Ly, vs. 40 for samples inoculated using the wild-type or abmdh mutants. Following host penetration, the fungus has to make necrotic factors to progress inside infected tissues. Brassicicolin A, a fungal metabolite thought of as becoming a mannitol derivative, represents a potent necrotic toxin developed by A. brassicicola (Pedras et al., 2009). We hypothesized that the weak virulence in mutants lacking mannitol might also be explained by the absence of brassicicolin A synthesis. To test this hypothesis, ethyl acetate extracts of culture filtrates from submerged cultures of A. brassicicola wild-type and abmpd-abmdh strains were analysed by HPLC-UV-MS. The resulting total ion chromatograms (TIC) of each extracts revealed a major metabolite at Rt = 39 min (Figures 12A,B) exhibiting the quasimolecular ion [M-H]- (m/z 683) of brassicicolin A (C32 H48 N2 O14 ), whereas typical MS/MS fragments (1 V) were recorded at m/z, 565 (lossFrontiers in Plant Science | Plant-Microbe InteractionMay 2013 | Volume four | Post 131 |Calmes et al.Function of mannitol metabolism in fungal pathogenicityFIGURE eight | Mannitol metabolism for the duration of plant colonization. (A) Important soluble carbohydrate concentrations (expressed in g/mg DF) measured by HPLC in the course of the infection kinetics in the A. brassicicola wild-type strain on Brassica oleracea. Undetected sugars are represented by the symbol ? 3 independent experiments were done. (B) Progression of symptoms on a B. oleracea leaf inoculated with the Abra43 strain and microscopic observations of your infection structures at two dpi. Inoculated plant tissue fragments had been collected at 2 dpi and stained with solophenyl flavine for fluorescence microscopy observations. Appressoria-like structures are indicated by white arrows. (C) QuantitativeRT-PCR benefits for the expression of AbMpd (white bars) and AbMdh (dark gray bars) during the infection kinetics of A. brassicicola wild-type strain on B. oleracea. For each gene, expression induction is represented as a ratio of its relative expression at two, 4, and six dpi (studied gene transcript abundance/actin transcript abundance) in every inoculated sample to its relative expression in free-living fungal manage cultures. The experiment was performed twice on biologically independent samples with three technical replicates. Error bars indicate standard deviations and asterisks indicate a relative expression considerably distinctive from 1 (Student test, P 0.01).of two acetyl units) and 473 (loss of one -hydroxyisovaleryl unit with each other with 1 -isocyanoisovaleryl unit) (information not shown).Methyl 5-bromo-2-formylbenzoate Chemscene The corresponding compound, which seems to accumulate inside the same amounts in both strains, was purified via preparative TLC from the wild-type extract and analyzed (1 H NMR, COSY and HMQC) inside a capillary NMR probe (500 MHz).4-(Methoxycarbonyl)nicotinic acid Price The resulting information (data not shown) have been also in complete agreement with formermeasurements obtained by Gloer et al.PMID:24238102 (1988) for this mixture of stereoisomers and confirmed that the isolated compound was brassicicolin A. We concluded that brassicicolin A was present in organic extracts in the culture broths of each the wild-type strain plus the abmpd-abmdh mutant and that the attenuated virulence of mannitol-deficient mutants could not be linked to the loss of brassicicolin A production.frontiersin.orgMay 2013 | Volume 4 | Post 131 |Calmes et al.Part of mannitol metabolism in fungal pathogenicityFIGURE 9 | Developmental phase-specific expression of AbMpd and AbMdh. All panels are micros.
