Nutrient profiling reveals potent inducers of trichothecene biosynthesis in Fusarium graminearum

Fungal Genetics and Biology 46 (2009) 604–613

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Nutrient profiling reveals potent inducers of trichothecene biosynthesis in Fusarium graminearum Donald M. Gardiner *, Kemal Kazan, John M. Manners CSIRO Plant Industry, Queensland Bioscience Precinct, 306 Carmody Road, St. Lucia, Queensland 4067, Australia

a r t i c l e

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Article history: Received 13 March 2009 Accepted 19 April 2009 Available online 3 May 2009 Keywords: Fusarium Deoxynivalenol DON Trichothecene Reporter Amine TRI5 Mycotoxin GFP Gibberella zeae

a b s t r a c t Fusarium head blight is one of the most important diseases of wheat worldwide due to crop losses and the contamination of grains with trichothecene mycotoxins. The biosynthesis of trichothecenes by Fusarium spp. is highest during infection, but relatively low levels are produced from saprophytic growth in axenic culture. A strain of Fusarium graminearum was constructed where the promoter from the TRI5 trichothecene biosynthesis gene was fused to GFP. Using this strain in large-scale nutrient profiling, a variety of amines were identified that significantly induce TRI5 expression. Analysis of trichothecene levels in the culture filtrates revealed accumulation of the toxin to over 1000 ppm in response to these inducers, levels either greater than or equivalent to those observed during infection. From this work, we propose that products of the arginine-polyamine biosynthetic pathway in plants may play a role in the induction of trichothecene biosynthesis during infection. Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved.

1. Introduction Fusarium head blight (FHB) is one of the most important diseases of wheat worldwide (Goswami and Kistler, 2004). Fusarium spp. that cause FHB on wheat are also capable of causing this disease on other small grain cereals such as barley and many also cause ear rot in maize (Bottalico and Perrone, 2002; Logrieco et al., 2002). Globally, members of the species complex containing Fusarium graminearum (teleomorph Gibberella zeae) are the most common cause of FHB (O’Donnell et al., 2004). Members of the F. graminearum species complex together with many other more distantly-related Fusarium species, such as Fusarium pseudograminearum and Fusarium culmorum, play significant roles in the incidence of head blight disease around the world (Bottalico and Perrone, 2002; Goswami and Kistler, 2004). Fusarium spp. produce a number of different trichothecene toxins, such as deoxynivalenol (DON) and nivalenol (NIV) produced by F. graminearum and T-2 toxin by F. sporotrichioides. Trichothecene biosynthesis has been well characterised in a number of Fusarium spp. and the different trichothecene products can be accounted for by genetic differences between producing strains (Desjardins, 2006). Despite the differing molecular structures of these toxins, the early steps of trichothecene biosyntheses are identical. The * Corresponding author. Fax: +61 (0)7 3214 2920. E-mail address: [email protected] (D.M. Gardiner).

genes encoding the biosynthetic enzymes are well conserved between species at the levels of gene sequence and genomic organisation, where they are located at three genomic locations in two clusters and a single lone gene (Alexander et al., 2004; Brown et al., 2004, 2003; Hohn et al., 1993; Kimura et al., 1998). The first committed step in the trichothecene biosynthesis pathway is the conversion of farnesyl pyrophosphate to trichodiene, catalysed by trichodiene synthase encoded by TRI5 (Hohn and Beremand, 1989). The expression of TRI5 and other genes in the gene clusters are correlated with the production of trichothecenes both in planta and in culture (Brown et al., 2004; Kimura et al., 2003). Mutants of F. graminearum unable to produce trichothecene toxins show reduced aggressiveness on wheat (Bai et al., 2002; Jansen et al., 2005; Proctor et al., 1995). Infection by F. graminearum occurs at the time of anthesis and is thought to be via the anthers, followed by colonisation of other parts of the floret before entering the vasculature and pith of the rachis from where it spreads up and down the head (reviewed in Trail et al., 2005). Although trichothecene non-producing mutants can cause initial infection, they are unable to spread throughout the head, and their growth is restricted at the rachis node between the rachis and floret (Jansen et al., 2005). Interestingly, although the toxins are produced during infection of barley and the development of FHB disease on this host (Boddu et al., 2006), trichothecene production does not appear to be necessary for spread of the infection throughout the barley head (Maier et al., 2006).

1087-1845/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2009.04.004

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In addition to the role of trichothecenes in the disease process, their presence in grain has important consequences for human and animal health. The concentration of these toxins in grain is coming under increasing scrutiny in both Europe and the United States of America, where legally enforceable limits on contamination by trichothecenes, such as DON, in food products are now in place (Anonymous, 2005; van Egmond et al., 2007). In mammalian systems, DON has been shown to act as a potent translational inhibitor and causes acute symptoms such as vomiting and feed refusal in pigs and also chronic symptoms such as growth retardation, reduced ovarian function and immunosuppression (Bennett and Klich, 2003; Rocha et al., 2005). Studies examining trichothecene synthesis by F. graminearum are often hampered by the low levels of toxin production in vitro. The highest production level of trichothecene toxins by F. graminearum appears to occur during the infection of host tissue. Autoclaved grain and axenic culture are often used for the analysis of trichothecene production, however, the levels of toxin observed in most in vitro culture conditions are orders of magnitude lower than those measured during the infection of live wheat heads and other tissues (Mudge et al., 2006; Voigt et al., 2007), suggesting that specific host signals are involved in eliciting toxin production in the pathogen. Identification of such signals is important for at least two reasons. Firstly, this can lead to a better understanding of the regulation of toxin biosynthesis genes in the pathogen. And secondly the genetic manipulation of host signals could help to reduce toxin accumulation and to increase pathogen resistance and improve food safety through the reduced presence of fungal toxins. In some instances, sequential nutritional regimes under highly controlled fermentation conditions have been reported to induce DON production in culture (Miller and Blackwell, 1986). There is also some evidence that hydrogen peroxide (H2O2), a product of the host oxidative burst elicited during plant infection, may enhance DON production in culture (Ponts et al., 2006) but the levels of DON induced by H2O2 in vitro are still very low when compared to those that accumulate in infected plants. Furthermore these authors also report that the induction of trichothecene production by H2O2 is chemotype dependent (Ponts et al., 2009). Jiao et al. (2008) report that carbon sources also have an effect on in vitro trichothecene production. However, the maximal toxin levels observed were less than 10 parts per million (ppm) in this latter study.

To find in vitro conditions that mimic the in planta environment with respect to the induction of trichothecene production, we have used nutritional profiling. The use of large-scale nutritional profiling has previously been applied to a comparison between wild type and mutant filamentous fungi (Druzhinina et al., 2006; Tanzer et al., 2003) and for studies of extracellular enzymatic activities in Trichoderma atroviride (Seidl et al., 2006). Here, we report on the application of a commercially available nutritional profiling platform in combination with a transgenic strain of F. graminearum that acts as a reporter for DON biosynthesis to assess the role different carbon and nitrogen sources play in the regulation of trichothecene synthesis. 2. Materials and methods 2.1. Fungal isolates and culturing

Hyg

TRI5 promoter

05T0020

05T0017

05T0015

05T0014

05T0005

11442 bp

05T0004

B

CS3005

A

05T0002

F. graminearum isolates CS3005 and PH-1 were used. Isolate CS3005 is an Australian isolate isolated in southern Queensland (Akinsanmi et al., 2006) and PH-1 is the isolate used for the F. graminearum genome sequence (Cuomo et al., 2007). Both isolates are defined as F. graminearum (O’Donnell et al., 2004) based on elongation factor 1 and phosphate permease sequence (data not shown). F. sporotrichioides isolate 10402002 (a kind gift from Ruth Dill-Macky, University of Minnesota) was used for experiments with this species. Isolates were grown under fluorescent lights (12 h/12 h light/dark) on synthetic nutrient agar for 7 days for spore production, before harvesting spores by flooding the plates with water. Liquid culture experiments were carried out in 96 well polypropylene plates in 100 lL defined media essentially as described by Correll et al. (1987) except 0.03% Phytagel (Sigma, St. Louis, MO, USA) was included and Fe(NH4)2(SO4)26H2O was omitted from the trace elements (Hamer et al., 2001). Per litre, the medium contained, 30 g sucrose, 2 g NaNO3, 1 g KH2PO4, 0.5 g MgSO47H2O, 0.5 g KCl, 10 mg FeSO47H2O, 0.03% Phytagel and 200 lL of trace element solution (per 100 mL, 5 g citric acid, 5 g ZnSO47H2O, 0.25 g CuSO45H2O, 50 mg MnSO4H2O, 50 mg H3BO3, 50 mg NaMoO42H2O) pH 6.5 with NaOH. For profiling carbon sources, the sucrose was omitted while for profiling nitrogen sources, NaNO3 was omitted. Nitrogen and carbon sources tested appear in Fig. 3, Supplementary Fig. 1 and the Supplementary Data file. Cultures were incubated in the dark without shaking at 28 °C for 7 days.

GFP

TRI5 promoter

HindIII (4774)

HindIII (2002)

HindIII (258)

14214 bp

TRI6

probe

TRI5

TRI5 promoter

TRI5 promoter

probe

HindIII (16216)

HindIII (2002)

HindIII (258)

5617 bp TRI6

1744 bp 17059 bp

Vector backbone GFP

2772 bp

Hyg

TRI5

Fig. 1. Insertion of TRI5–GFP at the TRI5 locus. (A) Vector, target locus and predicted resultant genomic locus. (B) Southern analysis of transformants for site specific integration of the construct. Genomic DNA was cut with HindIII. The position of the probe used is shown in (A).

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Nutrient profiling (phenotype microarrays, PM) plates were purchased from Biolog (Hayward, CA, USA). For carbon source profiling, PM1 and PM2 were used. For nitrogen source profiling, PM3 was used. Carbon sources in PM1 and PM2 are in the 5–50 mM range and nitrogen sources in PM3 are in the 2–20 mM range (Bochner et al., 2001). For subsequent experiments nitrogen sources were used at 5 mM unless stated otherwise. Fungi were inoculated into media at a final concentration of 1  104 conidia mL1. Growth (optical density) of fungal isolates was quantified using an iEMS Reader MF plate reader (Thermo Scientific, Waltham, MA, USA) at 595 nm. 2.2. Plant growth and infection Plants of the susceptible bread wheats (Triticum aestivum L.), cultivar Kennedy and Indian landrace INDIA44 (PBI 1190512) were grown in controlled environment rooms with photoperiod length of 14 h, 25 °C and 50% relative humidity at 500 mmol m2 s1. Night-time temperature was set at 15 °C, with 90% relative humidity. Three seeds were sown per pot (14 cm diameter, 17 cm height) containing autoclaved soil (50% peat and 50% sand by volume). FHB point inoculation was performed mid-anthesis as described by Akinsanmi et al. (2004). 2.3. Vector construction and fungal transformation The TRI5 promoter-GFP fusion vector was constructed by overlap PCR using primers to the TRI5 promoter (50 -GGCCTCGAGGCTCACGG

GCTACAGTGAAT-30 and 50 -TGCTCACCATGATGGCAAGGTTGTACTGG T-30 ) and eGFP (50 -TTGCCATCATGGTGAGCAAGGGCGAGGAG-30 and 50 -GGCCTCGAGCGCCTTAAGATACATTGATGAGTTT-30 ). The PCR construct consisted of 1.5 kb of TRI5 promoter amplified from F. graminearum CS3005, followed by eGFP and the SV40 terminator amplified from peGFP-N1 (Clontech, Mountain View, CA, USA). Individual fragments were amplified with Phusion DNA polymerase (Finnzymes, Espoo, Finland), gel purified, then fused using the same polymerase with the two external primers. The PCR product was digested with XhoI and ligated into the SalI site of pPZPhygHindX containing the hygromycin phosphotransferase gene under the Aspergillus nidulans TrpC promoter and terminator (Elliott and Howlett, 2006). One resultant clone was fully sequenced to check for potential errors introduced during PCR. No errors or differences between the TRI5 promoter sequences from isolate CS3005 and that of PH-1 were observed. Fungal protoplasts were generated from approximately 1  107 conidia germinated overnight in 200 mL of TB3 media (0.3% yeast extract, 0.3% casamino acids and 20% sucrose) at 20 °C with shaking at 150 rpm. Germlings were collected on miracloth and washed with 1 M sorbitol. Germlings were resuspended in 20 mL of 1 M sorbitol containing 5 U chitinase (Sigma–Aldrich, St. Louis, MO, USA), 500 mg Driselase (Sigma–Aldrich, St. Louis, MO, USA) and 200 mg lysing enzymes (Sigma–Aldrich, St. Louis, MO, USA), and incubated 1–2 h at 28 °C to release protoplasts. Protoplasts were collected by centrifugation at 2600g, 4 °C, washed three times with ice cold STC (20% sucrose, 50 mM Tris/HCl pH 8, 50 mM CaCl2). To a 200 lL aliquot of STC containing 2  107 protoplasts 10 lg circular

Fig. 2. In planta expression of GFP driven by the TRI5 promoter (transformant 05T0020). (A) Bright field of florets. The white material seen at the spikelet-rachis junction is fungal mycelia. The upper shown floret was the inoculated one close to the centre of the inflorescence. Hyphae can be seen emerging from the spikelet-rachis junction 4 dpi (bar = 5 mm). (B) Bright field close up (bar = 0.5 mm) of boxed region in (A), showing hyphae emerging from the rachis. (C) Fluorescent image of B showing strong GFP expression in the hyphae emerging from the rachis (bar = 0.5 mm). (D) Fluorescent view of a longitudinal hand section of the inside of the rachis two florets below inoculated floret 7 dpi (bar = 5 mm). (E) Fluorescent view of mycelia in the pith cavity of a longitudinal stem cross-section about 5 cm below stem head junction, 14 dpi (bar = 0.5 mm). (F) Fluorescent view of hyphae at the very front on the infection in the pith cavity about 5 cm below the stem head junction 14 dpi (bar = 0.5 mm). (A–C) cultivar INDIA44. (D– F) cultivar Kennedy.

D.M. Gardiner et al. / Fungal Genetics and Biology 46 (2009) 604–613 Time (days) 0 1 2 3 4 5 6 7 Negative Control Ammonia Nitrite Nitrate 0 Urea 20 Biuret L-Alanine 40 L-Arginine 60 L-Asparagine 80 L-Aspartic Acid 100 L-Cysteine L-Glutamic Acid L-Glutamine Glycine L-Histidine L-Isoleucine L-Leucine L-Lysine L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tryptophan L-Tyrosine L-Valine D-Alanine D-Asparagine D-Aspartic Acid D-Glutamic Acid D-Lysine D-Serine D-Valine L-Citrulline L-Homoserine L-Ornithine N-Acetyl-D,L-glutamic Acid N-Phthaloyl-L-Glutamic Acid L-Pyroglutamic Acid Hyroxylamine Methylamine N-Amylamine N-Butylamine Ethylamine Ethanolamine Ethylenediamine Putrescine Agmatine Histamine β-Phenylethylamine Tyramine Acetamide Formamide Glucuronamide D,L-Lactamide D-Glucosamine D-Galactosamine D-Mannosamine N-Acetyl-DGlucosamine N-Acetyl-DGalactosamine N-Acetyl-DMannosamine Adenine Adenosine Cytidine Cytosine Guanine Guanosine Thymine Thymidine Uracil Uridine Inosine Xanthine Xanthosine Uric Acid Alloxan Allantoin Parabanic Acid D,L-α-Amino-N-Butyric Acid γ-Amino-N-Butyric Acid ε-Amino-N-Caproic Acid D,L-α-Amino-Caprylic Acid δ-Amino-N-valeric Acid α-Amino-N-valeric Acid Ala-Asp Ala-Gln Ala-Glu Ala-Gly Ala-His Ala-Leu Ala-Thr Gly-Asn Gly-Gln Gly-Glu Gly-Met Met-Ala

% of max

0 20 40 60 80 100

% of max

Time (days) 0 1 2 3 4 5 6 7

Fig. 3. Growth and TRI5 induction during growth on 95 different nitrogen sources. Left panel (red) are optical density measurements over a 7 days time course. Right panel (green) are fluorescence values from the TRI5–GFP isolates less fluorescence measured from the untransformed parent strain CS3005 grown under the same conditions. All data points are the average of three biological replicates, expressed as a percentage of the maximum observed value during the time course. Full data files are available in the Supplementary Data File.

607

plasmid DNA was added, incubated for 20 min at room temperature, then 1 mL of 40% PEG4000, 10 mM Tris HCl pH 8, 50 mM CaCl2 was added, incubated for 20 min at room temperature and then 5 mL of TB3 media was added and the tubes incubated overnight at room temperature with gentle agitation. Three millilitre of the transformation mixture was embedded in 30 mL regeneration media (0.2% yeast extract, 0.2% casamino acids, 0.55% low melt agarose and 27% sucrose) and set in 14 cm Petri dishes, incubated overnight and then overlaid with 30 mL of regeneration media containing hygromycin (Roche, Penzberg, Germany) at 400 lg mL1. After 7–14 days of growth at room temperature in the dark, transformants were transferred to individual plates containing hygromycin (200 lg mL1), allowed to grow, single spored then checked for insertion of the construct by PCR and Southern hybridisation. The hybridisation probe was amplified using primers that flank the 50 end of the promoter region used for the reporter construct (50 -AGGTTTCAACCTCCGAGGAA-30 and 50 -TATCTGGCATGCCTGTCATC-30 ). 2.4. Toxin assays Deoxynivalenol was measured using the DON Plate Kit ELISA from Beacon Analytical Systems (Portland, Maine, USA) according to the manufacturer’s instructions, except an additional low concentration standard was used (0.02 ppm). The kit uses a monoclonal antibody (A. Trombley, Beacon Analytical Systems, personal communication) that is highly specific for the detection of DON and 15-ADON but not 3-ADON. Furthermore, the kit was cross-validated by GC–MS for quantification of DON using extracts from infected plant material. This revealed a high correlation (r = 0.88) with the ELISA assay but also suggested that the ELISA assay was underestimating DON content (slope = 0.7). Culture filtrates were initially diluted 1:25 in water, giving a detection range of 0.5– 12.5 ppm. Further sequential 1:10 dilutions were made to quantify toxin levels in samples that were above the detection range. T-2 toxin was measured using the Veratox T-2 ELISA kit from Neogen (Lansing, MI, USA) according to the manufacturer’s instructions, except an additional low concentration standard was used (0.0025 ppm). Culture filtrates were initially diluted 1:50 into 50% methanol to give a detection range of 0.125–2.5. Further dilutions were made as for DON detection. 2.5. Microscopy and fluorometry Hand sections and whole tissue mounts were examined using a Leica Microsystems MZ16FA fluorescent stereomicroscope (Wetzlar, Germany) fitted with GFP plus filter set. GFP fluorescence was quantified in microtitre plates using a Fluoroskan Ascent FL microplate fluorometer (Thermo Scientific, Waltham, MA, USA) using excitation at 485 nm and emission at 520 nm. All fluorescence values are the fluorescence of the TRI5–GFP isolate less the fluorescence measured from the parental strain grown under identical conditions. 2.6. RT-qPCR RNA extraction and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was carried out and data analysed as previously described (Mudge et al., 2006). 2.7. Putrescine quantification Putrescine quantification was carried out based on the method described by Marcé et al. (1995) with minor modifications. Plant material from wheat cultivar Kennedy was ground under liquid nitrogen in a mortar and pestle and a known mass (100 mg)

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was extracted in 600 lL of 5% ice cold perchloric acid. Extracts were centrifuged to pellet plant material and to 200 lL of the extract, 40 lL of 50 lM diaminoheptane was added, followed by 130 lL of a saturated sodium carbonate solution. Amines were derivatised with 260 lL of a 5 mg mL1 dansyl chloride solution in acetone (Sigma, St. Louise, MO, USA), incubated at 70 °C for 10 min before 65 lL of a 100 mg mL1 proline solution was added. To this solution, 325 lL of toluene was added, samples were mixed for 30 s and the phases allowed to separate. Two-hundred microlitres of the organic phase was dried under nitrogen and dissolved in 400 lL acetonitrile. Known volumes (and where required dilutions) of this solution was injected onto a Waters (Milford, MA, USA) HPLC apparatus (600 Controller, 717 Autosampler and 2475 Fluorescent detector) with a Phenomenex (Torrance, CA, USA) Luna 5l C18 100A column of 250 mm length and 4.6 mm internal diameter. The gradient used was identical to that described by Marcé et al. (1995). 2.8. Data analysis All data were collated into Microsoft Excel 2003. Heat maps were generated using the Heat Map Tool (VBA) downloadable from http://senorjosh.jblumenstock.com/archives/2003/04/heatmap_ tool_vba.shtml. The Heat Map Tool was modified to represent optical density and relative fluorescence by changing the Visual Basics code to represent a single colour on a linear scale between zero (no colour or black) and the maximum (red for optical density and green for fluorescence). Fluorescence values given are the fluorescent readout of the transformed strain less the background fluorescence of the untransformed isolate grown under identical conditions. Fluorescence is measured in the arbitrary relative fluorescence units (RFU). An estimate of total fluorescence over time was calculated by a crude integration of the fluorescence curve throughout the 7 days time course for each individual well. That is, each consecutive (daily) reading was assumed to be joined by a straight line, and the area under this curve calculated by summing the average of each consecutive time point. Graphs were generated in SigmaPlot version 10.

3. Results 3.1. Construction and testing of the TRI5–GFP isolate of F. graminearum The strategy adopted to identify conditions that would induce gene expression necessary for toxin production in the fungus was to first develop transgenic strains of F. graminearum that contained the reporter GFP gene regulated by the TRI5 promoter. The F. graminearum isolate CS3005 was transformed with the TRI5–GFP vector (Fig. 1A) and seven putative transformants were selected for further analysis. Six of these putative transformants showed integration at the TRI5 locus when tested by Southern blot analysis (Fig. 1B) and these were used in further experiments. The TRI5–GFP transformants showed no GFP fluorescence during culture on PDA whilst in autoclaved wheat grain culture they showed diffuse GFP throughout the cultures and strong GFP expression in the hyphae surrounding a limited number of grains (data not shown). Selected transformants were tested by point inoculations of flowering wheat cv Kennedy spikes. Transformants 05T0005, 05T0017 and 05T0020 were consistent in their expression of GFP during multiple independent infections. GFP expression in planta was strongest at sites of re-emergence from the rachis (Fig. 2A–C), inside the rachis (Fig. 2D) and during colonisation of the pith cavity of the stem (Fig. 2E), including at the very front of the infection (Fig. 2F). Weak fluorescence could also be seen in

the hyphae immediately in contact with the external surface of the spikelet-rachis junction (data not shown), although these were typically partially obscured by non-fluorescent hyphae, suggesting close physical contact with the plant is important for triggering TRI5 expression. The induction of TRI5 expression during infection and the infection patterns of the TRI5–GFP transformants generated in this study were consistent with previously published observations on the spatial location of trichothecene action (see Section 4) indicating that the TRI5–GFP strains may be used as a tool for in vitro screening of inducers of TRI5 expression and subsequently DON biosynthesis, as has been carried out here. 3.2. Nutrient profiling for the ability of F. graminearum to grow in various carbon and nitrogen sources Growth of F. graminearum in a variety of carbon and nitrogen sources was assessed using the phenotype microarray technology from Biolog. Amongst the carbon sources, D-fructose allowed the most growth but a number of other carbon sources, particularly the simple sugars, supported good growth (Supplementary Fig. 1). Similarly with nitrogen source profiling, F. graminearum was able to use a number of different compounds as sole nitrogen sources, including some that may be uncommon in nature such as D-enantiomers of amino acids (Fig. 3 and Supplementary Fig. 2). Both the Australian CS3005 and American PH-1 strains of F. graminearum were assayed for their ability to access nitrogen sources, and although generally these isolates were similar in their growth across the plate, some differences were observed between these strains. Most notable was the considerably poorer ability of the Australian CS3005 isolate to grow on biuret and methylamine (Supplementary Fig. 2). Neither isolate could grow on D-lysine, Hydroxylamine, ethylenediamine, N-acetyl-D-galactosamine, N-acetyl-D-mannosamine, thymine, thymidine, uracil, uridine or alloxan as a sole nitrogen source (Supplementary Fig. 2). 3.3. Nutrient profiling for induction of DON production in F. graminearum Using the TRI5–GFP reporter strain (05T0017) and parent (CS3005) as a control, the role of carbon and nitrogen sources were assessed for their ability to induce TRI5 promoter activity and GFP expression. To test carbon sources, the TRI5–GFP transformant 05T0017 was grown in the PM1 and PM2 carbon source plates but no fluorescence was observed with any of the 190 carbon sources tested in these plates (data not shown). Growth of the reporter strain in PM3 nitrogen source test plates revealed a key role for several specific nitrogen sources in regulating TRI5 expression. As shown in Fig. 3, even though many nitrogen sources supported growth of the fungus, most did not induce TRI5 expression. The exceptions were agmatine, putrescine, arginine and guanine, which promoted strong GFP fluorescence and to a lesser extent citrulline, ornithine, ethanolamine, b-phenylethylamine, tyramine and D-glucosamine. Expression of GFP seemed to appear after most of the growth of the fungus had occurred. For example in agmatine, fluorescence was maximal at 4 dpi, but at 3 dpi the well showed near maximal optical density and microscopically was essentially full of hyphae. The culture filtrate for 30 selected nitrogen sources was assayed for DON content at the end of the 7 days incubation. As shown in Fig. 4, the production of toxin (Fig. 4A) correlated (r = 0.82 ± 0.11) with the occurrence of GFP fluorescence (Fig. 4B). With some nitrogen sources, e.g. arginine, ornithine, putrescine and agmatine, the DON concentrations that were measured in the media were extremely high and either equal to, or greater than 1000 ppm. It should be noted the basal media used in the PM3 plates is very similar to that used in the two stage DON inducing media (Miller and Blackwell, 1986) except for the nitrogen source. Well A2 containing

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A

Ammonia Nitrate L-Alanine L-Arginine L-Cysteine L-Glutamine L-Histidine L-Leucine L-Lysine L-Methionine L-Phenylalanine L-Proline L-Tyrosine L-Valine L-Citrulline L-Ornithine Methylamine N-Amylamine N-Butylamine Ethanolamine Putrescine Agmatine β-Phenylethylamine Tyramine D-Glucosamine Adenine Adenosine Guanine Guanosine Gly-Met 0

500 1000 1500 DON concentration (ppm)

2000

B

Ammonia Nitrate L-Alanine L-Arginine L-Cysteine L-Glutamine L-Histidine L-Leucine L-Lysine L-Methionine L-Phenylalanine L-Proline L-Tyrosine L-Valine L-Citrulline L-Ornithine Methylamine N-Amylamine N-Butylamine Ethanolamine Putrescine Agmatine β-Phenylethylamine Tyramine D-Glucosamine Adenine Adenosine Guanine Guanosine Gly-Met

2500

0 20 40 60 80 100 Area under the GFP expression curve (RFUx time)

120

Fig. 4. (A) DON production in selected nitrogen sources from the phenotype microarray plates with the wild type isolate CS3005. Toxin was measured after 7 days culture. Data are the mean of three biological replicates. Error bars are standard errors of the mean. (B) Total area under the GFP expression curve for the same nitrogen sources as in (A). The correlation coefficient (r) between the DON concentration and total area under the expression curve was 0.82 (±0.11, standard error).

100 Glutamine Arginine Agmatine

80

60 RFU

ammonia as the nitrogen source is equivalent to the second stage media of Miller and Blackwell (1986), but relatively small amounts of DON were present in this media (44 ± 9 ppm) compared to other wells. The fluorescence read out for TRI5-GFP expression was in some circumstances dependent on the growth and biomass of the fungus and may not truly represent the ability of a compound to influence DON production by the fungus, particularly in cases where growth on these compounds was limited. This is exemplified by cultures using methylamine as a nitrogen source where growth was very limited and only a low fluorescence signal was obtained, however, analysis of media showed significant amounts of toxin was still produced (109 ± 9 ppm). The role of the polyamine agmatine in inducing trichothecene production appears to be a general phenomenon, as the production of T-2 toxin by F. sporotrichioides was also higher in agmatine (134 ± 14 ppm, n = 3) compared to glutamine (1.3 ± 0.3 ppm, n = 3) containing media. The experiments described above demonstrate a role for amines in promoting DON production when the amines are supplied as a sole nitrogen source at the start of culture. To further dissect whether the amines can stimulate DON production in the presence of an alternative nitrogen source, and in a growth stage independent fashion, the induction of TRI5–GFP was analysed after initiating growth in the non-inducing nitrogen source glutamine, followed by a further addition of glutamine, arginine or agmatine. As shown in Fig. 5, induction by arginine and agmatine occurred within 24 h of their application, but did not reach maximal levels until 48–72 h after addition of the inducing nitrogen source. The addition of additional glutamine did not have any effect on TRI5– GFP expression. Therefore, these experiments suggest that induction of DON production by amines is a dominant function and can over-ride alternative nitrogen sources like glutamine. Furthermore the induction is not growth stage dependent. This suggests that arginine and agmatine act as either cues for toxin synthesis or as preferred metabolic feedstocks.

40

20

0 0

24

48

72

96

Time (hours) post induction Fig. 5. Fluorescence from the TRI5–GFP isolate in cultures grown for 2 days in glutamine followed by either glutamine, arginine or agmatine addition to the media to a final equivalent concentration of 5 mM. Values are the mean relative fluorescence units (RFU) of 12 biological replicates, with error bars representing the standard error of the mean.

The identification of in vitro induction of TRI5–GFP by exogenous application of amines to pre-grown hyphae allowed further characterisation of the usefulness of the reporter. Strong correlation (r = 0.94) was observed between level of fluorescence from the TRI5–GFP strain and relative expression of the native TRI5 gene when these compounds were applied to the parental isolate

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3.4. Abolition and synergism of TRI5 induction by nitrogen source combinations

TRI5 expression relative to 18S rRNA

1e-2

1e-3

1e-4

1e-5

1e-6 Glutamine

Agmatine

In planta

Fig. 6. TRI5 gene expression relative to fungal 18S rRNA in culture 4 days postinoculation (dpi) in glutamine or agmatine as nitrogen sources and in planta. In planta expression is 4 dpi (the maximum level observed in a 7 days time course – data not shown). Error bars represent the standard error of the mean of four biological replicates.

(Supplementary Fig. 3). Quantification of DON in these samples also showed good correspondence with TRI5 gene expression, and the very low levels of DON detected in the early points of this time course, ruling out any possible cross-reactivity of amines such as agmatine with the monoclonal antibody used in ELISA quantification (Supplementary Fig. 3). Although DON production was strongly induced in vitro by the identified compounds, it is difficult to directly compare the levels of DON production in liquid culture to those produced in planta, due to the differences between these two growth conditions. However, on a fresh weight basis, DON levels in excess of 500 ppm in heavily infected heads have been observed (Mudge et al., 2006). Analysis of TRI5 gene expression relative to fungal reference gene in both culture and in planta provides an alternative comparison of fungal mycotoxin biosynthetic potential between these two growth conditions. Indeed, as shown in Fig. 6, the level of TRI5 gene expression, when the fungus was grown in agmatine as the nitrogen source, was very similar to that observed during infection. In addition, elevated levels of putrescine, one of the most potent inducers of DON production, could be detected in inoculated wheat heads (Supplementary Fig. 4), suggesting that these amine compounds might also have roles in inducing toxin biosynthesis in planta although further experiments are required for direct testing of this possibility.

The role of nitrogen in regulating the production of secondary metabolites is well documented in many other fungi. We were interested to see if there is a hierarchy of TRI5 activation when multiple nitrogen sources are present. Interactions between nitrogen sources and their effects on TRI5 transcript levels were investigated in media where two nitrogen sources were present during growth (Table 1). Nitrate and cysteine present in the media at the time of inoculation completely inhibited the induction of TRI5–GFP by nitrogen sources e.g. agmatine, putrescine, that when supplied alone, were highly inductive. Synergistic effects were also observed, particularly between methionine and agmatine. In this assay methionine alone also strongly supported TRI5 expression, whereas in the phenotype microarray plates methionine only supported intermediate levels of TRI5 expression and toxin production (Fig. 4). The reason for this is unknown. It should be noted that there was some concentration dependence observed with the inducing nitrogen sources. For example, the agmatine/control combination (where agmatine was at a concentration of 5 mM) showed considerably less fluorescence than the agmatine/agmatine combination (final concentration 10 mM). Concentration dependence was tested in hyphae initially grown in glutamine for 2 days followed by addition of either agmatine or arginine. At concentrations below 1 mM, these compounds no longer induced TRI5–GFP expression (data not shown). 4. Discussion The importance of trichothecene production by F. graminearum in the spread of the fungus from the site of inoculation during FHB of wheat is well described (Bai et al., 2002; Jansen et al., 2005; Proctor et al., 1995). Fungal strains where the TRI5 gene has been deleted and that are incapable of producing trichothecene toxins like DON are arrested at the rachis node and this is associated with modifications of the host cell wall, a potential defence response (Jansen et al., 2005). The expression of the TRI5 gene has not previously been studied at the histological level, but our results suggest that promoter reporter strains permit such analysis using the GFP reporter. The TRI5-promoter-GFP strains of the fungus developed here showed GFP production as the fungus entered the rachis from the inoculated floret and this is consistent with the role of DON proposed by Jansen et al. (2005) for fungal colonisation of the head. Our observations further indicated that TRI5 expression continues once the fungus is established in the rachis and as the fungus colonised the stem basipetally. Detection of TRI5 expression at the infection front in the stem pith cavity suggests that the production of toxin is one of the early events that occur during the physical interaction of leading hyphae at the infection front. Trichothecenes like deoxynivalenol have been shown to inhibit protein synthesis in plant cells and also induce programed cell death (Desmond

Table 1 Interaction of selected nitrogen sources in induction of TRI5–GFP. Cultures were grown in the presence of two nitrogen sources for 7 days with fluorescence measured daily. Data are total area under the expression curve expressed as a percentage of the maximal level observed (Methionine/Agmatine). The standard error of the mean shown in brackets (n = 4). The control is no nitrogen source.

Control (C) Ammonium (A) Nitrate (N) Agmatine (Ag) Putrescine (P) Glutamine (G) Methionine (M) Cysteine (Cy)

C

A

N

Ag

P

G

M

Cy

0(0) 0(0) 0(0) 4(1) 0(0) 0(0) 26(10) 0(0)

0(0) 0(0) 11(4) 6(3) 0(0) 29(4) 0(0)

0(0) 0(0) 0(0) 0(0) 0(0) 0(0)

22(12) 59(13) 36(5) 100(7) 4(4)

56(9) 32(11) 79(11) 4(3)

0(0) 73(6) 0(0)

35(4) 11(4)

0(0)

D.M. Gardiner et al. / Fungal Genetics and Biology 46 (2009) 604–613

611

Glutamate

Glutamine CPS

Ornithine

Carbamoyl-phosphate OCT Citrulline

CPS - carbamoyl phosphate synhtase OCT - ornithine carbamoyl transferase ASS - argininosuccinate synthase ASL - argininosuccinate lyase ADC - arginine decarboxylase ODC - ornithine decarboxylase ADA - agmatine deaminase CPA - carbamoylputrescine amidase AdoMetDC - adenosylmethionine decarboxylase SS - spermidine synthase

ASS Argininosuccinate ASL Arginine ADC

Arginase Ornithine

atin ase

Agmatine

Agm

ODC S-adenosylmethionine

ADA N-carbamoylputrescine CPA

AdoMetDC Putrescine S-adenosyl-methioninamine SS Spermidine

Fig. 7. Polyamine biosynthesis in plants. Pathway was constructed from multiple entries in MetaCyc (Caspi et al., 2006). Compounds identified that have TRI5 inducing activity have a grey background.

et al., 2008; Nishiuchi et al., 2006). Therefore, it has been hypothesised that DON production by F. graminearum may kill wheat cells during infection thereby promoting fungal colonisation (Nishiuchi et al., 2006) and our observations are consistent with its production in spreading infection hyphae. We are particularly interested in the cues that the fungus may respond to in the host to trigger the production of DON. To address this question, specific nutritional components that allowed maximal production of DON were sought and various amines and amino acids were identified as having strong inducing effects on TRI5 expression and DON production in culture. Using the T-2 toxin producing species F. sporotrichioides, we demonstrated that this induction by agmatine (and possibly other amines) extends to other trichothecene producing species. Many of the inducing compounds identified are biosynthetically-related as either products or precursors in polyamine biosynthetic pathways (Fig. 7). Polyamines are metabolites found in both plants and fungi and have frequently been associated with stress responses, particularly abiotic, in plants (Alcázar et al., 2006). Although the role of amines in biotic interactions is less well studied, altered expression of genes involved in polyamine biosynthesis is often observed during plant defence responses (Haggag and Abd-El-Kareem, 2009; Walters et al., 2002). During FHB, the level of free putrescine appears to increase at a time that coincides with DON production, giving support to the potential role of amines as the in planta signal for DON production. Therefore, it is tempting to speculate that F. graminearum has evolved to exploit this natural plant response for activating toxin production during colonisation of wheat, if indeed polyamines act as in planta signals for DON production. Both agmatine and arginine were shown to strongly induce TRI5 expression in cultures already supplied with growth-promoting nitrogen source glutamine, suggesting that these inducers may act more as either dominant molecular cues or preferred metabolic

feedstocks for toxin biosynthesis. However, the role of nitrogen containing compounds regulating DON production appears to be unrelated to precursor supply effects on DON biosynthesis because DON biosynthesis is not related metabolically to the inducing compounds, and indeed DON contains no nitrogen. Furthermore previous DON production or induction regimes appear extremely dependent upon complex manipulations (Miller et al., 1983) or in the case of oxidative stress inducing DON production, the growth stage at which the stress is applied (Ponts et al., 2006). Growth stage dependency was not observed with amine-mediated induction of DON biosynthesis. Taken together these studies suggest that amines act as specific cues for toxin synthesis. These cues could be generated either internally in the fungus as endogenous regulators or as exogenous signals derived from the host. Some significant interactions between nitrogen sources that may have relevance to the regulation of trichothecene biosynthesis during infection were also observed. For example, nitrate and cysteine abolished TRI5 induction by the amine and polyamine inducers whilst methionine had a synergistic effect on DON induction by other inducers such as putrescine and agmatine. Thus the regulation of trichothecene production in planta could be complex and determined as much by repressive metabolite levels as well as primary and synergistically inducing compounds. The identification of nitrate as a repressor of TRI5 induction is also strongly suggestive of a direct role for the global nitrogen regulatory system, common to many ascomycetous fungi (reviewed in Marzluf, 1997), in DON biosynthesis. Other authors have also noted that nitrate does not support DON production in F. culmorum (Covarelli et al., 2004). Nitrate in the media used for carbon source screening here could be responsible for the lack of induction observed by any carbon source. However, preliminary experiments using glutamine as a nitrogen source also failed to identify any GFP fluorescence (data not shown). Nitrogen supply also appears to be involved in the

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regulation of the biosynthesis of gibberellin, a virulence factor that also lacks nitrogen residues, in Gibberella fujikuroi. This fungus requires the global nitrogen regulator areA for normal gibberellin biosynthesis (Tudzynski et al., 1999). Previously a role for H2O2 in the induction of DON, but not NIV, has been characterised (Ponts et al., 2009, 2007). However, the absolute levels of DON induction using H2O2 appear low compared to, for example, the greater than 100-fold increase in production of DON with agmatine (compared to glutamine) observed here. Similarly the levels of fold gene expression (up to 20-fold) observed by Ponts et al. (2007) are relatively small compared to those changes seen here which were often greater than 1000-fold. Using H2O2 supplementation on the TRI5–GFP isolates we observed no induction of fluorescence (data not shown) which is probably a function of the lower sensitivity of the reporter assay compared to direct quantification of gene expression carried out by Ponts et al. (2007). Using a similar approach to that carried out here another group has identified a role for chemical stress in induction of trichothecene biosynthesis (Ochiai et al., 2007). These authors report the suppression of trichothecene production by NaCl and the induction by sub-lethal concentrations of triazole fungicides, but not other classes of fungicides. However, Ochiai et al. (2007) only reported relative inductions of up to about three-fold and use a different medium so it is difficult to compare the induction by amines to that of triazole fungicides. A similarity between the H2O2 and nitrogen containing metabolite induction of DON biosynthesis was the considerable lag time between application of the inducing compounds and induction of either the TRI5 gene expression or the detection of the toxin. In the experiment where the fungus was initially grown in glutamine and then agmatine or arginine added, the maximal fluorescence was not observed for another 48–72 h (Fig. 5). A similar time course was carried out using RT-qPCR measurement of TRI5 gene expression and the same delay in induction was observed (Supplementary Fig. 3), ruling out the possibility that a lag between reporter gene expression and GFP folding (and hence fluorescence) was the reason for the observed delay. In comparison, another gene involved in the pathogenicity of F. graminearum towards wheat, FGL1, is induced by 4 h after addition of wheat germ oil to the media (Voigt et al., 2005). This could indicate that the nitrogen compounds identified here may not be the primary inducing compounds of TRI5 gene expression but rather they induce the expression and synthesis of other endogenous regulators which in turn regulate TRI5 expression and DON biosynthesis. It is also possible that yet unknown breakdown products of the polyamine biosynthesis pathway are responsible for induction. The induction of mycotoxin production in Fusarium pathogens by commonly encountered metabolites is not without precedence. In the maize pathogen F. verticillioides, fumonisin production was observed to be dependent on the developmental state of the kernel, with the key inducer of fumonisin biosynthesis being the starch breakdown product amylopectin (Bluhm and Woloshuk, 2005). In this case, the inducers are clearly of host origin because, unlike the amines and polyamines, amylopectin is not known to be produced by fungi. Similarly, in the Aspergilli, oxylipids derived from plant membranes and lipid bodies are known to regulate aflatoxin production in plants (Burow et al., 1997; Tsitsigiannis and Keller, 2006). In Aspergilli, this is thought to be a receptor–ligand interaction, with the receptor likely to be a G-protein coupled receptor (Brodhagen and Keller, 2006). Considering the number of predicted genes for secondary metabolites in the genomes of filamentous fungi, the regulation of secondary metabolite production in these organisms is poorly understood. Indeed most known secondary metabolites from filamentous fungi are only known due to their contribution to colony pigmentation, roles in disease processes or pathologies

(e.g. aflatoxin and deoxynivalenol) or fortuitous discovery of bioactivities (e.g. penicillin). For example a genetic characterisation of all 15 polyketide synthase genes in F. graminearum did not identify any products that were not either already known, predicted based on homology, or easily phenotypically scorable (pigments) (Gaffoor et al., 2005). This type of approach is dependent on using culture conditions under which the metabolites are produced at a level high enough to detect. Coupling the use of reporter strains such as that used here, with large-scale nutritional screening or even more specific compound library screening, may allow the identification of defined conditions under which genes for the biosynthesis of secondary metabolites are optimally expressed. In turn this could provide a large number of previously unidentified compounds for use in medicine and agriculture. Acknowledgments We wish to thank Brendan Kidd and Anca Rusu for excellent technical assistance. We would also like to thank Sukumar Chakraborty and Louise Thatcher for critical reading of the manuscript. This work has been partly funded by Biogemma, France. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.fgb.2009.04.004. References Akinsanmi, O.A., Mitter, V., Simpfendorfer, S., Backhouse, D., Chakraborty, S., 2004. Identity and pathogenicity of Fusarium spp. isolated from wheat fields in Queensland and northern New South Wales. Aust. J. Agric. Res. 55, 97–107. Akinsanmi, O.A., Backhouse, D., Simpfendorfer, S., Chakraborty, S., 2006. Genetic diversity of Australian Fusarium graminearum and F. pseudograminearum. Plant Pathol. 55, 494–504. Alcázar, R., Marco, F., Cuevas, J.C., Patron, M., Ferrando, A., Carrasco, P., Tiburcio, A.F., Altabella, T., 2006. Involvement of polyamines in plant response to abiotic stress. Biotechnol. Lett. 28, 1867–1876. Alexander, N.J., McCormick, S.P., Larson, T.M., Jurgenson, J.E., 2004. Expression of Tri15 in Fusarium sporotrichioides. Curr. Genet. 45, 157–162. Anonymous, 2005. Commission Regulation (EC) No. 856/2005 of 6 June 2005 Amending Regulation (EC) No. 466/2001 as Regards Fusarium Toxins. Official Journal of the European Union. L143, 3-8. Bai, G.H., Desjardins, A.E., Plattner, R.D., 2002. Deoxynivalenol non-producing Fusarium graminearum causes initial infection, but does not cause disease spread in wheat spikes. Mycopathologia 153, 91–98. Bennett, J.W., Klich, M., 2003. Mycotoxins. Clin. Microbiol. Rev. 16, 497–516. Bluhm, B.H., Woloshuk, C.P., 2005. Amylopectin induces fumonisin B1 production by Fusarium verticillioides during colonization of maize kernals. Mol. Plant– Microbe Interact. 18, 1333–1339. Bochner, B.R., Gadzinski, P., Panomitros, E., 2001. Phenotype microarrays for highthroughput phenotypic testing and assay of gene function. Genome Res. 11, 1246–1255. Boddu, J., Cho, S., Kruger, W.M., Muehlbauer, G.J., 2006. Transcriptome analysis of the barley–Fusarium graminearum interaction. Mol. Plant-Microbe Interact. 19, 407–417. Bottalico, A., Perrone, G., 2002. Toxigenic Fusarium species and mycotoxins associated with head blight in small-grain cereals in Europe. Eur. J. Plant Pathol. 108, 611–624. Brodhagen, M., Keller, N.P., 2006. Signalling pathways connecting mycotoxin production and sporulation. Mol. Plant Pathol. 7, 285–301. Brown, D.W., Proctor, R.H., Dyer, R.B., Plattner, R.D., 2003. Characterization of a Fusarium 2-gene cluster involved in trichothecene C-8 modification. J. Agric. Food Chem. 51, 7936–7944. Brown, D.W., Dyer, R.B., McCormick, S.P., Kendra, D.F., Plattner, R.D., 2004. Functional demarcation of the Fusarium core trichothecene gene cluster. Fungal Genet. Biol. 41, 454–462. Burow, G.B., Nesbitt, T.C., Dunlap, J., Keller, N.P., 1997. Seed lipoxygenase products modulate Aspergillus mycotoxin biosynthesis. Mol. Plant–Microbe Interact. 10, 380–387. Caspi, R., Foerster, H., Fulcher, C.A., Hopkinson, R., Ingraham, J., Kaipa, P., Krummenacker, M., Paley, S., Pick, J., Rhee, S.Y., Tissier, C., Zhang, P., Karp, P.D., 2006. MetaCyc: a multiorganism database of metabolic pathways and enzymes. Nucleic Acids Res. 34, D511–516. Correll, J.C., Klittich, C.J.R., Leslie, J.F., 1987. Nitrate nonutilizing mutants of Fusarium oxysporum and their use in vegetative compatibility tests. Phytopathology 77, 1640–1646.

D.M. Gardiner et al. / Fungal Genetics and Biology 46 (2009) 604–613 Covarelli, L., Turner, A.S., Nicholson, P., 2004. Repression of deoxynivalenol accumulation and expression of Tri genes in Fusarium culmorum by fungicides in vitro. Plant Pathol. 53, 22–28. Cuomo, C.A., Guldener, U., Xu, J.-R., Trail, F., Turgeon, B.G., Di Pietro, A., Walton, J.D., Ma, L.-J., Baker, S.E., Rep, M., Adam, G., Antoniw, J., Baldwin, T., Calvo, S., Chang, Y.-L., DeCaprio, D., Gale, L.R., Gnerre, S., Goswami, R.S., Hammond-Kosack, K., Harris, L.J., Hilburn, K., Kennell, J.C., Kroken, S., Magnuson, J.K., Mannhaupt, G., Mauceli, E., Mewes, H.-W., Mitterbauer, R., Muehlbauer, G., Munsterkotter, M., Nelson, D., O’Donnell, K., Ouellet, T., Qi, W., Quesneville, H., Roncero, M.I.G., Seong, K.-Y., Tetko, I.V., Urban, M., Waalwijk, C., Ward, T.J., Yao, J., Birren, B.W., Kistler, H.C., 2007. The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science 317, 1400–1402. Desjardins, A.E., 2006. Fusarium Mycotoxins: Chemistry, Genetics, and Biology. The American Phytopathological Society, Minnesota. Desmond, O.J., Manners, J.M., Stephens, A.E., Maclean, D.J., Schenk, P.M., Gardiner, D.M., Munn, A.L., Kazan, K., 2008. The Fusarium mycotoxin deoxynivalenol elicits hydrogen peroxide production, programmed cell death and defence responses in wheat. Mol. Plant Pathol. 9, 435–445. Druzhinina, I.S., Schmoll, M., Seiboth, B., Kubicek, C.P., 2006. Global carbon utilization profiles of wild-type, mutant, and transformant strains of Hypocrea jecorina. Appl. Environ. Microbiol. 72, 2126–2133. Elliott, C.E., Howlett, B.J., 2006. Overexpression of a 3-ketoacyl-CoA thiolase in Leptosphaeria maculans causes reduced pathogenicity on Brassica napus. Mol. Plant–Microbe Interact. 19, 588–596. Gaffoor, I., Brown, D.W., Plattner, R., Proctor, R.H., Qi, W., Trail, F., 2005. Functional analysis of the polyketide synthase genes in the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum). Eukaryot. Cell. 4, 1926–1933. Goswami, R.S., Kistler, H.C., 2004. Heading for disaster: Fusarium graminearum on cereal crops. Mol. Plant Pathol. 5, 515–525. Haggag, W.M., Abd-El-Kareem, F., 2009. Methyl jasmonate stimulates polyamines biosynthesis and resistance against leaf rust in wheat plants. Arch. Phytopathol. Plant Protect. 42, 16–31. Hamer, L., Adachi, K., Montenegro-Chamorro, M.V., Tanzer, M.M., Mahanty, S.K., Lo, C., Tarpey, R.W., Skalchunes, A.R., Heiniger, R.W., Frank, S.A., Darveaux, B.A., Lampe, D.J., Slater, T.M., Ramamurthy, L., DeZwaan, T.M., Nelson, G.H., Shuster, J.R., Woessner, J., Hamer, J.E., 2001. Gene discovery and gene function assignment in filamentous fungi. Proc. Natl. Acad. Sci. USA 98, 5110–5115. Hohn, T.M., Beremand, P.D., 1989. Isolation and nucleotide sequence of a sesquiterpene cyclase gene from the trichothecene-producing fungus Fusarium sporotrichioides. Gene 79, 131–138. Hohn, T.M., McCormick, S.P., Desjardins, A.E., 1993. Evidence for a gene cluster involving trichothecene-pathway biosynthetic genes in Fusarium sporotrichioides. Curr. Genet. 24, 291–295. Jansen, C., von Wettstein, D., Schafer, W., Kogel, K.-H., Felk, A., Maier, F.J., 2005. Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum. Proc. Natl. Acad. Sci. USA 102, 16892–16897. Jiao, F., Kawakami, A., Nakajima, T., 2008. Effects of different carbon sources on trichothecene production and Tri gene expression by Fusarium graminearum in liquid culture. FEMS Microbiol. Lett. 285, 212–219. Kimura, M., Kaneko, I., Komiyama, M., Takatsuki, A., Koshino, H., Yoneyama, K., Yamaguchi, I., 1998. Trichothecene 3-O-acetyltransferase protects both the producing organism and transformed yeast from related mycotoxins. Cloning and characterization of Tri101. J. Biol. Chem. 273, 1654–1661. Kimura, M., Tokai, T., O’Donnell, K., Ward, T.J., Fujimura, M., Hamamoto, H., Shibata, T., Yamaguchi, I., 2003. The trichothecene biosynthesis gene cluster of Fusarium graminearum F15 contains a limited number of essential pathway genes and expressed non-essential genes. FEBS Lett. 539, 105–110. Logrieco, A., Mulè, G., Moretti, A., Bottalico, A., 2002. Toxigenic Fusarium species and mycotoxins associated with maize ear rot in Europe. Eur. J. Plant Pathol. 108, 597–609. Maier, F.J., Miedaner, T., Hadeler, B., Felk, A., Salomon, S., Lemmens, M., Kassner, H., Schafer, W., 2006. Involvement of trichothecenes in fusarioses of wheat, barley and maize evaluated by gene disruption of the trichodiene synthase (Tri5) gene in three field isolates of different chemotype and virulence. Mol. Plant Pathol. 7, 449–461. Marcé, M., Brown, D.S., Capell, T., Figueras, X., Tiburcio, A.F., 1995. Rapid highperformance liquid chromatographic method for the quantitation of

613

polyamines as their dansyl derivatives: application to plant and animal tissues. J. Chromatogr. B 666, 329–335. Marzluf, G.A., 1997. Genetic regulation of nitrogen metabolism in the fungi. Microbiol. Mol. Biol. Rev. 61, 17–32. Miller, J.D., Blackwell, B.A., 1986. Biosynthesis of 3-acetyldeoxynivalenol and other metabolites by Fusarium culmorum HLX 1503 in a stirred jar fermentor. Can. J. Bot. 64, 1–5. Miller, J.D., Taylor, A., Greenhalgh, R., 1983. Production of deoxynivalenol and related compounds in liquid culture by Fusarium graminearum. Can. J. Microbiol./Rev. Can. Microbiol. 29, 1171–1178. Mudge, A.M., Dill-Macky, R., Dong, Y., Gardiner, D.M., White, R.G., Manners, J.M., 2006. A role for the mycotoxin deoxynivalenol in stem colonisation during crown rot disease of wheat caused by Fusarium graminearum and Fusarium pseudograminearum. Physiol. Mol. Plant Pathol. 69, 73–85. Nishiuchi, T., Masuda, D., Nakashita, H., Ichimura, K., Shinozaki, K., Yoshida, S., Kimura, M., Yamaguchi, I., Yamaguchi, K., 2006. Fusarium phytotoxin trichothecenes have an elicitor-like activity in Arabidopsis thaliana, but the activity differed significantly among their molecular species. Mol. Plant– Microbe Interact. 19, 512–520. Ochiai, N., Tokai, T., Takahashi-Ando, N., Fujimura, M., Kimura, M., 2007. Genetically engineered Fusarium as a tool to evaluate the effects of environmental factors on initiation of trichothecene biosynthesis. FEMS Microbiol. Lett. 275, 53–61. O’Donnell, K., Ward, T.J., Geiser, D.M., Kistler, H.C., Aoki, T., 2004. Genealogical concordance between the mating type locus and seven other nuclear genes supports formal recognition of nine phylogenetically distinct species within the Fusarium graminearum clade. Fungal Genet. Biol. 41, 600–623. Ponts, N., Pinson-Gadais, L., Verdal-Bonnin, M.N., Barreau, C., Richard-Forget, F., 2006. Accumulation of deoxynivalenol and its 15-acetylated form is significantly modulated by oxidative stress in liquid cultures of Fusarium graminearum. FEMS Microbiol. Lett. 258, 102–107. Ponts, N., Pinson-Gadais, L., Barreau, C., Richard-Forget, F., Ouellet, T., 2007. Exogenous H2O2 and catalase treatments interfere with Tri genes expression in liquid cultures of Fusarium graminearum. FEBS Lett. 581, 443–447. Ponts, N., Couedelo, L., Pinson-Gadais, L., Verdal-Bonnin, M.-N., Barreau, C., RichardForget, F., 2009. Fusarium response to oxidative stress by H2O2 is trichothecene chemotype-dependent. FEMS Microbiol. Lett. 293, 255–262. Proctor, R.H., Hohn, T.M., McCormick, S.P., 1995. Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene. Mol. Plant–Microbe Interact. 8, 593–601. Rocha, O., Ansari, K., Doohan, F.M., 2005. Effects of trichothecene mycotoxins on eukaryotic cells: a review. Food Addit. Contam. 22, 369–378. Seidl, V., Druzhinina, I.S., Kubicek, C.P., 2006. A screening system for carbon sources enhancing beta-N-acetylglucosaminidase formation in Hypocrea atroviridis (Trichoderma atroviride). Microbiology 152, 2003–2012. Tanzer, M.M., Arst, H.N., Skalchunes, A.R., Coffin, M., Darveaux, B.A., Heiniger, R.W., Shuster, J.R., 2003. Global nutritional profiling for mutant and chemical modeof-action analysis in filamentous fungi. Funct. Integr. Genom. 3, 160–170. Trail, F., Gaffoor, I., Guenther, J.C., Hallen, H.E., 2005. Using genomics to understand the disease cycle of the Fusarium head blight fungus, Gibberella zeae (anamorph Fusarium graminearum). Can. J. Plant Pathol. 27, 486–498. Tsitsigiannis, D.I., Keller, N.P., 2006. Oxylipins act as determinants of natural product biosynthesis and seed colonization in Aspergillus nidulans. Mol. Microbiol. 59, 882–892. Tudzynski, B., Homann, V., Feng, B., Marzluf, G.A., 1999. Isolation, characterization and disruption of the areA nitrogen regulatory gene of Gibberella fujikuroi. Mol. Gen. Genet. 261, 106–114. van Egmond, H., Schothorst, R., Jonker, M., 2007. Regulations relating to mycotoxins in food. Anal. Bioanal. Chem. 389, 147–157. Voigt, C.A., Schäfer, W., Salomon, S., 2005. A secreted lipase of Fusarium graminearum is a virulence factor required for infection of cereals. Plant J. 42, 364–375. Voigt, C.A., Scheidt, B., Gácser, A., Kassner, H., Lieberei, R., Schäfer, W., Salomon, S., 2007. Enhanced mycotoxin production of a lipase-deficient Fusarium graminearum mutant correlates to toxin-related gene expression. Eur. J. Plant Pathol. 117, 1–12. Walters, D., Cowley, T., Mitchell, A., 2002. Methyl jasmonate alters polyamine metabolism and induces systemic protection against powdery mildew infection in barley seedlings. J. Exp. Bot. 53, 747–756.