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

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.

Keywords: Fusarium graminearum; HPLC/MS; malonichrome; sexual development; siderophores; virulence.

Figures

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.

Figure 2

LC-HR-MS spectra of malonichrome. (A) Isotope pattern of the [M+H]+ ion cluster of malonichrome (ferri-form). (B) LC-HR-MS spectrum of malonichrome (desferri-form), showing [M+H]+, [M+NH4]+ and [M+Na]+ ions. (C) LC-HR-MS/MS spectrum of malonichrome (desferri-form) at a collision energy of 35 eV.

Figure 3

Siderophores produced by wild-type and mutant strains. Siderophores produced by WT Gz3639 and mutant strains (see Table 1) cultivated in MM and subsequently starved in iron-free MM as described in Materials and Methods. Samples were taken 3, 6, 11, and 15 days after shift to iron-starvation medium. Supernatants were analyzed for siderophores by HPLC as described in the Materials and Methods. X-axis: upper row, days after shift to iron-free medium, lower row, strains investigated. Y-axis: relative concentrations (arbitrary units), R (TAFC, TAFB, DAFA): acetyl. Note, metabolites are missing in all strains with deletions of corresponding genes, as expected. Malonichrome is present in strains with a WT NPS1 gene and absent from strains deleted for this gene.

Figure 4

Sensitivity to iron depletion of WT and mutant strains. (A)nps2nps6, nps1nps6, and nps1nps2nps6 strains show further increased sensitivity to 2DP, compared to the nps6 strain. Average colony diameters of 4 day-old cultures on MM with/without 2DP are shown. Strains used are as in (B). Error bars indicate 95% confidence intervals. A statistically significant reduction in growth on MM with 100 μM 2DP was observed for the nps2nps6 and nps1nps6 strains, compared to the nps6 strain. The nps1nps2nps6 strains show further reduced growth on MM with 100 μM 2DP, compared to the nps2nps6 or nps1nps6 strains. No significant difference in growth was observed on MM among all strains. (B) Application of iron enhances growth of nps6 and nps2nps6, but not nps1nps6 and nps1nps2nps6 strains. Average colony diameters of 5 day-old cultures of WT (strain Gz3639), nps6 (strain Fgnps6G-1), nps2nps6 (Gznps2-6-1nps6-17), nps1nps6 (Gznps1-5-1nps6-3-1), and nps1nps2nps6 (Gznps2-6-1nps1-5-1nps6-3-2) strains on MM with/without iron are shown. Error bars indicate 95% confidence intervals. A statistically significant reduction in growth on MM was observed for all mutant strains compared to WT. Application of 200 or 400 μM ferric citrate restored WT growth to the nps6 and nps2nps6 strains, indicating that the growth defect of these strains on MM is due to iron deficiency. In contrast, application of iron did not enhance growth of the nps1nps6 strains. Application of 400 μM ferric citrate reduced growth of the nps1nps2nps6 strains, implying that they are hypersensitive to iron overload.

Figure 5

Nps2 is required for sexual reproduction of self-compatible strains but Nps6 plays a role when Nps2 is absent. (A)nps2nps6 and nps1nps6 strains form perithecia as well as WT strains do. Top and bottom, left, average number of perithecia per plate is shown for WT, nps2 (2), nps2nps6 (26), and nps1nps6 (16) selfs. Ten replicates were set up for each strain. Error bars indicate 95% confidence intervals. No significant difference was observed in the number of perithecia per plate between WT, and any of the mutant strains. Top, right, nps2nps6 strains fail to develop asci and ascospores. WT, nps2, and nps2nps6 selfs are shown. As observed in nps2 strains, nps2nps6 strains fail to develop asci and ascospores. Bottom, right, nps1nps6 strains are as fertile as WT. Average number of asci per perithecium is shown for WT and nps1nps6 selfs. Thirty perithecia were opened for each self. Error bars indicate 95% confidence intervals. No significant difference was observed in the number of asci per perithecium between WT and nps1nps6 selfs. (B)nps2 strains which develop immature ascus-like structures (II, arrows), however no ascus-like structures were found in perithecia developed in the nps2nps6 selfs (III, arrows), indicating that sexual development of the nps2nps6 strain is more severely affected than that of the nps2 strain. nps1nps6 strains are as fertile as WT (compare I and IV). Magnification is × 200, inserts × 500. (C)nps1nps2nps6 strains are more affected in sexual development than nps2nps6 strains. Application of iron restores ability to develop asci and ascospores to nps2, nps2nps6, and nps1nps2nps6 strains. Five replicates were set up for each self and for each condition. A statistically significant reduction in the number of perithecia per plate was observed in the nps1nps2nps6 selfs, compared to WT, nps2, and nps2nps6 selfs. Exogenous application of iron enhanced perithecium development of the nps1nps2nps6 selfs, indicating that reduced perithecium development is due to iron deficiency. When iron is applied, a reduction in perithecium development was observed in WT selfs. (D) Twenty perithecia were opened for each self and for each condition. Error bars indicate 95% confidence intervals. Although no statistically significant difference was observed in the number of asci per perithecium between the nps2nps6 and nps1nps2nps6 selfs, the number tended to be smaller in the nps1nps2nps6 compared to the nps2nps6 selfs. A statistically significant difference in the number of asci per perithecium was observed between the nps2 and nps2nps6 strains, further demonstrating that nps2nps6 strains are more affected in sexual development.

Figure 6

Virulence of WT, nps6, nps1nps6, nps2nps6, and nps1nps2nps6 strains to wheat. (A) Wheat spikes challenged by WT (strain Gz3639), nps6 (strain Fgnps6G-1), nps1nps6 (strain Gznps1-5-1nps6-3-1), nps2nps6 (strain Gznps2-6-1nps6-17), and nps1nps2nps6 (strain Gznps2-6-1nps1-5-1nps6-3-2), strains, 16 days after inoculation. Spikes inoculated with the nps2nps6 strain (second set from right) are less damaged than those inoculated with the nps6 strain (second set from left). Spikes inoculated with the nps1nps6 strain (middle set) were as severely damaged as those inoculated with nps6 strains. Spikes inoculated with the nps1nps2nps6 strain (right set) show further reduced virulence compared to the nps2nps6 strain and were almost intact. (B) Kernels taken from the spikes shown in (A). Relative positions of kernels correspond to those of the spikes shown in (A). Generally, spikes challenged by nps2nps6 strains contained more healthy, green kernels (note tops of spikes) than did those challenged by nps6 strains, demonstrating that the nps2nps6 strain is more reduced in virulence for systemic infection of wheat spikes, than the nps6 strain is. Kernels in the spikes infected by the nps1nps6 strains were as severely damaged as those in the spikes infected by the nps6 strain. Most of the kernels in the spikes infected by the nps1nps2nps6 strains appeared undamaged. Overall, these data demonstrate that the nps1nps6 strain is as virulent to wheat spikes as the nps6 strain is, and that the nps1nps2nps6 strain is further attenuated in virulence to wheat spikes, compared to the nps2nps6 strain.

Figure 7

Average time required for completion of local infection of wheat spikelets by WT, nps6 (6), nps1nps6 (16), and nps2nps6 (26) strains. Note that the nps1nps2nps6 strain was not included, as most of the spikelets inoculated with the nps1nps2nps6 strains were asymptomatic until the end of infection assays (i.e., 16 days after inoculation). No significant difference was observed in the time for local infection between the nps6 and nps1nps6 strains, indicating that the nps1nps6 strain is as virulent for local infection of wheat spikes as the nps6 strain is. Generally, it takes longer for the nps2nps6 strain to complete local infection, compared to the nps6 strain.

Figure 8

Structures of the malonichrome and ferricrocin siderophores and their associated biosynthetic Nps1 and Nps2 proteins, respectively. (A) Structural features of the desferri-forms of malonichrome and ferricrocin. Building blocks are highlighted in different colors. (B) Domain structures of the corresponding Nps1 and Nps2 proteins. Adenylation domains are colored according to the entities introduced into the respective molecules (A). Although the domain structures of F. graminearum (Fg) Nps1 and A. nidulans (An) SidC proteins are very similar, different products are generated (malonichrome and ferricrocin, respectively). In contrast, the domain structures of F. graminearum and C. heterostrophus (Ch) Nps2 proteins are different, but the same metabolite is formed (ferricrocin). White numbered boxes symbolize condensation domains. Arrows indicate orthologous domains (Bushley et al., 2008).