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J Exp Bot. Oct 2009; 60(14): 4105–4114.
Published online Sep 4, 2009. doi:  10.1093/jxb/erp240
PMCID: PMC2755027

Insect peptide metchnikowin confers on barley a selective capacity for resistance to fungal ascomycetes pathogens

Abstract

The potential of metchnikowin, a 26-amino acid residue proline-rich antimicrobial peptide synthesized in the fat body of Drosophila melanogaster was explored to engineer disease resistance in barley against devastating fungal plant pathogens. The synthetic peptide caused strong in vitro growth inhibition (IC50 value ~1 μM) of the pathogenic fungus Fusarium graminearum. Transgenic barley expressing the metchnikowin gene in its 52-amino acid pre-pro-peptide form under the control of the inducible mannopine synthase (mas) gene promoter from the Ti plasmid of Agrobacterium tumefaciens displayed enhanced resistance to powdery mildew as well as Fusarium head blight and root rot. In response to these pathogens, metchnikowin accumulated in plant apoplastic space, specifying that the insect signal peptide is functional in monocotyledons. In vitro and in vivo tests revealed that the peptide is markedly effective against fungal pathogens of the phylum Ascomycota but, clearly, less active against Basidiomycota fungi. Importantly, germination of the mutualistic basidiomycete mycorrhizal fungus Piriformospora indica was affected only at concentrations beyond 50 μM. These results suggest that antifungal peptides from insects are a valuable source for crop plant improvements and their differential activities toward different phyla of fungi denote a capacity for insect peptides to be used as selective measures on specific plant diseases.

Keywords: Antimicrobial peptides, ascomycete fungi, barley, disease resistance, metchnikowin

Introduction

Plant pathogens and pests account for severe and increasing crop losses worldwide, amounting to $30–$50 billion annually (Osusky et al., 2000; Cook, 2006). One of the crucial problems in modern plant production is the rapid adaptation of fungal pathogens to fungicides that, increasingly, imposes costs of chemical plant protection. On the other hand, there is growing public concern about the hazards of agrochemicals on environment and human health. Consequently, there is an urgent need to explore innovative approaches for plant protection (Moffat, 2001). Tuning of plant defence responses to microbial pathogens for rendering them disease-resistant has been recognized as a superior strategy in sustainable agriculture (Kogel and Langen, 2005).

Plants, insects, amphibians, and mammals including human defend themselves against pathogenic microorganisms using peptides or small proteins (Zasloff, 2002; Brogden, 2005). Numerous experiments have provided examples for improving the disease resistance of crops by incorporating antimicrobial peptides, thus providing novel potential traits for further breeding efforts (Marcos et al., 2008). Among these, transgenic expression of antimicrobial peptides from insects has emerged as a successful tool to create crops resistant to biotic stress (Vilcinskas and Gross, 2005). Antimicrobial peptides usually fall into two groups, distinguished by their primary and secondary structures. One group contains basic proteins, called defensins, with a structure containing an α-helix and two to five disulphide bonds connecting the helix to β-strands (Mygind et al., 2005; Selsted and Ouellete, 2005; Langen et al., 2006). Many of the defensin type antimicrobial peptides seem to interact with the plasma membrane and cause its permeabilization by either forming pores or blocking Ca2+ channels and, thus, disturbing the Ca2+ gradient essential for cellular activities. The other group of antimicrobial peptides is represented by the 52-amino acid residue proline-rich peptide metchnikowin (Mtk), identified as an immune inducible peptide in the fruit fly Drosophila melanogaster (Levashina et al., 1995), Pyrrhocoricin, and Drosocin with a less well-defined straight chain secondary structure (Brogden, 2005).

In a previous communication, it was described that tobacco plants transgenic for gallerimycin, an antifungal peptide from the greater wax moth Galleria mellonella, which shares structural similarity with plant defensins (Schuhmann et al., 2003), showed resistance to fungal pathogens Golovinomyces cichoracearum and Sclerotinia minor (Langen et al., 2006). In the present study, the potential of the proline-rich antifungal peptide Metchnikowin (Mtk) has been explored to engineer, in barley, disease resistance against microbial plant pathogens. Mtk was chosen, because the synthetic peptide could inhibit in vitro growth of the agronomically important pathogenic fungi Fusarium graminearum and F. culmorum, at low concentrations. These fungi not only cause considerable crop losses, but also affect crop quality by the secretion of mycotoxins into infected grains of barley, wheat, and other cereals. By means of in vitro and in vivo assays, it is shown here that metchnikowin possesses selective antimicrobial activities against various pathogenic fungi including the biotrophic Blumeria graminis, which causes powdery mildew, and the necrotrophic Fusarium graminearum that causes root rot and heat blight; whilst the basidiomycete fungi Piriformospora indica (Schäfer and Kogel, 2009) and Rhizoctonia solani are hardly impaired by this insect peptide at physiological concentrations.

Materials and methods

Fungal materials

Blumeria graminis f. sp. hordei (Bgh) race A6 (Wiberg, 1974) was propagated on barley cv. Golden Promise. Fusarium graminearum strain 8/1 (Miedaner et al., 2000) was grown on Nirenberg Synthetic Nutrient Agar (SNA) medium at 18 °C exposed to near-UV and white light (TLD 36 W-08, TL 40 W-33 RS, Philips, Hamburg, Germany) with a 12-h light photoperiod for 1–2 weeks (Babaeizad et al., 2009). Piriformospora indica (DSMZ 11827 from the German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) was propagated as described by Waller et al. (2005). Rhizoctonia solani isolate AG8 was kindly provided by Dr. Timothy Paulitz, Department of Plant Pathology, Washington State University, USA and maintained on plates of potato dextrose agar (PDA, Roth, Germany).

Generation of transgenic barley plants

Transformation was carried out with spring barley (Hordeum vulgare L.) cv. Golden Promise grown in a climate chamber at 18/14 °C (light/dark) with 60% relative humidity (RH) and a 16 h photoperiod and a photosynthetic photon flux density of 240 μmol m−2 s−1. To introduce the metchnikowin encoding sequence into the barley genome, the 156 bp Mtk intron-less gene (GenBank Accession no. AY226419; Lazzaro and Clark, 2003) was amplified from genomic DNA of wild-type D. melanogaster strain OregonR using Mtk specific primers (Mtk-Fwd 5′-GGATCCATGCAACTTAATCTTGGA-3′ and Mtk-Rev 5′-GTCGACTTAATAAATTGGACCCGGT-3′) flanked by BamHI/SalI restriction sites. The resulting amplicon was ligated into the same sites of the formerly manipulated expression vector p35S-BAM (Schmidt, DNA Cloning Service, Hamburg, Germany) for substitution of mas promoter (cut from pGE2-mas::gallerimycin, Langen et al., 2006) instead of CaMV 35S. The expression cassette of mas::metchnikowin::nos-T (in pMas::Mtk plasmid), cut by SfiI, was cloned into the SfiI-sites of binary vector pLH6000 (DNA Cloning Service, Hamburg, Germany; see Supplementary Fig. S1B at JXB online). For barley transformation, pLH6000::Mas::Mtk was introduced into the Agrobacterium strain AGL-1 (Lazo et al., 1991) through electroporation (E. coli Pulser, Bio-Rad, Munich, Germany). Agrobacterium-mediated transformation, selection, and regeneration of transformants were performed as described by Tingay et al. (1997). T0 plants were transferred into a greenhouse with temperatures ranging from 20–28 °C and uncontrolled humidity. Transformants were selected based on transgene integration test via genomic PCR with Mtk specific primers.

Bioassays for plant resistance

For the powdery mildew assay, barley seeds (surface-sterilized by 70% ethanol for 1 min, and a sodium hypochlorite solution containing 6% active chlorine for 2 h) were germinated on filter papers for 3 d, then transplanted into soil (Frühstorfer Erde, Hawita Gruppe, Vechta, Germany), and maintained in a climate chamber (Percival Scientific, Boone, Iowa, USA) with a 16 h photoperiod, 22/18 °C (light/dark) and 60% RH. After 7 d, first leaf segments were placed on 0.5% (w/v) water agar medium supplemented with 20 mg l−1 benzimidazole (Merck-Schuchardt, Munich, Germany) and, subsequently, inoculated with Bgh (5 conidia mm−2 density) using air current dispersal in an inoculation pillar. The plates were preserved in the same climate chamber for 5 d to, eventually, count Bgh colonies.

For the Fusarium root rot assay, roots of 3-d-old seedling were gently shake-incubated in F. graminearum inoculum (5×104 conidia ml−1; Deshmukh and Kogel, 2007) and, in parallel, in a 0.05% Tween-20 aqueous solution as a mock treatment for 2 h. Subsequently, seedlings were transplanted in a 2:1 mixture of expanded clay (Seramis; Asterfoods, Verden, Germany) and Oil Dri (Damolin, Mettmann, Germany) and maintained in a climate chamber with 60% RH and a 16 h photoperiod, 22/18 °C (light/dark), with 240 μmol m−2 s−1 photon flux density for 2 weeks. Later, shoot biomass was measured and root samples for quantitative measurement of fungal biomass were subjected to DNA isolation.

To perform the Fusarium head blight assay, spikes of 16-week-old plants were sprayed with F. graminearum inoculum (2×105 conidia) as described by McCallum and Tekauz (2002). Inoculated spikes were kept in plastic bags for 48 h for providing the required high humidity. After 3 weeks, the seeds were harvested and surface-sterilized by 70% ethanol and sodium hypochlorite solution containing 3% active chlorine for 1 min and 20 min, respectively, to eliminate any superficial contamination. Sterilized seeds were placed on PDA medium for 3–5 d. Fusarium resistance was scored based on Fusarium contamination outgrowing from the endosperm. The resistance assay was replicated twice for each transgenic line.

Effects of Mtk on P. indica–barley symbiosis

To assess the effect of Mtk on P. indica, surface-sterilized barley seeds were germinated for 3 d on filter paper. Roots of seedling were immersed and gently shake-incubated in an aqueous solution of 0.05% Tween-20 containing 5×105 P. indica chlamydospores ml−1 for 2 h (Deshmukh et al., 2006). Inoculated seedlings were transplanted in Seramis:OilDri (2:1) as described above. Shoot biomass was measured after 21 d. Colonization of plant roots by P. indica was detected by microscopy using wheat germ agglutinin stain conjugated with Alexa Fluor 488 (WGA-AF 488, Molecular Probes, Karlsruhe, Germany; Deshmukh et al., 2006).

In vitro antifungal assay

Fungicidal activity of a synthetic, active 26-amino acid Mtk peptide (purity more than 80%, Seqlab, Göttingen, Germany) and Mtk expressed in barley were determined using an inhibition assay (Cavallarin et al., 1998). Intercellular washing fluids (IWFs) of transgenic plants were extracted from leaves by vacuum infiltration of 10 mM phosphate buffer (pH 7.2). Infiltrated leaves were centrifuged (700 g, 5 min) and IWFs were obtained. For the in vitro antifungal activity assay, F. graminearum conidia (1×104 conidia) were incubated in IWF at room temperature for 24 h. Fungal growth was monitored on an inverse microscope (Olympus, Japan). Quantitative assay for inhibitory activity on pathogen development was performed after 24 h via staining the cultures in microtitre plate wells with Trypan blue (10 g phenol, 10 ml glycerol, 10 ml lactic acid, 10 ml distilled water, and 0.02 g of Trypan blue, Sigma-Aldrich Chemie, Steinheim, Germany). Absorbance (OD595nm) was measured using spectrophotometer (Tecan Deutschland GmbH, Crailsheim, Germany).

Pathogen/auxin inducibility of the mannopine synthase (mas) gene promoter

To analyse the inducibility of A. tumefaciens mas promoter by pathogens, transgene expression was monitored at 12, 24, and 48 h after Bgh (50 conidia mm−2) and 2, 4, and 6 d after F. graminearum (5×104 conidia ml−1) inoculations. Mock treatments were provided for each time point. To test auxin inducibility of the mas promoter, Mtk plants were treated with 1 mM indole-3-acetic acid (IAA) (Sigma-Aldrich Chemie) or water as the foliar spray. Transgene expression levels were checked at 0, 2, 4, 12, 18, and 24 h after spray. RNA extraction and RT-PCR were performed for each plant material as outlined below.

Determination of fungal biomass

The extents of root colonization by F. graminearum and P. indica were determined using quantitative PCR. Genomic DNA was isolated from 100 mg of root tissue by means of DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). To perform quantitative PCR, 5 ng of total DNA were used. Amplifications were performed in 5 μl of 2× SYBR green JumpStart mix (Sigma–Aldrich Chemie) with 10 pmol of the respective oligonucleotides using an Mx3000P thermal cycler (Stratagene, La Jolla, CA, USA). Relevant melting curves were determined at the end of cycling to ensure the amplification of just one PCR product. Cycle of threshold (Ct) values were ascertained with Mx3000P V2. Ct values were generated by deducting the raw Ct values of the F. graminearum Fg16N (Nicholson et al., 1998) and the P. indica β-tubulin gene (Serfling et al., 2007) from the respective raw Ct values of the plant-specific ubiquitin gene and relative fungal DNA abundance was determined as 2–ΔΔCt.

Transcript analyses

For quantitative two-step RT-PCR, 2 μg of total RNA, extracted from 100 mg of plant material using RNeasy Plant Mini Kit (Qiagen) and treated with DNase I (Fermentas, Sankt Leon-Rot, Germany) was reverse transcribed to first-strand cDNA using SuperScript™ II Reverse Trascriptase (Invitrogen, Carlsbad, CA). Five ng of first-strand cDNA were used for each quantitative PCR with gene-specific primers. Comparative expression levels (2–ΔΔCt; Livak and Schmittgen, 2001) were then calculated. Expression levels are shown relative to the level of ubiquitin expression steady in all RNA samples. Values are the means of four samples of two biological experiments assayed through quantitative PCR. Specific oligonucleotide pairs were used for ubiquitin (Accession no. M60175), Pathogenesis-related-1b (Accession no. Z21494), and Pathogenesis-related-5 (Accession no. AY839293) (Deshmukh and Kogel, 2007).

Histochemical analyses

To detect defence responses of barley plants to Bgh attack in epidermal cells, an endogenous peroxidase-dependent in situ staining procedure was performed using 3, 3-diaminobenzidine (DAB; Sigma-Aldrich Chemie, Steinheim, Germany; Thordal-Christensen et al., 1997) at 36 hai on the first leaves of 7-d-old plants detached and placed on 0.5% (w/v) water agar containing 20 mg l−1 benzimidazole. Leaf storage, fungal staining, and microscopy were done as described by Hückelhoven and Kogel (1998).

In vitro analysis of Mtk on protoplasts of fungi

Mycelia were produced by inoculating 100 ml modified Aspergillus minimal medium (Waller et al., 2005) with multiple agar plugs of 7-d-old R. solani and F. graminearum as well as 30-d-old P. indica cultures. The cultures were shake-incubated at RT for 7 d (100 rpm). Subsequently, mycelia were crushed using a blender and transferred to 100 ml of fresh CM medium (Waller et al., 2005). After 3 d, mycelia were harvested by filtration using sterile Miracloth filter and washed with 50 ml of 0.9% NaCl solution. Washed mycelia were incubated for 1 h at 37 °C in a cell wall-degrading enzyme mixture (0.2 g 10 ml−1 lysing enzymes from Trichoderma harzianum, Sigma). The reaction product was then filtered through sterile Miracloth and washed with 10 ml STC (1 M sorbitol, 10 mM TRIS-HCl, pH 7.5, 50 mM CaCl2). The eluted protoplasts were settled down by centrifugation at 4 °C for 10 min at 3000 rcf. The pellet was resuspended in 1 ml of STC (4 °C) and transferred to a microcentrifuge tube. Protoplasts were collected by centrifugation at 4 °C for 5 min at 3000 rcf. The pellet was washed in 1 ml STC and the number of protoplasts was determined using a Fuchs Rosenthal counting chamber. To determine the antifungal activity of Mtk, 400 protoplasts from each fungus were incubated in different concentrations of Mtk in microtitre plates at RT for 24 h (four replicates each). Protoplast numbers were recorded using an inverted contrasting microscope (Leica DM IL, Germany).

Results

Metchnikowin expressing barley shows resistance to pathogenic fungi

Metchnikowin is a 26-amino acid residue proline-rich peptide synthesized in the fat body of Drosophila melanogaster as a 52 amino acid pre-pro-peptide upon microbial challenges. The Mtk amino acid sequence is shown in Supplementary Fig. S1A at JXB online. According to initial studies, the peptide inhibits the growth of Gram-positive bacteria and the ascomycete fungus Neurospora crassa (Levashina et al., 1995). Custom synthesized 26-amino acid Mtk also inhibited the germination of conidia of the plant pathogenic fungus Fusarium graminearum with IC50 value of 1 μM (Fig. 1A). Encouraged by these results, transgenic barley plants expressing the Mtk gene encoding the pre-pro-peptide were produced. Agrobacterium-mediated transformation of immature embryos of the barley cv. Golden Promise (GP) was carried out with a plasmid (see Supplementary Fig. S1B at JXB online), containing the coding region of the Mtk gene under the control of the mannopine synthase (mas) gene promoter from Ti plasmid of Agrobacterium tumefaciens (Velten and Schell, 1985; Langridge et al., 1989). This promoter has proved to be responsive to wounding, plant growth hormones, as well as fungal infection in carrot (Imani et al., 2002) and tobacco (Langen et al., 2006). Integration of Mtk into the barley genome was verified by PCR using Mtk specific primers. One hundred transgenic lines with no visible abnormality in growth were regenerated from immature embryos. Out of these lines, 13 lines were analysed in more detail to confirm the level of Mtk transcription using RT-PCR. Four lines, namely, L4, L42, L50, and L57 showed particularly high transcription of the Mtk gene (see Supplementary Fig. S1C at JXB online) and were targeted for further studies. These lines were cultivated to obtain the T1 and T2 generations for subsequent bioassays. Intercellular washing fluids (IWFs) from leaves of Mtk-containing plants showed inhibitory effects on the growth of F. graminearum mycelia when compared with IWF from wild-type GP barley (Fig. 1B). This demonstrates the functionality of the insect origin pre-sequence for targeting the Mtk into the plant apoplast.

Fig. 1.
Inhibition of Fusarium graminearum macroconidia by metchnikowin (Mtk). (A) Inhibition of germination with increasing concentrations of Mtk in distilled water with IC50 of 1 μM. (B) Inhibition of mycelium growth by intercellular washing fluids ...

Mannopine synthase (mas) gene promoter is inducible by auxin and pathogens in monocots

It was shown earlier that the mas promoter from A. tumefaciens is auxin and pathogen responsive in dicotyledonous plants (Langridge et al., 1989; Imani et al., 2002; Langen et al., 2006). To test auxin inducibility of the mas promoter in the monocotyledonous barley, transcription of Mtk in transgenic line L4 was assessed by quantitative RT-PCR after foliar spray of synthetic Indol-3-acetic acid (IAA) and fungal inoculation (see Supplementary Table S1 at JXB online). As a result, the level of Mtk transcription increased 3-fold with a maximum at 2 h after treatment with 1 mM IAA. Upon inoculation of line L4 plants with conidia of the biotrophic powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh), Mtk mRNA abundance was amplified over 3-fold with a climax at 24 h after inoculation (hai) and levelled off, thereafter. Similarly, Mtk mRNA abundance in the roots of L4 plants was raised beyond 3-fold in response to necrotrophic F. graminearum at 144 hai. Overall, these observations demonstrate significant, albeit, sparse activity and inducibility of the bacterial mas promoter in different tissues of a monocotyledonous plant upon infection by fungi with both biotrophic and necrotrophic life styles.

Mtk plants show improved resistance to Fusarium root rot and head blight

To evaluate the effect of Mtk in barley plants on necrotrophic Fusarium species, Mtk plants were infected with F. graminearum that causes root rot (FRR) and head blight (FHB). Roots of 3-d-old seedlings of wild-type GP and Mtk expressing plants of line L4 were dip-inoculated into a solution of F. graminearum conidia for 2 h, transferred into pots and evaluated for disease symptoms 2 weeks later. At that stage, infected seedlings of the wild-type GP were retarded in growth compared with non-inoculated individuals, while those of transgenic line L4 were almost as tall and vigorous as the corresponding non-infected plants (see Supplementary Fig. S2 at JXB online). Shoot biomass of the four sets of plants is shown in Fig. 2A. The transgenic line L4 showed only a 10% cutback in biomass compared with 50% reduction in wild-type GP plants. The seed remnant and root bases of infected GP displayed discoloration, a typical symptom of F. graminearum root rot, whereas the inoculated Mtk plants developed a non-symptomatic root system (not shown). In order to quantify the infection more accurately, fungal biomass in roots was determined by fungal DNA using quantitative PCR and F. graminearum-specific primers. In agreement with the observed FRR symptoms, fungal DNA abundance was reduced by 80% in roots of line L4 plants compared with that in wild type (Fig. 2B).

Fig. 2.
Transgenic Mtk barley responses to inoculation with Fusarium graminearum (Fg). (A) Upon inoculation of the roots of 3-d-old wild type (WT) Golden Promise seedlings with Fg conidia, WT biomass production measured 14 d after inoculation was reduced, while ...

To test for resistance to head blight caused by F. graminearum (FHB), developing barley spikes of GP and transgenic Mtk plants were sprayed with 2×105 conidia inoculum and covered with plastic bags for 48 h for retaining high humidity for macroconidia germination. After 3 weeks, grains were harvested, surface-sterilized, placed on potato dextrose agar (PDA) medium, and assayed for the presence of fungus outgrowing from the endosperm. In Mtk-expressing lines L4 and L50, FHB severity was judged to be reduced to about 20% as compared to GP in two independent experiments. Fungal biomass in infected endosperm was also determined by quantitative PCR. Consistently, declines of fungal infection in grains by up to 38% and 46% were observed in respective transgenic lines L4 and L50 (Fig. 2C).

Mtk-producing plants demonstrate improved resistance to powdery mildew fungus

The development of powdery mildew fungus race Bgh-A6, which undergoes a compatible interaction with Golden Promise was examined on Mtk-expressing barley. Detached first leaves of 7-d-old wild type and Mtk plants were inoculated with fungal conidia and evaluated 5 d later for interaction phenotypes. All Mtk-containing lines showed a strong reduction (60% on average) in the frequency of the Bgh colony as well as in colony size compared with GP (Fig. 3A), which shows the enhanced resistance of Mtk plants to powdery mildew. Next, the effect of Mtk expression on the defence reactions typically exhibited by barley leaves in response to Bgh inoculation was determined. Microscopic inspection revealed no effect on fungal germination on barley leaves. However, the number of germinated conidia that penetrated successfully into epidermal cells of the transgenic lines was reduced significantly (Fig. 3B). Less than 10% of germinated conidia penetrated an epidermal cell, formed a functional haustorium, and elongated secondary hyphae. By contrast, more than 50% of germinated conidia produced functional haustoria in GP. As observed, the failure of haustorium formation in Mtk plants was associated with augmented frequencies of effective cell wall apposition (CWA) underneath the attempted penetration sites. Moreover, the incidence of the hypersensitive response (HR) resulting in the collapse of the attacked host cells was also higher (Fig. 3C). In order to test whether Mtk plants show an overall enhanced defence status, the transcript levels of pathogenesis-related (PR) genes were measured in transgenic and GP plants. Basal expression levels of genes PR-1b and osmotin (PR-5) in leaves from non-challenged Mtk and GP plants was indistinguishable (data not shown), suggesting that Mtk does not induce stress responses in barley. However, consistent with earlier reports, both genes were up-regulated upon inoculation with virulent Bgh (Fig. 4). At 24 hai, expression of PR-1b was induced 12-fold in GP and 35-fold in transgenic line L42. Correspondingly, PR-5 was induced 4-fold in GP compared with 6-fold in line L42.

Fig. 3.
Resistance of Mtk barley against Blumeria graminis f. sp. hordei. (A) The frequencies of Bgh colony formation on Mtk lines L4, L42, L50, and L57 at 5 dai are reduced, significantly, in comparison with that in wild-type Golden Promise (WT). Data are the ...
Fig. 4.
Induction of PR genes in Mtk plants and Golden Promise (WT) in response to Blumeria graminis f.sp. hordei (Bgh). PR-1b and PR-5 gene expression was monitored in 7-d-old Mtk plants of line L42 and in WT at 12, 24, and 48 h after Bgh inoculation. RNAs were ...

Metchnikowin shows differential activities against ascomycete and basidiomycete fungi

The effect of Mtk expression on P. indica, a beneficial basidiomycete colonizing roots of a broad plant spectrum including barley (Kogel et al., 2006), was analysed as well. P. indica belongs to the order Sebacinales that represents a huge range of symbiotically living, mycorrhizal fungi (except the arbuscular fungi of Glomeromycota) with major ecological relevance (Waller et al., 2005). Unexpectedly, in vitro tests revealed a lower sensitivity of P. indica to Mtk. No significant inhibition of chlamydospore germination was observed by up to 50 μM Mtk (Fig. 5A). This analysis reveals the tolerance of P. indica chlamydospores to high doses of Mtk, which is ten times stronger than the completely lethal dose to conidia of F. graminearum. Consistently, colonization of Mtk line L4 roots by P. indica was not impaired. As evidenced by quantitative PCR, fungal biomass in line L4 roots was even somewhat higher when compared with wild-type GP (Fig. 5B). Microscopic analysis corroborated an unrestricted colonization of transgenic barley roots by P. indica (see Supplementary Fig. S3 at JXB online). Furthermore, growth promotion elicited by P. indica in line L4 was in the same range as in wild-type GP (Fig. 5C), together indicating that the mutualistic potential of the fungus has not been disturbed by Mtk expression.

Fig. 5.
The effect of Mtk on Piriformospora indica development. (A) In vitro assay for Mtk antifungal activity on P. indica chlamydospore germination. Chlamydospores were incubated in different concentrations of synthetic Mtk and the percentage of chlamydospore ...

In order to substantiate the differential toxicity of Mtk, protoplast viability of various fungi was tested in the presence of the insect peptide. To this end, protoplasts isolated from axenic mycelia of the ascomycete F. graminearum as well as the basidiomycetes P. indica and Rhizoctonia solani were incubated in increasing concentrations of Mtk (2–50 μM). A reduced survival rate and, thus, a higher sensitivity of F. graminearum protoplasts (approximate IC50 of 1 μM) was found compared with those of P. indica and R. solani (IC50: ~20 μM; Fig. 6), which supports the hypothesis that the differential sensitivities of the fungi might be owing to the structural and/or biochemical differences in higher fungi.

Fig. 6.
Detrimental effect of synthetic Mtk peptide on fungal protoplast. Protoplasts of Fusarium graminearum, Rhizoctonia solani, and Piriformospora indica were incubated for 24 h in increasing concentrations of Mtk. Subsequently, the numbers of surviving protoplasts ...

Discussion

In this study, the potential of an antifungal peptide from Drosophila melanogaster was explored to engineer, in barley, disease resistance against devastating microbial plant pathogens. The proline-rich antifungal metchnikowin was chosen, because the peptide caused strong in vitro growth inhibition of pathogenic fungi of the genus Fusarium that are of high agronomic relevance in many crops.

As the instability of insect antimicrobial peptides in transgenic plants has been attributed to post-translational degradation by intracellular proteases (Mills et al., 1994), the sequence encoding the Mtk pre-pro-peptide was cloned into the plant transformation binary vector to target the mature peptide into the plant apoplastic space. This strategy has been successfully employed to confer resistance against fungal pathogens on tobacco by transgenic expression of the antifungal peptide gallerimycin from Galleria mellonella (Langen et al., 2006). Intercellular washing fluid of Mtk-expressing Golden Promise strongly inhibited the growth of F. graminearum in vitro, demonstrating that these transgenic plants produce a functional signal sequence, which is able to target the mature peptide into the apoplast correctly (Fig. 1B). Mtk was expressed under the control of the Agrobacterium tumefaciens mannopine synthase (mas) promoter that had been shown earlier to be responsive to phytohormones, wounding, and fungal infection in dicot plants (Langridge et al., 1989; Imani et al., 2002; Langen et al., 2006). It is shown here that the mas promoter is also inducible in leaves and roots of barley by IAA as well as fungal pathogens B. graminis and F. graminearum, which show biotrophic and necrotrophic life-styles, respectively. The overall advantage of the use of the mas promoter is its pathogen inducibility along with the fact that its basal activity in barley is much lower compared with the frequently used CaMV 35S or maize ubiquitin promoters (see Supplementary Table S1 at JXB online). Consistently, negative side-effects of Mtk were not detected in the transgenic plants. Interestingly, and in contrast to barley, the basal activity of the mas promoter in maize is much higher and similar to that of the CaMV 35S or maize ubiquitin promoters (Lee et al., 2007).

Assessment of Mtk effects on powdery mildew fungus during its interaction with Mtk barley provided evidence that the antifungal peptide impedes establishment of a functional haustorium, whose formation is a prerequisite for the commencement of the biotrophic phase. Microscopic analysis revealed that germination of conidia is not affected, and higher plant resistance is associated with higher frequencies of typical defence responses (Hückelhoven and Kogel, 2003), for example, hypersensitive response (HR) of attacked cells and the development of cell wall apposition (CWA) underneath attempted penetration sites. Thus, antifungal Mtk intensifies plant defence in epidermal cells, which leads to a diminished number of functional haustoria. Increased HR frequencies and accompanying cell death of attacked epidermal cells could be initiated by locally elevated Mtk levels reaching a toxic threshold for the plant cell. Although this cannot be excluded, it seems unlikely as Mtk transgenic plants never showed distinguishable detrimental effects on plant organs. Alternatively, and more likely, antifungal Mtk in the leaf apoplastic space may weaken the fitness of the attacking fungus and, in that way, disturb the pathogenic fungus’ essential capability actively to suppress the plant defence. Since defence suppression is brought about by effector molecules secreted by fungal microbes (Birch et al., 2009), it is anticipated that the fungal secretory pathway is indirectly affected by Mtk. Notably, active suppression of plant defence is a prerequisite for a successful fungal infection, a compatible interaction, and, eventually, pathogenesis (Kogel et al., 2006).

To elucidate in depth the impact of Mtk on the defence system, the expression of pathogenesis-related genes PR-1b and PR-5 were analysed. Without Bgh challenge, Mtk and GP plants showed equally low basal levels of PR genes expression. Upon fungal inoculation, PR genes expression significantly increased in GP and in Mtk plants (Fig. 4), which further supports the hypothesis that Mtk plants show a higher defence status upon challenges with powdery mildew compared with non-transgenic plants. Importantly, low PR gene expression in non-challenged Mtk plants suggests a low general stress status of the transgenic plants. Hence, molecular evidence is provided that expression of antifungal Mtk does not set off high fitness costs expected to affect yield in various other resistance strategies (Heil and Baldwin, 2002; Tian et al., 2003).

Consistent with in vitro inhibition of Fusarium spp. by synthetic Mtk, expression of Mtk in barley strikingly decreased Fusarium head blight and root rot damage (Fig. 2). Bioassays for disease resistance revealed that pathogen development in both roots and kernels was hampered. Thus, the lines generated in the present study might provide a promising source for breeding programmes aimed at improving the resistance to diseases caused by Fusarium spp.

There are a number of reports in which antimicrobial peptides of insect (Osusky et al., 2000; Langen et al., 2006), frog (Chakraborti et al., 2003; Yevtushenko and Misra, 2007) or human (Zakharchenko et al., 2005; Aerts et al., 2007) have been used to render the transformed plants more resistant to fungal pathogens. Beside the evaluation of Mtk effects on the development of pathogenic fungi, its possible adverse impact on the beneficial root endophyte Piriformospora indica (Waller et al., 2005) has been investigated, too. The results show that Mtk does not inhibit growth of this fungus in roots of transgenic plants. In order to substantiate the differential toxicity of Mtk, and since the ascomycetes Blumeria and Fusarium were affected by Mtk, the hypothesis was tested whether Mtk exerts differential activities on ascomycete and basidiomycete fungi. The results demonstrate that protoplasts isolated from axenic mycelia of ascomycete F. graminearum were much more sensitive to Mtk than those from the basidiomycetes P. indica and Rhizoctonia solani (Fig. 6), suggesting that the differential sensitivity of the fungi might be attributed to structural and/or biochemical differences of ascomycetes and basidiomycetes.

Supplementary data

Supplementary data are available at JXB online.

Supplementary Fig. S1. (A) Whole amino acid sequence of native metchnikowin peptide transformation; (B) T-DNA construct used for plant transformation; (C) relative quantification of Mtk expression in lines L4, L42, L50, and L57 by quantitative RT-PCR with cDNA obtained from total RNA as template.

Supplementary Fig. S2. Growth response of Golden Promise and Mtk seedlings L4 to Fusarium graminearum inoculation.

Supplementary Fig. S3. Microscopy of root colonization by P. indica in Golden Promise (WT) and transgenic barley L4.

Supplementary Table S1. Activity of mas promoter in barley after auxin treatment and during challenges with different pathogens.

[Supplementary Data]

Acknowledgments

This work was supported by a grant from Deutsche Forschungsgemeinschaft to KHK and a DAAD matching fund (STIBET) to MR and WK.

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