Induction of lcc2 expression and activity by Agaricus bisporus provides defence against Trichoderma aggressivum toxic extracts.

Laccases are used by fungi for several functions including defence responses to stresses associated with attack by other fungi. Laccase activity changes and the induction of two laccase genes, lcc1 and lcc2, in Agaricus bisporus were measured in response to toxic extracts of medium in which Trichoderma aggressivum, the cause of green mould disease, was grown. A strain of A. bisporus that shows resistance to the extracts showed higher basal levels and greater enzymatic activity after extract exposure than did a sensitive strain. Furthermore, pre-incubation of T. aggressivum extract with laccases reduced toxicity. Faster induction and greater numbers of lcc2 transcripts in response to the extract were noted in the resistant strain than in the sensitive strain. The timing and increase in lcc2 transcript abundance mirrored changes in total laccase activity. No correlation between resistance and lcc1 transcription was apparent. Transcript abundance in transformants with a siRNA construct homologous to both genes varied widely. A strong negative correlation between transcript abundance and sensitivity of the transformant to toxic extract was observed in plate assays. These results indicated that laccase activity and in particular that encoded by lcc2 contributes to toxin metabolism and by extension green mould disease resistance.

Characterization of gibberellin-signalling elements during plum fruit ontogeny defines the essentiality of gibberellin in fruit development.

Fruit growth is a coordinated, complex interaction of cell division, differentiation and expansion. Gibberellin (GA) involvement in the reproductive events is an important aspect of GA effects. Perennial fruit-trees such as plum (Prunus salicina L.) have distinct features that are economically important and provide opportunities to dissect specific GA mechanisms. Currently, very little is known on the molecular mechanism(s) mediating GA effects on fruit development. Determination of bioactive GA content during plum fruit ontogeny revealed that GA1 and GA4 are critical for fruit growth and development. Further, characterization of several genes involved in GA-signalling showed that their transcriptional regulation are generally GA-dependent, confirming their involvement in GA-signalling. Based on these results, a model is presented elucidating how the potential association between GA and other hormones may contribute to fruit development. PslGID1 proteins structure, Y2H and BiFC assays indicated that plum GA-receptors can form a complex with AtDELLA-repressors in a GA-dependent manner. Moreover, phenotypical-, molecular- and GA-analyses of various Arabidopsis backgrounds ectopically expressing PslGID1 sequences provide evidence on their role as active GA-signalling components that mediate GA-responsiveness. Our findings support the critical contribution of GA alone or in association with other hormones in mediating plum fruit growth and development.

Regulation of three genes encoding cell-wall-degrading enzymes of Trichoderma aggressivum during interaction with Agaricus bisporus.

Members of the genus Trichoderma are very effective competitors of a variety of fungi. Cell-wall-degrading enzymes, including proteinases, glucanases, and chitinases, are commonly secreted as part of the competitive process. Trichoderma aggressivum is the causative agent of green mould disease of the button mushroom, Agaricus bisporus. The structures of 3 T. aggressivum genes, prb1 encoding a proteinase, ech42 encoding an endochitinase, and a ß-glucanase gene, were determined. Promoter elements in the prb1 and ech42 genes suggested that transcription is regulated by carbon and nitrogen levels and by stress. Both genes had mycoparasitism-related elements indicating potential roles for the protein products in competition. The promoter of the ß-glucanase gene contained CreA and AreA binding sites indicative of catabolite regulation but contained no mycoparasitism elements. Transcription of the 3 genes was measured in mixed cultures of T. aggressivum and A. bisporus. Two A. bisporus strains, U1, which is sensitive to green mould disease, and SB65, which shows some resistance, were used in co-cultivation tests to assess possible roles of the genes in disease production and severity. prb1 and ech42 were coordinately upregulated after 5 days, whereas ß-glucanase transcription was upregulated from day 0 with both Agaricus strains. Upregulation was much less pronounced in mixed cultures of T. aggressivum with the resistant strain, SB65, than with the sensitive strain, U1. These observations suggested that the proteins encoded by these genes have roles in both nutrition and in severity of green mould disease.

Powerful bacterial killing by buckwheat honeys is concentration-dependent, involves complete DNA degradation and requires hydrogen peroxide.

Exposure of bacterial cells to honey inhibits their growth and may cause cell death. Our previous studies showed a cause-effect relationship between hydroxyl radical generated from honey hydrogen peroxide and growth arrest. Here we explored the role of hydroxyl radicals as inducers of bacterial cells death. The bactericidal effect of ·OH on antibiotic-resistant clinical isolates of MRSA and VRE and standard bacterial strains of E. coli and B. subtiles was examined using a broth microdilution assay supplemented with 3'-(p-aminophenyl) fluorescein (APF) as the ·OH trap, followed by colony enumeration. Bactericidal activities of eight honeys (six varieties of buckwheat, blueberry and manuka honeys) were analyzed. The MBC/MIC ratio ≤4 and the killing curves indicated that honeys exhibited powerful, concentration-dependent bactericidal effect. The extent of killing depended on the ratio of honey concentration to bacterial load, indicating that honey dose was critical for its bactericidal efficacy. The killing rate and potency varied between honeys and ranged from over a 6-log(10) to 4-log(10) CFU/ml reduction of viable cells, equivalent to complete bacterial eradication. The maximal killing was associated with the extensive degradation of bacterial DNA. Honey concentration at which DNA degradation occurred correlated with cell death observed in the concentration-dependent cell-kill on agar plates. There was no quantitative relationship between the ·OH generation by honey and bactericidal effect. At the MBC, where there was no surviving cells and no DNA was visible on agarose gels, the ·OH levels were on average 2-3x lower than at Minimum Inhibitory Concentration (MICs) (p < 0.0001). Pre-treatment of honey with catalase, abolished the bactericidal effect. This raised possibilities that either the abrupt killing prevented accumulation of ·OH (dead cells did not generate ·OH) or that DNA degradation and killing is the actual footprint of ·OH action. In conclusion, honeys of buckwheat origin exhibited powerful, concentration-dependent bactericidal effect. The killing and DNA degradation showed a cause-effect relationship. Hydrogen peroxide was an active part of honey killing mechanism.

Unraveling a mechanism of honey antibacterial action: polyphenol/H2O2-induced oxidative effect on bacterial cell growth and on DNA degradation.

Several compounds with antibacterial activities were identified in honey however, a mechanism by which they lead to bacterial growth inhibition and bacterial death remains still unknown. We recently found that honeys possess DNA degrading activity mediated by honey hydrogen peroxide and an unknown honey component(s). Here we provide evidence that active honeys (MIC90 of 6.25-12.5% v/v) possessed significantly higher levels of phenolics (p<0.02) of higher radical scavenging activities (p<0.005) than honeys of average activity. Removal of H2O2 by catalase eliminated bacteriostatic activities caused by both phenolics and H2O2 suggesting that the growth inhibition resulted from the coupling chemistry between these compounds. Both phenolics and H2O2 were involved in DNA degradation by honeys. Treatment of plasmid DNA with H2O2 alone did not affect the DNA integrity but H2O2 removal from honey by catalase prevented DNA degradation. Polyphenols extracted from honeys degraded plasmid DNA in the presence of H2O2 and Cu(II) in the Fenton-type reaction. The extent of DNA degradation was inversely related to the polyphenol concentration in this system as well as in honeys. At low content, honey polyphenols exerted pro-oxidant activity damaging to DNA. In conclusion, honey phenolics with pro-oxidant activities were necessary intermediates that conferred oxidative action of H2O2. Phenolic/H2O2-induced oxidative stress constituted the mechanism of honey bacteriostatic and DNA damaging activities.

Re-examining the role of hydrogen peroxide in bacteriostatic and bactericidal activities of honey.

The aim of this study was to critically analyze the effects of hydrogen peroxide on growth and survival of bacterial cells in order to prove or disprove its purported role as a main component responsible for the antibacterial activity of honey. Using the sensitive peroxide/peroxidase assay, broth microdilution assay and DNA degradation assays, the quantitative relationships between the content of H(2)O(2) and honey's antibacterial activity was established(.) The results showed that: (A) the average H(2)O(2) content in honey was over 900-fold lower than that observed in disinfectants that kills bacteria on contact. (B) A supplementation of bacterial cultures with H(2)O(2) inhibited E. coli and B. subtilis growth in a concentration-dependent manner, with minimal inhibitory concentrations (MIC(90)) values of 1.25 mM/10(7) cfu/ml and 2.5 mM/10(7) cfu/ml for E. coli and B. subtilis, respectively. In contrast, the MIC(90) of honey against E. coli correlated with honey H(2)O(2) content of 2.5 mM, and growth inhibition of B. subtilis by honey did not correlate with honey H(2)O(2) levels at all. (C) A supplementation of bacterial cultures with H(2)O(2) caused a concentration-dependent degradation of bacterial DNA, with the minimum DNA degrading concentration occurring at 2.5 mM H(2)O(2). DNA degradation by honey occurred at lower than ≤2.5 mM concentration of honey H(2)O(2) suggested an enhancing effect of other honey components. (D) Honeys with low H(2)O(2) content were unable to cleave DNA but the addition of H(2)O(2) restored this activity. The DNase-like activity was heat-resistant but catalase-sensitive indicating that H(2)O(2) participated in the oxidative DNA damage. We concluded that the honey H(2)O(2) was involved in oxidative damage causing bacterial growth inhibition and DNA degradation, but these effects were modulated by other honey components.