Publications (17)0 Total impact
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Article: Synthetic and structural studies on Pyrularia pubera thionin: a single-residue mutation enhances activity against Gram-negative bacteria
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ABSTRACT: The thionin from Pyrularia pubera (Pp-TH), a 47-residue peptide with four internal disulfide bonds, was efficiently produced by chemical synthesis. Its antimicrobial activity in vitro against several representative pathogens (EC50=0.3–3.0 μM) was identical to that of natural Pp-TH. This peptide has a unique Asp32 instead of the consensus Arg found in other thionins of the same family. In order to evaluate the effect of this mutation, the Arg32 analogue (Pp-TH(D32R)) was also synthesized and showed a significant increase in antibiotic activity against several Gram-negative bacteria, whereas it retained the same activity against other pathogens. The overall structure of Pp-TH(D32R) was maintained, though a slight decrease in the helical content of the peptide was observed. -
Article: Antibiotic activities of peptides, hydrogen peroxide and peroxynitrite in plant defence
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ABSTRACT: Genes encoding plant antibiotic peptides show expression patterns that are consistent with a defence role. Transgenic over-expression of defence peptide genes is potentially useful to engineer resistance of plants to relevant pathogens. Pathogen mutants that are sensitive to plant peptides in vitro have been obtained and a decrease of their virulence in planta has been observed, which is consistent with their hypothetical defence role. A similar approach has been followed to elucidate the potential direct anti-microbial role of hydrogen peroxide. Additionally, a scavenger of peroxynitrite has been used to investigate its involvement in plant defence -
Article: Novel defensin subfamily from spinach (Spinacia oleracea)
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ABSTRACT: Antimicrobial peptides (So-D1-7) were isolated from a crude cell wall preparation from spinach leaves (Spinacia oleracea cv. Matador) and, judged from their amino acid sequences, six of them (So-D2-7) represented a novel structural subfamily of plant defensins (group IV). Group-IV defensins were also functionally distinct from those of groups I–III. They were active at concentrations <20 μM against Gram-positive (Clavibacter michiganensis) and Gram-negative (Ralstonia solanacearum) bacterial pathogens, as well as against fungi, such as Fusarium culmorum, F. solani, Bipolaris maydis, and Colletotrichum lagenarium. Fungal inhibition occurred without hyphal branching. Group-IV defensins were preferentially distributed in the epidermal cell layer of leaves and in the subepidermal region of stems. -
Article: Enhanced tolerance to bacterial pathogens caused by the transgenic expression of barley lipid transfer protein LTP2
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ABSTRACT: Purified lipid transfer protein LTP2 from barley applied on tobacco leaves eliminated symptoms caused by infiltration of Pseudomonas syringae pv. tabaci 153. Growth of the pathogen in leaves of transgenic tobacco plants was retarded when compared with non-transformed controls. The percentage of inoculation points that showed necrotic lesions was greatly reduced in transgenic tobacco 17–38% versus 78%) and the average size of these lesions was 61–81% that of control. The average total lesion area (necrosis and chlorosis) in the transgenic plants was also reduced (38% of control). Arabidopsis thaliana transgenic plants inoculated with P. syringae pv. tomato DC3000 also had lower percentages of necrotic lesions (22–38% versus 76%), a reduced average area for each lesion (53–67% of control), and a smaller total lesion area per inoculation (43% of control). These results further support the assignment of a defense role for LTPs and highlight their biotechnological potential. -
Article: Differential effects of five types of antipathogenic plant peptides on model membranes
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ABSTRACT: he effects of five antipathogenic plant peptides, wheat α-thionin, potato PTH1 defensin, barley LTP2 lipid transfer protein, and potato tuber DL1 and DL2 defensins, have been tested against phospholipid vesicles (liposomes). Wheat thionin very actively induces aggregation and leakage of negatively charged vesicles. LTP2 displays the same activities, although to a limited extent. Under certain conditions PTH1 and DL2 induce vesicle aggregation, but not leakage. Potato defensin DL1 failed to show any effect on liposomes. The same peptides have been assayed against a plant pathogenic bacterium, both the membrane-active and -inactive compounds having efficient antibacterial action -
Article: Engineering plants against pathogens: A general strategy
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ABSTRACT: A general strategy to identify defense proteins and genes and to use them in transgenic plants to engineer enhanced resistance against pathogens is illustrated with specific experimental examples. A combinatorial model for the expression of defense genes in response to different pathogens is discussed. -
Article: Two cold inducible genes encoding lipid transfer protein LTP4 from barley show differential responses to bacterial pathogens
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ABSTRACT: The barley genesHvLtp4.2 andHvLtp4.3 both encode the lipid transfer protein LTP4 and are less than 1 kb apart in tail-to-tail orientation. They differ in their non-coding regions from each other and from the gene corresponding to a previously reportedLtp4 cDNA (nowLtp4.1). Southern blot analysis indicated the existence of three or moreLtp4 genes per haploid genome and showed considerable polymorphism among barley cultivars. We have investigated the transient expression of genesHvLtp4.2 andHvLtp4.3 following transformation by particle bombardment, using promoter fusions to the-glucuronidase reporter sequence. In leaves, activities of the two promoters were of the same order as those of the sucrose synthase (Ss1) and cauliflower mosaic virus 35S promoters used as controls. Their expression patterns were similar, except thatLtp4.2 was more active thanLtp4.3 in endosperm, andLtp4.3 was active in roots, whileLtp4.2 was not. The promoters of both genes were induced by low temperature, both in winter and spring barley cultivars. Northern blot analysis, using theLtp4-specific probe, indicated thatXanthomonas campestris pv.translucens induced an increase over basal levels ofLtp4 mRNA, whilePseudomonas syringae pv.japonica caused a decrease. TheLtp4.3-Gus promoter fusion also responded in opposite ways to these two compatible bacterial pathogens, whereas theLtp4.2-Gus construction did not respond to infection -
Article: Characterization and analysis of thionin genes
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ABSTRACT: The general designation of thionins has been proposed for a family of homologous proteins that have been isolated from different tissues in a wide range of plant taxa and have been variously named purothionins, viscotoxins, crambins, etc. (see Garcia-Olmedo et al., 1989). The possible involvement of thionins in plant defense was first suggested, on the basis of their in vitro toxicity to plant pathogens, by Fernandez de Caleya et al., (1972). Those observations had been prompted by earlier reports concerning the antimicrobial properties of these polypeptides (Stuart and Harris, 1942; Balls and Harris, 1944). Work on the thionins, which has been actively pursued over the past half-century, has been recently reviewed in detail (Garcia-Olmedo et al., 1989). For this reason, earlier work will only be partially summarized in the present chapter, which will focus on recent developments concerning thionin genes and their potential role in plant defense mechanisms. -
Article: Inhibition of bacterial and fungal plant pathogens by thionins of types I and II
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ABSTRACT: Thionins are cysteine-rich, 5 kDa polypeptides which are active against plant pathogens. Thionins of type I, from the endosperms of wheat (Wα1, Wα2, Wβ) and barley (Bα, Bβ), and of type II, from barley (BLa, BLb, BLc), have been purified to apparent homogeneity. For a given pathogen, the effective concentration giving 50% inhibition (EC-50) determined for the different thionins varied over a less than fivefold range. The ranking of the variants according to their activity differed among different pathogens, but certain variants, such as Wα1, Wβ or Bβ, tended to be more active than the others. Strains of some bacterial species, such as Clavibacter michiganensis subsp. sepedonicus or Pseudomonas solanacearum were sensitive in the 2–3 × 10−7M concentration rangem whereas the most sensitive fungal pathogens, such as Rosellinia necatrix, Colletotrichum lagenarium and Fusarium solani, had EC-50 values in the 1−4 × 10−6M range. Thionins, which were not particularly effective in liquid medium against Phytophthora infestans (EC-50=3.9 × 10−5M) were more effective than the fungicide Ridomil on a molar basis in a drop application assay on leaf discs from potato. -
Article: Developmental and pathogen-induced expression of three barley genes encoding lipid transfer proteins
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ABSTRACT: Clones for three barley non-specific lipid transfer proteins (LTP2, LTP3, and LTP4; formerly Cw18, Cw20 and Cw21, respectively) which had been previously shown to inhibit growth of plant pathogens, were selected and characterized from a cDNA library derived from young etiolated leaves. Genes Ltp2 and Ltp4 were located in chromosome 3H and gene Ltp3 was assigned to chromosome 7H by Southern blot analysis of wheat—barley disomic addition lines, using gene-specific probes (3′-ends of cDNAs). These assignments were confirmed by the polymerase chain reaction, using specific primers. The three genes were expressed in stem, shoot apex, leaves and roots (at low levels) throughout development. Genes Ltp3 and Ltp4 were expressed at high levels, and Lpt2 at low levels, in the spike (rachis, lemma plus palea and grain coats). Neither of the mRNAs was detected in endosperm. The proteins were localized by tissue-printing with polyclonal antibodies in the outer cell layer of the exposed surfaces of the plant, throughout the embryo, and in vascular tissues. Expression levels in leaves were moderately increased by 0.34 M NaCl and by 0.1 mM abscisic acid and were not affected by cold, drought, salicylate, 2,6-dichloro-isonicotinic acid, ethylene or ethephon. Methyl Jasmonate (10 µM) switched off all three genes. Inoculation with Av6 or vir6 isolates of the fungal pathogen Erysiphe graminis increased the three mRNAs, especially that of LTP4, which reached a maximum nine-fold increase 12–16 h after infection -
Article: Expression of the alpha-thionin gene from barley in tobacco confers enhanced resistance to bacterial pathogens
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ABSTRACT: Thionins are cysteine-rich, 5 kDa polypeptides which are toxic to plant pathogens in vitro. Expression of the gene encoding alpha-thionin from barley endosperm, under the 35S promoter from cauliflower mosaic virus, conferred to transgenic tobacco enhanced resistance to the bacterial plant pathogens Pseudomonas syringae pv. tabaci 153 and P. syringae pv. syringae. The barley alpha-thionin gene, which has two introns, was correctly spliced in tobacco. The alpha-thionin in transgenic plants had the expected mobility in the gradient, when separated by high- performance liquid chromatography, reacted with monospecific antibodies and showed the expected antibiotic properties in vitro -
Article: Leishmania donovani: thionins, plant antimicrobial peptides with leishmanicidal activity
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ABSTRACT: The leishmanicidal activity of plant antibiotic peptides (PAPs) from the principal families, such wheat thionins, a barley lipid transfer protein and potato defensins and snakins were tested in vitro against Leishmania donovani. Only thionins and defensins were active against this human pathogen at a low micromolar range of concentrations. Thionins resulted as the most active peptides tested until now. They collapsed ionic and pH gradients across the parasite plasma membrane together with a rapid depletion of intracellular ATP without affecting mitochondrial potential. Hence the lethal effect of thionins was mostly associated to permeabilization of the plasma membrane leading to an immediate death of the parasite. The present work is the first evidence for leishmanicidal activity in plant peptides. Future prospects for their development as new antiparasite agents on human diseases are considered -
Article: Plant defence peptides
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ABSTRACT: Eight families of antimicrobial peptides, ranging in size from 2 to 9 kD, have been identified in plants. These are thionins, defcnsins, so-called lipid iransfer proteins, hevein- and knottin-Iike peptides, MBPJ, lb AMP, and the recently reported snakins. All of them have compact structures that are stabilized by 2-6 disulfide bridges. They are part of both permanent and inducible defense barriers. Transgenic overe.xpression of the corresponding genes leads to enhanced tolerance to pathogens, and peptide-sensitive pathogen mutants have reduced virulence. -
Article: Snakin-2, an Antimicrobial Peptide from Potato WhoseGene Is Locally Induced by Wounding and Responds toPathogen Infection
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ABSTRACT: The thionin from Pyrularia pubera (Pp-TH), a 47-residue peptide with four internal disulfide bonds, was efficiently produced by chemical synthesis. Its antimicrobial activity in vitro against several representative pathogens (EC50=0.3–3.0 μM) was identical to that of natural Pp-TH. This peptide has a unique Asp32instead of the consensus Arg found in other thionins of the same family. In order to evaluate the effect of this mutation, the Arg32 analogue (Pp-TH(D32R)) was also synthesized and showed a significant increase in antibiotic activity against several Gram-negative bacteria, whereas it retained the same activity against other pathogens. The overall structure of Pp-TH(D32R) was maintained, though a slight decrease in the helical content of the peptide was observed. The peptide snakin-2 (StSN2) has been isolated from potato (Solanum tuberosum cv Jaerla) tubers and found to be active (EC50 = 1-20 µM) against fungal and bacterial plant pathogens. It causes a rapid aggregation of both Gram-positive and Gram-negative bacteria. The correspondingStSN2 cDNA encodes a signal sequence followed by a 15-residue acidic sequence that precedes the mature StSN2 peptide, which is basic (isoelectric point = 9.16) and 66 amino acid residues long (molecular weight of 7,025). The StSN2 gene is developmentally expressed in tubers, stems, flowers, shoot apex, and leaves, but not in roots, or stolons, and is locally up-regulated by wounding and by abscisic acid treatment. Expression of this gene is also up-regulated after infection of potato tubers with the compatible fungus Botritys cinerea and down-regulated by the virulent bacteriaRalstonia solanacearum and Erwinia chrysanthemi. These observations are congruent with the hypothesis that the StSN2 is a component of both constitutive and inducible defense barriers. -
Article: Expression of genes encoding thionins and lipid-transfer proteins. A combinatorial model for the responses of defense genes to pathogens.
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ABSTRACT: Seeds normally accumulate high levels of proteins that are either toxic or inhibitory towards heterologous systems, including pathogens, pests and predators. This is the case of cereal kernels, where a substantial fraction of the non-storage proteins is represented by different families of these proteins (for reviews, see Garcia-Olmedo et al.1989, 1992). The same types of proteins are often present in other tissues, either under normal development or under stress, including infection by pathogens. The thionins (THs) and the so-called non-specific lipid transfer proteins (LTPs) are two such families. Thionins were first reported in wheat endosperm (Balls et al., 1942) and their in vitro antipathogenic properties have been known for over 2 0 years (Fernadez de Caleya et al. 1972), while cereal LTPs were first reported in barley aleurone (Mundy and Rogers 1986) and we have recently shown that different members of this family can be isolated from other barley tissues and that they are potent growth inhibitors of bacterial and fungal plant pathogens (Molina and Garcia-Olmedo, 1991; Molina et al.1993; Molina and Garcia- Olmedo, submitted). In this report, we will update our knowledge of the molecular biology and inhibitory properties of barley THs and LTPs, and will describe the developmental and pathogeninduced expression of the different genes encoding these proteins. Also, a working model for the response of defense genes against infection by different pathogens will be proposed. -
Article: Repression of the auxin response pathway increases Arabidopsis susceptibility to necrotrophic fungi
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ABSTRACT: In plants, resistance to necrotrophic pathogens depends on the interplay between different hormone systems, such as those regulated by salicylic acid (SA), jasmonic acid (JA), ethylene, and abscisic acid. Repression of auxin signaling by the SA pathway was recently shown to contribute to antibacterial resistance. Here, we demonstrate that Arabidopsis auxin signaling mutants axr1, axr2, and axr6 that have defects in the auxin-stimulated SCF (Skp1¿Cullin¿ F-box) ubiquitination pathway exhibit increased susceptibility to the necrotrophic fungi Plectosphaerella cucumerina and Botrytis cinerea. Also, stabilization of the auxin transcriptional repressor AXR3 that is normally targeted for removal by the SCF-ubiquitin/proteasome machinery occurs upon P. cucumerina infection. Pharmacological inhibition of auxin transport or proteasome function each compromise necrotroph resistance of wild-type plants to a similar extent as in non-treated auxin response mutants. These results suggest that auxin signaling is important for resistance to the necrotrophic fungi P. cucumerina and B. cinerea. SGT1b (one of two Arabidopsis SGT1 genes encoding HSP90/HSC70 co-chaperones) promotes the functions of SCF E3-ubiquitin ligase complexes in auxin and JA responses and resistance conditioned by certain Resistance (R) genes to biotrophic pathogens. We find that sgt1b mutants are as resistant to P. cucumerina as wild-type plants. Conversely, auxin/SCF signaling mutants are uncompromised in RPP4-triggered resistance to the obligate biotrophic oomycete, Hyaloperonospora parasitica. Thus, the predominant action of SGT1b in R gene-conditioned resistance to oomycetes appears to be at a site other than assisting SCF E3-ubiquitin ligases. However, genetic additivity of sgt1b axr1 double mutants in susceptibility to H. parasitica suggests that SCF-mediated ubiquitination contributes to limiting biotrophic pathogen colonization once plant¿pathogen compatibility is established. -
Article: Snakin-1, a Peptide from Potato That Is Active Against Plant Pathogens
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ABSTRACT: A new type of antimicrobial peptide, snakin-1 (SN1), has been isolated from potato tubers and found to be active, at concentrations <10 uM, against bacterial and fungal pathogens from potato and other plant species. The action of SN1 and potato defensin PTH1 was synergistic against the bacterium Clavibacter michiganensis subsp. sepedonicus and additive against the fungus Botrytis cinérea. Snakin-1 causes aggregation of both gram-positive and gram-negative bacteria. The peptide has 63 amino acid residues (Mr 6,922), 12 of which are cysteines, and is unrelated to any previously isolated protein, although it is homologous to amino acid sequences deduced from cloned cDNAs that encode gibberellin-inducible mRNAs and has some sequence motifs in common with kistrin and other hemotoxic snake venoms. A degenerate oligonucleotide probé based on the internal sequence CCEECKC has been used to clone an SN1 cDNA. With the cDNA used as probé, one copy of the StSNl gene per haploid genome has been estimated and expression of the gene has been detected in tubers, stems, axillary buds, and young floral buds. Expression levéis in petáis and carpels from fully developed flowers were much higher than in sepáis and stamens. The expression pattern of gene StSNl suggests that protein SN1 may be a component of constitutive defense barriers, especially those of storage and reproductive plant organs.
