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Abstract: Background and aims: Prebiotics such as Arabinoxylooligosaccharides (AXOS) are non‐digestible, fermentable food ingredients stimulating growth/activity of colonic bacteria with enhanced carbohydrates fermentation (CF) in humans. The migrating motor complex (MMC) of the gastrointestinal tract has been recently identified as an important hunger signal, but no data are available yet on the role of acute CF on MMC activity and related hunger ratings. Thus, we aimed to study the effect of acute AXOS CF on MMC and hunger in humans. Methods: A total of 13 healthy volunteers were randomized in a single‐blind crossover placebo‐controlled study where 9.4 g of AXOS or 10 g of maltodextrin and 1 g of unlabelled lactose ureide (LU) were given 12 hours prior to the study and, in the next morning, together with a pancake containing 500 mg of 13C‐LU. In 10 hours after the meal, 13CO2 and hydrogen excretion were determined every 15 minutes while hunger/appetite ratings every 2 minutes through a VAS questionnaire. Five hours after the meal, antroduodenal motility was measured using HRM. Key Results: AXOS significantly increased CF (158 ± 81 vs 840 ± 464 H2 ppm*minute, placebo vs AXOS, P < .05) without affecting the orocecal transit time (OCTT). AXOS had no significant effect on the occurrence, origin, and duration of phase III and on the total number, origin, and duration of phases I and II. Hunger and appetite scores prior and after phase III were not affected by AXOS. Conclusions: AXOS acutely increases colonic fermentation, but this neither affects OCTT, activity of the MMC, nor interdigestive hunger scores in man. [ABSTRACT FROM AUTHOR]

Background Intestinal microbiota regulates gastrointestinal sensory-motor function. Prebiotics such as arabinoxylan-oligosaccharide ( AXOS) are non-digestible, fermentable food ingredients beneficially affecting intestinal microbiota, colon activity, and improving human health. We wanted to investigate whether acute AXOS or maltodextrin (placebo) administration may alter gastric sensitivity ( GS), accommodation ( GA), nutrient tolerance ( NT) in man. Methods Thirteen HV (6 M, 32.2 ± 1.8 years; BMI 22.3 ± 0.2) underwent two 48 h treatment periods with oral 4 × 9.4 g AXOS or 4 × 10 g maltodextrin (at least 1 week wash-out) for gastric barostat assessment of GS, gastric compliance ( GC), GA to a liquid test meal, on day 1, and NT drink test, on day 2. Oro-cecal transit-time ( OCTT), colonic fermentation ( CF) were assessed simultaneously with 13C-lactose ureide, H2 breath tests. Key Results Arabinoxylan-oligosaccharide significantly increased CF on day 1 and 2 (565 ± 272 vs 100 ± 24, 365 ± 66 vs 281 ± 25 H2 ppm/min, AXOS vs maltodextrin, both p < 0.05), not the OCTT. AXOS did not alter GC, sensitivity before and after the meal. Gastric accommodation was not significantly influenced by AXOS (volume increment: 171 ± 33 vs 130 ± 28 mL, AXOS vs maltodextrin, p = NS). On day 1, AXOS fermentation was associated with significantly higher postprandial bloating scores (960 ± 235 vs 396 ± 138 mm*min, AXOS vs maltodextrin, p < 0.05). On day 2, AXOS did not affect maximal NT (946 ± 102 vs 894 ± 97 mL, AXOS vs maltodextrin, p = NS), increased the bloating score (1236 ± 339 vs 675 ± 197 mm*min, AXOS vs maltodextrin, p < 0.05). Conclusions & Inferences Acute AXOS administration, associated with increased CF, does not affect GA, is not associated with increased meal-induced satiety or perception scores. [ABSTRACT FROM AUTHOR]

Arabinoxylan-oligosaccharides (AXOS) are a recently newly discovered class of candidate prebiotics as – depending on their structure – they are fermented in different regions of gastrointestinal tract. This can have an impact on the protein/carbohydrate fermentation balance in the large intestine and, thus, affect the generation of potentially toxic metabolites in the colon originating from proteolytic activity. In this study, we screened different AXOS preparations for their impact on the in vitro intestinal fermentation activity and microbial community structure. Short-term fermentation experiments with AXOS with an average degree of polymerization (avDP) of 29 allowed part of the oligosaccharides to reach the distal colon, and decreased the concentration of proteolytic markers, whereas AXOS with lower avDP were primarily fermented in the proximal colon. Additionally, prolonged supplementation of AXOS with avDP 29 to the Simulator of Human Intestinal Microbial Ecosystem (SHIME) reactor decreased levels of the toxic proteolytic markers phenol and p-cresol in the two distal colon compartments and increased concentrations of beneficial short-chain fatty acids (SCFA) in all colon vessels (25–48%). Denaturant gradient gel electrophoresis (DGGE) analysis indicated that AXOS supplementation only slightly modified the total microbial community, implying that the observed effects on fermentation markers are mainly caused by changes in fermentation activity. Finally, specific quantitative PCR (qPCR) analysis showed that AXOS supplementation significantly increased the amount of health-promoting lactobacilli as well as of Bacteroides–Prevotella and Clostridium coccoides– Eubacterium rectale groups. These data allow concluding that AXOS are promising candidates to modulate the microbial metabolism in the distal colon. [ABSTRACT FROM AUTHOR]

The objective of this in vivo experiment was to evaluate the influence of arabinoxylooligo-saccharides (AXOS) on shedding and colonization of Salmonella Enteritidis in broilers. Arabinoxylooligo-saccharides, which are oligosaccharides derived from arabinoxylans by partial hydrolysis, have a beneficial effect on feed conversion ratios when added to broiler diets. Additionally, AXOS have been shown to promote the growth of bifidobacteria in the ceco-colonic compartment of the gastrointestinal tract. To investigate the impact of AXOS on colonization of broilers with Salmonella, 224 one-day-old chicks were divided into 4 groups and given either unsupplemented feed or feed supplemented with 0.2% AXOS-3-0.25, 0.2% AXOS-9-0.25, or 0.4% AXOS-9-0.25 throughout the experiment. The AXOS-3-0.25 and AXOS-9-0.25 both have an arabinose-to-xylose ratio of 0.25 and have an average degree of polymerization of 3 and 9, respectively. At 14 d posthatch, each animal was orally inoculated with 2.5 x 10[sup9] cfu of Salmonella Enteritidis. Cloacal swabs, taken at regular times, showed a significant reduction of Salmonella presence in the group given 0.4% AXOS-9-0.25 compared with the control group. This reduction was observed in the 1- to 11-d postinfection period. Colonization of the ceca as well as the translocation of Salmonella to the spleen was significantly reduced at 3 and 7 d postinfection in the 0.4% AXOS-9-0.25 group. A similar, although more moderate, decrease in colonization was observed in the group given 0.2% AXOS-9-0.25. It was concluded that dietary addition of AXOS provides dose-dependent protection against oral infections with Salmonella Enteritidis in poultry. [ABSTRACT FROM AUTHOR]

Rs-AFPs are antifungal proteins, isolated from radish (Raphanus sativus) seed or leaves, which consist of 50 or 51 amino acids and belong to the plant defensin family of proteins. Four highly homologous Rs-AFPs have been isolated (Rs-AFP1-4). The structure of Rs-AFP1 consists of three beta-strands and an alpha-helix, and is stabilized by four cystine bridges. Small peptides deduced from the native sequence, still having biological activity, are not only important tools to study structure-function relationships, but may also constitute a commercially interesting target. In an earlier study, we showed that the antifungal activity of Rs-AFP2 is concentrated mainly in the beta2-beta3 loop. In this study, we synthesized linear 19-mer peptides, spanning the entire beta2-beta3 loop, that were found to be almost as potent as Rs-AFP2. Cysteines, highly conserved in the native protein, are essential for maintaining the secondary structure of the protein. Surprisingly, in the 19-mer loop peptides, cysteines can be replaced by alpha-aminobutyric acid, which even improves the antifungal potency of the peptides. Analogous cyclic 19-mer peptides, forced to adopt a hairpin structure by the introduction of one or two non-native disulfide bridges, were also found to possess high antifungal activity. The synthetic 19-mer peptides, like Rs-AFP2 itself, cause increased Ca2+ influx in pregerminated fungal hyphae.

Crude aqueous extracts from Arabidopsis leaves were subjected to chromatographic separations, after which the different fractions were monitored for antimicrobial activity using the fungus Neurospora crassa as a test organism. Two major fractions were obtained that appeared to have the same abundance in leaves from untreated plants versus leaves from plants challenge inoculated with the fungus Alternaria brassicicola. One of both major antimicrobial fractions was purified to homogeneity and identified by 1H nuclear magnetic resonance, gas chromatography/electron impact mass spectrometry, and gas chromatography/chemical ionization mass spectrometry as 4-methylsulphinylbutyl isothiocyanate (ITC). This compound has previously been described as a product of myrosinase-mediated breakdown of glucoraphanin, the predominant glucosinolate in Arabidopsis leaves. 4-Methylsulphinylbutyl ITC was found to be inhibitory to a wide range of fungi and bacteria, producing 50% growth inhibition in vitro at concentrations of 28 microM for the most sensitive organism tested (Pseudomonas syringae). A previously identified glucosinolate biosynthesis mutant, gsm1-1, was found to be largely deficient in either of the two major antimicrobial compounds, including 4-methylsulphinylbutyl ITC. The resistance of gsm1-1 was compared with that of wild-type plants after challenge with the fungi A. brassicicola, Plectosphaerella cucumerina, Botrytis cinerea, Fusarium oxysporum, or Peronospora parasitica, or the bacteria Erwinia carotovora or P. syringae. Of the tested pathogens, only F. oxysporum was found to be significantly more aggressive on gsm1-1 than on wild-type plants. Taken together, our data suggest that glucosinolate-derived antimicrobial ITCs can play a role in the protection of Arabidopsis against particular pathogens.

Not more than 10 years ago it was generally accepted that pathogen-inducible defense mechanisms in plants are triggered through a central signaling cascade that regulates a multicomponent defense response. Now we know that the plant defense system is regulated through a complex network of various signaling cascades.

We have previously identified a Saccharomyces cerevisiae mutant that is markedly more resistant than wild-type to Dahlia merckii antimicrobial peptide 1 (DmAMP1), an antifungal plant defensin isolated from seeds of dahlia (Dahlia merckii). A complementation approach was followed that consisted of the introduction of a genomic library of DmAMP1-sensitive wild-type yeast into the DmAMP1-resistant yeast mutant and screening for restored sensitivity to DmAMP1. The gene determining sensitivity of S. cerevisiae to DmAMP1 was identified as IPT1, a gene encoding an enzyme involved in the last step of the synthesis of the sphingolipid mannose-(inositol-phosphate)(2)-ceramide. Strains with a nonfunctional IPT1 allele lacked mannose-(inositol-phosphate)(2)-ceramide in their plasma membranes, bound significantly less DmAMP1 compared with wild-type strains, and were highly resistant to DmAMP1-mediated membrane permeabilization. All of these phenotypic deviations could be restored by reintroduction of a functional IPT1 gene. Our data support a model in which membrane patches containing sphingolipids act as binding sites for DmAMP1 or, alternatively, are required to anchor membrane or cell wall-associated proteins, which themselves interact with DmAMP1.

Dm-AMP1, an antifungal plant defensin from seeds of dahlia (Dahlia merckii), was radioactively labeled with t-butoxycarbonyl-[35S]-L-methionine N-hydroxy-succinimi-dylester. This procedure yielded a 35S-labeled peptide with unaltered antifungal activity. [35S]Dm-AMP1 was used to assess binding on living cells of the filamentous fungus Neurospora crassa and the unicellular fungus Saccharomyces cerevisiae. Binding of [35S]Dm-AMP1 to fungal cells was saturable and could be competed for by preincubation with excess, unlabeled Dm-AMP1 as well as with Ah-AMP1 and Ct-AMP1, two plant defensins that are highly homologous to Dm-AMP1. In contrast, binding could not be competed for by more distantly related plant defensins or structurally unrelated antimicrobial peptides. Binding of [35S]Dm-AMP1 to either N. crassa or S. cerevisiae cells was apparently irreversible. In addition, whole cells and microsomal membrane fractions from two independently obtained S. cerevisiae mutants selected for resistance to Dm-AMP1 exhibited severely reduced binding affinity for [35S]Dm-AMP1, compared with wild-type yeast. This finding suggests that binding of Dm-AMP1 to S. cerevisiae plasma membranes is required for antifungal activity of this protein.

Inoculation of wild-type Arabidopsis plants with the fungus Alternaria brassicicola results in systemic induction of genes encoding a plant defensin (PDF1.2), a basic chitinase (PR-3), and an acidic hevein-like protein (PR-4). Pathogen-induced induction of these three genes is almost completely abolished in the ethylene-insensitive Arabidopsis mutant ein2-1. This indicates that a functional ethylene signal transduction component (EIN2) is required in this response. The ein2-1 mutants were found to be markedly more susceptible than wild-type plants to infection by two different strains of the gray mold fungus Botrytis cinerea. In contrast, no increased fungal colonization of ein2-1 mutants was observed after challenge with avirulent strains of either Peronospora parasitica or A. brassicicola. Our data support the conclusion that ethylene-controlled responses play a role in resistance of Arabidopsis to some but not all types of pathogens.

We used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in fungi. Membrane permeabilization induced by plant defensins in Neurospora crassa was biphasic, depending on the plant defensin dose. At high defensin levels (10 to 40 microM), strong permeabilization was detected that could be strongly suppressed by cations in the medium. This permeabilization appears to rely on direct peptide-phospholipid interactions. At lower defensin levels (0.1 to 1 microM), a weaker, but more cation-resistant, permeabilization occurred at concentrations that correlated with the inhibition of fungal growth. Rs-AFP2(Y38G), an inactive variant of the plant defensin Rs-AFP2 from Raphanus sativus, failed to induce cation-resistant permeabilization in N. crassa. Dm-AMP1, a plant defensin from Dahlia merckii, induced cation-resistant membrane permeabilization in yeast (Saccharomyces cerevisiae) which correlated with its antifungal activity. However, Dm-AMP1 could not induce cation-resistant permeabilization in the Dm-AMP1-resistant S. cerevisiae mutant DM1, which has a drastically reduced capacity for binding Dm-AMP1. We think that cation-resistant permeabilization is binding site mediated and linked to the primary cause of fungal growth inhibition induced by plant defensins.

The phytoalexin-deficient Arabidopsis mutant pad3-1, which is affected in the production of the indole-type phytoalexin camalexin, has previously been shown not to display altered susceptibility to either the bacterium Pseudomonas syringae (Glazebrook & Ausubel 1994; Proc. Natl. Acad. Sci. USA, 91: 8955-8959) or the biotrophic fungi Peronospora parasitica (Glazebrook et al. 1997; Genetics, 146: 381-392) and Erysiphe orontii (Reuber et al. 1998; Plant J. 16: 473-485). We now show that this mutant is markedly more susceptible than its wild-type parental line to infection by the necrotrophic fungus Alternaria brassicicola, but not to Botrytis cinerea. A strong camalexin response was elicited in wild-type plants inoculated with either Alternaria brassicicola or Botrytis cinerea, whereas no camalexin could be detected in pad3-1 challenged with these fungi. Hence, PAD3 appears to be a key determinant in resistance to at least A. brassicicola. The induction of salicylate-dependent and jasmonate/ethylene-dependent defense genes was not reduced in Alternaria-challenged pad3-1 plants compared to similarly treated wild-type plants. Camalexin production could not be triggered by exogenous application of either salicylate, ethylene or jasmonate and was not, or not strongly, reduced in mutants with defects in perception of these defense-related signal molecules. Camalexin-production appears to be controlled by a pathway that exhibits little cross-talk with salicylate-, ethylene- and jasmonate-dependent signalling events.

The endogenous plant hormones salicylic acid (SA) and jasmonic acid (JA), whose levels increase on pathogen infection, activate separate sets of genes encoding antimicrobial proteins in Arabidopsis thaliana. The pathogen-inducible genes PR-1, PR-2, and PR-5 require SA signaling for activation, whereas the plant defensin gene PDF1.2, along with a PR-3 and PR-4 gene, are induced by pathogens via an SA-independent and JA-dependent pathway. An Arabidopsis mutant, coi1, that is affected in the JA-response pathway shows enhanced susceptibility to infection by the fungal pathogens Alternaria brassicicola and Botrytis cinerea but not to Peronospora parasitica, and vice versa for two Arabidopsis genotypes (npr1 and NahG) with a defect in their SA response. Resistance to P. parasitica was boosted by external application of the SA-mimicking compound 2, 6-dichloroisonicotinic acid [Delaney, T., et al. (1994) Science 266, 1247-1250] but not by methyl jasmonate (MeJA), whereas treatment with MeJA but not 2,6-dichloroisonicotinic acid elevated resistance to Alternaria brassicicola. The protective effect of MeJA against A. brassicicola was the result of an endogenous defense response activated in planta and not a direct effect of MeJA on the pathogen, as no protection to A. brassicicola was observed in the coi1 mutant treated with MeJA. These data point to the existence of at least two separate hormone-dependent defense pathways in Arabidopsis that contribute to resistance against distinct microbial pathogens.

The plant defensin PDF1.2 has previously been shown to accumulate systemically via a salicylic acid-independent pathway in leaves of Arabidopsis upon challenge by fungal pathogens. To further investigate the signalling and transcriptional processes underlying plant defensin induction, a DNA fragment containing 1184 bp and 1232 bp upstream of the transcriptional and translational start sites, respectively, was cloned by inverse PCR. To test for promoter activity this DNA fragment was linked to the beta-glucuronidase (GUS)-encoding region of the UidA gene as a translational fusion and introduced into Arabidopsis ecotype C-24. Challenge of the transgenic plants with the fungal pathogens Alternaria brassicicola and Botrytis cinerea resulted in both local and systemic induction of the reporter gene. Wounding of the transgenic plants had no effect on GUS activity. Treatment of the transgenic plants with either jasmonates or the active oxygen generating compound paraquat strongly induced the reporter gene. In contrast, neither salicylate nor its functional analogues 2,6-dichloroisonicotinic acid and 1,2,3-benzothiodiazole-7-carbothioic acid S-methyl ester resulted in reporter gene induction. These results are consistent with the existence of a salicylic acid-independent signalling pathway, possibly involving jasmonates as regulators, that is triggered by pathogen challenge but not by wounding. The transgenic plants containing the PDF1.2-based promoter-reporter construct will provide useful tools for future genetic dissection of this novel systemic signalling pathway.

Activation of the plant defensin gene PDF1.2 in Arabidopsis by pathogens has been shown previously to be blocked in the ethylene response mutant ein2-1 and the jasmonate response mutant coi1-1. In this work, we have further investigated the interactions between the ethylene and jasmonate signal pathways for the induction of this defense response. Inoculation of wild-type Arabidopsis plants with the fungus Alternaria brassicicola led to a marked increase in production of jasmonic acid, and this response was not blocked in the ein2-1 mutant. Likewise, A. brassicicola infection caused stimulated emission of ethylene both in wild-type plants and in coi1-1 mutants. However, treatment of either ein2-1 or coi1-1 mutants with methyl jasmonate or ethylene did not induce PDF1.2, as it did in wild-type plants. We conclude from these experiments that both the ethylene and jasmonate signaling pathways need to be triggered concomitantly, and not sequentially, to activate PDF1.2 upon pathogen infection. In support of this idea, we observed a marked synergy between ethylene and methyl jasmonate for the induction of PDF1.2 in plants grown under sterile conditions. In contrast to the clear interdependence of the ethylene and jasmonate pathways for pathogen-induced activation of PDF1.2, functional ethylene and jasmonate signaling pathways are not required for growth responses induced by jasmonate and ethylene, respectively.

Radish leaves contain two homologous 5-kDa plant defensins which accumulate systemically upon infection by fungal pathogens (F.R.G. Terras et al., 1995, Plant Cell 7: 573-588). Here we report on the molecular cloning of the cDNAs encoding the two pathogen-inducible plant defensin isoforms from radish (Raphanus sativus L.) leaves. Tissue-print and whole-leaf electroblot immunostaining showed that the plant defensin peptides not only accumulate at high levels at or immediately around the infection sites in leaves inoculated with Alternaria brassicicola, but also accumulate in healthy tissue further away from the infection sites and in non-infected leaves from injected plants. Gel blot analysis of RNA confirmed that expression of plant defensin genes is systemically triggered upon fungal infection whereas radish PR-1 gene expression is only activated locally. In contrast to the radish PR-1 gene(s), expression of the radish plant defensin genes was not induced by external application of salicylic acid. Activation of the plant defensin genes, but not that of PR-1 genes, occurred upon treatment with methyl jasmonate, ethylene and paraquat.

The three-dimensional solution structure of Ace-AMP1, an antifungal protein extracted from onion seeds, was determined using 1H NMR spectroscopy and molecular modeling. This cationic protein contains 93 amino acid residues and four disulfide bridges. Its structure was determined from 1260 NOE-derived distance restraints and 173 dihedral restraints derived from NOEs and 3JCaHNH coupling constants. The global fold involves four helical segments connected by three loops and a C-terminal tail without regular secondary structures, except for a 3(10)-helix turn and a beta-turn. The most striking feature is the absence of any continuous cavity running through the whole molecule as found in recently determined structures of nonspecific transfer proteins extracted from wheat and maize seeds, although their global folds are very similar. Consistent with the absence of a cavity in the core of Ace-AMP1, it was found that this protein, in contrast to ns-LTPs, does not bind fluorescently labeled phospholipids in solution. On the other hand, Ace-AMP1 is able to interact with phospholipid membranes as shown by the release of carboxyfluorescein from the lumen of artificial liposomes and by the induction of alterations in fluorescence polarization of fluorescently labeled phospholipids embedded in artificial liposomes.

Hs-AFP1, an antifungal plant defensin from seed of the plant Heuchera sanguinea, was radioactively labeled using t-butoxycarbonyl-[35S]L-methionine N-hydroxysuccinimidyl ester, resulting in a 35S-labeled peptide with unaltered antifungal activity. [35S]Hs-AFP1 was used to assess binding on living hyphae of the fungus Neurospora crassa. Binding of [35S]Hs-AFP1 was found to be competitive, reversible, and saturable with an apparent Kd of 29 nM and a Bmax of 1.4 pmol/mg protein. [35S]Hs-AFP1 also bound specifically and reversibly to microsomal membranes derived from N. crassa hyphae with a Kd of 27 nM and a Bmax of 102 pmol/mg protein. The similarity in Kd value between binding sites on hyphae and microsomes indicates that Hs-AFP1 binding sites reside on the plasma membrane. Binding of [35S]Hs-AFP1 to both hyphae and microsomal membranes could be competed to some extent by four different structurally related plant defensins but not by various structurally unrelated antimicrobial peptides. In addition, an inactive single amino acid substitution variant of the antifungal plant defensin Rs-AFP2 from Raphanus sativus seed was also unable to displace [35S]Hs-AFP1 from its binding sites, whereas Rs-AFP2 itself was able to compete with [35S]Hs-AFP1.

Four closely related peptides were isolated from seed of Impatiens balsamina and were shown to be inhibitory to the growth of a range of fungi and bacteria, while not being cytotoxic to cultured human cells. The peptides, designated Ib-AMP1, Ib-AMP2, Ib-AMP3, and Ib-AMP4, are 20 amino acids long and are the smallest plant-derived antimicrobial peptides isolated to date. The Ib-AMPs (I. balsamina antimicrobial peptides) are highly basic and contain four cysteine residues which form two intramolecular disulfide bonds. Searches of protein data bases have failed to identify any proteins with significant homology to the peptides described here. Characterization of isolated cDNAs reveals that all four peptides are encoded within a single transcript. The predicted Ib-AMP precursor protein consists of a prepeptide followed by 6 mature peptide domains, each flanked by propeptide domains ranging from 16 to 35 amino acids in length. Such a primary structure with repeated alternating basic mature peptide domains and acidic propeptide domains has, to date, not been reported in plants.

Mutational analysis of Rs-AFP2, a radish antifungal peptide belonging to a family of peptides referred to as plant defensins, was performed using polymerase chain reaction-based site-directed mutagenesis and yeast as a system for heterologous expression. The strategy followed to select candidate amino acid residues for substitution was based on sequence comparison of Rs-AFP2 with other plant defensins exhibiting differential antifungal properties. Several mutations giving rise to peptide variants with reduced antifungal activity against Fusarium culmorum were identified. In parallel, an attempt was made to construct variants with enhanced antifungal activity by substituting single amino acids by arginine. Two arginine substitution variants were found to be more active than wild-type Rs-AFP2 in media with high ionic strength. Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to be important for antifungal activity, namely the region around the type VI beta-turn connecting beta-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting beta-strand 1 and the alpha-helix and contiguous residues on the alpha-helix and beta-strand 3, on the other hand. When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca2+ uptake by up to 20-fold. An arginine substitution variant with enhanced antifungal activity caused increased Ca2+ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca2+ uptake.

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.

Plant defensins are a class of cysteine-rich peptides of which several members have been shown to be potent inhibitors of fungal growth. A series of overlapping 15-mer peptides based on the amino acid sequence of the radish antifungal protein Rs-AFP2 have been synthesized. Peptides 6, 7, 8 and 9, comprising the region from cysteine 27 to cysteine 47 of Rs-AFP2 showed substantial antifungal activity against several fungal species (minimal inhibitory concentrations of 30-60 micrograms/mL), but no activity towards bacteria (except peptide 6 at 100 micrograms/mL). The active peptides were shown to be sensitive to the presence of cations in the medium and to the composition and pH of the medium. When present at a subinhibitory concentration (20 micrograms/mL), peptides 1, 7, 8 and 10 potentiated the activity of Rs-AFP2 from 2.3-fold to 2.8-fold. By mapping the characteristics of the active peptide on the structure of Rs-AFP2 as determined by nuclear magnetic resonance, the active region of the antifungal protein appears to involve beta-strands 2 and 3 in combination with the loop connecting those strands. A cyclized synthetic mimic of the loop, cysteine 36 to cysteine 45, was shown to have antifungal activity. Substitution of tyrosine 38 by alanine in the cyclic peptide substantially reduced the antifungal activity, indicating the importance of this residue for the activity of Rs-AFP2 as demonstrated carrier by mutational analysis.

The cDNAs encoding the seed antimicrobial peptides (AMPs) from Mirabilis jalapa (Mj-AMP2) and Amaranthus caudatus (Ac-AMP2) have previously been characterized and it was found that Mj-AMP2 and Ac-AMP2 are processed from a precursor preprotein and preproprotein, respectively [De Bolle et al., Plant Mol Biol 28:713-721 (1995) and 22:1187-1190 (1993), respectively]. In order to study the processing, sorting and biological activity of these antimicrobial peptides in transgenic tobacco, four different gene constructs were made: a Mj-AMP2 wild-type gene construct, a Mj-AMP2 mutant gene construct which was extended by a sequence encoding the barley lectin carboxyl-terminal propeptide, a known vacuolar targeting signal [Bednarek and Raikhel, Plant Cell 3: 1195-1206 (1991)]; an Ac-AMP2 wild-type gene construct; and finally, an Ac-AMP2 mutant gene construct which was truncated in order to delete the sequence encoding the genuine carboxyl-terminal propeptide. Processing and localization analysis indicated that an isoform of Ac-AMP2 with a cleaved-off carboxyl-terminal arginine was localized in the intercellular fluid fraction of plants expressing either wild-type or mutant gene constructs. Mj-AMP2 was recovered extracellularly in plants transformed with Mj-AMP2 wild-type gene construct, whereas an Mj-AMP2 isoform with a cleaved-off carboxyl-terminal arginine accumulated intracellularly in plants expressing the mutant precursor protein with the barley lectin propeptide. The in vitro antifungal activity of the AMPs purified from transgenic tobacco expressing any of the four different precursor proteins was similar to that of the authentic proteins. However, none of the transgenic plants showed enhanced resistance against infection with either Botrytis cinerea or Alternaria longipes.

Treatment of hyphae of Neurospora crassa with antifungal plant defensins, i.e. Rs-AFP2 and Dm-AMP1 isolated from radish and dahlia seed, respectively, induced a rapid K+ efflux, Ca2+ uptake, and alkalinization of the incubation medium. The Rs-AFP2-induced alkalinization of the incubation medium could be inhibited with G-protein inhibitors. alpha-Hordothionin, an antifungal thionin from barley seed, caused a sustained increased Ca2+ uptake at subinhibitory concentrations but only a transient increased uptake at inhibitory concentrations. alpha-Hordothionin also caused increased K+ efflux and alkalinization of the medium, but these fluxes occurred more rapidly compared to those caused by plant defensins. Furthermore, alpha-hordothionin caused permeabilization of fungal hyphae to the non-metabolite alpha-aminoisobutyric acid and, in addition, altered the electrical properties of artificial lipid bilayers, consistently leading to rupture of the lipid bilayers. The plant defensins did not form ion-permeable pores in artificial membranes and did not exhibit substantial hyphal membrane permeabilization activity. Our results are consistent with the notion that thionins inhibit fungal growth as a result of direct protein-membrane interactions, whereas plant defensins might act via a different, possibly receptor-mediated, mechanism.

We have previously developed a protocol for efficient gene transfer and regeneration of transgenic calli following cocultivation of apple (cv. Jonagold) explants with Agrobacterium tumefaciens (De Bondt et al. 1994, Plant Cell Reports 13: 587-593). Now we report on the optimization of postcultivation conditions for efficient and reproducible regeneration of transgenic shoots from the apple cultivar Jonagold. Factors which were found to be essential for efficient shoot regeneration were the use of gelrite as a gelling agent and the use of the cytokinin-mimicing thidiazuron in the selective postcultivation medium. Improved transformation efficiencies were obtained by combining the hormones thidiazuron and zeatin and by using leaf explants from in vitro grown shoots not older than 4 weeks after multiplication. Attempts to use phosphinothricin acetyl transferase as a selectable marker were not successful. Using selection on kanamycin under optimal postcultivation conditions, about 2% of the leaf explants developed transgenic shoots or shoot clusters. The presence and expression of the transferred genes was verified by β-glucuronidase assays and Southern analysis. The transformation procedure has also been succesfully applied to several other apple cultivars.

From seeds of Aesculus hippocastanum, Clitoria ternatea, Dahlia merckii and Heuchera sanguinea five antifungal proteins were isolated and shown to be homologous to plant defensins previously characterised from radish seeds and gamma-thionins from Poaceae seeds. Based on the spectrum of their antimicrobial activity and the morphological distortions they induce on fungi the peptides can be divided into two classes. The peptides did not inhibit any of three different alpha-amylases.

We have isolated and characterized two cDNA clones (designated MJ1 and MJ2) encoding the two Mirabilis jalapa antimicrobial peptides (Mj-AMP1 and Mj-AMP2, respectively), which were previously purified from seeds of this plant species (Cammue et al. (1992), J Biol Chem 267: 2228-2233). In both cases, the deduced amino acid sequences reveal the presence of a putative signal sequence preceding the mature peptide, indicating that the Mj-AMPs are expressed as preproteins. The Mj-AMP1- and Mj-AMP2-encoding genes are interrupted in their coding sequences by a single intron (380 bp and 900 bp for Mj-AMP1 and Mj-AMP2 genes, respectively). Southern blot analysis indicates that the Mj-AMP-encoding genes belong to a gene family of low complexity. Northern blot analysis suggests seed-specific expression of Mj-AMPs since transcripts of the expected size could only be detected in near-mature and in mature seeds of M. jalapa.

Complementary DNA clones representing two alleles of the self-incompatibility (S) locus of apple (Malus x domestica Borkh.) have been isolated and characterised. One of the alleles corresponds to a 29 kDa ribonuclease (S-RNase) that was purified from pistil tissue. On northern blots, both cDNAs hybridized to a transcript that was only present in pistils and not in the other plant tissues analysed. Corresponding genomic sequences, amplified by PCR, were found to contain a single intron of 138 bp and 1100 bp respectively. Comparison of both sequences shows that the cDNAs encode mature proteins containing 65% of identical residues. Eight invariable cysteine residues, conserved regions around two histidines thought to play a role in RNA catalysis, and a number of other distinct residues are conserved between the apple S-RNases and similar proteins in the family Solanaceae. As this is the first report of sequences of S-alleles from a species belonging to a family that is not related with the Solanaceae, the structural features of S-RNases deduced from a comparison of their sequences are discussed.

In response to a septic injury (pricking with a bacteria-soaked needle) larvae and adults of Drosophila produce considerable amounts of a 44-residue peptide containing 8 cysteines engaged in intramolecular disulfide bridges. The peptide is synthesized in the fat body, a functional homologue of the mammalian liver, and secreted into the blood of the insect. It exhibits potent antifungal activity but is inactive against bacteria. This novel inducible peptide, which we propose to name drosomycin, shows a significant homology with a family of 5-kDa cysteine-rich plant antifungal peptides recently isolated from seeds of Brassicaceae. This finding underlines that plants and insects can rely on similar molecules in their innate host defense.

Rs-AFP2 is a 51 amino acid cysteine-rich peptide isolated from radish (Raphanus sativus) seeds that exhibits potent inhibitory activity against filamentous fungi. A cDNA clone encoding the Rs-AFP2 preprotein was modified by recombinant DNA methods to allow expression in the yeast Saccharomyces cerevisiae. This peptide was expressed in yeast as a fusion protein carrying at its N-terminus the prepro-sequences derived from the precursor of the yeast pheromone mating factor alpha 1. These sequences allow secretion of the biologically active peptide in a correctly processed form. Deletion of the mating factor alpha 1 pro-peptide drastically reduced the expression level of the peptide.

The factors influencing transfer of an intron - containing β-glucuronidase gene to apple leaf explants were studied during early steps of an Agrobacterium tumefaciens-mediated transformation procedure. The gene transfer process was evaluated by counting the number of β-glucuronidase expressing leaf zones immediately after cocultivation, as well as by counting the number of β-glucuronidase expressing calli developing on the explants after 6 weeks of postcultivation in the presence of 50 mg/l kanamycin. Of three different tested disarmed A. tumefaciens strains, EHA101(pEHA101) was the most effective for apple transformation. Cocultivation of leaf explants with A. tumefaciens on a medium with a high cytokinin level was more conducive to gene transfer than cocultivation on media with high auxin concentrations. Precultivation of leaf explants, prior to cocultivation, slightly increased the number of β-glucuronidase expressing zones measured immediately after cocultivation, but it drastically decreased the number of transformed calli appearing on the explants 6 weeks after infection. Other factors examined were: Agrobacterium cell density during infection, bacterial growth phase, nature of the carbon source, explant age, and explant genotype.

On the basis of an extensive screening of seeds from various plant species, we have isolated and characterized several different antimicrobial peptides. They were all typified by having a broad antifungal activity spectrum, a relatively low molecular weight (3-14 kDa), a high cysteine content and a high isoelectric point (pI > 10). With respect to their amino acid sequence, these peptides can be classified into six structural classes. Synergistic enhancement (up to 73-fold) of antimicrobial activity was demonstrated in some combinations of peptides belonging to different classes. cDNA clones corresponding to different antifungal peptides were isolated and used to transform tobacco plants. Extracts of these transgenic plants showed higher (up to 16-fold) antifungal activity than untransformed control plants. Such antimicrobial peptides may find applications in molecular breeding of plants with increased disease resistance.

Although thionins and 2S albumins are generally considered as storage proteins, both classes of seed proteins are known to inhibit the growth of pathogenic fungi. We have now found that the wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) thionin concentration required for 50% inhibition of fungal growth is lowered 2- to 73-fold when combined with 2S albumins (at sub- or noninhibitory concentrations) from radish (Raphanus sativus L.) or oilseed rape (Brassica napus L.). Furthermore, the thionin antifungal activity is synergistically enhanced (2- to 33-fold) by either the small subunit or the large subunit of the radish 2S albumins. Three other 2S albumin-like proteins, the barley trypsin inhibitor and two barley Bowman-Birk-type trypsin inhibitor isoforms, also act synergistically with the thionins (2- to 55-fold). The synergistic activity of thionins combined with 2S albumins is restricted to filamentous fungi and to some Gram-positive bacteria, whereas Gram-negative bacteria, yeast, cultured human cells, and erythrocytes do not show an increased sensitivity to thionin/albumin combinations (relative to the sensitivity to the thionins alone). Scanning electron microscopy and measurement of K+ leakage from fungal hyphae revealed that 2S albumins have the same mode of action as thionins, namely the permeabilization of the hyphal plasmalemma. Moreover, 2S albumins and thionins act synergistically in their ability to permeabilize fungal membranes.

A cDNA clone encoding an antimicrobial chitin-binding protein from amaranth (Amaranthus caudatus L.) was isolated using a cDNA library constructed from near-mature seed poly(A)+ mRNA. The deduced amino acid sequence of the cDNA clone encodes a predicted polypeptide of 86 amino acids. This polypeptide has three distinct domains: an amino-terminal putative signal peptide (25 amino acids), a domain corresponding to the mature protein (30 amino acids), and a carboxyl-terminal propeptide (31 amino acids) containing a putative N-glycosylation site. The encoded protein differs from all known members of the family of chitin-binding proteins. Transcripts of the expected size (650 bp) are present in developing seeds but not in roots, leaves or stressed leaves.

Out of seeds of 4 Brassicaceae species, 7 antifungal proteins were isolated which are nearly identical to 2 previously characterized radish seed antifungal proteins. These basic proteins, multimers of a 5 kDa polypeptide, specifically inhibit fungal growth. One of the antifungal proteins has decreased antifungal activity and an increased antibacterial activity. In addition, the previously described antifungal activity of the radish seed 2S albumins was extended to the 2S albumins of the seeds of the 4 other Brassicaceae species. A 2S albumin-like trypsin-inhibitor from barley seeds was found to have much less activity against fungi.

A basic 9-kD protein was purified from seeds of radish (Raphanus sativus L.). The 43 amino-terminal amino acids show extensive sequence identity with nonspecific lipid transfer proteins from other plant species. The radish seed nonspecific lipid transfer protein-like protein inhibits the growth of several fungi in vitro.

Two novel classes of antifungal proteins were isolated from radish seeds. The first class consists of two homologous proteins (Rs-AFP1 and Rs-AFP2) that were purified to homogeneity. They are highly basic oligomeric proteins composed of small (5-kDa) polypeptides that are rich in cysteine. Both Rs-AFPs have a broad antifungal spectrum and are among the most potent antifungal proteins hitherto characterized. In comparison with many other plant antifungal proteins, the activity of the Rs-AFPs is less sensitive to the presence of cations. Moreover, their antibiotic activity shows a high degree of specificity to filamentous fungi. The amino-terminal regions of the Rs-AFPs show homology with the derived amino acid sequences of two pea genes specifically induced upon fungal attack, to gamma-thionins and to sorghum alpha-amylase inhibitors. The radish 2S storage albumins were identified as the second novel class of antifungal proteins. All isoforms inhibit growth of different plant pathogenic fungi and some bacteria. However, their antimicrobial activities are strongly antagonized by cations.

Two antimicrobial peptides (Ac-AMP1 and Ac-AMP2) were isolated from seeds of amaranth (Amaranthus caudatus), and their physicochemical and biological properties were characterized. On the basis of fast atom bombardment mass spectroscopy, Ac-AMP1 and Ac-AMP2 have monoisotopic molecular masses of 3025 and 3181, respectively. Both proteins have pI values above 10. The amino acid sequence of Ac-AMP1 (29 residues) is identical to that of Ac-AMP2 (30 residues), except that the latter has 1 additional residue at the carboxyl terminus. The sequences are highly homologous to the cysteine/glycine-rich domain occurring in many chitin-binding proteins. Both Ac-AMP1 and Ac-AMP2 bind to chitin in a reversible way. Ac-AMP1 and Ac-AMP2 inhibit the growth of different plant pathogenic fungi at much lower doses than other known antifungal chitin-binding proteins. In addition, they show some activity on Gram-positive bacteria. The antimicrobial effect of Ac-AMP1 and Ac-AMP2 is strongly antagonized by cations.

We have isolated from seeds of Mirabilis jalapa L. two antimicrobial peptides, designated Mj-AMP1 and Mj-AMP2, respectively. These peptides are highly basic and consist of 37 and 36 residues for Mj-AMP1 and Mj-AMP2, respectively. Both peptides contain three disulfide bridges and differ from one another only by 4 amino acids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the reduced and unreduced peptides suggests that the peptides associate into dimers in their native form. The Mj-AMPs exhibit a broad spectrum of antifungal activity since they are active against all 13 tested plant pathogenic fungi. Concentrations required for 50% inhibition of fungal growth vary from 6 to 300 micrograms/ml for Mj-AMP1 and from 0.5 to 20 micrograms/ml for Mj-AMP2. These peptides were also active on two tested Gram-positive bacteria but were apparently nontoxic for Gram-negative bacteria and cultured human cells. Although the Mj-AMPs show sequence similarity to mu-agatoxins, a class of insecticidal neurotoxic peptides isolated from the venom of spiders, they do not affect pulse transmission in insect nerves.

Hevein is a chitin-binding protein of 43 amino acids found in the lutoid body-enriched fraction of rubber tree latex. A hevein cDNA clone (HEV1) (Broekaert, W., Lee, H.-i., Kush, A., Nam, C.-H., and Raikhel, N. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7633-7637) encodes a putative signal sequence of 17 amino acids followed by a polypeptide of 187 amino acids. Interestingly, this polypeptide has two distinct domains: an amino-terminal domain of 43 amino acids, corresponding to mature hevein, and a carboxyl-terminal domain of 144 amino acids. To investigate the mechanisms involved in processing of the protein encoded by HEV1, three domain-specific antisera were raised against fusion proteins harboring the amino-terminal domain (N domain), carboxyl-terminal domain (C domain), and both domains (NC domain). Translocation experiments using an in vitro translation system show that the first 17-amino acid sequence encoded by the cDNA functions as a signal peptide. Immunoblot analysis of proteins extracted from lutoid bodies demonstrates that a 5-kDa protein comigrated with purified mature hevein and cross-reacted with N domain- and NC domain-specific antibodies. A 14-kDa protein was recognized by C domain- and NC domain-specific antibodies. A 20-kDa protein was cross-reactive with all three antibodies. Microsequencing data further suggest that the 5-kDa (amino-terminal domain) and 14-kDa (carboxyl-terminal domain) proteins are post-translational cleavage products of the 20-kDa polypeptide (both domains) which corresponds to the proprotein encoded by HEV1. In addition, it was found that the amino-terminal domain could provide chitin-binding properties to a fusion protein bearing it either amino terminally or carboxyl terminally.

A technique was developed for the detection of antifungal activity of proteins after discontinuous polyacrylamide gel electrophoresis under native conditions. The antifungal activity is detected as growth inhibition zones in a homogeneous fungal lawn, grown in an agar layer spread on top of the polyacrylamide gel. The position of proteins with antifungal activity can be determined on a diffusion blot prepared from the same gel. The technique is illustrated for three antifungal plant proteins, i.e. alpha-purothionin, Urtica dioica agglutinin, and tobacco chitinase.

Several chitin-binding proteins were isolated from the "bottom fraction" of Hevea brasiliensis (Müll.) Arg. latex. One of these chitin-binding proteins is hevein, a small monomeric protein which strongly resembles the lectin from stinging nettle (Urtica dioica L.). Like the latter, hevein showed strong antifungal activity against several fungi in vitro. The possible involvement of this protein in the defense against invasion by potentially pathogenic fungi is discussed.

Treatment of wheat (Triticum aestivum L.) seedlings with elicitors originating from either plant or fungal cell walls induces about a 2-fold increase of wheat germ agglutinin (WGA) in the roots. While the WGA content in roots of healthy plants normally decreases as a function of germination time, a transient accumulation of WGA could be observed in plants challenged with different fungi, including Rhizoctonia solani, Fusarium culmorum, Pythium ultimum and Neurospora crassa. Peak levels in challenged roots were 2 to 5 times as high as in control plants. Most of this induced WGA could be released from the roots by soaking them in a solution of the hapten N-acetylglucosamine. On the basis of the results obtained it is postulated that WGA may be involved in the defence of wheat against fungal attack.

A new plant lectin from elderberry (Sambucus nigra L.) bark, which was shown by immunochemical techniques to bind specifically to terminal Neu5Ac(alpha 2-6)Gal/GalNAc residues of glycoconjugates, was immobilized onto Sepharose 4B (SNA-Sepharose) and its carbohydrate binding properties was determined using a series of standard compounds. Oligosaccharides, glycopeptides, or glycoproteins containing terminal Neu5Ac(alpha 2-6)Gal/GalNAc sequences bound to SNA-Sepharose and were eluted with 50-100 mM lactose, whereas those with Neu5Ac(alpha 2-3)Gal/GalNAc failed to bind to this column. Furthermore, the SNA-Sepharose column was capable of resolving two oligosaccharides/glycopeptides based on the number of Neu5Ac(alpha 2-6)Gal units present in each molecule. Application of this technique to two glycoproteins, fetuin and orosomucoid, revealed the presence of microheterogeneity. It was also shown that esterification of the carboxyl group of Neu5Ac units, or branching at the O-3 of the subterminal GalNAc (probably also Gal) destroyed the binding ability of the molecule.

A lectin has been isolated from rhizomes of ground elder (Aegopodium podagraria) using a combination of affinity chromatography on erythrocyte membrane proteins immobilized on cross-linked agarose and hydroxyapatite, and ion-exchange chromatography. The molecular structure of the lectin was determined by gelfiltration, sucrose density-gradient centrifugation and gel electrophoresis under denaturing conditions. It has an unusually high Mr (about 480000) and is most probably an octamer composed of two distinct types of subunits with slightly different Mr (about 60000). Hapten inhibition assays indicated that the Aegopodium lectin is preferentially inhibited by N-acetylgalactosamine. Nevertheless, it does not agglutinate preferentially blood-group-A erythrocytes. The ground-elder lectin is a typical non-seed lectin, which occurs virtually exclusively in the underground rhizomes. In this organ it is an abundant protein as it represents up to 5% of the total protein content. The lectin content of the rhizome tissue varies strongly according to its particular location along the organ. In addition, the lectin content changes dramatically as a function of the seasons. The ground-elder lectin differs from all other plant lectins by its unusually high molecular weight. In addition, it is the first lectin to be isolated from a species of the family Apiaceae.

Rhizomes of stinging nettle contain a small-sized lectin that exhibits binding specificity toward chitin. This lectin inhibits growth of several phytopathogenic and saprophytic chitin-containing fungi in vitro. The antifungal action of the nettle lectin differs from the action of chitinases, which are a ubiquitous class of antifungal plant proteins. Moreover, the nettle lectin acts synergistically with chitinase in inhibiting fungal growth. The nettle lectin may be a promising candidate for possible applications in the genetic engineering of disease-resistant crops.