Supporting InformationKoch et al. 10.1073/pnas.1306373110SI Materials and MethodsPreparation of Fungal Macroconidia and in Vitro Silencing RNATreatment. Both Fusarium graminearum (Fg) wild-type strainand GFP-expressing strain Fg strain WT-GFP (1) were routinelycultured on synthetic nutrient SNA-medium (2). Plates were in-cubated at room temperature under constant illumination fromone near-UV tube (Phillips TLD 36 W/08) and one white-lighttube (Phillips TLD 36 W/830HF). Conidia were harvested from1-wk-old cultures with a sterile glass rod and sterile water. Mac-roconidia were filtered through four layers of sterile mira-cloth,collected, and washed three times with sterile distilled water, andfinally diluted to 100 macroconidia in a volume of 100 μL liquidSNA medium, and plated in a 96-well microtiter plate. Differentconcentrations of dsRNA suspended in 2.5 μL of 50× annealingbuffer was added to fungal samples and equal volume of 50×annealing buffer was added to the control sample. Plates wereincubated at room temperature. Microscopic studies were per-formed 72 h posttransplantation (hpt) with an inverted micro-scope (Leica DM IL). Each experiment was done in triplicate.In parallel, experiments were conducted with tebuconazole(Sigma-Aldrich). Next, 15.4 mg of tebuconazole was suspendedin 1 mL absolute ethanol to obtain a stock solution of 50 μm.Control sample included addition of equal volume of solvent(sterile water and alcohol) without tebuconazole.

Staining and Microscopy of Infected Leaves. Infected leaves werecollected at 3 d postinfection (dpi), transferred into 2-mL tubescontaining 1 mL of Trypan blue solution (10 g of phenol, 10 mL ofglycerol, 10 mL of lactic acid, 10 mg Trypan blue, dissolved in 10mL of distilled water), followed by boiling for 3 min. The leaveswere then destained in 1 mL of chloral hydrate solution (2.5 g ofchloral hydrate dissolved in 1 mL distilled water) overnight, andanalyzed under a light microscope (Zeiss Axioplan 2 Imaging).GFP-tagged Fg was analyzed using a confocal microscope (LeicaTCS SP2).

Generation of Transgenic Arabidopsis and Barley Plants. The dsRNAconstruct CYP3RNA was amplified by gene-specific primersCYP51B-HindIII_F and CYP51C-XmaI_R (Table S2), andcloned into p7U10-RNAi binary vector (DNA cloning service)containing selectable marker gene bar under control of theArabidopsis Ubiquitin-10 promoter (UBQ10) and inverted con-

stitutive CaMV35s promoter (Fig. S1C). To transform barley,CYP3RNA was cloned into p6i-RNAi binary vector (DNAcloning service) containing selectable marker gene hpt and in-verted constitutive CaMV35s promoter (Fig. S1D). The resultingplasmids p7U10-CYP3RNA and p6i-CYP3RNA (Fig. S1 C and D)were introduced into the A. tumefaciens strain AGL1 (3) byelectroporation. Arabidopsis transformation and regenerationwas performed as described (4), and transformants were selectedby Basta (7 mg·L−1; Duchefa Biochemie). Barley transformationwas carried out with the spring barley (H. vulgare) cv. GoldenPromise grown in a climate chamber at 18 °C/ 14 °C (light/dark)with 65% relative humidity, with a 16-h photoperiod and aphoton flux density of 240 μmol·m−2·sec−1. Preparation of thescutellum and transformation of the immature barley embryoswas done as previously described (5).

Molecular Screening of Transformants. DNA extraction and PCRanalysis. Genomic DNA from young leaves of 5-wk-old trans-genic (T1) and nontransgenic A. thaliana lines was extractedusing the CTAB method (6). Integration of CYP3RNA into theArabidopsis genome was confirmed by PCR using gene-specificprimers CYP51B-HindIII_F and CYP51C-XmaI_R (Table S2).Detection of siRNA in transgenic Arabidopsis plants. Total RNA wasextracted from Arabidopsis lines using TRIzol reagent (In-vitrogen) following the manufacturer’s protocol. A total of 15 μgtotal RNA was separated in a 16% (vol/vol) polyacrylamide-7 Murea gel, and transferred onto Hybond Nylon membrane(Amersham) using a semidry blotter (Bio-Rad) at constant cur-rent (250 mA) for 3 h. The RNA was UV cross-linked to themembrane at 1,200 × 100 μJ (UV Stratalinker) energy. Themembrane was prehybridized for 45 min at 42 °C in 20 mLPerfectHyb-Puffer (Sigma). The complete 791-bp CYP3RNAPCR amplicon was used as a template for random labeling(Random Primer DNA Labeling with Kit, New England Biol-abs), added to the hybridization buffer, and hybridized overnightat 42 °C. The membrane was washed twice at room temperaturewith 5× SSC + 0.01% SDS. Membrane was exposed on phos-phorimaging screens (Bio-Rad) and signals were detected usinga molecular imager and the Quantity One software (Bio-Rad).Parallel loading of low range RNA marker (Fermentas) wereused as size marker.

1. Jansen C, et al. (2005) Infection patterns in barley and wheat spikes inoculated withwild-type and trichodiene synthase gene disrupted Fusarium graminearum. Proc NatlAcad Sci USA 102(46):16892–16897.

2. Nirenberg HI (1996) A simplified method for identifying Fusarium spp. occuring onwheat. Can J Bot 59(9):1599–1609.

3. Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsisgenomic library in Agrobacterium. Biotechnology (N Y) 9(10):963–967.

4. Bechtold N, Ellis J, Pelletier G (1993) In planta Agrobacterium-mediated gene transferby infiltration of adult Arabidopsis thaliana plants. Comptes Rendu de l’Academie desScience 316:1194–1199.

5. Imani J, Li L, Schäfer P, Kogel KH (2011) STARTS—A stable root transformation systemfor rapid functional analyses of proteins of the monocot model plant barley. Plant J67(4):726–735.

6. Chen DH, Ronald PC (1999) A rapid DNA minipreparation method suitable for AFLPand other PCR applications. Plant Mol Biol Rep 17:53–57.

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Fig. S1. Fragment combination strategy (stacking) for generating CYP3RNA, a construct designed from CYPA, -B, and -C partial sequences (A and B) to silenceall three cytochrome P450 lanosterol C-14α-demethylase (CYP51) genes; (C) p7U10-CYP3RNA and p6i-CYP3RNA; (D) generated by cloning the CYP3RNAfragment into binary vectors by replacing the GUS fragment using HindIII/XmaI restriction enzymes. Both vectors with inverted repeat CaMV35s promoters.

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Fig. S2. Growth inhibition and morphology of F. graminearum grown in axenic culture following treatment with CYP3RNA or tebuconazole. One-hundred Fgmacroconidia were suspended in 100 μL of liquid SNA medium treated with different concentrations of CYP3RNA, and microscopically evaluated at 48 hpt (A)or 72 hpt (B). (C) Treatment with tebuconazole and evaluated at 48 hpt. Control, (50× annealing buffer). (D) Quantification of fungal growth after treatmentwith increasing concentrations of CYP3RNA (i) and tebuconazole (ii).

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Fig. S3. Microscopic characterization of Fg-inoculated Arabidopsis leaves 3 dpi. (A, C, and E) Successful colonization of Col-0 ev. Profuse hyphal growth is seenoutside and inside the leaf, and is not restricted to the inoculation site. (A) Mycelium emerging from the inoculation site. (C) Successful fungal propagation,indicated by formation of loose sporodochia formed by branched conidiophores (white arrowheads). (B, D, and F) Hyphal formation is strongly reduced andconfined to the wounded leaf area (site of inoculation) on CYP3RNA-expressing plants. (B) Impaired fungal development occurs exclusively in the woundedarea around the inoculation site; the surrounding leaf tissue remained free of colonization. (D and F) Impaired fungal growth and the development of fungalstress symptoms, as indicated by the vast number of sporodochia at the inoculation site. (A and B) Black arrow heads mark the inoculation site. In A–D, leafsections were stained with Trypan blue and viewed under a light microscope (Zeiss Axioplan 2 Imaging). In E and F the GFP-expressing Fg strain was visualizedusing confocal microscopy (Leica TCS SP2).

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Fig. S4. Sporulation of the oomycete Hyaloperonospora arabidopsidis on CYP3RNA-expressing Arabidopsis leaves is not impaired, compared with the wild-type. Arabidopsis seeds were sown on a soil/sand mixture, stratified for 3 d at 4 °C, and then grown under a 12 h photoperiod in a growth chamber at 20 °C. Forinfection, 10-d-old plants were spray-inoculated to saturation with a spore suspension from the H. arabidopsidis isolate Noco2 at 40,000 spores per mL−1. Plantswere kept in a growth cabinet at 16 °C for 6 d with a 12-h photoperiod. Sporulation was induced by spraying plants with water and keeping them for 24 hunder high humidity, and the sporulation rate was determined with a hemocytometer. The bars represent mean values ± SD for 20 samples. Differencesbetween the lines were not statistically significant, as determined by Student’s two-tailed t test (P > 0.01).

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Table S1. Prediction of CYP3RNA off-target transcripts

Organism

Query name*

All hits† Efficient hits‡Gene Description

Fusarium graminearum FGSG_01000§ CYP51B 200 46FGSG_04092§ CYP51A 274 126FGSG_11024§ CYP51C 218 95

Arabidopsis thaliana AT2G17330 CYP51A1 0 0AT1G11680 CYP51A2 0 0

Hordeum vulgare Published database (1) 0 0Hyaloperonospora arabidopsidis Published database (2) 0 0Rhizophagus irregularis Published database (3) 0 0Piriformospora indica Published database (4) 0 0Homo sapiens Published database (5) 0 0Fusarium cerealis isolate NRRL13721 JN416614{ CYP51A 190 83Fusarium austroamericanum

isolate NRRL28718JN416607{ CYP51A 117 50

Fusarium vorosii isolate 67C1 JN416608{ CYP51A 116 50Fusarium acaciae-mearnsii isolate NRRL26752 JN416603{ CYP51A 94 43

F. graminearum gene sequence file (fusarium_graminearum_ph-1_3_genes.fasta) was obtained from BroadInstitute (www.broadinstitute.org) for off-targets prediction. No off-targets were found. A. thaliana genesequence file (TAIR9_seq_20090619) was obtained from TAIR(www.arabidopsis.org/) for off-targets prediction.No off-targets were found including in the two CYP51 genes (6). H. vulgare gene sequence files (barley_High-Conf_genes_MIPS_23Mar12_CDSSeq. and barley_LowConf_genes_MIPS_23Mar12_CDSSeq.) were obtained fromMIPS (www.helmholtz-muenchen.de/en/ibis) for off-targets prediction. No off-targets were found. H. arabidop-sidis gene sequence file (Hyaloperonospora_arabidopsidis.HyaAraEmoy2_2.0.20.cdna.all) was obtained fromENSEMBL (ftp://ftp.ensemblgenomes.org/pub/protists/release-20/fasta/hyaloperonospora_arabidopsidis/cdna/) foroff-targets prediction. No off-targets were found. R. irregularis gene sequence file (Gloin1_GeneCatalog_tran-scripts_20120510.nt.fasta) was obtained from JGI (www.jgi.doe.gov) for off-targets prediction. No off-targetswere found. P. indica gene sequence file (Pirin1_GeneCatalog_transcripts_20111203.nt) was obtained fromJGI (www.jgi.doe.gov) for off-targets prediction. No off-targets were found. H. sapiens gene sequence files(Homo_sapiens.GRCh37.73.cdna.abinitio.fa.gz and Homo_sapiens.GRCh37.73.cdna.all.fa.gz -both files modifiedon 8/16/13) were obtained from ENSEMBL (ftp://ftp.ensembl.org/pub/release-73/fasta/homo_sapiens/cdna/) foroff-targets prediction. No off-targets were found.*Simulations were run using Si-Fi software (v3.1) for predicting off-targets prediction (http://labtools.ipk-gatersleben.de).†Number of 21-mer siRNA sequences with perfect match to the query sequence.‡Number of 21-mer siRNA sequences with perfect match to the query sequence that fulfill additional criteria forefficient RNAi (See Si-Fi software).§Target genes in F. graminearum.{GenBank Accession ID.

1. Mayer KF, et al.; International Barley Genome Sequencing Consortium (2012) A physical, genetic and functional sequence assembly of the barley genome. Nature 491(7426):711–716.2. Baxter L, et al. (2010) Signatures of adaptation to obligate biotrophy in the Hyaloperonospora arabidopsidis genome. Science 330(6010):1549–1551.3. Tisserant E, et al. (2012) The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont. New Phytol

193(3):755–769.4. Zuccaro A, et al. (2011) Endophytic life strategies decoded by genome and transcriptome analyses of the mutualistic root symbiont Piriformospora indica. PLoS Pathog 7(10):e1002290.5. Venter JC, et al. (2001) The sequence of the human genome. Science 291(5507):1304–1351.6. Kim HB, et al. (2005b) Arabidopsis cyp51 mutant shows postembryonic seedling lethality associated with lack of membrane integrity. Plant Physiol 138(4):2033–2047.

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Table S2. List of primers used in this study for PCR and quantitative RT- PCR studies

No. Primer name Primer sequence Application

1 CYP51A_F CGGTCCATTGACAATCCCCGT Cloning CYP51ACYP51A_R GCAGCAAACTCGGCAGTGAG Cloning CYP51A

2 CYP51B(AatII)_F GACGTCCAGCAAGTTTGACGAGTC Cloning CYP51B, dsRNA synthesisCYP51B(NcoI)_R CCATGGAGAGTTCATAAGGTGCTTCA Cloning CYP51B

3 CYP51C(BcuI)_F ACTAGTATTGGAAGCACCGTACAAT Cloning CYP51CCYP51C(SacI)_R GAGCTCCATTGGAGCAGTCATAAACAA Cloning CYP51C, dsRNA synthesis

4 CYP51B (HindIII)_F AAGCTTCAGCAAGTTTGACGAGTC Plant transformationCYP51C (XmaI)_R CCCGGGCATTGGAGCAGTCATAAACAA Plant transformation

5 T7_F TAATACGACTCACTATAGGGCAGCAAGTTTGACGAGTC dsRNA synthesisT7_R TAATACGACTCACTATAGGGCATTGGAGCAGTCATAAACAA dsRNA synthesis

6 CYP51A4_F CCTTTGGTGCCGGTAGACAT Real-time RT-PCRCYP51A4_R CCCATCGAATAAACGCAGGC Real-time RT-PCR

7 QCYP51B_F TCTACACCGTTCTCACTACTCC Real-time RT-PCRQCYP51B_R GCTTCTCTTGAAGTAATCGC Real-time RT-PCR

8 CYP51C2_F CGAGTCCCTGGCACTGAATG Real-time RT-PCRCYP51C2_R GCTCATCACCCCAAAACCGT Real-time RT-PCR

9 qpcrBtubulin_F ATCTCGAGCCCGGTACCATGG Real-time RT-PCRqpcrBtubulin_R CTCGGTGTAATGACCCTTGGCC Real-time RT-PCR

dsRNA, double-stranded RNA.

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