Pisatin demethylation by fungal pathogens and nonpathogens of pea: Association with pisatin tolerance and virulence

Previous studies have indicated that detoxification of their hosts’ phytoalexins is a tolerance mechanism for some true fungi, but not the fungus-like Oomycota, and may be involved in determining the virulence of a pathogen. In the present study, the associations between demethylation of the pea phytoalexin pisatin, tolerance to pisatin, and viru­lence on pea were examined for 50 fungal isolates which represent 17 species of pathogens and nonpathogens of pea. All isolates of Pythium coloratum and P. irregulare failed to metabolize and were sensitive to pisatin, consistent with previous observations that members of the Oomycota generally lack the ability to metabolize and are sensitive to their hosts’ phytoalexins. Among true fungi tested, the ability to demethylate pisatin was common, regardless of whether the particular isolate was pathogenic on pea or not. However, when the rate of pisatin demethylation was compared to virulence, all but one of the moderate to highly virulent isolates rapidly demethylated pisatin. In addition, the more rapidly demethylating isolates were generally more tolerant of pisatin. These results suggest that a specialized en­zyme system for quickly detoxifying pisatin might be present in most pea pathogens. In previous studies a specific cy­tochrome P450 enzyme for demethylating pisatin was identified in the pea pathogen Nectria haematococca mating pop­ulation VI, and genes (PDA genes) encoding that enzyme have been cloned from this fungus. When DNA specific for these genes was used to probe genomic DNA from other fungi that demethylate pisatin, significant hybridization was detected with only one fungus, the pea pathogen Fusarium oxysporum f. sp. pisi. If the other pea pathogens possess a specific cytochrome P450 system for detoxification of pisatin, the genes encoding these enzymes apparently share lim­ited nucleotide similarity with N. haematococca PDA genes

Pisatin demethylation by fungal pathogens and nonpathogens of pea: Association with pisatin tolerance and virulence

Previous studies have indicated that detoxification of their hosts’ phytoalexins is a tolerance mechanism for some true fungi, but not the fungus-like Oomycota, and may be involved in determining the virulence of a pathogen. In the present study, the associations between demethylation of the pea phytoalexin pisatin, tolerance to pisatin, and viru­lence on pea were examined for 50 fungal isolates which represent 17 species of pathogens and nonpathogens of pea. All isolates of Pythium coloratum and P. irregulare failed to metabolize and were sensitive to pisatin, consistent with previous observations that members of the Oomycota generally lack the ability to metabolize and are sensitive to their hosts’ phytoalexins. Among true fungi tested, the ability to demethylate pisatin was common, regardless of whether the particular isolate was pathogenic on pea or not. However, when the rate of pisatin demethylation was compared to virulence, all but one of the moderate to highly virulent isolates rapidly demethylated pisatin. In addition, the more rapidly demethylating isolates were generally more tolerant of pisatin. These results suggest that a specialized en­zyme system for quickly detoxifying pisatin might be present in most pea pathogens. In previous studies a specific cy­tochrome P450 enzyme for demethylating pisatin was identified in the pea pathogen Nectria haematococca mating pop­ulation VI, and genes (PDA genes) encoding that enzyme have been cloned from this fungus. When DNA specific for these genes was used to probe genomic DNA from other fungi that demethylate pisatin, significant hybridization was detected with only one fungus, the pea pathogen Fusarium oxysporum f. sp. pisi. If the other pea pathogens possess a specific cytochrome P450 system for detoxification of pisatin, the genes encoding these enzymes apparently share lim­ited nucleotide similarity with N. haematococca PDA genes

Fermi-LAT Constraints on the Pulsar Wind Nebula Nature of HESS J1857+026

Since its launch, the Fermi satellite has firmly identified 5 pulsar wind nebulae plus a large number of candidates, all powered by young and energetic pulsars. HESS J1857+026 is a spatially extended gamma-ray source detected by H.E.S.S. and classified as a possible pulsar wind nebula candidate powered by PSR J1856+0245. Aims. We search for -ray pulsations from PSR J1856+0245 and explore the characteristics of its associated pulsar wind nebula. Methods. Using a rotational ephemeris obtained from the Lovell telescope at Jodrell Bank Observatory at 1.5 GHz, we phase.fold 36 months of gamma-ray data acquired by the Large Area Telescope (LAT) aboard Fermi. We also perform a complete gamma-ray spectral and morphological analysis. Results. No pulsation was detected from PSR J1856+0245. However, significant emission is detected at a position coincident with the TeV source HESS J1857+026. The gamma-ray spectrum is well described by a simple power law with a spectral index of Gamma = 1.53 +/- 0.11(sub stat) +/- 0.55(sub syst) and an energy flux of G(0.1 C100 GeV) = (2.71 +/- 0.52(sub stat) +/- 1.51(sub syst) X 10(exp -11) ergs/ sq cm/s. This implies a gamma.ray efficiency of approx 5 %, assuming a distance of 9 kpc, the gamma-ray luminosity of L(sub gamma) (sub PWN) (0.1 C100 GeV) = (2.5 +/- 0.5(sub stat) +/- 1.5(sub syst)) X 10(exp 35)(d/(9kpc))(exp 2) ergs/s and E-dot = 4.6 X 10(exp 36) erg /s, in the range expected for pulsar wind nebulae. Detailed multi-wavelength modeling provides new constraints on its pulsar wind nebula nature

Simultaneous Determination of Various Isothiocyanates by RP-LC Following Precolumn Derivatization with Mercaptoethanol

Numerous isothiocyanates (ITCs) are poorly soluble in water which causes their precipitation in aqueous mobile phases used in reversed phase liquid chromatography (RP-LC), thus impacting the accuracy of the quantification. By comparing the amounts of ITCs injected and released from the column, losses could be estimated at 5–32% depending on polarities and concentrations. Results could be dramatically improved in terms of separation and quantification using RP-LC with a mercaptoethanol precolumn derivatization aimed at avoiding ITCs precipitation. The cancer chemoprotective allyl-ITC and sulforaphane were found in cabbage extracts at 1.2 and 2.7 μg g−1 fresh weight, respectively

Cloning and characterization of the PDA6-1 gene encoding a fungal cytochrome P-450 which detoxifies the phytoalexin pisatin from garden pea

The ability to detoxify pisatin, a phytoalexin produced by garden pea (Pisum sativum), is controlled by a family of PDA (pisatin demethylating ability) genes in the phytopathogenic fungus Nectria haematococa, MP (mating population) VI. Six known PDA genes each encode characteristic levels of inducible enzyme activity and are associated with different degrees of virulence on pea. To elucidate the phenotypic differences associated with these genes, we have cloned and characterized the PDA6-1 gene which encodes a pisatin-detoxifying enzyme and we compare it to another PDA gene, PDAT9. Pisatin demethylation was measured in PDA6-1 transformants of Aspergillus nidulans and shown to be regulated by glucose. The deduced amino acid (aa) sequence of PDA6-1 was 90% identical to that of the cytochrome P-450 encoded by PDAT9, but lacked nine aa at the C terminus, which has been postulated to be a site involved in substrate binding. A 35-bp sequence present upstream of a third PDA gene, PDA1, which appears to be important for induction of PDA1 by pisatin, was conserved in PDAT9, but not in PDA6-1. We conclude that PDA6-1, which does not appear to contribute to the virulence of N. haematococa on pea, differs significantly from PDAT9, which is associated with high virulence