Supporting informationErnst et al. 10.1073/pnas.1202999109SI Materials and MethodsPrimers Used for Expression Analysis. Intron-spanning gene frag-ments were amplified using primer combinations 5′-ATT CACATG GCT TGG ATT TG-3′/5′-GGC ACA AAT CCT TTTTCT C-3′ (Nicotiana tabacum sieve element occlusion gene 1;NtSEO1), 5′-AAT ATG GTG AAT TTT GGC TAC-3′/5′-TTGGTA ACA TTG GTT TAG AAC-3′ (NtSEO2), and 5′-AGCTCA AGG TTA AGG ATG AC-3′/5′-TGG CCA AGG GAGCAA GGC AA-3′ (GAPDH). For real-time quantitative RT-PCR experiments, gene-specific fragments were amplified usingprimer combinations 5′-CAG CAC CAT GAA GCC AAC ATTGAA G-3′/5′-CCA TTG GTA GCC AGA GAG CAT AG-3′for NtSEO1 and 5′-CAA GAC ATC CCT GGA GAA ACAGAC-3′/5′-GAA GAG CAC GAA GTT GAC GGT C-3′ forNtSEO2.

Cloning of NtSEO Gene Promoter Constructs. The 1,450-bp NtSEO1promoter fragment was amplified from genomic DNA preparedaccording to established protocols (1) using primers 5′-AGAGGT ACC TAT CCG GTC AAA GGT TCG-3′ and 5′-AGACTC GAG TTT CTT GGG CTT TTT AAT TTG G-3′. Theunderlined bases are KpnI/XhoI restriction sites, which allowedthe 1,450-bp regulatory sequence upstream of NtSEO1, desig-nated “PNtSEO1,” to be inserted as a KpnI/XhoI restrictionfragment into the corresponding sites of a pUC18-based pAMvector (2). This insertion placed the promoter upstream of theEscherichia coli uidA gene encoding GUS. The promoter-GUScassette was isolated by digestion with KpnI and BamHI andwas inserted into the corresponding sites of binary planttransformation vector pBIN19 (3) to obtain the constructpBPNtSEO1GUS.Promoter-specific expression of endoplasmic reticulum-tar-

geted GFP (GFPER) was achieved using the NtSEO1 promoterelement described above inserted into the KpnI/XhoI sites ofvector pBSGFPER (4). The 1,464-bp NtSEO2 promoter frag-ment was amplified from genomic DNA using primers 5′-AGAGGA TCC CTA CTT ATA CTA GCT AAT CC-3′ and 5′-AGA CTC GAG TTG AAA GGA ATT AAA AAA TGG T-3′and, using the underlined BamHI/XhoI restriction sites, wasinserted into the corresponding sites of a pUC18-based pAMvector containing the coding sequence of GFPER fused tothe Cauliflower mosaic virus 35S terminator (CaMV, 35S-T).The promoter-GFPER constructs were transferred to pBIN19using the KpnI/BamHI (PNtSEO1GFPER) or BamHI/HindIII(PNtSEO2GFPER) sites to obtain constructs pBPNtSEO1GFPERand pBPNtSEO2GFPER. The integrity of all constructs was veri-fied by sequencing.

Cloning of SEO:Humanized Renilla GFP Fusion Constructs. Theregulatory sequence PNtSEO1 was isolated from plasmidpBPNtSEO1GFPER by KpnI/XhoI digestion, and PNtSEO2 wasamplified from genomic DNA using primers 5′-AGA GCGGCC GCC TAC TTA TAC TAG CTA ATC C-3′ and 5′-AGAACT AGT TTG AAA GGA ATT AAA AAA TGG T-3′. Bothpromoter fragments then were introduced into the appropriaterestriction sites of the pBS-humanized Renilla GFP (hrGFP)vectors. Coding sequences were amplified from cDNA forNtSEO1 (primers 5′-AGA CTC GAG ATG GCA AGT CGTGCT TTG G-3′ and 5′-AGA GTC GAC ATC AGT GCA GCAACG GTA C-3′), the NtSEO2 N-terminal fusion (primers 5′-AGA ACT AGT ATG GCT CAT GTT AAC CAA TTG-3′ and5′-AGA ACT AGT ATC AAT GCA GCA GCT GTA C-3′),

and the corresponding C-terminal fusion (primers 5′-AGAACT AGT ATG GCT CAT GTT AAC CAA TTG-3′ and 5′-AGA GGA TCC TTA ATC AAT GCA GCA GCT G-3′). Theresulting PCR fragments were digested with XhoI/SalI(NtSEO1), SpeI (N-terminal fusion of NtSEO2), or SpeI/BamHI (C-terminal fusion of NtSEO2) and were inserted intothe corresponding restriction sites of pBShrGFP vectors contain-ing the NtSEO promoters, as shown in Fig. S3. The entire ex-pression cassettes then were isolated with KpnI/XbaI (NtSEO1) orNotI/SalI (N- and C-terminal fusions of NtSEO2) and were in-serted either into the same restriction sites (NtSEO1) or, afterKlenow treatment, into the SmaI/SalI restriction sites of pBIN19(N- and C-terminal fusion of NtSEO2). The resulting constructswere designated “pBPNtSEO1NtSEO1:hrGFP,” “pBPNtSEO2NtSEO2:hrGFP,” and “pBPNtSEO2hrGFP:NtSEO2.”As a control, the expression cassette from vector

pBSPNtSEO1hrGFP (see above) was isolated with KpnI/XbaI andinserted into the corresponding restriction sites of pBIN19 toobtain the construct pBPNtSEO1hrGFP. The control, the Medicagotruncatula SEO-F1 (MtSEO-F1) gene, was constructed by iso-lating a 1,094-bp MtSEO-F1 promoter fragment from plasmidpBSPMtSEO-F1GFPER (4) using KpnI/XhoI and inserting the re-sulting fragment into pBShrGFP. The coding sequence ofMtSEO-F1 was amplified from plasmid MtSEO-F1/pENTR (5)using primers 5′-AGA CTC GAG ATG TCA TTG TCC AATGGA AC-3′ and 5′-AGA CTC GAG TAT CTT GCC ATT CTGTGG A-3′ (restriction sites are underlined), digested with XhoI,and inserted into the corresponding sites of pBSPMtSEO-F1hrGFP.The expression cassette PMtSEO-F1MtSEO-F1:hrGFP was isolatedby digestion with KpnI/SacI and inserted into pBIN19 to gen-erate construct pBPMtSEO-F1MtSEO-F1:hrGFP. The integrity ofall constructs was verified by sequencing.

Cloning of Fusion Constructs for Agroinfiltration. For the transientcoexpression of tagged and untagged NtSEO proteins in Nico-tiana benthamiana, NtSEO genes were needed with and withouttranslational stop codons (depending on the tag position in thedestination vectors). These genes were amplified from tobaccocDNA using primer combinations 5′-AGA CCA TGG CAAGTC GTG CTT TG-3′ and 5′-AGA GCG GCC GCT AATCAG TGC AGC AAC GGT-3′ or 5′-AGA CTC GAG CAATCA GTG CAG CAA CGG-3′ (NtSEO1) and 5′-AGA GTCGAC ATG GCT CAT GTT AAC CAA TTG-3′ and 5′-AGAGCG GCC GCT AAT CAA TGC AGC AGC TGT A-3′ or 5′-AGA GCG GCC GCC AAT CAA TGC AGC AGC TGT AC-3′(NtSEO2). The resulting variants of each gene (with or withouta translational stop codon) were digested (restriction sites areunderlined) and inserted into the corresponding sites of entryvector pENTR4 (Invitrogen).The construction of pBatTL binary expression vectors

encoding tagged proteins was achieved by the amplification ofN-terminal (5′-AGA AGA TCT ATG GTT AGC AAA GGAGAA GAA C-3′ and 5′-AGA AGA TCT AAG ATC CTC CTCAGA AAT CAA CTT TTG CTC TTT GTA TAG TTC ATCCAT GCC-3′) and C-terminal (5′-AGA ACT AGT GAG CAAAAG TTG ATT TCT GAG GAG GAT CTT ATG GTT AGCAAA GGA GAA GAA C-3′ and 5′-AGA TCT AGA TTA TTTGTA TAG TTC ATC CAT GCC-3′) Venus sequences (re-striction sites are underlined; the myc sequence is shown initalics). The reporter gene fragments were inserted into theBglII site (for N-terminal fusions) or SpeI/XbaI sites (for C-terminal fusions) of the GATEWAY (GW)-compatible vector

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pBatTL (kindly provided by Joachim Uhrig and Guido Jach,University of Cologne, Cologne, Germany) to obtain pBatTL-Venus-(GW) and pBatTL-(GW)-Venus. NtSEO genes wereintroduced into the three binary pBatTL destination vectors byrecombination (mediated by LR Clonase; Invitrogen) using thepENTR4 constructs (Fig. S4).To obtain a Venus control and a cytoplasmic monomeric RFP

(mRFP) marker, entry vector pENTR4 was digested with NcoI/XhoI and religated after Klenow treatment. The resulting vectorlacked the ccdB gene and was used in the subsequent re-combination reactions with destination vectors pBatTL-(GW)-Venus and pBatTL-(GW)-mRFP-Q66T (6).

Generation of NtSEO-Knockdown Mutants. To generate the hairpin(hp) RNAs required for NtSEO gene knockdown, 500-bpfragments with different restriction sites were amplified fromcDNA for NtSEO1 (primer combinations 5′-AGA CTC GAGAAG AAA AGG CAT CAC TTG CC-3′/5′- AGA GGT ACCAGT AAG TTG AGA GGC ACA AG-3′ and 5′-AGA TCTAGA AAG AAA AGG CAT CAC TTG CC-3′/5′-AGA GGATCC AGT AAG TTG AGA GGC ACA AG-3′) and NtSEO2(primer combinations 5′-AGA CTC GAG AAG AGA AGGTTC CCC ACA G-3′/5′-AGA GGT ACC TGT ACA CATGAC AGC GGC-3′ and 5′-AGA TCT AGA AAG AGA AGGTTC CCC ACA G-3′/5′-AGA GGA TCC TGT ACA CATGAC AGC GGC-3′). These fragments were inserted into theXhoI/KpnI sites (sense orientation) and XbaI/BamHI sites(antisense orientation) of the RNAi vector pHANNIBAL (7)(kindly provided by CSIRO Plant Industry) to createhpNtSEO1/pHANNIBAL and hpNtSEO2/pHANNIBAL. Toensure strong transgene expression in sieve elements, we re-moved the CaMV 35S promoter from each vector by digestionwith SacI, followed by Klenow treatment, followed by digestionwith XhoI, and then inserted the NtSEO1 and NtSEO2 pro-moter sequences carrying appropriate restriction sites, whichwere prepared by amplification using primers 5′-AGA CCCGGG GTC GAG CGG CCG CTA TCC GGT CAA AGG TTCG-3′/5′-AGA CTC GAG TTT CTT GGG CTT TTT AAT TTGG-3′ for PNtSEO1 and 5′-AGA CCC GGG GTC GAG CGG

CCG CCT ACT TAT ACT AGC TAA TCC-3′/5′-AGA CTCGAG TTG AAA GGA ATT AAA AAA TGG-3′ for PNtSEO2.The NotI sites were introduced for the second cloning step.After digestion with SmaI/XhoI, the regulatory sequences wereintroduced into the corresponding pHANNIBAL vectors, yield-ing PNtSEO1hpNtSEO1/pHANNIBAL and PNtSEO2hpNtSEO2/pHANNIBAL. The binary vectors pBIN19 (kanamycin selec-tion) and the modified version pBINHyg (hygromycin selection)were linearized with EcoICRI, whereas the pHANNIBAL-de-rived hpRNA cassettes were isolated with NotI. After Klenowtreatment, the cassettes were inserted into the binary vectorsresulting in constructs pBPNtSEO1hpNtSEO1 and pBPNtSEO2hpNtSEO2. The integrity of all constructs was verified by sequencing.

CmSEO1:Venus Fusion Construct for P-Protein Visualization inTransgenic Squash Roots. A 1,476-bp regulatory sequence fromthe Cucurbita maxima SEO1 (CmSEO1) gene, designated“PCmSEO1,” was amplified from genomic C. maxima DNA usingprimers 5′-AGA GGT ACC GCA ACG ACT TCG TGT ACAC-3′ and 5′-AGA CTC GAG GGT TTG GGT ATT GAG AGAG-3′. The promoter element was digested with KpnI/XhoI andwas inserted into vector pBSGFPER, and the resultingPCmSEO1GFPER fragment was transferred into the KpnI/BamHIsites of pBIN19 to produce construct pBPCmSEO1GFPER. Con-struct pBPCmSEO1CmSEO1:Venus was generated by amplifyingthe PCmSEO1 sequence from pBPCmSEO1GFPER using primers 5′-AGA GGT ACC GCA ACG ACT TCG TGT ACA C-3′ and 5′-AGA GTC GAC GGT TTG GGT ATT GAG AGA G-3′ andthe CmSEO1 coding sequence lacking a translational stop codonfrom squash cDNA (primers 5′-AGA GTC GAC ATG GCCACT ACA CTC AAG-3′ and 5′-AGA GTC GAC CAT ATGAGC ACC TCC GTG-3′). The corresponding sequences wereassembled in a pBSVenus vector containing the Venus codingsequence fused to the CaMV 35S terminator, using the restrictionsites underlined in the primer sequences. The whole expressioncassette then was isolated from pBSPCmSEO1CmSEO1:Venus by di-gestion with NotI, followed by Klenow treatment and a subsequentdigestion with KpnI, and then was inserted into the KpnI/SmaIsites of pBIN19, resulting in construct pBPCmSEO1CmSEO1:Venus.

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phloem during plant development. Plant Mol Biol 65:285–294.

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Fig. S1. Alignment of tobacco and M. truncatula SEO proteins. Protein sequences of NtSEO1, NtSEO2, and all MtSEO proteins were aligned using T-Coffee (1).The typical SEO domains are shaded in yellow (SEO-NTD), blue (potential thioredoxin fold), and gray (SEO-CTD).

1. Notredame C, Higgins DG, Heringa J (2000) T-Coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol 302:205–217.

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Fig. S2. NtSEO expression in true leaves of different ages. (A) NtSEO-specific fragments were amplified from cDNA generated from the total RNA of leaves at10%, 60% (sections indicated in B), and 100% of final leaf size. The tobacco GAPDH gene was used as a control. Expression was strong in leaves at 10% finalsize but was not detectable in fully mature leaves (100% final size). In leaves at 60% final size, NtSEO expression mirrored the course of basipetal secondaryvein development. (B) Tobacco leaves as used for expression profiling. (C) When hrGFP was expressed under the control of PNtSEO1, reporter fluorescence couldbe detected only in immature sieve elements and was absent from mature phloem. (Scale bars: 5 cm in B; 20 μm in C.)

Fig. S3. Schematic representation of hrGFP-tagged NtSEO expression cassettes and controls used for stable transformation of tobacco. All constructs wereplaced under the control of a sieve element-specific SEO promoter and the CaMV 35S terminator (T). White arrowheads indicate the position of the AUGtranslation initiation codon, and asterisks mark the location of the translational stop codons. Restriction sites used for cloning are shown.

Fig. S4. Schematic representation of NtSEO, Venus-tagged NtSEO, and control expression cassettes used for transient expression in N. benthamiana andexpression patterns detected for Venus fusions. (A) All constructs were placed under the control of the constitutive double-enhanced CaMV 35S promoter(d35S-P) and CaMV 35S terminator (T). The Ω sequence (TE) from Tobacco mosaic virus was used for optimal initiation of translation at the AUG start codon(white arrowheads). The att-recombination sites are represented by dark gray boxes; asterisks indicate the location of the translational stop codons. (B) Theexpression of NtSEO1:Venus alone always resulted in cytoplasmic fluorescence. (C) Heteromorphic structural agglomerates were detected when Venus:NtSEO2was expressed without an untagged partner. (Scale bars = 30 μm.)

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1 2

3

exuda�on

3 leaves of each plant were

successively placed into the same vial for 10 min, respec�vely

sample prepara�on

exudates were lyophilized and subsequently

dissolved in 200 μl H2O

enzyma�c sugar assay

25 μl

25 μl

determina�on of total D-glucose content a�er enzyma�c

hydrolysis of sucrose (β-fructosidase)

determina�on of total D-glucose content

(without addi�on of β-fructosidase)

M1

M2

D-glucose content of measurement 2 (M2) was substracted from D-glucose

content of measurement 1 (M1)

calculated difference corresponds to sucrose content

(main transport sugar of tobacco)

Fig. S5. Schematic overview of tobacco exudation studies.

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Fig. S6. Graphical presentation of glucose concentrations and calculated relative sucrose contents of all tobacco plants included in the exudation studies.Exudate samples were prepared for selected plants representing NtSEO-RNAi lines N (N01–N48) and T (T01–T57) and wild-type tobacco (WT01–WT57). (A) D-glucose concentrations were measured after (total bars) and before (hatched sections of bars) hydrolysis of sucrose, enabling us to differentiate betweensugars derived from damaged cells at cutting sites and phloem-derived photoassimilates. (B) Exudation rates were compared as relative values by setting themean sucrose concentrations of wild-type plants to 1. The exudation rates of RNAi lines N and T are significantly higher than those determined for wild-typeplants.

Table S1. Summary of the major parameters analyzed in the exudation experiments

Table S1

Exudation was characterized in representative plants from NtSEO-RNAi lines N (N01–N48) and T (T01–T57) and wild-type tobacco (WT01–WT57). Threeleaves from each plant were exuded consecutively into the same vial for 10 min. After lyophilization, samples were dissolved in 200 μL water, and equivalentaliquots were used to determine D-glucose concentrations before and after the hydrolysis of sucrose.

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Table S2. Sucrose concentration in petioles of wild-type plants and NtSEO-RNAi lines

Table S2

Sucrose concentrations were determined for total petioles. Petiole sections were harvested from wild-type tobacco and corresponding NtSEO-RNAi plants(lines N and T). After sections were shredded, total sugars were extracted, and D-glucose concentrations were measured before and after hydrolysis of sucroseto determine sucrose contents, which were consistent in all analyzed samples. Mean ± SD, nWT = nN = nT = 6.

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