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.

Flower-specific jasmonate signaling regulates constitutive floral defenses in wild tobacco

“…Plant defensins are small, cysteine-rich proteins with antimicrobial activity. Floral defensins were first identified in Nicotiana alata and Petunia plants and are thought to have antifungal activity in vitro (62,63). The regulation of floral defensin remains unclear.…”

Expression of apoplast-targeted plant defensin MtDef4.2 confers resistance to leaf rust pathogen Puccinia triticina but does not affect mycorrhizal symbiosis in transgenic wheat

“…Certain cationic plant defensins exhibit growth inhibitory activity against various fungi and oomycetes at micromolar (μM) concentrations and this potent antifungal activity translates into disease resistance against these pathogens in transgenic plants (Kaur et al 2011). In case of rust pathogens, it has been reported that Nicotiana alata defensins, NaD1 and NaD2, inhibit germination of urediniospores and mitigate germ tube growth and differentiation of Puccinia species, and confer resistance to crown rust in oat seedlings when applied as a foliar spray (Dracatos et al 2013). …”

Defensins from the tick Ixodes scapularis are effective against phytopathogenic fungi and the human bacterial pathogen Listeria grayi

“…The expression of radish defensin in trangenic wheat confers resistance to F. graminearum [54]. In addition, defensins isolated from the venom of the snake Bothrops jararaca [55], from the plant Nicotiana alata [56] and from mice [57] have been shown to be effective against some species of Fusarium. In our study, the IC50 against F. culmorum and F. graminearum of both Scapularisin-3 and Scapularisin-6 are considerably low (1 μM).…”