Summary: 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 N icotiana 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 N t‐thionin from N . tabacum (96% identical) and shares 81% identity with M t D ef4 from alfalfa. The concentration required to inhibit in vitro fungal growth by 50% ( IC ₅₀ ) was assessed for both NaD1 and NaD2 for the biotrophic basidiomycete fungi P uccinia coronata f. sp. avenae ( P ca ) and P . sorghi ( P s ), the necrotrophic pathogenic ascomycetes F usarium oxysporum f. sp. vasinfectum ( F ov ), F . graminearum ( F gr ), V erticillium dahliae ( V d ) and T hielaviopsis basicola ( T b ), and the saprobe A spergillus 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 P uccinia 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 P uccinia species tested. NaD1 and NaD2 were fungicidal on P uccinia 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 P uccinia spp. This study provides evidence that both secreted ( NaD2 ) and nonsecreted ( NaD1 ) defensins may be useful for broad‐spectrum resistance to pathogens.

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