ORCID Profile
0000-0002-8257-5128
Current Organisation
La Trobe University - Melbourne Campus
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Biochemistry and Cell Biology | Biochemistry And Cell Biology Not Elsewhere Classified | Proteomics and Intermolecular Interactions (excl. Medical Proteomics) | Mycology | Crop and Pasture Protection (Pests, Diseases and Weeds) | Biological And Medical Chemistry | Medicinal and Biomolecular Chemistry | Biotechnology Not Elsewhere Classified | Plant Cell and Molecular Biology | Microbiology | Medical Biotechnology | Plant Protection (Pests, Diseases And Weeds) | Receptors and Membrane Biology | Biochemistry and Cell Biology not elsewhere classified | Animal Protection (Pests And Pathogens) | Biological Physics | Medical Biotechnology | Macromolecular Chemistry Not Elsewhere Classified | Membrane Biology | Cellular Interactions (Incl. Adhesion, Matrix, Cell Wall) | Infectious Agents | Medical Parasitology | Characterisation of Biological Macromolecules | Agriculture, Land and Farm Management | Crop and Pasture Production | Animal Production | Plant Protection (Pests, Diseases And Weeds) | Medical Biochemistry and Metabolomics not elsewhere classified | Global Change Biology | Enzymes | Protein Trafficking | Plant Pathology | Plant Physiology | Farm Management, Rural Management and Agribusiness | Bioprocessing, Bioproduction and Bioproducts
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Cotton | Field crops | Control of Plant Pests, Diseases and Exotic Species in Farmland, Arable Cropland and Permanent Cropland Environments | Treatments (e.g. chemicals, antibiotics) | Crop and animal protection chemicals | Infectious Diseases | Environmentally Sustainable Plant Production not elsewhere classified | Plant Production and Plant Primary Products not elsewhere classified | Horticultural crops | Wheat | Biological sciences | Livestock | Prevention—biologicals (e.g. vaccines) | Diagnostics | Health not elsewhere classified | Cotton lint and cotton seed | Maize | Plant Extracts (e.g. Pyrethrum, Alkaloids, Jojoba Oil) | Infectious diseases | Industrial Crops not elsewhere classified | Climate and Climate Change not elsewhere classified | Expanding Knowledge in the Chemical Sciences | Expanding Knowledge in the Physical Sciences |
Related Links
Publications
Arabinogalactan-proteins are localized extracellularly in the transmitting tissue of Nicotiana alata link and otto, an ornamental tobacco
Publisher: Elsevier BV
Date: 1985
Characterization of the Protease Processing Sites in a Multidomain Proteinase Inhibitor Precursor from Nicotiana Alata
Publisher: Wiley
Date: 05-1995
Quantitative analysis of backbone-cyclised peptides in plants
Publisher: Elsevier BV
Date: 09-2008
DOI: 10.1016/J.JCHROMB.2008.07.023
Abstract: Progress in understanding the biosynthetic pathway of the cyclotides has been h ered as this unique family of cyclic plant peptides are notoriously difficult to analyse by standard proteomic approaches such as gel electrophoresis. We have developed a simple, rapid and robust strategy for the quantification of cyclotides in crude plant extracts using MALDI-TOF MS making use of generic peptides similar in mass to the analyte as internal standards for calibration. Linearity (r(2)>0.99) over two orders of magnitude (down to femtomole levels) was achieved in plant extracts, allowing quantitative analysis of transgenic and endogenous peptide expression.
High-affinity Cyclic Peptide Matriptase Inhibitors
Publisher: Elsevier BV
Date: 05-2013
Action of the style product of the self-Incompatibility gene of Nicotiana alata (S-RNase) on in vitro-grown pollen tubes
Publisher: JSTOR
Date: 03-1991
DOI: 10.2307/3869367
Resistance to the Plant Defensin NaD1 Features Modifications to the Cell Wall and Osmo-Regulation Pathways of Yeast
Publisher: Frontiers Media SA
Date: 24-07-2018
Antibacterial and antifungal activity of defensins from the Australian paralysis tick, Ixodes holocyclus
Publisher: Elsevier BV
Date: 10-2019
DOI: 10.1016/J.TTBDIS.2019.101269
Abstract: Tick innate immunity involves humoral and cellular responses. Among the humoral effector molecules in ticks are the defensins which are a family of small peptides with a conserved γ-core motif that is crucial for their antimicrobial activity. Defensin families have been identified in several hard and soft tick species. However, little is known about the presence and antimicrobial activity of defensins from the Australian paralysis tick Ixodes holocyclus. In this study the I. holocyclus transcriptome was searched for the presence of defensins. Unique and non-redundant defensin sequences were identified and designated as holosins 1 - 5. The antimicrobial activity of holosins 2 and 3 and of the predicted γ-cores of holosins 1-4 (HoloTickCores 1-4), was assessed using Gram-negative and Gram-positive bacteria as well as the fungus Fusarium graminearum and the yeast Candida albicans. All holosins had molecular features that are conserved in other tick defensins. Furthermore holosins 2 and 3 were very active against the Gram-positive bacteria Staphylococcus aureus and Listeria grayi. Holosins 2 and 3 were also active against F. graminearum and C. albicans and 5 μM of peptide abrogate the growth of these microorganisms. The activity of the synthetic γ-cores was lower than that of the mature defensins apart from HoloTickCore 2 which had activity comparable to mature holosin 2 against the Gram-negative bacterium Escherichia coli. This study reveals the presence of a multigene defensin family in I. holocyclus with wide antimicrobial activity.
Molecular basis for the resistance of an insect chymotrypsin to a potato type II proteinase inhibitor
Publisher: Proceedings of the National Academy of Sciences
Date: 09-08-2010
Abstract: Plants produce a variety of proteinase inhibitors (PIs) that have a major function in defense against insect herbivores. In turn, insects have developed strategies to minimize the effect of dietary PIs on digestion. We have discovered that Helicoverpa larvae that survive consumption of a multidomain serine PI from Nicotiana alata (NaPI) contain high levels of a chymotrypsin that is not inhibited by NaPI. Here we describe the isolation of this NaPI-resistant chymotrypsin and an NaPI-susceptible chymotrypsin from Helicoverpa larvae, together with their corresponding cDNAs. We investigated the mechanism of resistance by mutating selected positions of the NaPI-susceptible chymotrypsin using the corresponding amino acids of the NaPI-resistant chymotrypsin. Four critical residues that conferred resistance to NaPI were identified. Molecular modeling revealed that a Phe→Leu substitution at position 37 in the chymotrypsin results in the loss of important binding contacts with NaPI. Identification of the molecular mechanisms that contribute to PI resistance in insect digestive proteases will enable us to develop better inhibitors for the control of lepidopteran species that are major agricultural pests worldwide.
Insecticidal plant cyclotides and related cystine knot toxins
Publisher: Elsevier BV
Date: 03-2007
DOI: 10.1016/J.TOXICON.2006.11.018
Abstract: Cyclotides are small disulphide-rich peptides found in plants from the violet (Violaceae), coffee (Rubiaceae) and cucurbit (Cucurbitaceae) families. They have the distinguishing structural features of a macrocyclic peptide backbone and a cystine knot made up of six conserved cysteine residues, which makes cyclotides exceptionally stable. In idual plants express a suite of cyclotides in a wide range of tissue types, including leaves, flowers, stems and roots and it is thought that their natural function in plants is as defence agents. This proposal is supported by their high expression levels in plants and their toxic and growth retardant activity in feeding trials against Helicoverpa spp. insect pests. This review describes the structures and activities of cyclotides with specific reference to their insecticidal activity and compares them with structurally similar cystine knot proteins from peas (Pisum sativum) and an amaranthus crop plant (Amaranthus hypocondriancus). More broadly, cystine knot proteins are common in a wide range of organisms from fungi to mammals, and it appears that this interesting structural motif has evolved independently in different organisms as a stable protein framework that has a variety of biological functions.
Extracellular vesicles secreted by Saccharomyces cerevisiae are involved in cell wall remodelling
Publisher: Springer Science and Business Media LLC
Date: 09-08-2019
DOI: 10.1038/S42003-019-0538-8
Abstract: Extracellular vesicles (EVs) are membranous vesicles that are released by cells. In this study, the role of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in the biogenesis of yeast EVs was examined. Knockout of components of the ESCRT machinery altered the morphology and size of EVs as well as decreased the abundance of EVs. In contrast, strains with deletions in cell wall biosynthesis genes, produced more EVs than wildtype. Proteomic analysis highlighted the depletion of ESCRT components and enrichment of cell wall remodelling enzymes, glucan synthase subunit Fks1 and chitin synthase Chs3, in yeast EVs. Interestingly, EVs containing Fks1 and Chs3 rescued the yeast cells from antifungal molecules. However, EVs from fks1 ∆ or chs3 ∆ or the vps23 ∆ chs3 ∆ double knockout strain were unable to rescue the yeast cells as compared to vps23 ∆ EVs. Overall, we have identified a potential role for yeast EVs in cell wall remodelling.
Recent developments in the molecular genetics and biology of self-incompatibility
Publisher: Springer Science and Business Media LLC
Date: 09-1989
DOI: 10.1007/BF00025314
Abstract: Tumor rupture and pulmonary metastasis in patients with hepatocellular carcinoma are both associated with poor prognosis and treatment strategies are controversial. Here we report a 50-year-old man with survival of over 90 months after undergoing an extended right lobectomy for a ruptured hepatocellular carcinoma and then repeated resections for pulmonary metastasis during the followup period. This case report shows that surgical resection can be an effective treatment for patients with both ruptured hepatocellular carcinoma and pulmonary recurrences.
Hydrolysis of α-d-glucans and α-d-gluco-oligosaccharides by cladosporium resinae glucoamylases
Publisher: Elsevier BV
Date: 11-1980
DOI: 10.1016/S0008-6215(00)84583-8
Abstract: Culture filtrates of Cladosporium resinae ATCC 20495 contain a mixture of enzymes able to convert starch and pullulan efficiently into D-glucose. Culture conditions for optimal production of the pullulan-degrading activity have been established. The amylolytic enzyme preparation was fractionated by ion-exchange and molecular-sieve chromatography, and shown to contain alpha-D-glucosidase, alpha-amylase, and two glucoamylases. The glucoamylases have been purified to homogeneity and their substrate specificities investigated. One of the glucoamylases (termed P) readily hydrolyses the (1 leads to 6)-alpha-D linkages in pullulan, amylopectin, isomaltose, panose, and 6(3)-alpha-D-glucosylmaltotriose. Each of the glucoamylases cleaves the (1 leads to 6)-alpha-D linkage in panose much more readily than that in isomaltose.
Self-incompatibility in Nicotiana alata involves degradation of pollen rRNA
Publisher: Springer Science and Business Media LLC
Date: 10-1990
DOI: 10.1038/347757A0
The plant defensin NaD1 introduces membrane disorder through a specific interaction with the lipid, phosphatidylinositol 4,5 bisphosphate
Publisher: Elsevier BV
Date: 06-2016
DOI: 10.1016/J.BBAMEM.2016.02.016
Abstract: Plant defensins interact with phospholipids in bilayers as part of their cytotoxic activity. Solanaceous class II defensins with the loop 5 sequence pattern "S-[KR]-[ILVQ]-[ILVQ]-[KR]-[KR]" interact with PI(4,5)P2. Here, the prototypical defensin of this class, NaD1, is used to characterise the biophysical interactions between these defensins and phospholipid bilayers. Binding of NaD1 to bilayers containing PI(4,5)P2 occurs rapidly and the interaction is very strong. Dual polarisation interferometry revealed that NaD1 does not dissociate from bilayers containing PI(4,5)P2. Binding of NaD1 to bilayers with or without PI(4,5)P2 induced disorder in the bilayer. However, permeabilisation assays revealed that NaD1 only permeabilised liposomes with PI(4,5)P2 in the bilayer, suggesting a role for this protein-lipid interaction in the plasma membrane permeabilising activity of this defensin. No defensins in the available databases have the PI(4,5)P2 binding sequence outside the solanaceous class II defensins, leading to the hypothesis that PI(4,5)P2 binding co-evolved with the C-terminal propeptide to protect the host cell against the effects of the tight binding of these defensins to their cognate lipid as they travel along the secretory pathway. This data has allowed us to develop a new model to explain how this class of defensins permeabilises plasma membranes to kill target cells.
Co-expression of a cyclizing asparaginyl endopeptidase enables efficient production of cyclic peptides in planta
Publisher: Oxford University Press (OUP)
Date: 22-12-2018
DOI: 10.1093/JXB/ERX422
Discovery of Cyclotides in the Fabaceae Plant Family Provides New Insights into the Cyclization, Evolution, and Distribution of Circular Proteins
Publisher: American Chemical Society (ACS)
Date: 20-01-2011
DOI: 10.1021/CB100388J
Abstract: Cyclotides are plant proteins whose defining structural features are a head-to-tail cyclized backbone and three interlocking disulfide bonds, which in combination are known as a cyclic cystine knot. This unique structural motif confers cyclotides with exceptional resistance to proteolysis. Their endogenous function is thought to be as plant defense agents, associated with their insecticidal and larval growth-inhibitory properties. However, in addition, an array of pharmaceutically relevant biological activities has been ascribed to cyclotides, including anti-HIV, anthelmintic, uterotonic, and antimicrobial effects. So far, >150 cyclotides have been elucidated from members of the Rubiaceae, Violaceae, and Cucurbitaceae plant families, but their wider distribution among other plant families remains unclear. Clitoria ternatea (Butterfly pea) is a member of plant family Fabaceae and through its usage in traditional medicine to aid childbirth bears similarity to Oldenlandia affinis, from which many cyclotides have been isolated. Using a combination of nanospray and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) analyses, we examined seed extracts of C. ternatea and discovered cyclotides in the Fabaceae, the third-largest family of flowering plants. We characterized 12 novel cyclotides, thus expanding knowledge of cyclotide distribution and evolution within the plant kingdom. The discovery of cyclotides containing novel sequence motifs near the in planta cyclization site has provided new insights into cyclotide biosynthesis. In particular, MS analyses of the novel cyclotides from C. ternatea suggest that Asn to Asp variants at the cyclization site are more common than previously recognized. Moreover, this study provides impetus for the examination of other economically and agriculturally significant species within Fabaceae, now the largest plant family from which cyclotides have been described.
Discovery and structures of the cyclotides: novel macrocyclic peptides from plants
Publisher: Springer Science and Business Media LLC
Date: 05-2001
DOI: 10.1007/BF02446507
Synergistic Activity between Two Antifungal Proteins, the Plant Defensin NaD1 and the Bovine Pancreatic Trypsin Inhibitor.
Publisher: American Society for Microbiology
Date: 25-10-2017
Abstract: This work describes the increased activity of a natural antifungal peptide in the presence of another antifungal peptide from a different family. This is termed antifungal synergy. Synergy is important for decreasing the amount of antifungal molecule needed to control the disease. Traditionally, naturally occurring antifungal molecules are assayed in isolation. Identification of synergistic interactions between antifungal peptides means that their activities in a complex biological system are likely to be different from what we observe when examining them in idually. This study identified synergy between an antifungal peptide and a group of peptides that do not affect fungal growth in vitro . This provides the foundation for generation of transgenic plants with increased resistance to fungal disease and identification of antifungal accessory factors that enhance the activity of innate immune molecules but do not have an antifungal effect on their own.
Bovine pancreatic trypsin inhibitor is a new antifungal peptide that inhibits cellular magnesium uptake
Publisher: Wiley
Date: 08-05-2014
DOI: 10.1111/MMI.12621
Abstract: Antimicrobial peptides (AMPs) are promising agents for control of bacterial and fungal infections. Traditionally, AMPs were thought to act through membrane disruption but recent experiments have revealed a ersity of mechanisms. Here we describe a novel antifungal activity for bovine pancreatic trypsin inhibitor (BPTI). BPTI has several features in common with a subset of antimicrobial proteins in that it is small, cationic and stabilized by disulphide bonds. BPTI inhibits growth of Saccharomyces cerevisiae and the human pathogen Candida albicans. Screening of the yeast heterozygous essential deletion collection identified the magnesium transporter Alr1p as a potential BPTI target. BPTI treatment of wild type cells resulted in a lowering of cellular Mg(2+) levels. Populations treated with BPTI had fewer cells in S-phase of the cell cycle and a corresponding increase of cells in G(0)/G(1) and G(2) phases. The same patterns of cell cycle arrest obtained with BPTI were also obtained with the magnesium channel inhibitor hexamine(III)cobalt chloride. Analysis of the growth inhibition of C. albicans revealed that BPTI is inhibiting growth via the same mechanism in the two yeast species. Inhibition of magnesium uptake by BPTI represents a novel mechanism of action for AMPs.
Chemical synthesis and biosynthesis of the cyclotide family of circular proteins
Publisher: Wiley
Date: 09-2006
DOI: 10.1080/15216540600889532
Abstract: Cyclotides are a recently discovered class of proteins that have a characteristic head-to-tail cyclized backbone stabilized by a knotted arrangement of three disulfide bonds. They are exceptionally resistant to chemical, enzymatic and thermal treatments because of their unique structural scaffold. Cyclotides have a range of bio-activities, including uterotonic, anti-HIV, anti-bacterial and cytotoxic activity but their insecticidal properties suggest that their natural physiological role is in plant defense. They are genetically encoded as linear precursors and subsequently processed to produce mature cyclic peptides but the mechanism by which this occurs remains unknown. Currently most cyclotides are obtained via direct extraction from plants in the Rubiaceae and Violaceae families. To facilitate the screening of cyclotides for structure-activity studies and to exploit them in drug design or agricultural applications a convenient route for the synthesis of cyclotides is vital. In this review the current chemical, recombinant and biosynthetic routes to the production of cyclotides are discussed.
Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae
Publisher: Proceedings of the National Academy of Sciences
Date: 29-01-2008
Abstract: 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 erse 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.
Effects of microstructure and growth conditions on quantum emitters in gallium nitride
Publisher: AIP Publishing
Date: 08-2019
DOI: 10.1063/1.5098794
Abstract: Single-photon emitters in gallium nitride (GaN) are gaining interest as attractive quantum systems due to the well-established techniques for growth and nanofabrication of the host material, as well as its remarkable chemical stability and optoelectronic properties. We investigate the nature of such single-photon emitters in GaN with a systematic analysis of various s les produced under different growth conditions. We explore the effect that intrinsic structural defects (dislocations and stacking faults), doping, and crystal orientation in GaN have on the formation of quantum emitters. We investigate the relationship between the position of the emitters—determined via spectroscopy and photoluminescence measurements—and the location of threading dislocations—characterized both via atomic force microscopy and cathodoluminescence. We find that quantum emitters do not correlate with stacking faults or dislocations instead, they are more likely to originate from point defects or impurities whose density is modulated by the local extended defect density.
A Centipede Toxin Family Defines an Ancient Class of CSαβ Defensins
Publisher: Elsevier BV
Date: 02-2019
DOI: 10.1016/J.STR.2018.10.022
Abstract: Disulfide-rich peptides (DRPs) play erse physiological roles and have emerged as attractive sources of pharmacological tools and drug leads. Here we describe the 3D structure of a centipede venom peptide, U-SLPTX
A radiochemical approach to the determination of carboxylic acid groups in polysaccharides
Publisher: Elsevier BV
Date: 1985
The Three-dimensional Solution Structure of NaD1, a New Floral Defensin from Nicotiana alata and its Application to a Homology Model of the Crop Defense Protein alfAFP
Publisher: Elsevier BV
Date: 2003
DOI: 10.1016/S0022-2836(02)01103-8
Abstract: NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of NaD1, a novel antifungal and insecticidal protein isolated from the flowers of Nicotiana alata. NaD1 is a basic, cysteine-rich protein of 47 residues and is the first ex le of a plant defensin from flowers to be characterized structurally. Its three-dimensional structure consists of an alpha-helix and a triple-stranded antiparallel beta-sheet that are stabilized by four intramolecular disulfide bonds. NaD1 features all the characteristics of the cysteine-stabilized alphabeta motif that has been described for a variety of proteins of differing functions ranging from antibacterial insect defensins and ion channel-perturbing scorpion toxins to an elicitor of the sweet taste response. The protein is biologically active against insect pests, which makes it a potential candidate for use in crop protection. NaD1 shares 31% sequence identity with alfAFP, an antifungal protein from alfalfa that confers resistance to a fungal pathogen in transgenic potatoes. The structure of NaD1 was used to obtain a homology model of alfAFP, since NaD1 has the highest level of sequence identity with alfAFP of any structurally characterized antifungal defensin. The structures of NaD1 and alfAFP were used in conjunction with structure-activity data for the radish defensin Rs-AFP2 to provide an insight into structure-function relationships. In particular, a putative effector site was identified in the structure of NaD1 and in the corresponding homology model of alfAFP.
Dual location of a family of proteinase inhibitors within the stigmas of Nicotiana alata
Publisher: Springer Science and Business Media LLC
Date: 20-10-2006
DOI: 10.1007/S00425-006-0418-6
Abstract: Reproductive and storage tissues of many plants produce large amounts of serine proteinase inhibitors (PIs). The ornamental tobacco, Nicotiana alata, produces a series of 6 kDa chymotrypsin and trypsin inhibitors that accumulate to up to 30% of soluble protein in the stigma. These inhibitors are derived by proteolytic processing of two closely related multidomain precursor proteins. Using immunogold electron microscopy, we find that the stigmatic PIs accumulate in both the central vacuole and in the extracellular mucilage. Labelling with antibodies specific for the C-terminal vacuolar targeting peptide (VTS) of each precursor confirms earlier biochemical data showing that the VTS is removed during passage through the secretory pathway. We have isolated and characterised the extracellular population of PIs, which are largely identical to PIs isolated from whole stigmas and are functional inhibitors of serine proteases. Subcellular fractionation of immature stigmas reveals that a sub-population of the PI precursor protein is proteolytically processed within the endoplasmic reticulum. This proteolysis results in the removal of the vacuolar sorting information, causing secretion of this PI population. We propose a novel mechanism whereby a single gene product may be simultaneously trafficked to two separate compartments mediated by proteolysis early in the secretory pathway.
Transgenic tobacco and peas expressing a proteinase inhibitor from Nicotiana alata have increased insect resistance
Publisher: Springer Science and Business Media LLC
Date: 1999
Bacitracin significantly reduces degradation of peptides in plant cell cultures
Publisher: Wiley
Date: 20-01-1997
DOI: 10.1002/(SICI)1097-0290(19970120)53:2<226::AID-BIT14>3.0.CO;2-I
Identification and mechanism of action of the plant defensin NaD1 as a new member of the antifungal drug arsenal against Candida albicans
Publisher: American Society for Microbiology
Date: 08-2013
DOI: 10.1128/AAC.00365-13
Abstract: In recent decades, pathogenic fungi have become a serious threat to human health, leading to major efforts aimed at characterizing new agents for improved treatments. Promising in this context are antimicrobial peptides produced by animals and plants as part of innate immune systems. Here, we describe an antifungal defensin, NaD1, with activity against the major human pathogen Candida albicans , characterize the mechanism of killing, and identify protection strategies used by the fungus to survive defensin treatment. The mechanism involves interaction between NaD1 and the fungal cell surface followed by membrane permeabilization, entry into the cytoplasm, hyperproduction of reactive oxygen species, and killing induced by oxidative damage. By screening C. albicans mutant libraries, we identified that the high-osmolarity glycerol (HOG) pathway has a unique role in protection against NaD1, while several other stress-responsive pathways are dispensable. The involvement of the HOG pathway is consistent with induction of oxidative stress by NaD1. The HOG pathway has been reported to have a major role in protection of fungi against osmotic stress, but our data indicate that osmotic stress does not contribute significantly to the adverse effects of NaD1 on C. albicans . Our data, together with previous studies with human beta-defensins and salivary histatin 5, indicate that inhibition of the HOG pathway holds promise as a broad strategy for increasing the activity of antimicrobial peptides against C. albicans .
A Novel Plant Protein-disulfide Isomerase Involved in the Oxidative Folding of Cystine Knot Defense Proteins
Publisher: Elsevier BV
Date: 07-2007
Nicotiana alata Defensin Chimeras Reveal Differences in the Mechanism of Fungal and Tumor Cell Killing and an Enhanced Antifungal Variant.
Publisher: American Society for Microbiology
Date: 10-2016
DOI: 10.1128/AAC.01479-16
Abstract: The plant defensin NaD1 is a potent antifungal molecule that also targets tumor cells with a high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity against fungi and no activity against tumor cells. All plant defensins have a common tertiary structure known as a cysteine-stabilized α-β motif which consists of an α helix and a triple-stranded β-sheet stabilized by four disulfide bonds. The chimeras were produced by replacing loops 1 to 7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] and is essential for the potent cytotoxic effect of NaD1 on tumor cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P 2 binding and the tumor cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules, even though they bound poorly to PI(4,5)P 2 , suggesting that additional mechanisms operate against fungal cells. Unexpectedly, the loop 1B swap chimera was 10 times more active than NaD1 against filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to that of the natural variants.
Plant Defensins NaD1 and NaD2 Induce Different Stress Response Pathways in Fungi
Publisher: MDPI AG
Date: 03-09-2016
DOI: 10.3390/IJMS17091473
Identification and Characterization of a Prevacuolar Compartment in Stigmas of Nicotiana alata
Publisher: JSTOR
Date: 08-1999
DOI: 10.2307/3870978
Phosphoinositide-mediated oligomerization of a defensin induces cell lysis
Publisher: eLife Sciences Publications, Ltd
Date: 04-2014
DOI: 10.7554/ELIFE.01808
Abstract: Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the crystal structure of a NaD1:PIP2 complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP2 molecules through a unique ‘cationic grip’ configuration. Site-directed mutagenesis of NaD1 confirms that PIP2-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP2 that permeabilizes cells via a novel membrane disrupting mechanism.
Solution structures by 1 H NMR of the novel cyclic trypsin inhibitor SFTI-1 from sunflower seeds and an acyclic permutant 1 1Edited by M. F. Summers
Publisher: Elsevier BV
Date: 08-2001
Proteinase inhibitors from Nicotiana alata enhance plant resistance to insect pests
Publisher: Elsevier BV
Date: 09-1997
DOI: 10.1016/S0022-1910(97)00026-7
Abstract: The ornamental tobacco (Nicotiana alata) produces one 6-kDa chymotrypsin inhibitor and four 6-kDa trypsin inhibitors from a single 40.3-kDa precursor protein. Three different approaches have been used to assess the potential of these proteinase inhibitors (PIs) in insect control. The first was an in-vitro approach in which all five inhibitors, the single chymotrypsin inhibitor or three of the four trypsin inhibitors were tested for their ability to inhibit gut protease activity in insects from four orders. The second approach was to incorporate the N. alata PIs in the artificial diet of the native budworm (Helicoverpa punctigera) and the black field cricket (Teleogryllus commodus). H. punctigera larvae and T. commodus nymphs had a significant (P<0.01) reduction in growth after ingestion of the PI and were more lethargic than insects on the control diet. Several of the H. punctigera larvae also failed to complete moulting at the third or fourth instar. The third approach was to express the N. alata PIs in transgenic tobacco under the control of the 35S CaMV promoter. When H. punctigera larvae were fed tobacco leaves expressing the N. alata PIs at 0.2% soluble protein, significant (P<0.01) differences in mortality and/or growth rate were observed.
The N‐terminal pro‐domain of the kalata B1 cyclotide precursor is intrinsically unstructured
Publisher: Wiley
Date: 11-2016
DOI: 10.1002/BIP.22977
Abstract: Cyclotides are plant-derived, gene-encoded, circular peptides with a range of host-defense functions, including insecticidal activity. They also have potential as pharmaceutical scaffolds and understanding their biosynthesis is important to facilitate their large-scale production. Insights into the biosynthesis of cyclotides are emerging but there are still open questions, particularly regarding the influence of the structure of the precursor proteins on processing/biosynthetic pathways. The precursor protein of kalata B1, encoded by the plant Oldenlandia affinis, contains N- and C-terminal propeptides that flank the mature cyclotide domain. The C-terminal region (ctr) is important for the cyclization process, whereas the N-terminal repeat (ntr) has been implicated in vacuolar targeting. In this study we examined the structure and folding of various truncated constructs of the ntr coupled to the mature domain of kalata B1. Despite the ntr having a well-defined helical structure in isolation, once coupled to the natively folded mature domain there is no evidence of an ordered structure. Surprisingly, the ntr appears to be highly disordered and induces self-association of the precursor. This self-association might be associated with the role of the ntr as a vacuolar-targeting signal, as previously shown for unrelated storage proteins.
Evolutionary Origins of a Bioactive Peptide Buried within Preproalbumin
Publisher: Oxford University Press (OUP)
Date: 03-2014
Abstract: The de novo evolution of proteins is now considered a frequented route for biological innovation, but the genetic and biochemical processes that lead to each newly created protein are often poorly documented. The common sunflower (Helianthus annuus) contains the unusual gene PawS1 (Preproalbumin with SFTI-1) that encodes a precursor for seed storage albumin however, in a region usually discarded during albumin maturation, its sequence is matured into SFTI-1, a protease-inhibiting cyclic peptide with a motif homologous to unrelated inhibitors from legumes, cereals, and frogs. To understand how PawS1 acquired this additional peptide with novel biochemical functionality, we cloned PawS1 genes and showed that this dual destiny is over 18 million years old. This new family of mostly backbone-cyclic peptides is structurally erse, but the protease-inhibitory motif was restricted to peptides from sunflower and close relatives from its subtribe. We describe a widely distributed, potential evolutionary intermediate PawS-Like1 (PawL1), which is matured into storage albumin, but makes no stable peptide despite possessing residues essential for processing and cyclization from within PawS1. Using sequences we cloned, we retrodict the likely stepwise creation of PawS1's additional destiny within a simple albumin precursor. We propose that relaxed selection enabled SFTI-1 to evolve its inhibitor function by converging upon a successful sequence and structure.
Gametophytic Self-Incompatibility Systems
Publisher: JSTOR
Date: 10-1993
DOI: 10.2307/3869784
Properties and mechanisms of action of naturally occurring antifungal peptides
Publisher: Springer Science and Business Media LLC
Date: 05-02-2013
DOI: 10.1007/S00018-013-1260-1
Abstract: Antimicrobial peptides are a vital component of the innate immune system of all eukaryotic organisms and many of these peptides have potent antifungal activity. They have potential application in the control of fungal pathogens that are a serious threat to both human health and food security. Development of antifungal peptides as therapeutics requires an understanding of their mechanism of action on fungal cells. To date, most research on antimicrobial peptides has focused on their activity against bacteria. Several antimicrobial peptides specifically target fungal cells and are not active against bacteria. Others with broader specificity often have different mechanisms of action against bacteria and fungi. This review focuses on the mechanism of action of naturally occurring antifungal peptides from a erse range of sources including plants, mammals, hibians, insects, crabs, spiders, and fungi. While antimicrobial peptides were originally proposed to act via membrane permeabilization, the mechanism of antifungal activity for these peptides is generally more complex and often involves entry of the peptide into the cell.
The Plant Defensin NaD1 Enters the Cytoplasm of Candida albicans via Endocytosis
Publisher: MDPI AG
Date: 06-02-2018
DOI: 10.3390/JOF4010020
Abstract: Antimicrobial peptides are widespread in nature and are produced by many organisms as a first line of defence against pathogens. These peptides have a broad range of biological activities, such as antibacterial or antifungal activities and act with varied mechanisms of action. A large number of the peptides are hipathic α-helices which act by disrupting plasma membranes and allowing leakage of intracellular contents. However, some peptides have more complex mechanisms of action that require internalisation into the target organisms’ cytoplasm. The method by which these peptides enter the cytoplasm varies, with some requiring the energy dependent processes of endocytosis or polyamine transport and others entering via passive transport. Here we describe the mechanism that the antimicrobial peptide, the plant defensin NaD1, uses to transverse the fungal membrane and gain access to the fungal cytoplasm. By inhibiting ATP synthesis and using an inhibitor of actin polymerisation, we show that NaD1 is internalised into C. albicans yeast cells by the energy-dependent process of endocytosis.
Activation of stress signalling pathways enhances tolerance of fungi to chemical fungicides and antifungal proteins
Publisher: Springer Science and Business Media LLC
Date: 14-02-2014
DOI: 10.1007/S00018-014-1573-8
Abstract: Fungal disease is an increasing problem in both agriculture and human health. Treatment of human fungal disease involves the use of chemical fungicides, which generally target the integrity of the fungal plasma membrane or cell wall. Chemical fungicides used for the treatment of plant disease, have more erse mechanisms of action including inhibition of sterol biosynthesis, microtubule assembly and the mitochondrial respiratory chain. However, these treatments have limitations, including toxicity and the emergence of resistance. This has led to increased interest in the use of antimicrobial peptides for the treatment of fungal disease in both plants and humans. Antimicrobial peptides are a erse group of molecules with differing mechanisms of action, many of which remain poorly understood. Furthermore, it is becoming increasingly apparent that stress response pathways are involved in the tolerance of fungi to both chemical fungicides and antimicrobial peptides. These signalling pathways such as the cell wall integrity and high-osmolarity glycerol pathway are triggered by stimuli, such as cell wall instability, changes in osmolarity and production of reactive oxygen species. Here we review stress signalling induced by treatment of fungi with chemical fungicides and antifungal peptides. Study of these pathways gives insight into how these molecules exert their antifungal effect and also into the mechanisms used by fungi to tolerate sub-lethal treatment by these molecules. Inactivation of stress response pathways represents a potential method of increasing the efficacy of antifungal molecules.
Extracellular Vesicles From the Cotton Pathogen Fusarium oxysporum f. sp. vasinfectum Induce a Phytotoxic Response in Plants
Publisher: Frontiers Media SA
Date: 10-01-2020
Fungal Extracellular Vesicles with a Focus on Proteomic Analysis
Publisher: Wiley
Date: 04-2019
Abstract: Extracellular vesicles (EVs) perform crucial functions in cell-cell communication. The packaging of biomolecules into membrane-enveloped vesicles prior to release into the extracellular environment provides a mechanism for coordinated delivery of multiple signals at high concentrations that is not achievable by classical secretion alone. Most of the understanding of the biosynthesis, composition, and function of EVs comes from mammalian systems. Investigation of fungal EVs, particularly those released by pathogenic yeast species, has revealed erse cargo including proteins, lipids, nucleic acids, carbohydrates, and small molecules. Fungal EVs are proposed to function in a variety of biological processes including virulence and cell wall homeostasis with a focus on host-pathogen interactions. EVs also carry signals between fungal cells allowing for a coordinated attack on a host during infection. Research on fungal EVs in still in its infancy. Here a review of the literature thus far with a focus on proteomic analysis is provided with respect to techniques, results, and prospects.
Selective Removal of Individual Disulfide Bonds within a Potato Type II Serine Proteinase Inhibitor from Nicotiana alata Reveals Differential Stabilization of the Reactive-Site Loop
Publisher: Elsevier BV
Date: 2010
DOI: 10.1016/J.JMB.2009.11.031
Abstract: The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4-C41 and C8-C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were in idually removed and replaced by alanine residues. Trypsin inhibition assays show that wild-type T1 has a K(i) of <5 nM, C4A/C41A-T1 has a weaker K(i) of approximately 350 nM, and the potency of the C8A/C37A variant is further decreased to a K(i) of approximately 1.8 microM. To assess the influence of the disulfide bonds on the structure of T1, we determined the structure and dynamics of both disulfide variants by NMR spectroscopy. The structure of C4A/C41A-T1 and the litude of intrinsic flexibility in the reactive-site loop resemble that of the wild-type protein closely, despite the lack of the C4-C41 disulfide bond, whereas the timescale of motions is markedly decreased. The rescue of the structure despite loss of a disulfide bond is due to a previously unrecognized network of interactions, which stabilizes the structure of the reactive-site loop in the region of the missing disulfide bond, while allowing intrinsic motions on a fast (picosecond-nanosecond) timescale. In contrast, no comparable interactions are present around the C8-C37 disulfide bond. Consequently, the reactive-site loop becomes disordered and highly flexible in the structure of C8A/C37A-T1, making it unable to bind to trypsin. Thus, the reactive-site loop of T1 is stabilized differently by the C8-C37 and C4-C41 disulfide bonds. The C8-C37 disulfide bond is essential for the inhibitory activity of T1, whereas the C4-C41 disulfide bond is not as critical for maintaining the three-dimensional structure and function of the molecule but is responsible for maintaining flexibility of the reactive-site loop on a microsecond-nanosecond timescale.
Structure of Petunia hybrida Defensin 1, a Novel Plant Defensin with Five Disulfide Bonds
Publisher: American Chemical Society (ACS)
Date: 20-06-2003
DOI: 10.1021/BI034379O
Abstract: The structure of a novel plant defensin isolated from the flowers of Petunia hybrida has been determined by (1)H NMR spectroscopy. P. hybrida defensin 1 (PhD1) is a basic, cysteine-rich, antifungal protein of 47 residues and is the first ex le of a new subclass of plant defensins with five disulfide bonds whose structure has been determined. PhD1 has the fold of the cysteine-stabilized alphabeta motif, consisting of an alpha-helix and a triple-stranded antiparallel beta-sheet, except that it contains a fifth disulfide bond from the first loop to the alpha-helix. The additional disulfide bond is accommodated in PhD1 without any alteration of its tertiary structure with respect to other plant defensins. Comparison of its structure with those of classic, four-disulfide defensins has allowed us to identify a previously unrecognized hydrogen bond network that is integral to structure stabilization in the family.
The Tomato Defensin TPP3 Binds Phosphatidylinositol (4,5)-Bisphosphate via a Conserved Dimeric Cationic Grip Conformation To Mediate Cell Lysis
Publisher: Informa UK Limited
Date: 06-2015
DOI: 10.1128/MCB.00282-15
Ptychographic imaging of NaD1 induced yeast cell death
Publisher: The Optical Society
Date: 06-09-0026
A Pollen-Specific RALF from Tomato That Regulates Pollen Tube Elongation
Publisher: Oxford University Press (OUP)
Date: 13-04-2010
Abstract: Rapid Alkalinization Factors (RALFs) are plant peptides that rapidly increase the pH of plant suspension cell culture medium and inhibit root growth. A pollen-specific tomato (Solanum lycopersicum) RALF (SlPRALF) has been identified. The SlPRALF gene encodes a preproprotein that appears to be processed and released from the pollen tube as an active peptide. A synthetic SlPRALF peptide based on the putative active peptide did not affect pollen hydration or viability but inhibited the elongation of normal pollen tubes in an in vitro growth system. Inhibitory effects of SlPRALF were detectable at concentrations as low as 10 nm, and complete inhibition was observed at 1 μ m peptide. At least 10-fold higher levels of alkSlPRALF, which lacks disulfide bonds, were required to see similar effects. A greater effect of peptide was observed in low-pH-buffered medium. Inhibition of pollen tube elongation was reversible if peptide was removed within 15 min of exposure. Addition of 100 nm SlPRALF to actively growing pollen tubes inhibited further elongation until tubes were 40 to 60 μm in length, after which pollen tubes became resistant to the peptide. The onset of resistance correlated with the timing of the exit of the male germ unit from the pollen grain into the tube. Thus, exogenous SlPRALF acts as a negative regulator of pollen tube elongation within a specific developmental window.
Dimerization of Plant Defensin NaD1 Enhances Its Antifungal Activity
Publisher: Elsevier BV
Date: 06-2012
Novel insights on the mechanism of action of α‐amylase inhibitors from the plant defensin family
Publisher: Wiley
Date: 22-05-2008
DOI: 10.1002/PROT.22086
Abstract: Plant defensins are small cysteine-rich proteins commonly synthesized in plants, encoded by large multigene families. Most plant defensins that have been characterized to date show potent antifungal and/or bactericidal activities. This report describes VuD1, an unusual defensin that is able to inhibit insect-pest alpha-amylases. VuD1 was cloned from cowpea (Vigna unguiculata) seeds and expressed in a heterologous system. Inhibitory enzyme assays showed that VuD1 efficiently inhibits alpha-amylases from the weevils Acanthoscelides obtectus and Zabrotes subfasciatus, caused low inhibition toward mammalian enzymes and was unable to inhibit the alpha-amylases from Callosobruchus maculatus and Aspergillus fumigatus. To shed some light over the mechanism of action of VuD1, molecular modeling analyses were performed, revealing that the N-terminus of the molecule is responsible for binding with the active site of weevil enzymes. Moreover, models of VuD1 and mammalian enzymes were also generated to elucidate the specificity mechanisms. The data presented herein suggests that this defensin has potential application in the development of transgenic plants for insect pest control.
Self-compatibility in aLycopersicon peruvianum variant (LA2157) is associated with a lack of style S-RNase activity
Publisher: Springer Science and Business Media LLC
Date: 08-1994
DOI: 10.1007/BF01253997
Enzymic degradation of chemically modified extracellular polysaccharides from Rhizobia
Publisher: Elsevier BV
Date: 03-1978
Structural Refinement of Insecticidal Plant Proteinase Inhibitors from Nicotiana alata
Publisher: Bentham Science Publishers Ltd.
Date: 09-2008
DOI: 10.2174/092986608785849326
Abstract: Ornamental tobacco (Nicotiana alata) produces a series of 6 kDa proteinase inhibitors belonging to the potato type II inhibitor family. These proteins inhibit trypsin and chymotrypsin, the main digestive enzymes of predatory insects, thus leading to starvation, impaired larval development or death. In this context, the three-dimensional structures of these inhibitors are important for developing novel strategies for pest control. The solution structures of C1 and T1, the two main prototypes of the N. alata inhibitors, were originally determined more than a decade ago (J. Mol. Biol. 242, 231-243 (1994) and Biochemistry 34, 14304-14311 (1995)). Since then methods for NMR structure calculations have evolved considerably. Here we report the refinement of the structures of C1 and T1 with state-of-the-art protocols for NMR structure calculations. This refinement leads to an improved quality of the structures, making them a more reliable basis for the development of novel pesticides and modeling applications.
Uncoating the mechanisms of vacuolar protein transport
Publisher: Elsevier BV
Date: 02-1999
Circular Permutation of the Native Enzyme-Mediated Cyclization Position in Cyclotides
Publisher: American Chemical Society (ACS)
Date: 23-03-2020
Plant defensins: Common fold, multiple functions
Publisher: Elsevier BV
Date: 2013
Molecular characterisation of a cDNA sequence encoding the backbone of a style-specific 120 kDa glycoprotein which has features of both extensins and arabinogalactan proteins
Publisher: Springer Science and Business Media LLC
Date: 1997
Abstract: Nicotiana alata has a style-specific hydroxyproline-rich glycoprotein (the 120 kDa glycoprotein) which has properties of both extensins and AGPs [19, 20]. The 120 kDa glycoprotein is a soluble component in the extracellular matrix of the transmitting tract of styles where it accounts for ca. 9% of the total buffer-soluble protein. Here we describe the molecular cloning of a cDNA representing the gene NaPRP5 which encodes the backbone of the 120 kDa glycoprotein. Expression of mRNA is restricted to styles, consistent with observations on the distribution of the 120 kDa glycoprotein. Levels of accumulation of the transcript encoding the 120 kDa protein backbone are not altered significantly by pollination with either compatible or incompatible pollen. The protein backbone of the 120 kDa glycoprotein, as predicted by the cDNA sequence, is composed of three distinct domains. The sequence of these domains, together with linkage analysis of the carbohydrate component of the 120 kDa glycoprotein, allows predictions of the likely distribution of substituent glycosyl chains along the protein backbone. The similarity of the C-terminal domains of the 120 kDa glycoprotein and GaRSGP, the galactose-rich style glycoprotein of N. alata, is consistent with the two molecules sharing a common antigenic domain in their backbones [31]. The sharing of domains between distinct hydroxyproline-rich glycoproteins suggests that identification of a glycoprotein of this class solely by its protein or carbohydrate epitope is not valid.
Extracellular peptidases of the cereal pathogen Fusarium graminearum
Publisher: Frontiers Media SA
Date: 06-11-2015
Molecular Aspects of Fertilization in Flowering Plants
Publisher: Annual Reviews
Date: 11-1988
DOI: 10.1146/ANNUREV.CB.04.110188.001233
Abstract: Cystine-knot miniproteins are characterized by a similar molecular structure. Some cystine-knot miniproteins display therapeutically useful biological activities, as antithrombotic agents or tumour growth inhibitors. A critical event in the progression of tumours is the formation of new blood vessels. The aim of this work was to test two tomato cystine-knot miniproteins for their effects on endothelial cell proliferation and angiogenesis in vitro. Two tomato cystine-knot miniproteins (TCMPs) were expressed and purified either as recombinant or as native proteins from tomato fruits. The Matrigel assay was used to investigate the effects of TCMPs on in vitro angiogenesis. Viability and proliferation of endothelial cells were tested. Extracellular signal-regulated kinase (ERK)1/2 phosphorylation was assayed in either HUVEC or A431 epidermal growth factor receptor (EGFR)-overexpressing cells treated with TCMPs. EGFR phosphorylation was tested in A431 cells. Both recombinant and native TCMPs inhibited in vitro angiogenesis of HUVEC cells at concentrations of 15-100 nM. The anti-angiogenic effect of TCMPs was associated with the inhibition of ERK phosphorylation. The two miniproteins did not alter the viability and proliferation of the endothelial cells. The anti-angiogenetic properties of TCMPs are of potential pharmacological interest because they are common and natural components of the human diet, they possess low toxicity, they are active at submicromolar concentrations, they share a common molecular structure that can be used as a molecular platform for the design of molecules with enhanced biological activity.
Sequence Variability of Three Alleles of the Self-Incompatibility Gene of Nicotiana alata
Publisher: JSTOR
Date: 05-1989
DOI: 10.2307/3868968
Genetic polymorphism of self-incompatibility in flowering plants
Publisher: Elsevier BV
Date: 1989
Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase.
Publisher: Springer Science and Business Media LLC
Date: 18-12-2015
DOI: 10.1038/NCOMMS10199
Abstract: Cyclotides are erse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Backbone cyclization is a key enzyme-mediated step of cyclotide biosynthesis and confers a measure of stability on the resultant cyclotide. Furthermore, cyclization would be desirable for engineered peptides. Here we report the identification of four asparaginyl endopeptidases (AEPs), proteases implicated in cyclization, from the cyclotide-producing plant Oldenlandia affinis. We recombinantly express Oa AEP1 b and find it functions preferably as a cyclase by coupling C-terminal cleavage of propeptide substrates with backbone cyclization. Interestingly, Oa AEP1 b cannot cleave at the N-terminal site of O. affinis cyclotide precursors, implicating additional proteases in cyclotide biosynthesis. Finally, we demonstrate the broad utility of this enzyme by cyclization of peptides unrelated to cyclotides. We propose that recombinant Oa AEP1 b is a powerful tool for use in peptide engineering applications where increased stability of peptide products is desired.
An Enzyme-Linked Immunosorbent Assay (ELISA) for in Vitro Pollen Growth Based on Binding of a Monoclonal Antibody to the Pollen Tube Surface
Publisher: Oxford University Press (OUP)
Date: 07-1987
DOI: 10.1104/PP.84.3.851
Delaying surgery for patients with a previous SARS-CoV-2 infection
Publisher: Oxford University Press (OUP)
Date: 25-09-2020
DOI: 10.1002/BJS.12050
Protein markers for Candida albicans EVs include claudin‐like Sur7 family proteins
Publisher: Wiley
Date: 16-04-2020
Molecular genetics of self‐incompatibility in flowering plants
Publisher: Wiley
Date: 1988
Structure of a putative ancestral protein encoded by a single sequence repeat from a multidomain proteinase inhibitor gene fromNicotiana alata
Publisher: Elsevier BV
Date: 07-1999
DOI: 10.1016/S0969-2126(99)80103-8
Abstract: The ornamental tobacco Nicotiana alata produces a series of proteinase inhibitors (PIs) that are derived from a 43 kDa precursor protein, NaProPI. NaProPI contains six highly homologous repeats that fold to generate six separate structural domains, each corresponding to one of the native PIs. An unusual feature of NaProPI is that the structural domains lie across adjacent repeats and that the sixth PI domain is generated from fragments of the first and sixth repeats. Although the homology of the repeats suggests that they may have arisen from gene duplication, the observed folding does not appear to support this. This study of the solution structure of a single NaProPI repeat (aPI1) forms a basis for unravelling the mechanism by which this protein may have evolved. The three-dimensional structure of aPI1 closely resembles the triple-stranded antiparallel beta sheet observed in each of the native PIs. The five-residue sequence Glu-Glu-Lys-Lys-Asn, which forms the linker between the six structural domains in NaProPI, exists as a disordered loop in aPI1. The presence of this loop in aPI1 results in a loss of the characteristically flat and disc-like topography of the native inhibitors. A single repeat from NaProPI is capable of folding into a compact globular domain that displays native-like PI activity. Consequently, it is possible that a similar single-domain inhibitor represents the ancestral protein from which NaProPI evolved.
The Three-dimensional Solution Structure by 1H NMR of a 6-Kda Proteinase Inhibitor Isolated from the Stigma of Nicotiana alata
Publisher: Elsevier BV
Date: 09-1994
Abstract: The three-dimensional structure and disulfide connectivities of a 6-kDa protein isolated from the stigma of the ornamental tobacco Nicotiana alata has been determined by 1H NMR spectroscopy combined with simulated annealing calculations. The protein, termed C1, is a chymotrypsin inhibitor and is one of five homologous proteinase inhibitors that are proteolytically cleaved from a 40.3-kDa precursor protein. The other four proteinase inhibitors (T1 to T4) contain reactive sites for trypsin. The three-dimensional structure of C1 is generally well defined and contains a triple stranded beta-sheet as the dominant secondary structural feature. Several turns and a short region of 3(10) helix are also present. The putative chymotrypsin reactive site is present on an exposed loop which is less defined than the rest of the protein. The overall shape of C1 is disc-like and the N and C termini are exposed, supporting the proposal that this protein results from post-translational processing of the 40.3-kDa precursor protein.
The Plant Defensin, NaD1, Enters the Cytoplasm of Fusarium Oxysporum Hyphae
Publisher: Elsevier BV
Date: 05-2008
Isolation, Solution Structure, and Insecticidal Activity of Kalata B2, a Circular Protein with a Twist: Do Möbius Strips Exist in Nature?,
Publisher: American Chemical Society (ACS)
Date: 24-12-2004
DOI: 10.1021/BI047837H
Abstract: A large number of macrocyclic miniproteins with erse biological activities have been isolated from the Rubiaceae, Violaceae, and Cucurbitaceae plant families in recent years. Here we report the three-dimensional structure determined using (1)H NMR spectroscopy and demonstrate potent insecticidal activity for one of these peptides, kalata B2. This peptide is one of the major components of an extract from the leaves of the plant Oldenlandia affinis. The structure consists of a distorted triple-stranded beta-sheet and a cystine knot arrangement of the disulfide bonds and is similar to those described for other members of the cyclotide family. The unique cyclic and knotted nature of these molecules makes them a fascinating ex le of topologically complex proteins. Examination of the sequences reveals that they can be separated into two subfamilies, one of which contains a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-peptidyl-proline bond and may conceptually be regarded as a molecular Mobius strip. Kalata B2 is the second putative member of the Mobius cyclotide family to be structurally characterized and has a cis-peptidyl-proline bond, thus validating the suggested name for this subfamily of cyclotides. The observation that kalata B2 inhibits the growth and development of Helicoverpa armigera larvae suggests a role for the cyclotides in plant defense. A comparison of the sequences and structures of kalata B1 and B2 provides insight into the biological activity of these peptides.
Agp2p, the plasma membrane transregulator of polyamine uptake, regulates the antifungal activities of the plant defensin nad1 and other cationic peptides
Publisher: American Society for Microbiology
Date: 05-2014
DOI: 10.1128/AAC.02087-13
Abstract: Cationic antifungal peptides (AFPs) act through a variety of mechanisms but share the common feature of interacting with the fungal cell surface. NaD1, a defensin from Nicotiana alata , has potent antifungal activity against a variety of fungi of both hyphal and yeast morphologies. The mechanism of action of NaD1 occurs via three steps: binding to the fungal cell surface, permeabilization of the plasma membrane, and internalization and interaction with intracellular targets to induce fungal cell death. The targets at each of these three stages have yet to be defined. In this study, the screening of a Saccharomyces cerevisiae deletion collection led to the identification of Agp2p as a regulator of the potency of NaD1. Agp2p is a plasma membrane protein that regulates the transport of polyamines and other molecules, many of which carry a positive charge. Cells lacking the agp2 gene were more resistant to NaD1, and this resistance was accompanied by a decreased uptake of defensin. Agp2p senses and regulates the uptake of the polyamine spermidine, and competitive inhibition of the antifungal activity of NaD1 by spermidine was observed in both S. cerevisiae and the plant pathogen Fusarium oxysporum . The resistance of agp2 Δ cells to other cationic antifungal peptides and decreased binding of the cationic protein cytochrome c to agp2 Δ cells compared to that of wild-type cells have led to a proposed mechanism of resistance whereby the deletion of agp2 leads to an increase in positively charged molecules at the cell surface that repels cationic antifungal peptides.
Subcellular distribution of arabinogalactan proteins in pollen grains and tubes as revealed with a monoclonal antibody raised against stylar arabinogalactan proteins
Publisher: Springer Science and Business Media LLC
Date: 03-1999
DOI: 10.1007/BF01279257
A High Proportion of Hybridomas Raised to a Plant Extract Secrete Antibody to Arabinose or Galactose
Publisher: Oxford University Press (OUP)
Date: 08-1984
DOI: 10.1104/PP.75.4.1013
Molecular Genetics and Biology of Self-Incompatibility in Nicotiana alata, an Ornamental Tobacco
Publisher: CSIRO Publishing
Date: 1990
DOI: 10.1071/PP9900345
Abstract: Glycoproteins are present in the styles of several self-incompatible species within the Solanaceae which segregate with particular alleles of the S-(self-incompatibility) gene. The amino acid sequences of style glycoproteins corresponding to different S-alleles of N. alata are homologous in some regions and variable in others. Homologous regions include N-terminal sequences as well as most of the glycosylation sites and cysteine residues. The isolated style S-glycoproteins inhibit in vitro growth of pollen tubes of several S-genotypes, with some specificity in the interaction. The isolated S-glycoproteins have ribonuclease activity which may be involved in their action in arrest of growth of incompatible (self) pollen.
Novel insecticidal proteins from ferns resemble insecticidal proteins from Bacillus thuringiensis
Publisher: Proceedings of the National Academy of Sciences
Date: 23-10-2023
Synthesis and Structure Determination by NMR of a Putative Vacuolar Targeting Peptide and Model of a Proteinase Inhibitor from Nicotiana alata,
Publisher: American Chemical Society (ACS)
Date: 1996
DOI: 10.1021/BI952228I
A style-specific 120-kDa glycoprotein enters pollen tubes ofNicotiana alata in vivo
Publisher: Springer Science and Business Media LLC
Date: 03-1996
DOI: 10.1007/BF02153054
A new substrate for investigating the specificity of β‐glucan hydrolases
Publisher: Wiley
Date: 04-1975
DOI: 10.1016/0014-5793(75)80806-4
Abstract: Some isolates of Trichomonas vaginalis carry a double-stranded RNA virus (TVV) and undergo phenotypic variation between surface expression and cytoplasmic expression of a prominent immunogen (P270). Only trichomonads with TVV express P270 on the surface. Northern (RNA) blots using a specific cDNA encoding the repeat element of the phenotypically varying P270 immunogen as a probe showed accumulation of P270 transcript only in isolates with TVV (V+) in contrast to isolates without the virus (V-). To test further the influence of virus infection on P270 mRNA expression, V- progeny, derived from the parental V+ isolates, were tested. Trichomonads of V- progeny, like the fresh clinical V- isolates, also showed no accumulation of P270 mRNA. By immunoblotting with a monoclonal antibody to the key repeated epitope of P270, it was found that V- organisms had quantitatively less immunoreactive protein than the parental V+ isolates. Although V+ and V- isolates contained proteins immunoreactive with the monoclonal antibody to P270, it was necessary to test for the presence of the P270 gene among all the isolates. As expected, Southern blots demonstrated that V+ and V- trichomonads possessed the gene encoding P270. These data suggest that the double-stranded RNA virus of T. vaginalis is involved in the regulation of P270 mRNA accumulation.
A style‐specific hydroxyproline‐rich glycoprotein with properties of both extensins and arabinogalactan proteins
Publisher: Wiley
Date: 10-1994
DOI: 10.1046/J.1365-313X.1994.6040491.X
Abstract: A basic, hydroxyproline-rich glycoprotein (molecular mass 120 kDa) has been purified from the styles of Nicotiana alata. An antibody, specific for the protein backbone (molecular mass 78 kDa) of the glycoprotein, was used to demonstrate that the glycoprotein is a soluble, style-specific component and that related molecules are present in the styles of other solanaceous species. Linkage analysis of the carbohydrate portion of the glycoprotein, together with antibody binding studies, indicates that the glycoprotein contains both extensin-like and arabinogalactan-protein (AGP)-like side chains. Furthermore, the AGP-like side-chains contain a style-specific epitope that is also present on AGPs from N. alata styles and glycoconjugates from the styles of other members of the Solanaceae. The abundance of this 120 kDa glycoprotein, its location in the extracellular matrix of the transmitting tract and its conservation in several species within the Solanaceae suggests a role in pistil function.
Screening the Saccharomyces cerevisiae Nonessential Gene Deletion Library Reveals Diverse Mechanisms of Action for Antifungal Plant Defensins
Publisher: American Society for Microbiology
Date: 11-2019
DOI: 10.1128/AAC.01097-19
Abstract: Plant defensins are a large family of proteins, most of which have antifungal activity against a broad spectrum of fungi. However, little is known about how they exert their activity. The mechanisms of action of only a few members of the family have been investigated and, in most cases, there are still a number of unknowns.
The solution structure of C1-T1, a two-domain proteinase inhibitor derived from a circular precursor protein from Nicotiana alata11Edited by P. E. Wright
Publisher: Elsevier BV
Date: 02-2001
Crystal structure of a subtilisin-like autotransporter passenger domain reveals insights into its cytotoxic function
Publisher: Springer Science and Business Media LLC
Date: 03-2023
DOI: 10.1038/S41467-023-36719-2
Abstract: Autotransporters (ATs) are a large family of bacterial secreted and outer membrane proteins that encompass a wide range of enzymatic activities frequently associated with pathogenic phenotypes. We present the structural and functional characterisation of a subtilase autotransporter, Ssp, from the opportunistic pathogen Serratia marcescens . Although the structures of subtilases have been well documented, this subtilisin-like protein is associated with a 248 residue β-helix and itself includes three finger-like protrusions around its active site involved in substrate interactions. We further reveal that the activity of the subtilase AT is required for entry into epithelial cells as well as causing cellular toxicity. The Ssp structure not only provides details about the subtilase ATs, but also reveals a common framework and function to more distantly related ATs. As such these findings also represent a significant step forward toward understanding the molecular mechanisms underlying the functional ergence in the large AT superfamily.
Enzyme Mechanism and Function of a Novel Plant PDI Involved in the Oxidative Folding of Cystine Knot Defense Peptides
Publisher: Springer New York
Date: 2009
The C-terminal propeptide of a plant defensin confers cytoprotective and subcellular targeting functions
Publisher: Springer Science and Business Media LLC
Date: 05-02-2014
In Vitro and In Planta Cyclization of Target Peptides Using an Asparaginyl Endopeptidase from Oldenlandia affinis
Publisher: Springer New York
Date: 2019
DOI: 10.1007/978-1-4939-9546-2_12
Abstract: Cyclization of the peptide backbone by connecting the N- and C-terminus can endow target peptides with favorable properties, such as increased stability or potential oral bioavailability. However, there are few tools available for carrying out this modification. Asparaginyl endopeptidases (AEPs) are a class of enzymes that typically work as proteases, but a subset is highly efficient at cyclization of the peptide backbone. In this chapter we describe how to utilize a cyclizing AEP (OaAEP1
The interaction with fungal cell wall polysaccharides determines the salt tolerance of antifungal plant defensins.
Publisher: Elsevier BV
Date: 12-2019
Biosynthesis of circular proteins in plants
Publisher: Wiley
Date: 30-10-2007
Transforming growth factor(s) production enables cells to grow in the absence of serum: an autocrine system.
Publisher: Proceedings of the National Academy of Sciences
Date: 1982
Abstract: Kirsten murine sarcoma virus (KiMSV)-transformed rat-1, normal rat kidney (NRK), and BALB/c 3T3 cells are capable of continual growth in a serum-free medium supplemented with transferrin and insulin but with no exogenous mitogenic growth factors. Cells transformed by a mutant of KiMSV that is temperature sensitive for the maintenance of transformation grow in this medium at the permissive temperature only. At the nonpermissive temperature, growth is dependent upon the presence of serum-free conditioned medium from the transformed cells. Normal rat-1 cells are also dependent upon factors from the transformed cells for growth in this serum-free/mitogen-free medium. The serum-derived growth factors, epidermal growth factor, and fibroblast growth factor have no effect on the transformed cells, although epidermal growth factor can replace transforming growth factors produced by KiMSV-transformed cells for the growth of rat-1 cells. Growth of the transformed cells in serum-free medium at clonal densities is dependent upon the presence of conditioned medium collected from the same cells grown to high densities. These results show that (i) growth in serum-free/mitogen-free medium is a general property of KiMSV-transformed cells and (ii) growth of the transformed cells in this medium is dependent upon the presence of growth factors known to be produced by the cells, and they provide support for the hypothesis that serum-free growth of KiMSV-transformed cells is dependent upon ectopically produced growth factors.
Proteinase Inhibitors in Nicotiana alata Stigmas Are Derived from a Precursor Protein Which Is Processed into Five Homologous Inhibitors
Publisher: JSTOR
Date: 02-1993
DOI: 10.2307/3869586
Biosynthesis of Cyclotides
Publisher: Elsevier
Date: 2015
Rapid and scalable plant-based production of a potent plasmin inhibitor peptide
Publisher: Frontiers Media SA
Date: 15-05-2019
The evolution, function and mechanisms of action for plant defensins
Publisher: Elsevier BV
Date: 04-2018
DOI: 10.1016/J.SEMCDB.2018.02.004
Abstract: Plant defensins are an extensive family of small cysteine rich proteins characterised by a conserved cysteine stabilised alpha beta protein fold which resembles the structure of insect and vertebrate defensins. However, secondary structure and disulphide topology indicates two independent superfamilies of defensins with similar structures that have arisen via an extreme case of convergent evolution. Defensins from plants and insects belong to the cis-defensin superfamily whereas mammalian defensins belong to the trans-defensin superfamily. Plant defensins are produced by all species of plants and although the structure is highly conserved, the amino acid sequences are highly variable with the exception of the cysteine residues that form the stabilising disulphide bonds and a few other conserved residues. The majority of plant defensins are components of the plant innate immune system but others have evolved additional functions ranging from roles in sexual reproduction and development to metal tolerance. This review focuses on the antifungal mechanisms of plant defensins. The activity of plant defensins is not limited to plant pathogens and many of the described mechanisms have been elucidated using yeast models. These mechanisms are more complex than simple membrane permeabilisation induced by many small antimicrobial peptides. Common themes that run through the characterised mechanisms are interactions with specific lipids, production of reactive oxygen species and induction of cell wall stress. Links between sequence motifs and functions are highlighted where appropriate. The complexity of the interactions between plant defensins and fungi helps explain why this protein superfamily is ubiquitous in plant innate immunity.
A proteinase inhibitor from Nicotiana alata inhibits the normal development of light-brown apple moth, Epiphyas postvittana in transgenic apple plants
Publisher: Springer Science and Business Media LLC
Date: 05-01-2007
DOI: 10.1007/S00299-006-0281-8
Abstract: Insecticidal proteins are a potential resource to enhance resistance to insect pests in transgenic plants. Here, we describe the generation and analysis of the apple cultivar 'Royal Gala' transgenic for Nicotiana alata (N. alata) proteinase inhibitor (PI) and the impact of this PI on the growth and development of the Epiphyas postvittiana (light-brown apple moth). A cDNA clone encoding a proteinase inhibitor precursor from N. alata (Na-PI) under the control of either a double 35S promoter or a promoter from a ribulose-1,5-bisphosphate carboxylase small sub-unit gene (rbcS-E9 promoter) was stably incorporated into 'Royal Gala' apple using Agrobacterium-mediated transformation. A 40.3 kDa Na-PI precursor protein was expressed and correctly processed into 6-kDa proteinase inhibitors in the leaves of transgenic apple lines. The 6-kDa polypeptides accumulated to levels of 0.05 and 0.1% of the total soluble protein under the control of the rbc-E9 promoter and the double 35S promoter, respectively. Light-brown apple moth larvae fed with apple leaves expressing Na-PI had significantly reduced body weight after 7 days of feeding and female pupae were 19-28% smaller than controls. In addition, morphological changes such as pupal cases attached to the wing, deformed wings, deformed body shape, and pupal cases and curled wings attached to a deformed body were observed in adults that developed from larvae fed with apple leaves expressing Na-PI, when compared to larvae fed with the non-transformed apple leaves.
Molecular basis for the production of cyclic peptides by plant asparaginyl endopeptidases
Publisher: Springer Science and Business Media LLC
Date: 20-06-2018
DOI: 10.1038/S41467-018-04669-9
Abstract: Asparaginyl endopeptidases (AEPs) are proteases that have crucial roles in plant defense and seed storage protein maturation. Select plant AEPs, however, do not function as proteases but as transpeptidases (ligases) catalyzing the intra-molecular ligation of peptide termini, which leads to peptide cyclization. These ligase-type AEPs have potential biotechnological applications ranging from in vitro peptide engineering to plant molecular farming, but the structural features enabling these enzymes to catalyze peptide ligation/cyclization rather than proteolysis are currently unknown. Here, we compare the sequences, structures, and functions of erse plant AEPs by combining molecular modeling, sequence space analysis, and functional testing in planta. We find that changes within the substrate-binding pocket and an adjacent loop, here named the “marker of ligase activity”, together play a key role for AEP ligase efficiency. Identification of these structural determinants may facilitate the discovery of more ligase-type AEPs and the engineering of AEPs with tailored catalytic properties.
Reply: The Role of BP-80 in Sorting to the Vacuole in Stigmas
Publisher: Oxford University Press (OUP)
Date: 11-1999
Insights into Processing and Cyclization Events Associated with Biosynthesis of the Cyclic Peptide Kalata B1
Publisher: Elsevier BV
Date: 08-2012
Style proteins of a wild tomato (Lycopersicon peruvianum) associated with expression of self-incompatibility
Publisher: Springer Science and Business Media LLC
Date: 10-1986
DOI: 10.1007/BF00392312
Antimicrobial host defence peptides: functions and clinical potential
Publisher: Springer Science and Business Media LLC
Date: 27-02-2020
An Asparaginyl Endopeptidase Mediates in Vivo Protein Backbone Cyclization
Publisher: Elsevier BV
Date: 10-2007
Identification of a novel four-domain member of the proteinase inhibitor II family from the stigmas of Nicotiana alata
Publisher: Springer Science and Business Media LLC
Date: 2000
Abstract: Proteinase inhibitors (PIs) of the potato type II family have been identified in a number of solanaceous species. Most family members have two PI domains which are specific for either chymotrypsin or trypsin. More recently family members have been described with three or six repeated PI domains. Here we describe a novel four-domain family member produced in the stigmas and leaves of the ornamental tobacco, Nicotiana alata, which has high sequence identity with a six-domain member from the same species. Both proteins are produced as precursors that enter the secretory pathway and are subsequently processed into a series of 6 kDa Pis. The four- and six-domain precursor proteins were isolated from immature stigmas and characterised by mass spectrometry which revealed that both proteins had been trimmed at the N-terminus, at a position corresponding to the predicted signal peptide cleavage site. Furthermore, no post-translational modifications were apparent.
Defensins - Components of the Innate Immune System in Plants
Publisher: Bentham Science Publishers Ltd.
Date: 02-2005
Abstract: 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 erse 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.
Isolation and Properties of Floral Defensins from Ornamental Tobacco and Petunia
Publisher: Oxford University Press (OUP)
Date: 03-2003
Abstract: 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.
Subcellular targeting and biosynthesis of cyclotides in plant cells
Publisher: Wiley
Date: 12-2011
DOI: 10.3732/AJB.1100382
Abstract: The cyclotide kalata B1 is found in the leaves of Oldenlandia affinis and is a potent insecticidal and nematocidal molecule. This peptide is cleaved from a precursor protein, Oak1, and ligation of the N- and C-termini occurs to form a continuous peptide backbone. The subcellular location of the excision and cyclization reactions is unknown, and there is debate as to which enzyme catalyzes the event. To determine where in the plant cell Oak1 is processed, we prepared constructs encoding GFP (green fluorescent protein) linked to the cyclotide precursor Oak1. The GFP constructs were transiently expressed in the leaves of Nicotiana benthamiana, and GFP fluorescence was observed in living cells using confocal microscopy. A Fei Mao (FM) styryl dye was infiltrated into whole leaves that were still growing and expressing GFP constructs, enabling the plasma membrane and the tonoplast to be highlighted for visualization of the vacuole in living cells. The full length Oak1 precursor directed GFP to the vacuole, suggesting that excision and cyclization of the cyclotide domain occurs in the vacuole where the cyclotides are then stored. The N-terminal propeptide and N-terminal repeat of Oak1 were both sufficient to target GFP to the vacuole, although the C-terminal propeptide, which is essential for cyclization, was not a targeting signal. The vacuolar location of cyclotides supports our hypothesis that the vacuolar processing enzyme, asparaginyl endoproteinase, has a pivotal role in excision and cyclization from cyclotide precursors.
S-RNase Gene of Nicotiana alata Is Expressed in Developing Pollen
Publisher: JSTOR
Date: 12-1993
DOI: 10.2307/3869693
Cloning of cDNA for a stylar glycoprotein associated with expression of self-incompatibility in Nicotiana alata
Publisher: Springer Science and Business Media LLC
Date: 05-1986
DOI: 10.1038/321038A0
Extracellular vesicles including exosomes in cross kingdom regulation: a viewpoint from plant-fungal interactions
Publisher: Frontiers Media SA
Date: 23-09-2015
A radish seed antifungal peptide with a high amyloid fibril-forming propensity
Publisher: Elsevier BV
Date: 08-2013
DOI: 10.1016/J.BBAPAP.2013.04.030
Abstract: The amyloid fibril-forming ability of two closely related antifungal and antimicrobial peptides derived from plant defensin proteins has been investigated. As assessed by sequence analysis, thioflavin T binding, transmission electron microscopy, atomic force microscopy and X-ray fiber diffraction, a 19 amino acid fragment from the C-terminal region of Raphanus sativus antifungal protein, known as RsAFP-19, is highly amyloidogenic. Further, its fibrillar morphology can be altered by externally controlled conditions. Freezing and thawing led to amyloid fibril formation which was accompanied by loss of RsAFP-19 antifungal activity. A second, closely related antifungal peptide displayed no fibril-forming capacity. It is concluded that while fibril formation is not associated with the antifungal properties of these peptides, the peptide RsAFP-19 is of potential use as a controllable, highly amyloidogenic small peptide for investigating the structure of amyloid fibrils and their mechanism of formation.
A relic S-RNase is expressed in the styles of self-compatible Nicotiana sylvestris.
Publisher: Wiley
Date: 12-1998
DOI: 10.1046/J.1365-313X.1998.00331.X
Abstract: We surveyed ribonuclease activity in the styles of Nicotiana spp. and found little or no activity in self-compatible species and in a self-compatible accession of a self-incompatible species. All self-incompatible species had high levels of ribonuclease activity in their style. Interestingly, one self-compatible species, N. sylvestris, had a level of stylar ribonuclease activity comparable to that of some self-incompatible Nicotiana species. A ribonuclease with biochemical properties similar to those of the self-incompatibility (S-)RNases of N. alata was purified from N. sylvestris styles. The N-terminal sequence of this protein was used to confirm the identity of a cDNA corresponding to the stylar RNase. The amino acid sequence deduced from the cDNA was related to those of the S-RNases and included the five conserved regions characteristic of these proteins. It appears that the N. sylvestris RNase may have evolved from the S-RNases and is an ex le of a 'relic S-RNase'. A number of features distinguish the N. sylvestris RNase from the S-RNases, and the role these may have played in the presumed loss of the self-incompatibility response during the evolution of this species are discussed.
Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis
Publisher: Proceedings of the National Academy of Sciences
Date: 04-09-2001
Abstract: 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.
Salt-Tolerant antifungal and antibacterial activities of the corn defensin ZmD32
Publisher: Frontiers Media SA
Date: 12-04-2019
Inhibition of cereal rust fungi by both class I and II defensins derived from the flowers of Nicotiana alata
Publisher: Wiley
Date: 10-09-2014
DOI: 10.1111/MPP.12066
Immuno-gold localization of ?-L-arabinofuranosyl residues in pollen tubes of Nicotiana alata Link et otto
Publisher: Springer Science and Business Media LLC
Date: 08-1987
DOI: 10.1007/BF00392290
X-ray structure of a carpet-like antimicrobial defensin-phospholipid membrane disruption complex
Publisher: Springer Science and Business Media LLC
Date: 17-05-2018
DOI: 10.1038/S41467-018-04434-Y
Abstract: Defensins are cationic antimicrobial peptides expressed throughout the plant and animal kingdoms as a first line of defense against pathogens. Membrane targeting and disruption is a crucial function of many defensins, however the precise mechanism remains unclear. Certain plant defensins form dimers that specifically bind the membrane phospholipids phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate, thereby triggering the assembly of defensin–lipid oligomers that permeabilize cell membranes. To understand this permeabilization mechanism, here we determine the crystal structure of the plant defensin NaD1 bound to PA. The structure reveals a 20-mer that adopts a concave sheet- or carpet-like topology where NaD1 dimers form one face and PA acyl chains form the other face of the sheet. Furthermore, we show that Arg39 is critical for PA binding, oligomerization and fungal cell killing. These findings identify a putative defensin–phospholipid membrane attack configuration that supports a longstanding proposed carpet mode of membrane disruption.
Pest and disease protection conferred by expression of barley β - hordothionin and Nicotiana alata proteinase inhibitor genes in transgenic tobacco
Publisher: CSIRO Publishing
Date: 2005
DOI: 10.1071/FP04105
Abstract: Proteinase inhibitors and thionins are among the many proteins thought to have a role in plant defence against pests and pathogens. Complementary DNA clones encoding the precursors of a multi-domain proteinase inhibitor from Nicotiana alata Link et Otto (NA-PI) (Mr approximately 43 000) and a β-hordothionin (β-HTH) (Mr approximately 13 000) from barley, were linked to constitutive promoters and subsequently transferred by Agrobacterium-mediated transformation into tobacco. The NA-PI and β-HTH precursor proteins were synthesised and post-translationally processed in transgenic tobacco and accumulated as polypeptides of apparent size Mr approximately 6000 and Mr approximately 8500, respectively. The na-pi and β-hth genes were stably inherited for at least two generations. Transgenic tobacco plants containing the highest amounts of NA-PI and β-HTH were crossed to produce plants containing both genes. Helicoverpa armigera (tobacco budworm) larvae that ingested transgenic tobacco leaves expressing both NA-PI and β-HTH, exhibited higher mortality and slower development relative to larvae fed on non-transgenic tobacco. NA-PI and β-HTH, either alone, or in combination, also conferred protection against the fungal pathogen, Botrytis cinerea (grey mould) and the bacterial pathogen, Pseudomonas solanacearum (bacterial wilt). The effect of the two proteins depended upon the organism tested and the contribution of each gene to the protective effects was not necessarily equal. The genetic engineering of plants with proteinase inhibitors or thionins, therefore, has potential for improving crop productivity by simultaneously increasing resistance to both pests and pathogens.
The Defensins Consist of Two Independent, Convergent Protein Superfamilies
Publisher: Oxford University Press (OUP)
Date: 13-06-2016
Abstract: The defensin and defensin-like proteins are an extensive group of small, cationic, disulfide-rich proteins found in animals, plants, and fungi and mostly perform roles in host defense. The term defensin was originally used for small mammalian proteins found in neutrophils and was subsequently applied to insect proteins and plant γ-thionins based on their perceived sequence and structural similarity. Defensins are often described as ancient innate immunity molecules and classified as a single superfamily and both sequence alignments and phylogenies have been constructed. Here, we present evidence that the defensins have not all evolved from a single ancestor. Instead, they consist of two analogous superfamilies, and extensive convergent evolution is the source of their similarities. Evidence of common origin necessarily gets weaker for distantly related genes, as is the case for defensins, which are both ergent and small. We show that similarities that have been used as evidence for common origin are all expected by chance in short, constrained, disulfide-rich proteins. Differences in tertiary structure, secondary structure order, and disulfide bond connectivity indicate convergence as the likely source of the similarity. We refer to the two evolutionarily independent groups as the cis-defensins and trans-defensins based on the orientation of the most conserved pair of disulfides.
Field resistance to Fusarium oxysporum and Verticillium dahliae in transgenic cotton expressing the plant defensin NaD1
Publisher: Oxford University Press (OUP)
Date: 06-02-2014
DOI: 10.1093/JXB/ERU021
Cell wall carbohydrate dynamics during the differentiation of infection structures by the apple scab fungus, Venturia inaequalis
Publisher: Cold Spring Harbor Laboratory
Date: 21-09-2022
DOI: 10.1101/2022.09.21.508768
Abstract: Scab, caused by the biotrophic fungal pathogen Venturia inaequalis , is the most economically important disease of apples. During infection, V. inaequalis colonizes the subcuticular host environment, where it develops specialized infection structures called runner hyphae and stromata. These structures are thought to be involved in nutrient acquisition and effector (virulence factor) delivery, but also give rise to conidia that further the infection cycle. Despite their importance, very little is known about how these structures are differentiated. Likewise, nothing is known about how these structures are protected from host defences or recognition by the host immune system. To better understand these processes, we first performed a glycosidic linkage analysis of sporulating tubular hyphae from V. inaequalis developed in culture. This analysis revealed that the V. inaequalis cell wall is mostly composed of glucans (44%) and mannans (37%), whereas chitin represents a much smaller proportion (4%). Next, we used transcriptomics and confocal laser scanning microscopy to provide insights into the cell wall carbohydrate composition of runner hyphae and stromata. These analyses revealed that, during subcuticular host colonization, genes of V. inaequalis putatively associated with the biosynthesis of immunogenic carbohydrates, such as chitin and β-1,6-glucan, are down-regulated relative to growth in culture, while on the surface of runner hyphae and stromata, chitin is deacetylated to the less immunogenic carbohydrate, chitosan. These changes are anticipated to enable the subcuticular differentiation of runner hyphae and stromata by V. inaequalis , as well as to protect these structures from host defences and recognition by the host immune system. Plant-pathogenic fungi are a major threat to food security. Among these are subcuticular pathogens, which often cause latent asymptomatic infections, making them difficult to control. A key feature of these pathogens is their ability to differentiate specialized subcuticular infection structures that, to date, remain largely understudied. This is typified by Venturia inaequalis , which causes scab, the most economically important disease of apples. In this study, we show that, during subcuticular host colonization, V. inaequalis down-regulates genes associated with the biosynthesis of two immunogenic cell wall carbohydrates, chitin and β-1,6-glucan, and coats its infection structures with a less-immunogenic carbohydrate, chitosan. These changes are anticipated to enable subcuticular host colonization by V. inaequalis and provide a foundation for understanding subcuticular host colonization by other plant-pathogenic fungi. Such an understanding is important, as it may inform the development of novel control strategies against subcuticular plant-pathogenic fungi.
Circular proteins and mechanisms of cyclization
Publisher: Wiley
Date: 2010
DOI: 10.1002/BIP.21422
Abstract: Cyclization via head-to-tail linkage of the termini of a peptide chain occurs in only a small percentage of proteins, but engenders the resultant cyclic proteins with exceptional stability. The mechanisms involved are poorly understood and this review attempts to summarize what is known of the events that lead to cyclization. Cyclic proteins are found in both prokaryotic and eukaryotic species. The prokaryotic circular proteins include the bacteriocins and pilins. The eukaryotic circular proteins in mammals include the theta defensins, found in rhesus macaques, and the retrocyclins. Two types of cyclic proteins have been found in plants, the sunflower trypsin inhibitor and the larger, more prolific, group known as cyclotides. The cyclotides from Oldenlandia affinis, the plant in which these cyclotides were first discovered, are processed by an asparaginyl endopeptidase which is a cysteine protease. Cysteine proteases are commonly associated with transpeptidation reactions, which, for suitable substrates can lead to cyclization events. These proteases cleave an amide bond and form an acyl enzyme intermediate before nucleophilic attack by the amine group of the N-terminal residue to form a peptide bond, resulting in a cyclic peptide.
Structures of a Series of 6-kDa Trypsin Inhibitors Isolated from the Stigma of Nicotiana alata
Publisher: American Chemical Society (ACS)
Date: 11-1995
DOI: 10.1021/BI00044A007
Abstract: The three-dimensional structures of a series of 6-kDa trypsin inhibitors isolated from the stigma of the ornamental tobacco Nicotiana alata have been determined by 1H NMR spectroscopy combined with simulated annealing calculations. The proteins, T1-T4, are proteolytically cleaved from a 40.3-kDa precursor protein, NA-proPI, together with a chymotrypsin inhibitor, C1, the structure of which was reported recently [Nielsen, K.J., Heath, R.L., Anderson, M.A., & Craik, D.J. (1994) J. Mol. Biol. 242, 231-243]. Each of the proteinase inhibitors comprises 53 amino acids, including 8 cysteine residues which are linked to form 4 disulfide bridges. The proteins have a high degree of sequence identity and differ mainly in residues around the putative reactive sites. The structure of T1 was determined using a set of 533 interproton distance restraints derived from NOESY spectra, combined with 33 dihedral restraints derived from 3JNH-H alpha coupling constants and 16 hydrogen bonds. The structures of the remaining inhibitors (T2-T4) were deduced to be almost identical to T1, on the basis of their similar chemical shifts and 2D spectra. The current study demonstrates that the structures of the trypsin inhibitors (T1-T4) are similar to that previously found for the chymotrypsin inhibitor, C1. Despite differences in sequence, there is conservation in backbone geometry between the reactive site loops of the two classes of inhibitors. From this, it is clear that the nature of the side chain on the primary binding residue, rather than the backbone fold, is the main determinant of the enzyme specificities of these proteinase inhibitors.
Permeabilization of Fungal Hyphae by the Plant Defensin NaD1 Occurs through a Cell Wall-dependent Process
Publisher: Elsevier BV
Date: 11-2010
Cell Wall Carbohydrate Dynamics during the Differentiation of Infection Structures by the Apple Scab Fungus, Venturia inaequalis
Publisher: American Society for Microbiology
Date: 15-06-2023
DOI: 10.1128/SPECTRUM.04219-22
Abstract: Plant-pathogenic fungi are a major threat to food security. Among these are subcuticular pathogens, which often cause latent asymptomatic infections, making them difficult to control.
A suite of kinetically superior AEP ligases can cyclise an intrinsically disordered protein
Publisher: Springer Science and Business Media LLC
Date: 25-07-2019
DOI: 10.1038/S41598-019-47273-7
Abstract: Asparaginyl endopeptidases (AEPs) are a class of enzymes commonly associated with proteolysis in the maturation of seed storage proteins. However, a subset of AEPs work preferentially as peptide ligases, coupling release of a leaving group to formation of a new peptide bond. These “ligase-type” AEPs require only short recognition motifs to ligate a range of targets, making them useful tools in peptide and protein engineering for cyclisation of peptides or ligation of separate peptides into larger products. Here we report the recombinant expression, ligase activity and cyclisation kinetics of three new AEPs from the cyclotide producing plant Oldenlandia affinis with superior kinetics to the prototypical recombinant AEP ligase OaAEP1 b . These AEPs work preferentially as ligases at both acidic and neutral pH and we term them “canonical AEP ligases” to distinguish them from other AEPs where activity preferences shift according to pH. We show that these ligases intrinsically favour ligation over hydrolysis, are highly efficient at cyclising two unrelated peptides and are compatible with organic co-solvents. Finally, we demonstrate the broad scope of recombinant AEPs in biotechnology by the backbone cyclisation of an intrinsically disordered protein, the 25 kDa malarial vaccine candidate Plasmodium falciparum merozoite surface protein 2 (MSP2).
Convergent evolution of defensin sequence, structure and function
Publisher: Springer Science and Business Media LLC
Date: 24-08-2017
DOI: 10.1007/S00018-016-2344-5
Abstract: Defensins are a well-characterised group of small, disulphide-rich, cationic peptides that are produced by essentially all eukaryotes and are highly erse in their sequences and structures. Most display broad range antimicrobial activity at low micromolar concentrations, whereas others have other erse roles, including cell signalling (e.g. immune cell recruitment, self/non-self-recognition), ion channel perturbation, toxic functions, and enzyme inhibition. The defensins consist of two superfamilies, each derived from an independent evolutionary origin, which have subsequently undergone extensive ergent evolution in their sequence, structure and function. Referred to as the cis- and trans-defensin superfamilies, they are classified based on their secondary structure orientation, cysteine motifs and disulphide bond connectivities, tertiary structure similarities and precursor gene sequence. The utility of displaying loops on a stable, compact, disulphide-rich core has been exploited by evolution on multiple occasions. The defensin superfamilies represent a case where the ensuing convergent evolution of sequence, structure and function has been particularly extreme. Here, we discuss the extent, causes and significance of these convergent features, drawing ex les from across the eukaryotes.
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Amount: $346,439.00
Funder: Australian Research Council
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