Inhibition of cereal rust fungi by both class I and II defensins derived from the flowers of Nicotiana alata

Abstract

Defensins are a large family of small, cysteine-rich, basic proteins, produced by most plants and plant tissues. They have a primary function in defence against fungal disease, although other functions have been described. This study reports the isolation and characterization of a class I secreted defensin (NaD2) from the flowers of Nicotiana alata, and compares its antifungal activity with the class II defensin (NaD1) from N. alata flowers, which is stored in the vacuole. NaD2, like all other class I defensins, lacks the C-terminal pro-peptide (CTPP) characteristic of class II defensins. NaD2 is most closely related to Nt-thionin from N. tabacum (96% identical) and shares 81% identity with MtDef4 from alfalfa. The concentration required to inhibit in vitro fungal growth by 50% (IC50 ) was assessed for both NaD1 and NaD2 for the biotrophic basidiomycete fungi Puccinia coronata f. sp. avenae (Pca) and P. sorghi (Ps), the necrotrophic pathogenic ascomycetes Fusarium oxysporum f. sp. vasinfectum (Fov), F. graminearum (Fgr), Verticillium dahliae (Vd) and Thielaviopsis basicola (Tb), and the saprobe Aspergillus nidulans. NaD1 was a more potent antifungal molecule than NaD2 against both the biotrophic and necrotrophic fungal pathogens tested. NaD2 was 5-10 times less effective at killing necrotrophs, but only two-fold less effective on Puccinia species. A new procedure for testing antifungal proteins is described in this study which is applicable to pathogens with spores that are not amenable to liquid culture, such as rust pathogens. Rusts are the most damaging fungal pathogens of many agronomically important crop species (wheat, barley, oats and soybean). NaD1 and NaD2 inhibited urediniospore germination, germ tube growth and germ tube differentiation (appressoria induction) of both Puccinia species tested. NaD1 and NaD2 were fungicidal on Puccinia species and produced stunted germ tubes with a granular cytoplasm. When NaD1 and NaD2 were sprayed onto susceptible oat plants prior to the plants being inoculated with crown rust, they reduced the number of pustules per leaf area, as well as the amount of chlorosis induced by infection. Similar to observations in vitro, NaD1 was more effective as an antifungal control agent than NaD2. Further investigation revealed that both NaD1 and NaD2 permeabilized the plasma membranes of Puccinia spp. This study provides evidence that both secreted (NaD2) and nonsecreted (NaD1) defensins may be useful for broad-spectrum resistance to pathogens.

Figure 1

(A) Diagram of precursors encoding class I and class II defensins. Both precursors have endoplasmic reticulum (ER) signal sequences that direct the protein into the secretory pathway. Class I defensins have no additional targeting information and are secreted. Class II defensins have a C‐terminal pro‐peptide (CTPP). In the solonaceous class II defensins, this pro‐peptide targets the defensin to the vacuole, where the CTPP is removed to release the mature domain. Processing sites are indicated by black arrows. (B) Full‐length amino acid sequence alignment of NaD2 with the class I defensins Nt‐thionin (Accession No AB034956) and NTS13 (Accession No X99403) (Nicotiana tabacum) and the class II defensin NaD1 (N. alata) (van der Weerden and Anderson, 2013). ER signals are indicated in italics and the C‐terminal prodomain of NaD1 is underlined. Processing sites are indicated by an arrow. (C) Amino acid alignment of NaD2 with mature class I defensin sequences from N. tabacum (Nt‐thionin and NTS13), Capsicum annum (CaDef2), Medicago truncatula (MtDef4), Helianthus annuus (JI‐2), Elaeis guineensis (EGAD1), Vigna unguiculata (Cpthio1), Raphanus sativus (RsAFP2) and Dahliae merckii (DmAMP1), and mature class II defensin sequences from N. alata (NaD1), N. tabacum (FST), Petunia hybrida (PhD1) and Solanum lycopersicum (TPP3). Disulphide bonds between the eight conserved cysteines are shown by connecting lines. Loops (L1–L7) are classified as the regions between cysteine residues (van der Weerden and Anderson, 2013).

Figure 2

Expression of defensins in Nicotiana alata. Proteins extracted from flowers (F), stems (S), leaves (L) and roots (R) and separated by sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Defensins were detected by immunoblotting with antibodies raised against NaD2 (A) or NaD1 (B). M, SeeBlue^® plus two prestained protein standard (Life Technologies) size markers. The NaD2 and NaD1 defensins were only detected in flowers. Recombinant defensins (rNaD1 and rNaD2) were used as positive controls.

Figure 3

The inhibitory effects of defensin on urediniospore germination. (A) Micrographs of the effect of double‐distilled water (ddH₂O) (i) on uredinospore germination of Puccinia coronata f. sp avenae (Pca) compared with treatment with 10 μm NaD1 (ii). Scale bar, 20 μm. (B) Graph representation of the effect of NaD1 and NaD2 on urediniospore germination of two rust pathogens Pca and Puccinia sorghi (Ps). Full and broken lines represent Pca and Ps, respectively, and squares, triangles and diamonds represent assays using NaD1, NaD2 and NaPI protein treatments, respectively. All experiments were repeated three times and at least 100 urediniospores were scored on each agar plug. (C) Graphic representation of germ tube growth (μm) on 1% water agar plates versus time (min) after germination. In all cases, error bars represent the standard error of the mean of four biological replicates.

Figure 4

(A) Micrograph images of 10 μm NaD1‐treated germlings of Puccinia coronata f. sp avenae (Pca) (iii), Puccinia sorghi (Ps) (iv) and their corresponding water controls (i) and (ii). Arrows denote examples of stunted germlings with granular cytoplasm. Scale bar, 20 μm. (B) The effect of 10 μm NaD1 treatment on germ tube growth from germinated Pca and Ps (white) urediniospores relative to untreated controls (black). Germlings were classified as stunted if, 4 h after defensin treatment, their length was less than twice the diameter of the urediniospore. The percentage of stunted germ tubes was calculated as the ratio of stunted to total germ tubes multipled by 100. In all cases, error bars represent the standard error of the mean of four replicates. All experiments were repeated three times and at least 100 urediniospores were scored on each agar plug. (C) The effect of NaD1 treatment, applied 4 h after urediniospore germination, on the differentiation of Pca germlings, as measured by appressorium production on artificial media. Induction of appressoria was performed by treatment with 0.5 mm trans‐2‐hexen‐1‐ol together with heating at 30 °C for 2 h. Appressorium production was the most reproducible measurement of differentiation for experimental purposes using this method.

Figure 5

Micrographs of Puccinia sorghi (Ps) germlings treated with SYTOX green dye and double‐distilled water (ddH₂O) (i and ii), 10 μm NaD1 (iii and iv) and 10 μm NaD2 (v and vi). Note that fluorescence caused by SYTOX green uptake is only visible in Ps germ tubes that have been exposed to both SYTOX green and NaD1 or NaD2 treatment. Images were viewed under white light (i, iii and v) and under a 460–490‐nm MWIB filter (ii, iv and vi). Scale bar, 20 μm.

Figure 6

Images of whole oat plants (A) and oat leaves (B) sprayed with NaD2 (left and top), NaD1 (centre) or water (right and bottom) prior to inoculation with Puccinia coronata spores. (C) Percentage reduction in pustules per square centimetre of infected leaves for NaD2‐treated (black) and NaD1‐treated (white) plants relative to water‐treated leaves (control) (grey). NaD1 and NaD2 reduced the number of pustules per square centimetre by 70% and 37%, respectively.