scispace - formally typeset
Search or ask a question
Journal Article•DOI•

Small cysteine-rich antifungal proteins from radish: their role in host defense.

Franky R. G. Terras1, Kristel Eggermont1, Kovaleva1, Natasha V. Raikhel1, Rupert W. Osborn1, Kester A1, Rees Sb1, Sophie Torrekens1, Van Leuven F1, Jozef Vanderleyden1 •
Katholieke Universiteit Leuven1
01 May 1995-The Plant Cell (American Society of Plant Biologists)-Vol. 7, Iss: 5, pp 573-588
TL;DR: It is demonstrated that two homologous, 5-kD cysteine-rich proteins designated Raphanus sativus-antifungal protein 1 and Rs-AFP2 are located in the cell wall and occur predominantly in the outer cell layers lining different seed organs, and are preferentially released during seed germination after disruption of the seed coat.
Abstract: Radish seeds have previously been shown to contain two homologous, 5-kD cysteine-rich proteins designated Raphanus sativus-antifungal protein 1 (Rs-AFP1) and Rs-AFP2, both of which exhibit potent antifungal activity in vitro. We now demonstrate that these proteins are located in the cell wall and occur predominantly in the outer cell layers lining different seed organs. Moreover, Rs-AFPs are preferentially released during seed germination after disruption of the seed coat. The amount of released proteins is sufficient to create a microenvironment around the seed in which fungal growth is suppressed. Both the cDNAs and the intron-containing genomic regions encoding the Rs-AFP preproteins were cloned. Transcripts (0.55 kb) hybridizing with an Rs-AFP1 cDNA-derived probe were present in near-mature and mature seeds. Such transcripts as well as the corresponding proteins were barely detectable in healthy uninfected leaves but accumulated systemically at high levels after localized fungal infection. The induced leaf proteins (designated Rs-AFP3 and Rs-AFP4) were purified and shown to be homologous to seed Rs-AFPs and to exert similar antifungal activity in vitro. A chimeric Rs-AFP2 gene under the control of the constitutive cauliflower mosaic virus 35S promoter conferred enhanced resistance to the foliar pathogen Alternaria longipes in transgenic tobacco. The term "plant defensins" is proposed to denote these defense-related proteins.
Citations
More filters
Journal Article•DOI•
Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway.

[...]

Iris A. M. A. Penninckx1, Kristel Eggermont1, Franky R. G. Terras1, Bart P. H. J. Thomma1, G.W. de Samblanx1, Antony Buchala1, Jean-Pierre Métraux1, John Michael Manners1, Willem F. Broekaert1 •
Katholieke Universiteit Leuven1
01 Dec 1996-The Plant Cell
TL;DR: The results indicate that systemic pathogen-induced expression of the plant defensin gene in Arabidopsis is independent of salicylic acid but requires components of the ethylene and jasmonic acid response.
Abstract: A 5-kD plant defensin was purified from Arabidopsis leaves challenged with the fungus Alternaria brassicicola and shown to possess antifungal properties in vitro. The corresponding plant defensin gene was induced after treatment of leaves with methyl jasmonate or ethylene but not with salicylic acid or 2,6-dichloroisonicotinic acid. When challenged with A. brassicicola, the levels of the plant defensin protein and mRNA rose both in inoculated leaves and in nontreated leaves of inoculated plants (systemic leaves). These events coincided with an increase in the endogenous jasmonic acid content of both types of leaves. Systemic pathogen-induced expression of the plant defensin gene was unaffected in Arabidopsis transformants (nahG) or mutants (npr1 and cpr1) affected in the salicylic acid response but was strongly reduced in the Arabidopsis mutants eln2 and col1 that are blocked in their response to ethylene and methyl jasmonate, respectively. Our results indicate that systemic pathogen-induced expression of the plant defensin gene in Arabidopsis is independent of salicylic acid but requires components of the ethylene and jasmonic acid response.

983 citations

Cites background or methods from "Small cysteine-rich antifungal prot..."

  • ...PDF1.1 is expressed predominantly in seed and is considered to be the analog of the radish Rs-AFPI and Rs-AFP2 genes, whereas PDF1.2 is expressed in leaves upon pathogen-induced stress and can be considered the analog of the Rs-AFP3 and RS-AFP4 genes from radish ( Terras et al., 1995 )....

    [...]

  • ...Extracts were prepared from 20 g of either water-treated or inoculated leaves and subjected to the purification procedure, exactly as previously described in Terras et al. (1995) ....

    [...]

  • ...The rabbit anti-Rs-AFPI antiserum ( Terras et al., 1995 ) was diluted twofold in ImmunoPure Gentle Ag/Ab binding buffer (Pierce Chemical Company, Rockford, IL) and passed several times over the affinity column....

    [...]

  • ...The induced accumulation of plant defensins in radish leaves is not restricted to the infected leaves but also occurs in noninfected leaves ( Terras et al., 1995 )....

    [...]

  • ...Protein analysis, in vitro antifungal activity analysis, and SDS-PAGE on precast PhastGel High Density gels (Pharmacia) were performed as previously described ( Terras et al., 1995 )....

    [...]

Journal Article•DOI•
Plant defensins: novel antimicrobial peptides as components of the host defense system.

[...]

Willem F. Broekaert1, Franky R. G. Terras, Bruno P. A. Cammue, R. W. Osborn•
Katholieke Universiteit Leuven1
01 Aug 1995-Plant Physiology
TL;DR: A novel class of plant peptides whose structural and functional properties resemble those of insect and mammalian defensins are characterized, which are one class among the numerous types of Cys-rich antimicrobial peptides.
Abstract: Various mechanisms to fend off microbial invaders have been devised by all living organisms, including microorganisms themselves. The most sophisticated of these mechanisms relies on the synthesis of immunoglobulins directed against specific microbial targets. However, immunoglobulin-based immunity operates only in a relatively minor subset of living species, namely the higher vertebrates. A much more ancient and widespread defense strategy involves the production of small peptides that exert antimicrobial properties. As products of single genes, antimicrobial peptides can be synthesized in a swift and flexible way, and because of their small size they can be produced by the host with a minimal input of energy and biomass. Wellknown examples of antimicrobial peptides are the cecropins that accumulate in the hemolymph of many invertebrates in response to injury or infection (reviewed by Boman and Hultmark, 1987) and the magainins that are secreted by glands in the skin of amphibians (reviewed by Bevins and Zasloff, 1990). Cecropins and magainins are small (20-40 residues) basic peptides displaying an amphipathic a-helical structure that can integrate in microbial membranes to form ion channels (Duclohier, 1994). Another class of antimicrobial peptides is formed by the Cys-rich peptides, which in contrast to cecropins and magainins, have a complex cystine-stabilized three-dimensional folding pattern often involving antiparallel ,3-sheets. Defensins are one class among the numerous types of Cys-rich antimicrobial peptides, which differ in length, number of cystine, bonds, or folding pattern (reviewed by Boman, 1995). Insect defensins (34-43 residues, three disulfide bridges) are, like cecropins, produced in a pathogeninducible manner by the insect fat body and secreted in the hemolymph (reviewed by Hoffmann and Hetru, 1992). Mammalian defensins (29-34 amino acids, three disulfide bridges) are produced by various specialized cells in the mammalian body (reviewed by Lehrer et al., 1993; Ganz and Lehrer, 1994). For example, they are very abundant in granules of phagocytic blood cells. These granules fuse with phagocytosis vesicles containing microorganisms, where the defensins are thought to contribute, together with other antimicrobial proteins and active oxygen species, to killing of the engulfed microorganisms. Defensins are also secreted by epithelial cells of the intestines and airways, where they may help maintain the normal microbial flora in a steady state. In addition, the expression of defensins in the airway epithelium has been shown to be up-regulated after exposure to bacterial lipopolysaccharides (Diamond et al., 1993). The importance of defensins in innate immunity of humans is underscored by the observation that certain disorders characterized by recurrent infections are associated with a lack of defensins in blood phagocytes (Ganz et al., 1988). Moreover, transposon mutants of a pathogenic Salmonella strain known to infect and grow inside phagocytes simultaneously lost their resistance to defensins (and other antimicrobial peptides) and their virulence (Groisman et al., 1992). Recently, we characterized a novel class of plant peptides whose structural and functional properties resemble those of insect and mammalian defensins. Hence, we termed this family of peptides "plant defensins" (Terras et al., 1995).

764 citations

Journal Article•DOI•
The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance.

[...]

Scott A. Bowling1, Joseph D. Clarke1, Yidong Liu1, Daniel F. Klessig1, Xinnian Dong1 •
Duke University1
01 Sep 1997-The Plant Cell
TL;DR: Identification of the cpr5 mutation indicates that these pathways are connected in early signal transduction steps and that they have overlapping functions in providing resistance.
Abstract: The cpr5 mutant was identified from a screen for constitutive expression of systemic acquired resistance (SAR). This single recessive mutation also leads to spontaneous expression of chlorotic lesions and reduced trichome development. The cpr5 plants were found to be constitutively resistant to two virulent pathogens, Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2; to have endogenous expression of the pathogenesis-related gene 1 (PR-1); and to have an elevated level of salicylic acid (SA). Lines homozygous for cpr5 and either the SA-degrading bacterial gene nahG or the SA-insensitive mutation npr1 do not express PR-1 or exhibit resistance to P. s. maculicola ES4326. Therefore, we conclude that cpr5 acts upstream of SA in inducing SAR. However, the cpr5 npr1 plants retained heightened resistance to P. parasitica Noco2 and elevated expression of the defensin gene PDF1.2, implying that NPR1-independent resistance signaling also occurs. We conclude that the cpr5 mutation leads to constitutive expression of both an NPR1-dependent and an NPR1-independent SAR pathway. Identification of this mutation indicates that these pathways are connected in early signal transduction steps and that they have overlapping functions in providing resistance.

676 citations

Journal Article•DOI•
Signal perception and transduction in plant defense responses.

[...]

Yinong Yang1, Jyoti Shah1, Daniel F. Klessig1•
Rutgers University1
01 Jul 1997-Genes & Development

660 citations

Journal Article•DOI•
Plant pathogenesis-related (PR) proteins: a focus on PR peptides.

[...]

Jan Sels1, Janick Mathys1, Barbara De Coninck1, Bruno P. A. Cammue1, Miguel F.C. De Bolle1 •
Katholieke Universiteit Leuven1
01 Nov 2008-Plant Physiology and Biochemistry
TL;DR: This review specifically focuses on these pathogenesis-related peptides, including proteinase inhibitors, plant defensins, thionins,Thionins and lipid transfer proteins, including novel peptide families, including PR-6 family, which are identified during the last decade.

656 citations

Cites background from "Small cysteine-rich antifungal prot..."

  • ...[102] discovered two antifungal radish (Raphanus sativus) defensins (Rs-AFP3, Rs-AFP4) that were barely detectable in healthy uninfected leaves but accumulated at high levels after fungal infection....

    [...]

  • ...[102] showed that transgenic tobacco overexpressing the Rs-AFP2 gene was more resistant to the fungal pathogen Alternaria longipes....

    [...]

  • ...Table 1 Main properties of classified families of PR proteins Family Type member Typical size (kDa) Properties Proposed microbial target Original reference PR-1 Tobacco PR-1a 15 Antifungal Unknown [3] PR-2 Tobacco PR-2 30 b-1,3-Glucanase b-1,3-Glucan [3] PR-3 Tobacco P, Q 25–30 Chitinase (class I,II, IV,V,VI,VI) Chitin [117] PR-4 Tobacco ‘R’ 15–20 Chitinase class I,II Chitin [117] PR-5 Tobacco S 25 Thaumatin-like Membrane [117] PR-6 Tomato Inhibitor I 8 Proteinase-inhibitor –a [43] PR-7 Tomato P69 75 Endoproteinase – a [123] PR-8 Cucumber chitinase 28 Chitinase class III Chitin [77] PR-9 Tobacco ‘lignin-forming peroxidase’ 35 Peroxidase –a [62] PR-10 Parsley ‘PR1’ 17 ‘Ribonuclease-like’ –a [95] PR-11 Tobacco ‘class V’ chitinase 40 Chitinase class I Chitin [74] PR-12 Radish Rs-AFP3 5 Defensin Membrane [102] PR-13 Arabidopsis THI2....

    [...]

  • ...Later, because of their antimicrobial properties and their structural similarity to mammalian and insect defensins, g-thionins were renamed ‘‘plant defensins’’ [102]....

    [...]

  • ...in seed [102]) whereas others are developmentally regulated [122] or induced by different abiotic and biotic stress factors, including cold [59], drought [30], heavy metals [78], potassium starvation [4], or microbial pathogens (e....

    [...]

References
More filters
Journal Article•DOI•
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

[...]

Marion M. Bradford1•
University of Georgia1
07 May 1976-Analytical Biochemistry
TL;DR: This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr with little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose.

225,085 citations

"Small cysteine-rich antifungal prot..." refers methods in this paper

  • ...Determination of total protein was performed by the Bradford protein assay (Bradford, 1976) using BSA as a standard....

    [...]

  • ...Total protein levels were determined by the Coomassie blue dye binding method (Bradford, 1976)....

    [...]

  • ...Total protein levels were determined by the Coomassie blue dye binding method (Bradford, 1976). b In vitro antifungal activity was measured on partially purified leaf extracts using A. longipes as a test fungus (see Methods for additional experimental details)....

    [...]

  • ...…imbibition s o b tion or seed extract was done by densitometry scanning of an x-ray film obtained after chemiluminescent detection of RsAFPs on an immunoblot (see the following section) and standardized to a twofold performed by the Bradford protein assay (Bradford, 1976) using BSA as a standard....

    [...]

Book•
Molecular Cloning: A Laboratory Manual

[...]

Joseph Sambrook, Edward F. Fritsch, Tom Maniatis
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol

215,169 citations

Journal Article•DOI•
A revised medium for rapid growth and bio assays with tobacco tissue cultures

[...]

Toshio Murashige1, Folke Skoog1•
University of Wisconsin-Madison1
01 Jul 1962-Physiologia Plantarum
TL;DR: In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provide a basis for understanding how compartment-specific redox dynamics may operate in retrograde signaling and stress 67 acclimation in plants.
Abstract: In experiments with tobacco tissue cultured on White's modified medium (basal meditmi hi Tnhles 1 and 2) supplemenk'd with kiticthi and hidoleacctic acid, a slrikin^' fourlo (ive-told intTease iu yield was ohtaitu-d within a three to Tour week j^rowth period on addition of an aqtteotis exlrarl of tobacco leaves (Fi^'ures 1 and 2). Subse(iueutly it was found Ihiit this jnoniotiou oi' f^rowih was due mainly though nol entirely to inorj^auic rather than organic con.stitttenls in the extract. In the isolation of Rrowth factors from plant tissues and other sources inorj '̂anic salts are fre(|uently carried along with fhe organic fraclioits. When tissue cultures are used for bioassays, therefore, il is necessary lo lake into account increases in growth which may result from nutrient elements or other known constituents of the medium which may he present in the te.st materials. To minimize interference trom rontaminaitis of this type, an altempt has heen made to de\\eh)p a nieditmi with such adequate supplies of all re(iuired tnineral nutrients and cotntnott orgattic cottslitueitls that no apprecial»le change in growth rate or yield will result from the inlroduclion of additional amounts in the range ordinarily expected to be present in tnaterials to be assayed. As a point of referetice for this work some of the culture media in mc)st common current use will he cotisidered briefly. For ease of comparis4)n Iheir mineral compositions are listed in Tables 1 and 2. White's nutrient .solution, designed originally for excised root cultures, was based on Uspeuski and Uspetiskaia's medium for algae and Trelease and Trelease's micronutrieni solution. This medium also was employed successfully in the original cttltivation of callus from the tobacco Iiybrid Nicotiana gtauca x A', tanijadorffii, atitl as further modified by White in 194̂ ^ and by others it has been used for the

63,098 citations

"Small cysteine-rich antifungal prot..." refers methods in this paper

  • ...Germination assays for checking the segregation of the chimeric nptil gene in transgenic tobacco were performed on half-strength Murashige and Skoog medium (Murashige and Skoog, 1962) containing 100 mg/L kanamycin and 3% (w/v) sucrose as described by Deblaere et al....

    [...]

Journal Article•DOI•
DNA sequencing with chain-terminating inhibitors

[...]

Frederick Sanger, S Nicklen, Alan Coulson
01 Dec 1977-Proceedings of the National Academy of Sciences of the United States of America
TL;DR: A new method for determining nucleotide sequences in DNA is described, which makes use of the 2',3'-dideoxy and arabinon nucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase.
Abstract: A new method for determining nucleotide sequences in DNA is described. It is similar to the “plus and minus” method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2′,3′-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage ϕX174 and is more rapid and more accurate than either the plus or the minus method.

62,728 citations

"Small cysteine-rich antifungal prot..." refers methods in this paper

  • ...Nucleotide sequence determinations were performed with an automatic sequencer (A.L.F; Pharmacia) based on the dideoxynucleotide chain termination method ( Sanger et al., 1977 )....

    [...]

  • ...F; Pharmacia) based on the dideoxynucleotide chain termination method (Sanger et al., 1977)....

    [...]

Book•
Antibodies: A Laboratory Manual

[...]

Ed Harlow, David P. Lane
01 Jan 1988
TL;DR: A second edition of Antibodies: A Laboratory Manual is being published in September 2013, Revised, extended and updated by Edward Greenfield of the Dana-Farber Cancer Center, the material has been recast with extensive new information and new chapters have been added.
Abstract: ince its publication in 1988, Antibodies: A Laboratory Manual, by Harlow and Lane, has become a classic, an essential resource for molecular biology, immunology, and cell culture labs. In order to keep the book in print, Cold Spring Harbor Laboratory Press eventually produced the paperback edition currently available for sale. Now, after 25 years, a second edition is being published in September 2013. Revised, extended and updated by Edward Greenfield of the Dana-Farber Cancer Center, the material has been recast with extensive new information and new chapters have been added. The new edition provides clear, authoritative, current and up-to-date protocols with background information and troubleshooting advice. The book is an invaluable resource for all those engaged in antibody research and development.

22,254 citations

Related Papers (5)
Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds.

[...]

05 Aug 1992-Journal of Biological Chemistry
Franky R. G. Terras, Hilde M. E. Schoofs, M. F. C. De Bolle, F. Van Leuven, S B Rees, Jozef Vanderleyden, Bruno P. A. Cammue, Willem F. Broekaert 
Plant defensins: novel antimicrobial peptides as components of the host defense system.

[...]

01 Aug 1995-Plant Physiology
Willem F. Broekaert, Franky R. G. Terras, Bruno P. A. Cammue, R. W. Osborn 
Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae

[...]

17 Jul 1995-FEBS Letters
Rupert W. Osborn, Genoveva W. De Samblanx, Karin Thevissen, Inge J.W.M. Goderis, Sophie Torrekens, Fred Van Leuven, Sheila Attenborough, Sarah Bronwen Rees, Willem F. Broekaert 
Fungal pathogen protection in potato by expression of a plant defensin peptide.

[...]

01 Dec 2000-Nature Biotechnology
A G Gao, Salim Hakimi, C A Mittanck, Yonnie Shun Wu, B M Woerner, David M. Stark, Dilip M. Shah, Jihong Liang, Caius M. T. Rommens 
Antimicrobial Peptides from Plants

[...]

01 Jan 1997-Critical Reviews in Plant Sciences
Willem F. Broekaert, Bruno P. A. Cammue, M. F. C. De Bolle, Karin Thevissen, G.W. de Samblanx, R. W. Osborn