Evidence that the role of plant defensins in radish defense responses is independent of salicylic acid.

Radish leaves contain two homologous 5-kDa plant defensins which accumulate systemically upon infection by fungal pathogens (F.R.G. Terras et al., 1995, Plant Cell 7: 573-588). Here we report on the molecular cloning of the cDNAs encoding the two pathogen-inducible plant defensin isoforms from radish (Raphanus sativus L.) leaves. Tissue-print and whole-leaf electroblot immunostaining showed that the plant defensin peptides not only accumulate at high levels at or immediately around the infection sites in leaves inoculated with Alternaria brassicicola, but also accumulate in healthy tissue further away from the infection sites and in non-infected leaves from injected plants. Gel blot analysis of RNA confirmed that expression of plant defensin genes is systemically triggered upon fungal infection whereas radish PR-1 gene expression is only activated locally. In contrast to the radish PR-1 gene(s), expression of the radish plant defensin genes was not induced by external application of salicylic acid. Activation of the plant defensin genes, but not that of PR-1 genes, occurred upon treatment with methyl jasmonate, ethylene and paraquat.

Mutational analysis of a plant defensin from radish (Raphanus sativus L.) reveals two adjacent sites important for antifungal activity.

Mutational analysis of Rs-AFP2, a radish antifungal peptide belonging to a family of peptides referred to as plant defensins, was performed using polymerase chain reaction-based site-directed mutagenesis and yeast as a system for heterologous expression. The strategy followed to select candidate amino acid residues for substitution was based on sequence comparison of Rs-AFP2 with other plant defensins exhibiting differential antifungal properties. Several mutations giving rise to peptide variants with reduced antifungal activity against Fusarium culmorum were identified. In parallel, an attempt was made to construct variants with enhanced antifungal activity by substituting single amino acids by arginine. Two arginine substitution variants were found to be more active than wild-type Rs-AFP2 in media with high ionic strength. Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to be important for antifungal activity, namely the region around the type VI beta-turn connecting beta-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting beta-strand 1 and the alpha-helix and contiguous residues on the alpha-helix and beta-strand 3, on the other hand. When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca2+ uptake by up to 20-fold. An arginine substitution variant with enhanced antifungal activity caused increased Ca2+ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca2+ uptake.

Antimicrobial peptides from Mirabilis jalapa and Amaranthus caudatus: expression, processing, localization and biological activity in transgenic tobacco.

The cDNAs encoding the seed antimicrobial peptides (AMPs) from Mirabilis jalapa (Mj-AMP2) and Amaranthus caudatus (Ac-AMP2) have previously been characterized and it was found that Mj-AMP2 and Ac-AMP2 are processed from a precursor preprotein and preproprotein, respectively [De Bolle et al., Plant Mol Biol 28:713-721 (1995) and 22:1187-1190 (1993), respectively]. In order to study the processing, sorting and biological activity of these antimicrobial peptides in transgenic tobacco, four different gene constructs were made: a Mj-AMP2 wild-type gene construct, a Mj-AMP2 mutant gene construct which was extended by a sequence encoding the barley lectin carboxyl-terminal propeptide, a known vacuolar targeting signal [Bednarek and Raikhel, Plant Cell 3: 1195-1206 (1991)]; an Ac-AMP2 wild-type gene construct; and finally, an Ac-AMP2 mutant gene construct which was truncated in order to delete the sequence encoding the genuine carboxyl-terminal propeptide. Processing and localization analysis indicated that an isoform of Ac-AMP2 with a cleaved-off carboxyl-terminal arginine was localized in the intercellular fluid fraction of plants expressing either wild-type or mutant gene constructs. Mj-AMP2 was recovered extracellularly in plants transformed with Mj-AMP2 wild-type gene construct, whereas an Mj-AMP2 isoform with a cleaved-off carboxyl-terminal arginine accumulated intracellularly in plants expressing the mutant precursor protein with the barley lectin propeptide. The in vitro antifungal activity of the AMPs purified from transgenic tobacco expressing any of the four different precursor proteins was similar to that of the authentic proteins. However, none of the transgenic plants showed enhanced resistance against infection with either Botrytis cinerea or Alternaria longipes.

A potent antimicrobial protein from onion seeds showing sequence homology to plant lipid transfer proteins.

An antimicrobial protein of about 10 kD, called Ace-AMP1, was isolated from onion (Allium cepa L.) seeds. Based on the near-complete amino acid sequence of this protein, oligonucleotides were designed for polymerase chain reaction-based cloning of the corresponding cDNA. The mature protein is homologous to plant nonspecific lipid transfer proteins (nsLTPs), but it shares only 76% of the residues that are conserved among all known plant nsLTPs and is unusually rich in arginine. Ace-AMP1 inhibits all 12 tested plant pathogenic fungi at concentrations below 10 micrograms mL-1. Its antifungal activity is either not at all or is weakly affected by the presence of different cations at concentrations approximating physiological ionic strength conditions. Ace-AMP1 is also active on two Gram-positive bacteria but is apparently not toxic for Gram-negative bacteria and cultured human cells. In contrast to nsLTPs such as those isolated from radish or maize seeds, Ace-AMP1 was unable to transfer phospholipids from liposomes to mitochondria. On the other hand, lipid transfer proteins from wheat and maize seeds showed little or no antimicrobial activity, whereas the radish lipid transfer protein displayed antifungal activity only in media with low cation concentrations. The relevance of these findings with regard to the function of nsLTPs is discussed.

A molecular method for S-allele identification in apple based on allele-specific PCR.

cDNA sequences corresponding to two self-incompatibility alleles (S-alleles) of the apple cv 'Golden Delicious' have previously been described, and now we report the identification of three additional S-allele cDNAs of apple, one of which was isolated from a pistil cDNA library of cv 'Idared' and two of which were obtained by reverse transcription-PCR (RT-PCR) on pistil RNA of cv 'Queen's Cox'. A comparison of the deduced amino acid sequences of these five S-allele cDNAs revealed an average homology of 69%. Based on the nucleotide sequences of these S-allele cDNAs, we developed a molecular technique for the diagnostic identification of the five different S-alleles in apple cultivars. The method used consists of allele-specific PCR amplification of genomic DNA followed by digestion of the amplification product with an allele-specific restriction endonuclease. Analysis of a number of apple cultivars with known S-phenotype consistently showed coincidence of phenotypic and direct molecular data of the S-allele constitution of the cultivars. It is concluded that the S-allele identification approach reported here provides a rapid and useful method to determine the S-genotype of apple cultivars.

In Vitro Antifungal Activity of a Radish (Raphanus sativus L.) Seed Protein Homologous to Nonspecific Lipid Transfer Proteins.

A basic 9-kD protein was purified from seeds of radish (Raphanus sativus L.). The 43 amino-terminal amino acids show extensive sequence identity with nonspecific lipid transfer proteins from other plant species. The radish seed nonspecific lipid transfer protein-like protein inhibits the growth of several fungi in vitro.

A technique for detecting antifungal activity of proteins separated by polyacrylamide gel electrophoresis.

A technique was developed for the detection of antifungal activity of proteins after discontinuous polyacrylamide gel electrophoresis under native conditions. The antifungal activity is detected as growth inhibition zones in a homogeneous fungal lawn, grown in an agar layer spread on top of the polyacrylamide gel. The position of proteins with antifungal activity can be determined on a diffusion blot prepared from the same gel. The technique is illustrated for three antifungal plant proteins, i.e. alpha-purothionin, Urtica dioica agglutinin, and tobacco chitinase.