Antibiotic activities of peptides, hydrogen peroxide and peroxynitrite in plant defence.

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.

Effects of urate, a natural inhibitor of peroxynitrite-mediated toxicity, in the response of Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae.

Urate, a natural peroxynitrite scavenger, has been used to investigate the possible role of peroxynitrite during plant-pathogen interactions. Urate greatly reduced lesion formation in Arabidopsis leaves treated with an abiotic peroxynitrite-generating system or with a peroxynitrite solution, indicating that it can act as an effective scavenger in planta. In the interaction with the avirulent Pseudomonas syringae pv. phaseolicola (avrRPM1+), cell death in the inoculated area was strongly reduced by urate, without compromising disease resistance. In contrast, urate promoted discrete cell death in response to an isogenic Pseudomonas syringae (avrRPM1-), which did not trigger an HR when inoculated alone, and it induced resistance and arrest of pathogen growth. Scavenging of peroxynitrite did not modify the response of Arabidopsis to an avirulent strain of Xanthomonas campestris pv campestris, that showed a high resistance to NO and peroxynitrite. Our data indicate that peroxynitrite plays a significant role in the responses of plants to Pseudomonas syringae.

Relative effects on virulence of mutations in the sap, pel, and hrp loci of Erwinia chrysanthemi.

We constructed strains of Erwinia chrysanthemi EC16 with multiple mutations involving three virulence systems in this bacterium, namely pel (coding for the major pectate lyases pelABCE), hrp (hypersensitive response and pathogenicity), and sap (sensitivity to antimicrobial peptides). The relative effects on virulence of those mutations have been analyzed on potato tubers and chicory leaves. In potato tubers, the sap mutation (BT105) had a greater effect in the reduction of the virulence than the pel (CUCPB5006) and hrp (CUCPB5039) mutations. This reduction was similar to that observed in the pel-hrp double mutant (CUCPB5037). The analysis of the strains affected in Pel-Sap (BT106), Hrp-Sap (BT107), and Pel-Hrp-Sap (BT108) suggested that the effects of these mutations are additive. In chicory leaves, the mutation in the sap locus appeared to have a greater effect than in potato tubers. The competitive indices of strains BT105, UM1005 (Pel-), CUCPB5039, and CUCPB5037 have been estimated in vivo and in vitro. These results indicate that the mutation in the hrp locus can be complemented in vivo by coinfection, whereas the mutations in pel and sap cannot.

Evidence against a direct antimicrobial role of H2O2 in the infection of plants by Erwinia chrysanthemi.

We have investigated the role of bacterial resistance to oxidative stress in pathogenesis. The oxyR gene from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It is closely related to that found in Escherichia coli (88% overall amino acid identity). An E. chrysanthemi oxyR mutant strain was constructed by marker exchange. After induction with a sublethal dose of H2O2, this mutant was more sensitive to H2O2 and showed reduced levels of catalase and glutathione reductase activities, compared with the wild type. The oxyR mutant was unable to form individual colonies on agar plates unless catalase was added exogenously. However, it retained full virulence in potato tubers and tobacco leaves. These results suggest that the host-produced H2O2 has no direct antimicrobial effect on the interaction of E. chrysanthemi with the two plant species.

Snakin-1, a peptide from potato that is active against plant pathogens.

A new type of antimicrobial peptide, snakin-1 (SN1), has been isolated from potato tubers and found to be active, at concentrations < 10 microM, 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 cinerea. Snakin-1 causes aggregation of both gram-positive and gram-negative bacteria. The peptide has 63 amino acid residues (M(r) 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 probe based on the internal sequence CCEECKC has been used to clone an SN1 cDNA. With the cDNA used as probe, one copy of the StSN1 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 levels in petals and carpels from fully developed flowers were much higher than in sepals and stamens. The expression pattern of gene StSN1 suggests that protein SN1 may be a component of constitutive defense barriers, especially those of storage and reproductive plant organs.

Novel defensin subfamily from spinach (Spinacia oleracea).

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 microM 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.

Inactivation of the sapA to sapF locus of Erwinia chrysanthemi reveals common features in plant and animal bacterial pathogenesis.

We investigated the role in pathogenesis of bacterial resistance to plant antimicrobial peptides. The sapA to sapF (for sensitive to antimicrobial peptides) operon from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It has five open reading frames that are closely related (71% overall amino acid identity) and are in the same order as those of the sapA to sapF operon from Salmonella typhimurium. An E. chrysanthemi sap mutant strain was constructed by marker exchange. This mutant was more sensitive than was the wild type to wheat alpha-thionin and to snakin-1, which is the most abundant antimicrobial peptide from potato tubers. This mutant was also less virulent than was the wild-type strain in potato tubers: lesion area was 37% that of the control, and growth rate was two orders of magnitude lower. These results indicate that the interaction of antimicrobial peptides from the host with the sapA to sapF operon from the pathogen plays a similar role in animal and in plant bacterial pathogenesis.

Plant defense peptides.

Eight families of antimicrobial peptides, ranging in size from 2 to 9 kD, have been identified in plants. These are thionins, defensins, so-called lipid transfer proteins, hevein- and knottin-like peptides, MBP1, IbAMP, 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 overexpression of the corresponding genes leads to enhanced tolerance to pathogens, and peptide-sensitive pathogen mutants have reduced virulence.

Mutants of Ralstonia (Pseudomonas) solanacearum sensitive to antimicrobial peptides are altered in their lipopolysaccharide structure and are avirulent in tobacco.

Ralstonia solanacearum K60 was mutagenized with the transposon Tn5, and two mutants, M2 and M88, were isolated. Both mutants were selected based on their increased sensitivity to thionins, and they had the Tn5 insertion in the same gene, 34 bp apart. Sequence analysis of the interrupted gene showed clear homology with the rfaF gene from Escherichia coli and Salmonella typhimurium (66% similarity), which encodes a heptosyltransferase involved in the synthesis of the lipopolysaccharide (LPS) core. Mutants M2 and M88 had an altered LPS electrophoretic pattern, consistent with synthesis of incomplete LPS cores. For these reasons, the R. solanacearum gene was designated rfaF. The mutants were also sensitive to purified lipid transfer proteins (LTPs) and to an LTP-enriched, cell wall extract from tobacco leaves. Mutants M2 and M88 died rapidly in planta and failed to produce necrosis when infiltrated in tobacco leaves or to cause wilting when injected in tobacco stems. Complemented strains M2* and M88* were respectively obtained from mutants M2 and M88 by transformation with a DNA fragment harboring gene rfaF. They had a different degree of wild-type reconstituted phenotype. Both strains retained the rough phenotype of the mutants, and their LPS electrophoretic patterns were intermediate between those of the wild type and those of the mutants.

Enhanced tolerance to bacterial pathogens caused by the transgenic expression of barley lipid transfer protein LTP2.

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.

Differential effects of five types of antipathogenic plant peptides on model membranes.

The effects of five antipathogenic plant peptides, wheat alpha-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.

Differential expression of pathogen-responsive genes encoding two types of glycine-rich proteins in barley.

Gene-specific probes (3' ends of cDNAs) were obtained from barley cDNAs encoding two types of glycine-rich proteins: HvGRP2, characterized by a cytokeratin-like and a cysteine-rich domain, and HvGRP3, whose main feature was an RNA-binding domain. Expression of genes Hvgrp2 and Hvgrp3, which are present at one (or two) copies per haploid genome, was ubiquitous and gene Hvgrp3 was under light/darkness modulation. Cold treatment increased Hvgrp2 and Hvgrp3 mRNA levels. Methyl jasmonate (10 microM) switched off the two genes. Expression of Hvgrp2, but not that of Hvgrp3, was induced by ethylene treatment (100 ppm). Fungal pathogens Erysiphe graminis and Rhynchosporium secalis increased the mRNAs levels of the two genes, both in compatible and in incompatible interactions, while bacterial pathogens did not.

Processing of thionin precursors in barley leaves by a vacuolar proteinase.

Thionins are synthesized as precursors with a signal peptide and a long C-terminal acidic peptide that is post-translationally processed. A fusion protein including the maltose-binding protein from Escherichia coli (MalE), thionin DG3 from barley leaves, and its acidic C-terminal peptide has been used to obtain antibodies that recognize both domains of the precursor. In barley leaf sections, mature thionins accumulated in the vacuolar content, while the acidic peptide was not detected in any cell fraction. Brefeldin A and monensin inhibited processing of the precursor but its export from the microsomal fraction was not inhibited. Both purified vacuoles and an acid (pH 5.5) extract from leaves processed the fusion protein into a MalE-thionin and an acidic peptide fragment. A 70-kDa proteinase that effected this cleavage was purified from the acid extract. Processing of the fusion protein by both lysed vacuoles and the purified proteinase was inhibited by Zn2+ and by Cu2+, but not by inhibitors of the previously described vacuolar processing thiol or aspartic proteinases. In vivo processing of the thionin precursor in leaf sections was also inhibited by Zn2+ and Cu2+. Variants of the fusion protein with altered processing sites that represented those of thionin precursors from different taxa were readily processed by the proteinase, whereas changing the polarity of either the C-terminal or N-terminal residues of the processing site prevented cleavage by the proteinase.

Two cold-inducible genes encoding lipid transfer protein LTP4 from barley show differential responses to bacterial pathogens.

The barley genes HvLtp4.2 and HvLtp4.3 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 reported Ltp4 cDNA (now Ltp4.1). Southern blot analysis indicated the existence of three or more Ltp4 genes per haploid genome and showed considerable polymorphism among barley cultivars. We have investigated the transient expression of genes HvLtp4.2 and HvLtp4.3 following transformation by particle bombardment, using promoter fusions to the beta-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 that Ltp4.2 was more active than Ltp4.3 in endosperm, and Ltp4.3 was active in roots, while Ltp4.2 was not. The promoters of both genes were induced by low temperature, both in winter and spring barley cultivars. Northern blot analysis, using the Ltp4-specific probe, indicated that Xanthomonas campestris pv. translucens induced an increase over basal levels of Ltp4 mRNA, while Pseudomonas syringae pv. japonica caused a decrease. The Ltp4.3-Gus promoter fusion also responded in opposite ways to these two compatible bacterial pathogens, whereas the Ltp4.2-Gus construction did not respond to infection.

Selective disulphide linkage of plant thionins with other proteins.

Thionins are shown to form disulphide linkages with other proteins. The reaction with bacterial enzymes beta-glucuronidase and neomycin phosphotransferase II could be prevented and reversed with dithiothreitol and blocked with N-ethylmaleimide. Other cysteine-rich low-molecular-weight toxic peptides from plants (LTP-3 from barley and P19 from potato) did not react as the thionins. Certain cysteine-containing proteins, such bovine serum albumin, ovalbumin and cytochrome c, reacted with thionins, while others, including carbonic anhydrase, soybean trypsin inhibitor, bovine-lung trypsin inhibitor and phosphorylase B did not. Selectivity of the reaction with a periplasmic component of the phytopathogenic bacterium Pseudomonas solanacearum was also shown.

The defensive role of nonspecific lipid-transfer proteins in plants.

Plant nonspecific lipid-transfer proteins stimulate the transfer of a broad range of lipids between membranes in vitro. In view of their ability to inhibit bacterial and fungal pathogens, their distribution at high concentrations over exposed surfaces and in the vascular system, and the response of Ltp-gene expression to infection with pathogens, they are now thought to be active plant-defense proteins.

Pseudothionin-St1, a potato peptide active against potato pathogens.

A 5-kDa polypeptide, pseudothionin Solanum tuberosum 1 (Pth-St1), which was active against Clavibacter michiganensis subspecies sepedonicus, a bacterial pathogen of potatoes, has been purified from the buffer-insoluble fraction of potato tubers by salt extraction and HPCL. Pth-St1 was also active against other potato pathogens tested (Pseudomonas solanacearum and Fusarium solani). The N-terminal amino acid sequence of this peptide was identical (except for a N/H substitution at position 2) to that deduced from a previously reported cDNA sequence (EMBL accession number X-13180), which had been misclassified as a Browman-Birk protease inhibitor. Pth-St1 did not inhibit either trypsin or insect alpha-amylase activities, and, in contrast with true thionins, did not affect cell-free protein synthesis or beta-glucuronidase activity. Northern-blot and tissue-print analyses showed that steady-state mRNA levels were highest in flowers (especially in petals), followed by tubers (especially in the epidermal cell layers and in leaf primordia), stems and leaves. Infection of leaves with a bacterial pathogen suspended in 10 mM MgCl2 switched off the gene, whereas mock inoculation with 10 mM MgCl2 alone induced higher mRNA levels.