The multifunctional beta-oxidation enzyme is required for full symptom development by the biotrophic maize pathogen Ustilago maydis

Abstract

The transition from yeast-like to filamentous growth in the biotrophic fungal phytopathogen Ustilago maydis is a crucial event for pathogenesis. Previously, we showed that fatty acids induce filamentation in U. maydis and that the resulting hyphal cells resemble the infectious filaments observed in planta. To explore the potential metabolic role of lipids in the morphological transition and in pathogenic development in host tissue, we deleted the mfe2 gene encoding the multifunctional enzyme that catalyzes the second and third reactions in beta-oxidation of fatty acids in peroxisomes. The growth of the strains defective in mfe2 was attenuated on long-chain fatty acids and abolished on very-long-chain fatty acids. The mfe2 gene was not generally required for the production of filaments during mating in vitro, but loss of the gene blocked extensive proliferation of fungal filaments in planta. Consistent with this observation, mfe2 mutants exhibited significantly reduced virulence in that only 27% of infected seedlings produced tumors compared to 88% tumor production upon infection by wild-type strains. Similarly, a defect in virulence was observed in developing ears upon infection of mature maize plants. Specifically, the absence of the mfe2 gene delayed the development of teliospores within mature tumor tissue. Overall, these results indicate that the ability to utilize host lipids contributes to the pathogenic development of U. maydis.

FIG. 1.

Organization of the peroxisomal multifunctional enzymes (type 2). (A) Organization of the catalytic domains of U. maydis, S. cerevisiae, and human peroxisomal Mfe2 enzymes. (B) Sequence alignment of the conserved catalytic domains from the U. maydis Mfe2 protein with fungal (C. tropicalis [CANTR, P22414], S. cerevisiae [SACCE, CAA82079], N. crassa [NEUCR, CAA56355], and Y. lipolytica [YARLI, AAF82684]), and human (P51659) homologs. HD, (3R)-hydroxyacyl-CoA dehydrogenase domain; H2, 2-enoyl-CoA hydratase 2 domain.

FIG. 2.

RNA blot analysis of mfe2 transcript levels in the presence of fatty acids. (A) Total RNA was isolated from wild-type (a2b2) cells grown in minimal medium containing either glucose, fatty acid, or fatty acid and glucose. Total RNA was also isolated from the Δmfe2 a2b2 cells grown in minimal medium containing fatty acids. The RNA blot was hybridized with a probe for the mfe2 gene. (B) The RNA blot stained with 0.04% methylene blue to show total RNA loading.

FIG. 3.

Morphology and growth of mfe2 mutant strains on fatty acids differing in carbon chain length and saturation state. (A) Cellular morphology of the wild-type (a2b2) and mutant (Δmfe2 a2b2) strains in response to glucose, lipids (corn oil), LCFA (palmitic, oleic, and linoleic), VLCFA (erucic, arachidic, and arachidonic), SCFA (caproic), and MCFA (lauric and myristic). The cells were visualized by DIC optics (left) and by epifluorescence after staining cell walls with calcofluor (right). (B) The ability of the wild-type and mutant strains to grow on fatty acid-containing agar medium. The cells were spotted in decreasing concentration from 10⁶ to 10². All of the agar plates contain tergitol to facilitate the solubility of the fatty acids. (C) Total number of the wild-type (a2b2, black bars) and mutant (Δmfe2 a2b2, white bars) cells in culture supplemented with glucose, corn oil, and fatty acids as a sole carbon source. The bars represent the average number of cells from three independent experiments.

FIG. 4.

Intracellular lipids in U. maydis. (A) Cellular lipid accumulation in U. maydis wild-type and mfe2 mutant strains grown on glucose and various fatty acids. The wild-type and mfe2 mutant strains were grown in minimal medium supplemented with either glucose, myristic (C_14:0), oleic (C_18:1), or linoleic acid (C_18:2) as a sole carbon source. The internal lipids accumulated in lipid bodies were stained using the lipid-specific fluorescent dye Nile red and visualized using epifluorescence. The fungal cells produced large (arrowhead) to small (arrow) lipid bodies that varied in number depending on carbon source. Bar, 10 μm. (B) An abundance of lipid bodies produced in the mfe2 mutant strain grown on oleic acid as a sole carbon source. TEM observation of lipid bodies in wild-type cells (a2b2) and mfe2 mutant (Δmfe2 a2b2) cells grown on glucose and oleic acid. Cells were grown on glucose medium overnight and then transferred into oleic acid medium. After 18 h, the oleic acid-grown cells were fixed and processed for TEM. Bar, 500 nm. L, lipid body.

FIG. 5.

Mating filaments produced by compatible mfe2 mutant strains during mating. (A) Mating test of wild-type (a1b1 and a2b2) and mfe2 mutant strains. Each strain was spotted on charcoal-containing medium, and the compatible strains were mixed in the center of the plate to assess the mating reaction. The positive mating reaction is represented by the production of white filaments that are visible on the dark medium. (B) Dikaryotic filaments formed by a cross of the wild-type and mfe2 mutant strains on charcoal-containing medium after 48 h. Both wild-type and mutant strains produced unbranched mating and dikaryotic filaments with collapsed sections of hyphal cells. The images were captured using DIC optics (left panel) or epifluorescence (right panel) to visualize calcofluor-stained cell walls. Bar, 10 μm.

FIG. 6.

Hyphal morphology in planta. (A) Wild-type filaments growing within plant tissue 7 days after inoculation with compatible wild-type strains (a1b1 × a2b2). The filaments branched (arrowheads) and could penetrate epidermal cells through a stoma (arrow). The tip of the penetrating hypha is out of the focal plane. (B) Yeast-like cells of compatible mfe2 mutant strains on the epidermal surface of a maize leaf. Some of the cells were elongated and started to produce conjugation tubes (ct), possibly in response to a mating partner. The cells often clustered around stomata, and once a dikaryotic filament was formed, it sometimes penetrated through the stomata (arrow). (C) mfe2 filament growing within plant tissue. The mutant filaments were often observed on the epidermal surface or within plant tissue without branching, exhibiting typical straight mating-like hyphal morphology. Epidermal peels from maize leaves were examined using DIC (top panel) and epifluorescence (bottom panel) to visualize the calcofluor-stained cells. Bar, 10 μm.

FIG. 7.

Teliospore production of mfe2 mutants is compromised in mature tumors. (A) Tumors collected from infected mature maize plants 14 days postinoculation. The cross of compatible wild-type strains resulted in production of mature tumors in a 14-day period in contrast to the immature tumors produced by the cross of mfe2 mutant strains. (B) Subset of tumors collected from mature maize plants 14 days postinoculation. All mfe2 mutant tumors were white in appearance and contained sporogenic hyphae, indicating that fungal development was not yet complete. (C) The cross section of the tumors shown in panel A. The wild-type “black” tumors were filled with melanized teliospores. The mutant “white” tumors were filled mostly with sporogenic hyphae and immature sexual spores that were not yet melanized and therefore had not completed development. Bar, 10 μm.