Rupert W. Osborn

Antimicrobial peptides from plants

Abstract Peptides with antimicrobial properties are present in most if not all plant species. All plant antimicrobial peptides isolated so far contain even numbers of cysteines (4, 6, or 8), which are all pairwise connected by disulfide bridges, thus providing high stability to the peptides. Based on homologies at the primary structure level, plant antimicrobial peptides can be classified into distinct families including thionins, plant defensins, lipid transfer proteins, and he vein- and knottin-type antimicrobial peptides. Detailed three-dimensional structure information has been obtained for one or more members of these peptide families. All antimicrobial peptides studied thus far appear to exert their antimicrobial effect at the level of the plasma membrane of the target microorganism, but the different peptide types are likely to act via different mechanisms. Antimicrobial peptides can occur in all plant organs. In unstressed organs, antimicrobial peptides are usually most abundant in the outer cell ...

Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae

From seeds of Aesculus hippocastanum, Clitoria ternatea, Dahlia merckii and Heuchera sanguinea five antifungal proteins were isolated and shown to be homologous to plant defensins previously characterised from radish seeds and γ‐thionins from Poaceae seeds. Based on the spectrum of their antimicriobial activity and the morphological distortions they induce on fungi the peptides can be divided into two classes. The peptides did not inhibit any of three different α‐amylases.

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 [mu]g 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.

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) fromMirabilis jalapa (Mj-AMP2) andAmaranthus 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 Bolleet 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-AMP2wild-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-AMP2wild-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 eitherwild-type or mutant gene constructs. Mj-AMP2 was recovered extracellularly in plants transformed with Mj-AMP2wild-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. Thein 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 eitherBotrytis einerea orAlternaria longipes.

A new family of basic cysteine-rich plant antifungal proteins from Brassicaceae species

Out of seeds of 4 Brassicaceae species, 7 antifungal proteins were isolated which are nearly identical to 2 previously characterized radish seed antifungal proteins. These basic proteins, multimers of a 5 kDa polypeptide, specifically inhibit fungal growth. One of the antifungal proteins has decreased antifungal activity and an increased antibacterial activity. In addition, the previously described antifungal activity of the radish seed 2S albumins was extended to the 2S albumins of the seeds of the 4 other Brassicaceae species. A 2S albumin‐like trypsin‐inhibitor from barley seeds was found to have much less activity against fungi.

Synergistic Enhancement of the Antifungal Activity of Wheat and Barley Thionins by Radish and Oilseed Rape 2S Albumins and by Barley Trypsin Inhibitors.

Although thionins and 2S albumins are generally considered as storage proteins, both classes of seed proteins are known to inhibit the growth of pathogenic fungi. We have now found that the wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) thionin concentration required for 50% inhibition of fungal growth is lowered 2- to 73-fold when combined with 2S albumins (at sub- or noninhibitory concentrations) from radish (Raphanus sativus L.) or oilseed rape (Brassica napus L.). Furthermore, the thionin antifungal activity is synergistically enhanced (2- to 33-fold) by either the small subunit or the large subunit of the radish 2S albumins. Three other 2S albumin-like proteins, the barley trypsin inhibitor and two barley Bowman-Birk-type trypsin inhibitor isoforms, also act synergistically with the thionins (2- to 55-fold). The synergistic activity of thionins combined with 2S albumins is restricted to filamentous fungi and to some Gram-positive bacteria, whereas Gram-negative bacteria, yeast, cultured human cells, and erythrocytes do not show an increased sensitivity to thionin/albumin combinations (relative to the sensitivity to the thionins alone). Scanning electron microscopy and measurement of K+ leakage from fungal hyphae revealed that 2S albumins have the same mode of action as thionins, namely the permeabilization of the hyphal plasmalemma. Moreover, 2S albumins and thionins act synergistically in their ability to permeabilize fungal membranes.

A Novel Family of Small Cysteine-rich Antimicrobial Peptides from Seed of Impatiens balsamina Is Derived from a Single Precursor Protein

Four closely related peptides were isolated from seed of Impatiens balsamina and were shown to be inhibitory to the growth of a range of fungi and bacteria, while not being cytotoxic to cultured human cells. The peptides, designated Ib-AMP1, Ib-AMP2, Ib-AMP3, and Ib-AMP4, are 20 amino acids long and are the smallest plant-derived antimicrobial peptides isolated to date. The Ib-AMPs (I. balsamina antimicrobial peptides) are highly basic and contain four cysteine residues which form two intramolecular disulfide bonds. Searches of protein data bases have failed to identify any proteins with significant homology to the peptides described here. Characterization of isolated cDNAs reveals that all four peptides are encoded within a single transcript. The predicted Ib-AMP precursor protein consists of a prepeptide followed by 6 mature peptide domains, each flanked by propeptide domains ranging from 16 to 35 amino acids in length. Such a primary structure with repeated alternating basic mature peptide domains and acidic propeptide domains has, to date, not been reported in plants.

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 β-turn connecting β-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting β-strand 1 and the α-helix and contiguous residues on the α-helix and β-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.

Specific binding sites for an antifungal plant defensin from dahlia (Dahlia merckii) on fungal cells are required for antifungal activity.

Dm-AMP1, an antifungal plant defensin from seeds of dahlia (Dahlia merckii), was radioactively labeled with t-butoxycarbonyl-[35S]-L-methionine N-hydroxy-succinimi-dylester. This procedure yielded a 35S-labeled peptide with unaltered antifungal activity. [35S]Dm-AMP1 was used to assess binding on living cells of the filamentous fungus Neurospora crassa and the unicellular fungus Saccharomyces cerevisiae. Binding of [35S]Dm-AMP1 to fungal cells was saturable and could be competed for by preincubation with excess, unlabeled Dm-AMP1 as well as with Ah-AMP1 and Ct-AMP1, two plant defensins that are highly homologous to Dm-AMP1. In contrast, binding could not be competed for by more distantly related plant defensins or structurally unrelated antimicrobial peptides. Binding of [35S]Dm-AMP1 to either N. crassa or S. cerevisiae cells was apparently irreversible. In addition, whole cells and microsomal membrane fractions from two independently obtained S. cerevisiae mutants selected for resistance to Dm-AMP1 exhibited severely reduced binding affinity for [35S]Dm-AMP1, compared with wild-type yeast. This finding suggests that binding of Dm-AMP1 to S. cerevisiae plasma membranes is required for antifungal activity of this protein.

Specific, high affinity binding sites for an antifungal plant defensin on Neurospora crassa hyphae and microsomal membranes

Hs-AFP1, an antifungal plant defensin from seed of the plant Heuchera sanguinea, was radioactively labeled usingt-butoxycarbonyl-[35S]l-methionineN-hydroxysuccinimidyl ester, resulting in a35S-labeled peptide with unaltered antifungal activity. [35S]Hs-AFP1 was used to assess binding on living hyphae of the fungus Neurospora crassa. Binding of [35S]Hs-AFP1 was found to be competitive, reversible, and saturable with an apparent K d of 29 nmand a B max of 1.4 pmol/mg protein. [35S]Hs-AFP1 also bound specifically and reversibly to microsomal membranes derived from N. crassa hyphae with aK d of 27 nm and aB max of 102 pmol/mg protein. The similarity inK d value between binding sites on hyphae and microsomes indicates that Hs-AFP1 binding sites reside on the plasma membrane. Binding of [35S]Hs-AFP1 to both hyphae and microsomal membranes could be competed to some extent by four different structurally related plant defensins but not by various structurally unrelated antimicrobial peptides. In addition, an inactive single amino acid substitution variant of the antifungal plant defensin Rs-AFP2 fromRaphanus sativus seed was also unable to displace [35S]Hs-AFP1 from its binding sites, whereas Rs-AFP2 itself was able to compete with [35S]Hs-AFP1.