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 (IC(50) value approximately 1 muM) 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 T(i) 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 muM. 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.

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 (IWFs) extracted from transgenic barley Mtk lines L4, L42, L50, and L57 compared with that from the wild type (WT) Golden Promise. Conidia were incubated in IWF for 24 h. Growth of mycelia was determined by measuring absorbance at 595 nm after Trypan blue staining. Values are means of two replications and error bars show the standard errors of the experiments. *, ** Significant differences from the control at P <0.05 and P <0.01, respectively (t test).

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 seedlings of transgenic line L4 developed almost like the non-inoculated control individuals. The experiment was replicated once and the bars indicate the standard errors of two separate experiments. *, ** Differences from the control at P <0.05 and P <0.01, respectively (t test). (B, C) Quantitative measurement of fungal DNA abundance in roots affected by Fusarium root rot (FRR, B) and in kernels affected by Fusarium head blight (FHB, C), respectively. Fungal DNA was sampled from roots of Fusarium infected WT and L4 plants as well as from seeds of WT, L4, and L50 plants 14 d after root and 21 d after spike inoculation, respectively. Results were normalized using the plant ubiquitin gene. Data are the means of values from two independent experiments and error bars indicate the corresponding standard errors.

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 means of values from 20 individuals per line. Error bars indicate standard errors. (B) Penetration efficiency of Bgh on WT, L4, and L42. Data were obtained via microscopic evaluation using DAB staining of leaf segments and are the means of successful penetration events associated with mature haustoria and the formation of elongated secondary hyphae in a total of 100 interaction sites in 12 individuals per line at 36 hai. Error bars represent standard errors. (C) Defence responses of barley to Bgh; frequencies of cells showing a non-penetrated cell wall apposition (CWA) or a hypersensitive response (HR) in response to Bgh attack are higher in Mtk lines L4 and L42 as compared with Golden Promise (WT). Data were obtained as described in section (B). Error bars show the standard errors. ** The difference from the control at P <0.01 (t test).

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 extracted from first leaves of three plants for each time point and transcript levels were quantified using RT-PCR. Values were normalized with constitutively expressed ubiquitin as the internal control and are presented relative to the expression level of the zero time point. Data are the means of values from two independent experiments and error bars show the standard errors.

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 germination was measured after 24 h. (B) Relative fungal biomass in P. indica-colonized roots of WT and transgenic plants of line L4. Values were normalized by the plant ubiquitin gene. Error bars illustrate the standard errors of two independent experiments. (C) Growth promoting effect elicited by P. indica. Shoot weight of transgenic line L4 plants was measured 21 d after root colonization by P. indica, and compared with that of corresponding non-colonized plants. Data are the means of values out of two independent experiments and 25 plants each. Error bars show the standard errors.

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 were determined by microscopy.