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Robust Cre recombinase activity in the biotrophic smut fungus Ustilago maydis enables efficient conditional null mutants in planta.

ABSTRACT: Site-specific recombinases have been used in higher eukaryotes, especially in animals, for a broad range of applications, including chromosomal translocations, large deletions, site-specific integration, and tissue-specific as well as conditional knock-outs. The application of site-specific recombination has also been demonstrated in simple eukaryotes like fungi and protozoa. However, its use in fungal research, especially in phytopathogenic fungi, has often been limited to "recycle" the marker genes used in transformation experiments. We show that Cre recombinase can be used for conditional gene deletions in the phytopathogenic fungus Ustilago maydis. Conditional gene knock-outs can be generated via the transcriptional control of the recombinase by U. maydis promoters specifically activated during the biotrophic phase of fungal growth, enabling gene deletions at defined developmental stages inside the plant tissue. Also, we show that a tamoxifen-activated Cre-recombinase allows the tight control necessary for the induced deletion of essential genes by the addition of tamoxifen. These tools will be helpful to address the function of genes under both axenic and in planta conditions for the U. maydis-maize pathosystem and should pave the way for similar approaches in other plant pathosystems.

SUBMITTER: de la Torre A

PROVIDER: S-EPMC8733456 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Plant surface cues prime Ustilago maydis for biotrophic development.

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Septation of infectious hyphae is critical for appressoria formation and virulence in the smut fungus Ustilago maydis.

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Characterization of ApB73, a virulence factor important for colonization of Zea mays by the smut Ustilago maydis.

Project description:The biotrophic fungus Ustilago maydis, the causal agent of corn smut disease, uses numerous small secreted effector proteins to suppress plant defence responses and reshape the host metabolism. However, the role of specific effectors remains poorly understood. Here, we describe the identification of ApB73 (Apathogenic in B73), an as yet uncharacterized protein essential for the successful colonization of maize by U. maydis. We show that apB73 is transcriptionally induced during the biotrophic stages of the fungal life cycle. The deletion of the apB73 gene results in cultivar-specific loss of gall formation in the host. The ApB73 protein is conserved among closely related smut fungi. However, using virulence assays, we show that only the orthologue of the maize-infecting head smut Sporisorium reilianum can complement the mutant phenotype of U. maydis. Although microscopy shows that ApB73 is secreted into the biotrophic interface, it seems to remain associated with fungal cell wall components or the fungal plasma membrane. Taken together, the results show that ApB73 is a conserved and important virulence factor of U. maydis that localizes to the interface between the pathogen and its host Zea mays.

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

Project description: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.

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Opsin 1 and Opsin 2 of the Corn Smut Fungus Ustilago maydis Are Green Light-Driven Proton Pumps.

Project description:In fungi, green light is absorbed by rhodopsins, opsin proteins carrying a retinal molecule as chromophore. The basidiomycete Ustilago maydis, a fungal pathogen that infects corn plants, encodes three putative photoactive opsins, called ops1 (UMAG_02629), ops2 (UMAG_00371), and ops3 (UMAG_04125). UmOps1 and UmOps2 are expressed during the whole life cycle, in axenic cultures as well as in planta, whereas UmOps3 was recently shown to be absent in axenic cultures but highly expressed during plant infection. Here we show that expression of UmOps1 and UmOps2 is induced by blue light under control of white collar 1 (Wco1). UmOps1 is mainly localized in the plasma membrane, both when expressed in HEK cells and U. maydis sporidia. In contrast, UmOps2 was mostly found intracellularly in the membranes of vacuoles. Patch-clamp studies demonstrated that both rhodopsins are green light-driven outward rectifying proton pumps. UmOps1 revealed an extraordinary pH dependency with increased activity in more acidic environment. Also, UmOps1 showed a pronounced, concentration-dependent enhancement of pump current caused by weak organic acids (WOAs), especially by acetic acid and indole-3-acetic acid (IAA). In contrast, UmOps2 showed the typical behavior of light-driven, outwardly directed proton pumps, whereas UmOps3 did not exhibit any electrogenity. With this work, insights were gained into the localization and molecular function of two U. maydis rhodopsins, paving the way for further studies on the biological role of these rhodopsins in the life cycle of U. maydis.

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