Rs-AFP2

Transgenic rice (Oryza sativa L. cv. Pusa basmati 1), overexpressing the Rs-AFP2 defensin gene from the Raphanus sativus was generated by Agrobacterium tumefaciens-mediated transformation. Expression levels of Rs-AFP2 ranged from 0.45 to 0.53% of total soluble protein in transgenic plants. It was observed that constitutive expression of Rs-AFP2 suppresses the growth of Magnaporthe oryzae and Rhizoctonia solani by 77 and 45%, respectively. No effect on plant morphology was observed in the Rs-AFP2 expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of Rs-AFP2 plants on the in vitro growth of M. oryzae indicated that the Rs-AFP2 protein produced by transgenic rice plants was biologically active. Transgene expression of Rs-AFP2 was not accompanied by an induction of pathogenesis-related (PR) gene expression, suggesting that the expression of Rs-AFP2 directly inhibits the pathogens. Here, we demonstrate that transgenic rice plants expressing the Rs-AFP2 gene show enhanced resistance to M. oryzae and R. solani, two of the most important pathogens of rice.

Plant defensins are a class of cysteine-rich peptides of which several members have been shown to be potent inhibitors of fungal growth. A series of overlapping 15-mer peptides based on the amino acid sequence of the radish antifungal protein Rs-AFP2 have been synthesized. Peptides 6, 7, 8 and 9, comprising the region from cysteine 27 to cysteine 47 of Rs-AFP2 showed substantial antifungal activity against several fungal species (minimal inhibitory concentrations of 30-60 micrograms/mL), but no activity towards bacteria (except peptide 6 at 100 micrograms/mL). The active peptides were shown to be sensitive to the presence of cations in the medium and to the composition and pH of the medium. When present at a subinhibitory concentration (20 micrograms/mL), peptides 1, 7, 8 and 10 potentiated the activity of Rs-AFP2 from 2.3-fold to 2.8-fold. By mapping the characteristics of the active peptide on the structure of Rs-AFP2 as determined by nuclear magnetic resonance, the active region of the antifungal protein appears to involve beta-strands 2 and 3 in combination with the loop connecting those strands. A cyclized synthetic mimic of the loop, cysteine 36 to cysteine 45, was shown to have antifungal activity. Substitution of tyrosine 38 by alanine in the cyclic peptide substantially reduced the antifungal activity, indicating the importance of this residue for the activity of Rs-AFP2 as demonstrated carrier by mutational analysis.

Drosomycin is the first strictly antifungal protein isolated from an insect (Drosophila melanogaster). The solution structure of this 44-residue protein has been reported previously. It involves a three-stranded beta-sheet and an alpha-helix, the protein global fold being maintained by four disulfide bridges. Rs-AFP2 is a plant antifungal protein exhibiting 41% sequence similarity with drosomycin. Mutational analysis of Rs-AFP2 showed the importance of some residues in the antifungal activity of the protein against the fungus target. In order to determine the structural features responsible for antifungal activity in both drosomycin and Rs-AFP2, we modeled the three-dimensional structure of Rs-AFP2, and of other antifungal proteins, using the solution structure of drosomycin as a template. Structure analysis of drosomycin and Rs-AFP2, and comparisons with the other modeled antifungal structures, revealed that the two proteins shared a hydrophobic cluster located at the protein surface in which a lysine residue is embedded. Based on these close structural similarities and the experimental data available for Rs-AFP2 mutants, an antifungal active site of the insect protein is proposed.