Marilyn A. Anderson

Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis

Several members of the Rubiaceae and Violaceae families produce a series of cyclotides or macrocyclic peptides of 29–31 amino acids with an embedded cystine knot. We aim to understand the mechanism of synthesis of cyclic peptides in plants and have isolated a cDNA clone that encodes the cyclotide kalata B1 as well as three other clones for related cyclotides from the African plant Oldenlandia affinis. The cDNA clones encode prepropeptides with a 20-aa signal sequence, an N-terminal prosequence of 46–68 amino acids and one, two, or three cyclotide domains separated by regions of about 25 aa. The corresponding cyclotides have been isolated from plant material, indicating that the cyclotide domains are excised and cyclized from all four predicted precursor proteins. The exact processing site is likely to lie on the N-terminal side of the strongly conserved GlyLeuPro or SerLeuPro sequence that flanks both sides of the cyclotide domain. Cyclotides have previously been assigned an antimicrobial function; here we describe a potent inhibitory effect on the growth and development of larvae from the Lepidopteran species Helicoverpa punctigera.

Defensins - components of the innate immune system in plants.

Plant defensins are small (c.a. 5 kDa), basic, cysteine-rich proteins with antimicrobial activities. They are ubiquitous in plants and form part of the innate immunity arsenal. Plant defensins are encoded by small multigene families and are expressed in various plant tissues, but are best characterized in seeds. They are typically produced as preproteins, however, a small subset are produced as larger precursors with C-terminal prodomains. To date, the three-dimensional solution structures of seven seed- and two floral-derived defensins have been elucidated by (1)H-NMR spectroscopy. Despite limited amino acid sequence identities, these defensins have comparable global folds with features that are characteristic of the cysteine-stabilized alphabeta (CSalphabeta) motif. Interestingly, their structures are remarkably similar to those of insect defensins and scorpion toxins. Functionally, these proteins exhibit a diverse array of biological activities, although they all serve a common function as defenders of their hosts. This review describes the distribution, biosynthesis, structure, function and mode of action of plant defensins and reflects on their potential in agribiotechnological applications.

Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors.

A cDNA clone, NA-PI-II, encoding a protein with partial identity to proteinase inhibitor (PI) II of potato and tomato has been isolated from a cDNA library constructed from Nicotiana alata stigma and style mRNA. The cDNA encodes a polypeptide of 397 amino acids with a putative signal peptide of 29 amino acids and six repeated domains, each with a potential reactive site. Domains 1 and 2 have chymotrypsin-specific sites and domains 3, 4, 5, and 6 have sites specific for trypsin. In situ hybridization experiments demonstrated that expression of the gene is restricted to the stigma of both immature and mature pistils. Peptides with inhibitory activity toward chymotrypsin and trypsin have been isolated from stigmas of N. alata. The N-terminal amino acid sequence obtained from this protein preparation corresponds to six regions in the cDNA clone NA-PI-II. The purified PI protein preparation is likely to be composed of a mixture of up to five similar peptides of approximately 6 kD, produced in vivo by proteolytic processing of a 42-kD precursor. The PI may function to protect the reproductive tissue against potential pathogens.

An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.

Isolation and Properties of Floral Defensins from Ornamental Tobacco and Petunia

The flowers of the solanaceous plants ornamental tobacco (Nicotiana alata) and petunia (Petunia hybrida) produce high levels of defensins during the early stages of development. In contrast to the well-described seed defensins, these floral defensins are produced as precursors with C-terminal prodomains of 27 to 33 amino acids in addition to a typical secretion signal peptide and central defensin domain of 47 or 49 amino acids. Defensins isolated from N. alata and petunia flowers lack the C-terminal domain, suggesting that it is removed during or after transit through the secretory pathway. Immunogold electron microscopy has been used to demonstrate that the N. alata defensin is deposited in the vacuole. In addition to the eight canonical cysteine residues that define the plant defensin family, the two petunia defensins have an extra pair of cysteines that form a fifth disulfide bond and hence define a new subclass of this family of proteins. Expression of the N. alata defensinNaD1 is predominantly flower specific and is most active during the early stages of flower development. NaD1transcripts accumulate in the outermost cell layers of petals, sepals, anthers, and styles, consistent with a role in protection of the reproductive organs against potential pathogens. The floral defensins inhibit the growth of Botrytis cinerea andFusarium oxysporum in vitro, providing further support for a role in protection of floral tissues against pathogen invasion.

Gametophytic Self-Incompatibility Systems.

is one of the mechanisms that have evolved to encourage outbreeding in flowering plants and is defined as “the inability of a fertile hermaphrodite seed plant to produce zygotes after self-pollination” (de Nettancourt, 1977). The effectiveness of SI in promoting outbreeding is believed to be one of the most important factors that ensured the evolu- tionary success of flowering plants (Whitehouse, 1951). It is a genetically controlled phenomenon, and in many cases, the control is by a single locus (known as the S locus) with a large number of alleles, up to severa1 hundred in some species (Ockendon, 1974; de Nettancourt, 1977). SI has been a favor- ite topic for botanists and geneticists since Darwin (1877) first discussed the phenomenon and suggested the idea of its cen- tral significance during the evolution of flowering plants. During the century or more of work on the subject, there have been a number of key reviews, the most significant

Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae

Several members of the Rubiaceae and Violaceae plant families produce a series of cyclotides or macrocyclic peptides of 28–37 aa with an embedded cystine knot. The cyclic peptide backbone together with the knotted and strongly braced structure confers exceptional chemical and biological stability that has attracted attention for potential pharmaceutical applications. Cyclotides display a diverse range of biological activities, such as uterotonic action, anti-HIV activity, and neurotensin antagonism. In plants, their primary role is probably protection from insect attack. Ingestion of the cyclotide kalata B1 severely retards the growth of larvae from the Lepidopteran species Helicoverpa armigera. We examined the gut of these larvae after consumption of kalata B1 by light, scanning, and transmission electron microscopy. We established that kalata B1 induces disruption of the microvilli, blebbing, swelling, and ultimately rupture of the cells of the gut epithelium. The histology of this response is similar to the response of H. armigera larvae to the Bacillus thuringiensis delta-endotoxin, which is widely used to control these insect pests of crops such as cotton.

A new substrate for investigating the specificity of β-glucan hydrolases

The tine distinctions between the substrate speciflcities of the /3-glucan endo-hydrolases [ 1 ] can only be defined precisely by the use of substrates with known, and preferably regular, structures. A new 1,3; 1,4-/Iglucan with these properties has been prepared and its use in studies on the specificity of /3-glucan endohydrolases is described. the proportions of 1,3and 1,4linkages in the mixedlinked substrates and in the hydrolysis products, that for the Streptomyces enzyme, x can only be a 1,4&kage. However, unequivocal proof was not provided by these experiments; An Aspergillus niger fi-glucan hydrolase [5] and an enzyme from Trichoderma uiride [6] appear to have the same specificity as the Streptomyces enzyme.

The plant defensin, NaD1, enters the cytoplasm of Fusarium Oxysporum hyphae.

The plant defensin, NaD1, from the flowers of Nicotiana alata displays potent antifungal activity against a variety of agronomically important filamentous fungi including Fusarium oxysporum f. sp. vasinfectum (Fov). To understand the mechanism of this antifungal activity, the effect of NaD1 on Fov fungal membranes and the location of NaD1 in treated hyphae was examined using various fluorescence techniques. NaD1 permeabilized fungal plasma membranes via the formation of an aperture with an internal diameter of between 14 and 22Å. NaD1 bound to the cell walls of all treated hyphae and entered several hyphae, resulting in granulation of the cytoplasm and cell death. These results suggest that the activity of antifungal plant defensins may not be restricted to the hyphal membrane and that they enter cells and affect intracellular targets.

Permeabilization of Fungal Hyphae by the Plant Defensin NaD1 Occurs through a Cell Wall-dependent Process

The antifungal activity of the plant defensin NaD1 involves specific interaction with the fungal cell wall, followed by permeabilization of the plasma membrane and entry of NaD1 into the cytoplasm. Prior to this study, the role of membrane permeabilization in the activity of NaD1, as well as the relevance of cell wall binding, had not been investigated. To address this, the permeabilization of Fusarium oxysporum f. sp. vasinfectum hyphae by NaD1 was investigated and compared with that by other antimicrobial peptides, including the cecropin-melittin hybrid peptide CP-29, the bovine peptide BMAP-28, and the human peptide LL-37, which are believed to act largely through membrane disruption. NaD1 appeared to permeabilize cells via a novel mechanism that required the presence of the fungal cell wall. NaD1 and Bac2A, a linear variant of the bovine peptide bactenecin, were able to enter the cytoplasm of treated hyphae, indicating that cell death is accelerated by interaction with intracellular targets.