Supplemental Materials and Methods
Authors and contact information
Stephanie Walter1, Josephine M. Brennan1, Chanemougasoundharam. Arunachalam1, Khairul I. Ansari1, Xuejun Hu1, Mojibur R. Khan1, Friederike Trognitz2, Bodo Trognitz2, Damian Egan1 and Fiona M. Doohan1
1Molecular Plant-Microbe Interactions Laboratory, School of Biology and Environmental Sciences, College of Life Sciences, University College Dublin, Belfield, Dublin 4, Ireland, 2Austrian Research Centers GmbH (ARC), Biogenetics-Natural Resources Division, Bioresources Department, 2444 Seibersdorf, Donau City Str. 1, 1220 Vienna, Austria. *Author for correspondence (Phone: +353-1-7162248; E-mail: )
Plant Material
‘Frontana’ carries a major QTL on chromosome 3A and other minor QTLs associated with FHB resistance (Steiner et al. 2004). Cultivar (cv.) CM82036 carries a major QTL on chromosome 3BS associated with FHB resistance and DON tolerance (i.e. Fhb1; syn. Qfhs.ndsu-3BS). Remus is susceptible to FHB and intolerant of DON (Buerstmayr et al. 2003; Lemmens et al. 2005). The 14 DH lines used in this study had or had not inherited this QTL (see Supplemental Table S2). Phenotyping of spikelets and genotyping of these lines confirmed that the DH lines segregated for this QTL and associated DON tolerance (Buerstmayr et al. 2003; Lemmens et al. 2005). Genotyping of the DH lines was performed using RFLPs, AFLPS, SSRs, storage proteins and the molecular markers Barc75, Gwm389, Gwm1034, Gwm533, Barc133, Gwm493, Barc141, Barc40, Gwm304, Gwm293, Barc117, Barc186 and Barc1 (Buerstmayr et al. 2003; Lemmens et al. 2005).
For production of callus of the cv. CM82036 x cv. Remus DH line E2-24T (that inherited Fhb1 from cv. CM82036) (Buerstmayr et al. 2003) plants were grown in the greenhouse and immature embryos were cultivated on solid Murashige and Skoog (MS) medium (Sigma-Aldrich) including 2.8g l-1 Gelrite (Duchefa Biochemie), 20g l-1 sucrose (Sigma-Aldrich), 2mg l-1 2,4-dichloro-phenoxyacetic acid (Sigma-Aldrich) for 4 weeks at 27°C under dark conditions.
Seeds of wheat cv. Chinese Spring (accession no. Cltr 14108) and its five chromosome 3BS deletion mutants derivatives TA4524L1, TA4524L4, TA4524L2, TA4524L7 and TA4524L3 were obtained from the Wheat Genetics Resource Centre of Kansas State University (http://www.k-state.edu/wgrc/). For these deletion mutants, the fragment length of the retained part of the distal region of chromosome 3BS is 0.33, 0.55, 0.56, 0.75 and 0.87, respectively (Endo and Gill 1996; Liu and Anderson 2003). The wheat QTL Fhb1 was fine-mapped to the chromosome bin 3BS 0.78-0.87 (Liu and Anderson 2003). Seeds of a 3BS Chinese Spring ditelosomic line (Dt3BS) (accession no. GSTR36) and of a 3B nullisomic-tetrasomic line (N3B-T3D) (accession no. GSTR73) were obtained from USDA-ARS National Germplasm Resources Laboratory (http://www.ars-grin.gov/npgs/). The ditelosomic line lacks 3BS and in the nullisomic-tetrasomic line chromosome 3B has been replaced with 3D (Endo and Gill 1996).
Adult plant DON tolerance trials
DON tolerance trials were conducted under both glasshouse and contained environment conditions [with a 16h photoperiod, 75% relative humidity and a day/night temperature of 20/12°C]. Plants were grown (2 per pot) as described by Doohan et al. (1999) and each of the three florets of four central spikelets per head were treated with 15ml of either 5mg ml-1 0.2% v v-1 Tween20 DON (Sigma-Aldrich) or 0.2% v v-1 Tween20 (controls) at mid-anthesis, applied between the palea and the lemma, as described by Lemmens et al. (2005) and Ansari et al. (2007). Treated spikelets were harvested at 4h post-treatment, flash-frozen in liquid N2, freeze-dried and stored at -70°C prior to RNA extraction (Ansari et al. 2007). Each DON tolerance trial included four heads (one per plant) per treatment per wheat cultivar and was conducted twice.
Construction of a wheat cDNA library from DON-treated roots
Germinating seeds of ‘Frontana’ were treated with water or DON (20mg ml-1) (Sigma-Aldrich) and harvested 24h post-treatment, as previously described by Ansari et al. (2007). The experiment was conducted twice (12 seedlings per plate and two plates per experiment). Replicate samples from both experiments were bulked and 1mg of extracted RNA (Ansari et al. 2007) was used to construct a phagemid lTriplEx2 vector library using the SMARTTM cDNA Library Construction kit (Clontech). Escherichia coli strain BM25.8 was used to convert lTriplEx2 to pTriplEx2 plasmid clones. The library was normalized as described by Reddy et al. (2002) and E. coli strain DH10B was transformed with the plasmid DNA from the normalized library and selected on Luria Bertani (LB) agar supplemented with 100mg l-1 ampicillin. The resulting 3066 clones were picked and sub-cultured overnight in LB broth supplemented with 100mg l -1 ampicillin (37°C, 180rpm); glycerol was added (15% v v-1) and cultures were stored at -70°C.
Construction of a suppressive subtractive hybridisation (SSH) cDNA library from wheat callus following treatment with F. graminearum culture filtrate
Cultures of the DON-producing Fusarium graminearum strain IFA65 were produced by inoculating MS medium (250ml) with a mycelial plug (1cm diam., derived from a 7-day-old SNA culture). After 5 days cultures were centrifuged (10000g, 10min) and three ml of collected culture filtrate or of fresh MS medium (control) was used to inoculate calli of the resistant wheat DH line E2-24T, a progeny of cv. CM82036 x cv. Remus carrying Fhb1 from CM82036. Callus was harvested 10h post-inoculation, flash frozen and stored at -70°C. Total RNA was extracted using the RNeasy Plant Mini kit and RLT buffer (Qiagen). The SSH library was constructed using the BD PCR-Select™ cDNA Subtraction Kit (Clontech). Wheat callus inoculated with F. graminearum culture filtrate was used as the ‘tester’ and wheat callus inoculated with MS medium as the ‘driver’. PCR products from differentially expressed cDNAs were cloned into the pCR®4-TOPO cloning vector using the TOPO® Cloning Kit (Invitrogen). The product was transformed into E. coli strain TOP10 (Invitrogen) and the resulting 297 clones were selected and stored as described above for the wheat root cDNA library.
Sequencing, sequence processing, sequence annotation and phylogenetic analysis
The SSH clones were sequenced by the Austrian Research Centers GmbH (Seibersdorf, Austria). Clones from the ‘Frontana’ cDNA library were sequenced by MWG Biotech (Ebersberg, Germany). Vector sequences were removed from the sequencing outputs after BLAST search against the UniVec database (NCBI, Bethesda, MD, USA; release 4.0). Repeat masking, clustering and assembling of revealed 3′ end EST sequences were performed with the program EGassembler (Masoudi-Nejad et al. 2006). For transcript annotation and further sequence analyses one representative sequence (the longest) of a contig was selected.
EST sequences were queried by BLASTn search against nucleotides sequences in the TIGR Plant Transcript Assembly (TA) database (release 2.0) (http://plantta.tigr.org/). Protein functions were ascribed based on those described for the associated homologs in the database and/or based on those described for homologs identified by BLASTx analyses against the UniProt Knowledgebase using the WU BLAST tool (release 2.0MP-WashU) of the European Bioinformatics Institute (http://www.ebi.ac.uk/blast2/index.html). Based on putative protein functions, transcripts were allocated to functional categories according to the Munich Information Centre for Protein Sequences (MIPS) Functional Catalogue (FunCat) scheme (release 2.1) (http://mips.gsf.de). The EST/TA homologs identified for the clones listed in Table I were used to identify the corresponding Arabidopsis protein homolog using the NCBI Arabidopsis thaliana BLASTx tool (release 2.2.17) (http://www.ncbi.nlm.nih.gov/blast/). Transcripts were also BLASTn queried against the database of GenBank+EMBL+DDBJ EST sequences NCBI (release 2.2.17) (http://www.ncbi.nlm.nih.gov/blast/) in order to determine if sequence homologs from Fusarium-infected or Fusarium culture filtrate-treated wheat or barley had previously been described (> 90% identity and E ≤ 1e-48). Transcript homology to ESTs previously mapped to the wheat cv. Chinese Spring 3BS chromosome bin 0.78 – 1.00 (http://wheat.pw.usda.gov/wEST/binmaps/wheat3 _ rice.html) was determined by BLAST analysis.
A phylogenetic tree was constructed with aligned plant cytochrome P450 (CYP) protein sequences from this study and the Arabidopsis genome using MEGA version 4 software (http://www.megasoftware.net/index.html) (Tamura et al. 2007) based on the neighbour-joining method with the following parameters: p-distance model, pairwise deletion, and bootstrap (1000 replicates; random seed).
Microarray analysis: Minimum information about a Microarray Experiment (MIAME)
Goal of the experiment:
Identification of mycotoxin-responsive transcripts associated with the quantitative trait locus (QTL) Fhb1 (on the short arm of chromosome 3B) that confers DON tolerance to cv. CM82036.
Brief description of the experiment:
Deoxynivalenol (DON) is a mycotoxin produced by Fusarium fungi and it facilitates fungal pathogenesis of wheat heads and the development of Fusarium head blight disease. Wheat genotypes vary in their response to this toxin; unlike susceptible cultivars, tolerant cultivars do not display the premature bleaching of spikelets in response to toxin treatment. This trait is associated with a quantitative trait locus, Fhb1, located on the short arm of chromosome 3B. Gene expression profiling (conducted using custom-made 3K wheat cDNA microarray chips) identified transcripts that accumulate in spikelets of the DON-tolerant wheat cultivar CM82036 as an early response to toxin treatment (at 4h post-treatment). This included transcripts encoding proteins involved in energy production, metabolism, transport, cell rescue, defence and virulence and some of unknown function. Gene expression profiling of DON-treated spikelets of progeny derived from a cross between this DON-tolerant and a DON-susceptible cultivar (Remus) discriminated 10 toxin-responsive transcripts associated with Fhb1. These transcripts are most likely not encoded by genes exclusively located within Fhb1, based on their lack of homology to ESTs previously mapped to this QTL and the results of gene mapping studies. Proteins encoded by these DON-responsive Fhb1-associated transcripts are involved in the protection against oxidative stress and programmed cell death, and in the biotransformation and compartmentalization of endogenous and/or exogenous metabolites. We discuss how classical detoxification pathways, alleviation of oxidative stress and promotion of cell survival might contribute to DON tolerance.
Keywords:
Spotted_ss_PCR_amplicon_features (MGED identifier MO_921), compound treatment design (MGED identifier MO_555), individual genetic characteristics design (MGED identifier: MO_527).
I. Experimental factors:
Comparative effect of DON on wheat lines that inherited the Fusarium head blight resistance-/DON tolerance-associated quantitative trait locus Fhb1 from cv CM82036 [individual_genetic_characteristics_design (MGED identifier: MO_527)].
II. Experimental design:
Microarray analysis was conducted to compare gene expression DON-treated (4h, 5mg DON ml-1 0.2% Tween20) wheat spikelets of DH lines inheriting QTL Fhb1 vs. DH lines that did not inherit this QTL [individual_genetic_characteristics_design (MGED identifier: MO_527)]. A bulk RNA sample from 4 DH lines that did not contain Fhb1 was compared with two different bulk RNA samples from 4 DH lines that contained Fhb1 (comparisons A and B). See Supplemental Table S3 for experimental design used for microarray analyses.
Quality related indicators: biological replicates: 2; technical replicates (dye swaps): 1.
Links: Accession numbers for genes of interest are listed in Tables I.
III. Samples used, extract preparation and labelling:
1. Plant samples used, extract preparation and labelling
1.1. Biosource properties
Plant species: Triticum aestivum
Germplasm: See supplemental Table S2
Starting material: Seed, Source: Dr. Hermann Buerstmayr (IFA-Tulln,
Austria).
Developmental stage: 7.02-anthesis half-way; GRO:0007103
Organism part (tissue): Wheat head spikelets (4 central spikelets)
1.2. Biomaterial manipulations
Growth substrate: John Innes No. 2 Potting-on Compost (Westland Horticulture Ltd., Dungannon, Ireland) (The compost contains a balanced fertilizer complete with trace elements and has a pH of 6.0-7.0.)
Growth environment: Conventional greenhouse without artificial light
Light conditions: Sun light
Watering conditions: Daily watering
Density of plants: 2 plants per 7’’ pot
Pot size: 7’’ plastic pots (National Agrochemical Distributors Ltd., Lusk, Ireland)
Experimental factor value for each experimental factor and sample:
Time: 4h post treatment
Treatment: 5mg deoxynivalenol ml-1 0.2% (v v-1) Tween20
1.3. Extraction method
Extraction source: Freeze-dried
Extraction method: Manual (Ansari et al. 2007)
1.4. RNA labelling procedure
Labelled first-strand cDNA was prepared from 8mg RNA using the SuperscriptTM Direct cDNA Labelling System (Invitrogen) and 25nmol of Cy3-dCTP or Cy5-dCTP (Amersham Biosciences) according to the manufacturer’s instructions. The dye-labelled cDNA was purified from RNA strands and unincorporated nucleotides using the SuperscriptTM Direct cDNA Labelling System Purification Module (Invitrogen). Then, Cy3- and Cy5-labeled cDNAs were pooled together and concentrated using a Microcon YM-30 filter device (Millipore) to obtain 11.5ml of labelled cDNA.
IV. Hybridization procedures and parameters:
Microarray slides were incubated for 30min at 42°C in sodium borohydride [0.25% (w v-1) in 2x SSC] (Sigma-Aldrich) to reduce the auto-fluorescence of the spots (Martinez et al. 2003). Subsequently slides were washed twice with 1x SSC for 5min at room temperature, rinsed three times with 0.1x SSC and washed once with 0.1% N-lauroylsarcosine (w v-1) (Sigma-Aldrich) in 5x SSC. The slides were blocked in 1.79% (w v-1) succinic anhydride/ 4.48% (w v-1) sodium borate pH 8/ 10.37M 1-methyl-2-pyrrolidinone (Sigma-Aldrich) for 15min in the dark. After 5 washes with sterile ultra pure water, the slides were immersed in 95°C water for 1min and immediately placed in pre-cooled (-20°C) 95% ethanol until being dried under a stream of compressed air.
For hybridization 1 volume of 4x hybridization buffer (GE Healthcare Ltd) and 2 volumes of formamide (Sigma-Aldrich) were added per volume of labelled cDNA and the hybridization probe was loaded onto the blocked microarray slide under a glass lifterslip (Erie Scientific Co.). Microarray hybridization was performed overnight at 42°C in a sealed hybridization chamber (Camlab Ltd). Following hybridization, the microarray slides were washed once in 1x SSC (0.15M sodium chloride, 0.015M sodium citrate)/ 0.1% (w v-1) SDS for 15 min at 50°C, 0.1x SSC/ 0.1% (w v-1) SDS for 10min at room temperature, then twice in 0.2x SSC for 5min at room temperature. Finally the slides were briefly rinsed with sterile ultra pure water and dried using compressed air.
V. Measurement data and specifications:
Image scanning hardware:
Microarray slides were scanned using a GenePix 4000B scanner (Axon Instruments). The microarray slides were scanned with dual lasers at wavelengths of 532nm and 635nm to excite Cy3 and Cy5 fluorescence emittance, respectively. The photo multiplier tube values for both wavelengths were adjusted to capture a similar signal for each wavelength channel.
Image scanning software:
Genepix 5.0 software (Axon Instruments) was used to quantify spot intensities and integrate the gene array list file in order to identify spots.
Normalization, transformation and data selection procedures and parameters:
For statistical analysis the data were imported into the R software (http://www.R-project.org) and analyzed using the Bioconductor (http://www.bioconductor.org/pub/docs/mogr/) package LIMMA (Smyth 2004). The data within arrays were normalized by loess normalization (Yang et al. 2001) and then across replicate arrays using scale normalization (Yang et al. 2002). The linear modelling and empirical Bayes methods implemented in LIMMA were applied to identify differential expression. Genes with significantly differential expression (P ≤ 0.05) were selected after the P values were adjusted for multiple testing using the false discovery rate method of Benjamini and Hochberg (1995). Microarray data presented is the inverse of the M value (log2-fold change) of normalized data.