Recombinant Wheat Antifungal PR4 Proteins Expressed in Escherichia coli

Constitutive over-expression of two wheat pathogenesis-related genes enhances resistance of tobacco plants to Phytophthora nicotianae

“…Besides, two highly homologous PR4 proteins (Wheatwin1 and Wheatwin2) were purified from wheat kernel and characterised thoroughly (Caruso et al 1993;Caruso et al 1996). Both Wheatwin1 and Wheatwin2 are induced by infection with Fusarium culmorum in wheat seedlings (Caruso et al 1999a) and revealed a strong in vitro antimicrobial activity against both wide host-range pathogens such as Botrytis cinerea and wheat-specific pathogenic fungi like F. culmorum and F. graminearum (Caruso et al 1996;Caruso et al 2001b). The reported ribonuclease activity of Wheatwin1 (Caporale et al 2004) may, at least in part, be responsible for its antimicrobial properties.…”

“…Equal protein concentrations were used for SDS-PAGE and immunoblotting following the procedures of Laemmli (1970) and Towbin et al (1979), respectively. Recombinant Wheatwin2 purified from E. coli heterologously expressing the protein (Caruso et al 2001b) was used as a western blot standard. Polyclonal antibodies raised in rabbit against purified Wheatwin1 expressed in the same heterologous system (Caruso et al 2001b) were used as first antibody and goat anti-rabbit horseradish peroxidaseconjugated immunoglobulins (MP Biomedicals, Irvine, CA, USA) were used as second antibody.…”

“…Recombinant Wheatwin2 purified from E. coli heterologously expressing the protein (Caruso et al 2001b) was used as a western blot standard. Polyclonal antibodies raised in rabbit against purified Wheatwin1 expressed in the same heterologous system (Caruso et al 2001b) were used as first antibody and goat anti-rabbit horseradish peroxidaseconjugated immunoglobulins (MP Biomedicals, Irvine, CA, USA) were used as second antibody. 4-Chloro-1-naphthol (Sigma-Aldrich) was used as a chromogen for detecting positive bands.…”

“…Four PR4 proteins from wheat kernels were isolated and sequenced, named Wheatwin1 to Wheatwin4 (Caruso et al 1993;Caruso et al 1996;Caruso et al 2001a) and the cDNAs encoding Wheatwin1 and Wheatwin2 were cloned (Caruso et al 1999b). The isolated Wheatwins were demonstrated to be specifically induced in wheat seedlings upon fungal infection (Caruso et al 1999a) and to be effective in inhibiting hyphal growth and spore germination of pathogens in vitro (Caruso et al 1996;Caruso et al 2001b), although their action mechanism and interaction with pathogen molecular targets are still unclear. Caporale et al (2004) reported that Wheatwin1 shows ribonuclease activity in in vitro experiments, which cannot be related to the action mechanism reported for other pathogeninduced ribonucleases grouped in the PR10 family, but rather to the classical acid-base mechanism of ribonuclease A and T1 involving two histidine residues.…”

Functional roles of the pepper leucine-rich repeat protein and its interactions with pathogenesis-related and hypersensitive-induced proteins in plant cell death and immunity

“…The in vitro antifungal activity against plant pathogenic fungi has been demonstrated for some plant PR4 proteins (Zhu et al 2006;Mukherjee et al 2010). Recombinant Wheatwin1, a wheat PR protein of class 4, exhibits antifungal activity against Fusarium culmorum, F. graminearum, and B. cinerea (Caruso et al 2001;Bertini et al 2009). Purified OsPR4b and Lycoris radiata LrPR4 have antifungal activity against R. solani (Zhu et al 2006) and M. oryzae ), respectively.…”