.

Supplemental Table S2. Accession numbers and CYP nomenclature forthe phylogenetic tree.

Supplemental Table S3. Primers used.

ACKNOWLEDGMENTS

We thank Dr. David R. Nelson (University of Tennessee) for naming therowan and apple B4H sequences according to the standardized CYP nomen-clature system and Dr. Boris Voigt (University of Bonn) for providing the 35S::HDEL-DsRED construct.

Received January 19, 2015; accepted April 9, 2015; published April 10, 2015.

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