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Individual and combined roles of malonichrome, ferricrocin, and TAFC siderophores in Fusarium graminearum pathogenic and sexual development.

Oide S, Berthiller F, Wiesenberger G, Adam G, Turgeon BG - Front Microbiol (2015)

Bottom Line: Deletion of the NPS6 gene, required for extracellular siderophore biosynthesis, does not affect sexual reproduction but results in sensitivity to iron starvation and oxidative stress and leads to reduced virulence to the host.Using comparative biochemical analysis of wild-type and mutant strains, we show that NPS1, a third gene associated with siderophore biosynthesis, is responsible for biosynthesis of a second extracellular siderophore, malonichrome. nps1 mutants fail to produce this metabolite.Thus, combinatorial mutants lacking key iron-associated genes uncovered malonichrome function.

View Article: PubMed Central - PubMed

Affiliation: Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University Ithaca, NY, USA ; The Research Institute of Innovative Technology for the Earth (RITE) Kizugawa-Shi, Japan.

ABSTRACT
Intra- and extracellular iron-chelating siderophores produced by fungal non-ribosomal peptide synthetases have been shown to be involved in reproductive and pathogenic developmental processes and in iron and oxidative stress management. Here we report individual and combined contributions of three of these metabolites to developmental success of the destructive cereal pathogen Fusarium graminearum. In previous work, we determined that deletion of the NPS2 gene, responsible for intracellular siderophore biosynthesis, results in inability to produce sexual spores when mutants of this homothallic ascomycete are selfed. Deletion of the NPS6 gene, required for extracellular siderophore biosynthesis, does not affect sexual reproduction but results in sensitivity to iron starvation and oxidative stress and leads to reduced virulence to the host. Building on this, we report that double mutants lacking both NPS2 and NPS6 are augmented in all collective phenotypes of single deletion strains (i.e., abnormal sexual and pathogenic development, hypersensitivity to oxidative and iron-depletion stress), which suggests overlap of function. Using comparative biochemical analysis of wild-type and mutant strains, we show that NPS1, a third gene associated with siderophore biosynthesis, is responsible for biosynthesis of a second extracellular siderophore, malonichrome. nps1 mutants fail to produce this metabolite. Phenotypic characterization reveals that, although single nps1 mutants are like wild-type with respect to sexual development, hypersensitivity to ROS and iron-depletion stress, and virulence to the host, triple nps1nps2nps6 deletion strains, lacking all three siderophores, are even more impaired in these attributes than double nps2nps6 strains. Thus, combinatorial mutants lacking key iron-associated genes uncovered malonichrome function. The intimate connection between presence/absence of siderophores and resistance/sensitivity to ROS is central to sexual and pathogenic development.

No MeSH data available.


Related in: MedlinePlus

LC-UV chromatogram of wild-type F. graminearum. The fungus was cultivated in liquid MM without iron for 6 days. FeCl3 was added before measurement of the supernatant at 435 nm. Siderophores present are DAFA, malonichrome, TAFB, and TAFC. * denotes a small interfering peak, also visible in pure solvent.
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Figure 1: LC-UV chromatogram of wild-type F. graminearum. The fungus was cultivated in liquid MM without iron for 6 days. FeCl3 was added before measurement of the supernatant at 435 nm. Siderophores present are DAFA, malonichrome, TAFB, and TAFC. * denotes a small interfering peak, also visible in pure solvent.

Mentions: A big advantage of iron-chelating compounds is their UV absorbance at 435 nm, which renders analytical detection straightforward. A LC-UV chromatogram of the culture supernatant of WT F. graminearum, grown under iron starvation for 6 days, is provided as Figure 1. Concurrent MS analyses of the peak at 8.00 min identified compounds with the mass and sum formula corresponding to malonichrome (after iron supplementation) and desferri-malonichrome (in the absence of iron) (Figures 2A,B). The enhanced resolution scan of the compound with the mass of malonichrome shows clearly that this compound is able to bind iron. The signal with m/z 885 originates from binding of 54Fe (making up 6% in naturally occurring isotope mixtures) whereas the main peak with m/z 887 is derived from 56Fe (92%) and the peak with m/z 888 from 57Fe (2%). LC-HR-MS measurements yielded the highest intensity at m/z 887.2244 for the [M+H]+ ion of the ferri-form. This confirms the sum formula of C31H44FeN9O18 with a deviation from the theoretical mass of +1.4 ppm (Figure 2A). For the desferri-form m/z values of 834.3105 (Δ+0.9 ppm), 851.3375 (Δ+0.2 ppm) and 856.2925 (Δ +0.7 ppm) have been found, which match the protonated ion, the ammonium adduct and the sodium adduct of C31H47N9O18, respectively (Figure 2B). Also the collision induced dissociation pattern in LC-MS/MS experiments (Figure 2C) is consistent with the structure proposed by Emery (1980). Cleavage of carbon dioxide from the three malonic acid ester (MA) groups, cleavage of the respective MA groups as well as cleavage of one or both AHO (including MA) groups from the molecule can be explained from the MS/MS spectrum. We therefore conclude that the compound identified in F. graminearum is malonichrome. As evident from Figure 3, malonichrome is missing in samples of all mutants lacking NPS1 (nps1, nps1nps2, nps1nps6 and the triple mutant nps1nps2nps6), demonstrating, formally, that the NPS1 gene is required for malonichrome biosynthesis.


Individual and combined roles of malonichrome, ferricrocin, and TAFC siderophores in Fusarium graminearum pathogenic and sexual development.

Oide S, Berthiller F, Wiesenberger G, Adam G, Turgeon BG - Front Microbiol (2015)

LC-UV chromatogram of wild-type F. graminearum. The fungus was cultivated in liquid MM without iron for 6 days. FeCl3 was added before measurement of the supernatant at 435 nm. Siderophores present are DAFA, malonichrome, TAFB, and TAFC. * denotes a small interfering peak, also visible in pure solvent.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4290682&req=5

Figure 1: LC-UV chromatogram of wild-type F. graminearum. The fungus was cultivated in liquid MM without iron for 6 days. FeCl3 was added before measurement of the supernatant at 435 nm. Siderophores present are DAFA, malonichrome, TAFB, and TAFC. * denotes a small interfering peak, also visible in pure solvent.
Mentions: A big advantage of iron-chelating compounds is their UV absorbance at 435 nm, which renders analytical detection straightforward. A LC-UV chromatogram of the culture supernatant of WT F. graminearum, grown under iron starvation for 6 days, is provided as Figure 1. Concurrent MS analyses of the peak at 8.00 min identified compounds with the mass and sum formula corresponding to malonichrome (after iron supplementation) and desferri-malonichrome (in the absence of iron) (Figures 2A,B). The enhanced resolution scan of the compound with the mass of malonichrome shows clearly that this compound is able to bind iron. The signal with m/z 885 originates from binding of 54Fe (making up 6% in naturally occurring isotope mixtures) whereas the main peak with m/z 887 is derived from 56Fe (92%) and the peak with m/z 888 from 57Fe (2%). LC-HR-MS measurements yielded the highest intensity at m/z 887.2244 for the [M+H]+ ion of the ferri-form. This confirms the sum formula of C31H44FeN9O18 with a deviation from the theoretical mass of +1.4 ppm (Figure 2A). For the desferri-form m/z values of 834.3105 (Δ+0.9 ppm), 851.3375 (Δ+0.2 ppm) and 856.2925 (Δ +0.7 ppm) have been found, which match the protonated ion, the ammonium adduct and the sodium adduct of C31H47N9O18, respectively (Figure 2B). Also the collision induced dissociation pattern in LC-MS/MS experiments (Figure 2C) is consistent with the structure proposed by Emery (1980). Cleavage of carbon dioxide from the three malonic acid ester (MA) groups, cleavage of the respective MA groups as well as cleavage of one or both AHO (including MA) groups from the molecule can be explained from the MS/MS spectrum. We therefore conclude that the compound identified in F. graminearum is malonichrome. As evident from Figure 3, malonichrome is missing in samples of all mutants lacking NPS1 (nps1, nps1nps2, nps1nps6 and the triple mutant nps1nps2nps6), demonstrating, formally, that the NPS1 gene is required for malonichrome biosynthesis.

Bottom Line: Deletion of the NPS6 gene, required for extracellular siderophore biosynthesis, does not affect sexual reproduction but results in sensitivity to iron starvation and oxidative stress and leads to reduced virulence to the host.Using comparative biochemical analysis of wild-type and mutant strains, we show that NPS1, a third gene associated with siderophore biosynthesis, is responsible for biosynthesis of a second extracellular siderophore, malonichrome. nps1 mutants fail to produce this metabolite.Thus, combinatorial mutants lacking key iron-associated genes uncovered malonichrome function.

View Article: PubMed Central - PubMed

Affiliation: Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University Ithaca, NY, USA ; The Research Institute of Innovative Technology for the Earth (RITE) Kizugawa-Shi, Japan.

ABSTRACT
Intra- and extracellular iron-chelating siderophores produced by fungal non-ribosomal peptide synthetases have been shown to be involved in reproductive and pathogenic developmental processes and in iron and oxidative stress management. Here we report individual and combined contributions of three of these metabolites to developmental success of the destructive cereal pathogen Fusarium graminearum. In previous work, we determined that deletion of the NPS2 gene, responsible for intracellular siderophore biosynthesis, results in inability to produce sexual spores when mutants of this homothallic ascomycete are selfed. Deletion of the NPS6 gene, required for extracellular siderophore biosynthesis, does not affect sexual reproduction but results in sensitivity to iron starvation and oxidative stress and leads to reduced virulence to the host. Building on this, we report that double mutants lacking both NPS2 and NPS6 are augmented in all collective phenotypes of single deletion strains (i.e., abnormal sexual and pathogenic development, hypersensitivity to oxidative and iron-depletion stress), which suggests overlap of function. Using comparative biochemical analysis of wild-type and mutant strains, we show that NPS1, a third gene associated with siderophore biosynthesis, is responsible for biosynthesis of a second extracellular siderophore, malonichrome. nps1 mutants fail to produce this metabolite. Phenotypic characterization reveals that, although single nps1 mutants are like wild-type with respect to sexual development, hypersensitivity to ROS and iron-depletion stress, and virulence to the host, triple nps1nps2nps6 deletion strains, lacking all three siderophores, are even more impaired in these attributes than double nps2nps6 strains. Thus, combinatorial mutants lacking key iron-associated genes uncovered malonichrome function. The intimate connection between presence/absence of siderophores and resistance/sensitivity to ROS is central to sexual and pathogenic development.

No MeSH data available.


Related in: MedlinePlus