Maydis Strains Research Articles

A New Method to Obtain Infective Ustilago maydis Binucleate Conidia for Corn Smut Production

The fungus Ustilago maydis produces galls or tumors on corn ears called corn smut or huitlacoche. Used for human consumption in several countries for its nutritional and sensory traits, huitlacoche is considered a delicacy in Mexican cuisine and has a significant economic value. Hybrid U. maydis strains are regularly used for the large-scale production of huitlacoche; however, depending on the genetic characteristics of the parent strains, the pathogenicity and infection rate of hybrid fungi are often suboptimal due to compatibility issues between different strains. Using double-loaded organisms is common in agriculture to improve product characteristics, performance, and shelf-life. A methodology to obtain unicellular U. maydis strains with a double genetic load (n + n) capable of producing galls on corn ears without mating (hybridization) is reported herein. This methodology resulted in 206 U. maydis isolates. Screening showed that 147 corn plants (>70%) underwent infection and gall production. Of the 147 gall-producing U. maydis strains, those with the highest field performance were selected. Three strains, Um-UAEMor-78 (yielding 21.65 ton/ha), Um-UAEMor-120 (22.31 ton/ha), and Um-UAEMor-187 (22.99 ton/ha), showed higher yields than the control strain, CP-436(a1b1) × CP-437(a2b2) (17.80 ton/ha). A specific methodology to obtain unicellular U. maydis strains with a double genetic load capable of infecting baby corn ears and forming galls is described for the first time, providing a novel alternative for producing huitlacoche and helping to improve the yields and morphological traits of galls.

Novel Secreted Effectors Conserved Among Smut Fungi Contribute to the Virulence of Ustilago maydis.

Fungal pathogens deploy a set of molecules (proteins, specialized metabolites, and sRNAs), so-called effectors, to aid the infection process. In comparison to other plant pathogens, smut fungi have small genomes and secretomes of 20 Mb and around 500 proteins, respectively. Previous comparative genomic studies have shown that many secreted effector proteins without known domains, i.e., novel, are conserved only in the Ustilaginaceae family. By analyzing the secretomes of 11 species within Ustilaginaceae, we identified 53 core homologous groups commonly present in this lineage. By collecting existing mutants and generating additional ones, we gathered 44 Ustilago maydis strains lacking single core effectors as well as 9 strains containing multiple deletions of core effector gene families. Pathogenicity assays revealed that 20 of these 53 mutant strains were affected in virulence. Among the 33 mutants that had no obvious phenotypic changes, 13 carried additional, sequence-divergent, structurally similar paralogs. We report a virulence contribution of seven previously uncharacterized single core effectors and of one effector family. Our results help to prioritize effectors for understanding U. maydis virulence and provide genetic resources for further characterization. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Enhanced oxidative stress resistance in Ustilago maydis and its implications on the virulence

The phytopathogenic fungus Ustilago maydis causes corn smut by suppressing host plant defenses, including the oxidative burst response. While many studies have investigated how U. maydis responds to oxidative stress during infection, the consequences of heightened resistance to oxidative stress on virulence remain understudied. This study aimed to identify the effects on virulence in U. maydis strains exhibiting enhanced resistance to hydrogen peroxide (H2O2).To achieve this, we exposed U. maydis SG200 to 20 escalating H2O2 shocks, resulting in an adapted strain resistant to concentrations as high as 60 mM of H2O2, a lethal dose for the initial strain. Genetic analysis of the adapted strain revealed five nucleotide substitutions, two minor copy number variants, and a large amplification event on chromosome nine (1–149 kb) encompassing the sole catalase gene. Overexpressing catalase increased resistance to H2O2; however, this resistance was lower than that observed in the adapted strain. Additionally, virulence was reduced in both strains with enhanced H2O2 resistance.In summary, enhanced H2O2 resistance, achieved through either continuous exposure to the oxidative agent or through catalase overexpression, decreased virulence. This suggests that the response to the oxidative stress burst in U. maydis is optimal and that increasing the resistance to H2O2 does not translate into increased virulence. These findings illuminate the intricate relationship between oxidative stress resistance and virulence in U. maydis, offering insights into its infection mechanisms.

Anti-fungal activity of a novel succinate dehydrogenase inhibitor pydiflumetofen against Bipolaris maydis

Southern corn leaf blight (SCLB) is a disease caused by Bipolaris maydis. Pydiflumetofen is a novel succinate dehydrogenase inhibitor (SDHI) fungicide. However, its inhibitory activity against B. maydis has not been investigated in depth. In this study, mycelial growth rate and spore germination methods were used to establish a baseline for the sensitivity of 102 B. maydis strains collected from five regions of China to pydiflumetofen. The results showed that pydiflumetofen exhibited strong inhibitory activity against B. maydis. The EC50 values ranged from 0.0292 to 0.5207 μg/mL and from 0.0115 to 0.4883 μg/mL, with the average EC50 values of 0.2050 ± 0.1779 μg/mL and 0.1858 ± 0.0994 μg/mL for inhibition of mycelial growth and spore germination, respectively. After treatment with pydiflumetofen, the apical branching of mycelia increased significantly, and morphological changes such as twisting, deformation, rupture, and shriveling of mycelia and spores were observed. In addition, it significantly increased cell membrane permeability and decreased succinate dehydrogenase (SDH) activity and adenosine triphosphate (ATP) content. Moreover, the SDH gene expressions were changed, in which MbSdhB, MbSdhC and MbSdhD expressions were significantly upregulated, except for MbSdhA expression, which was significantly downregulated. In detached leaf experiments, pydiflumetofen showed strong protective and curative effects against B. maydis, superior to the same concentration of prochloraz and pyraclostrobin, and its protective effect was better than its curative effect. The field trials showed that the control efficacy of pydiflumetofen at 225 g/hm2 was >70% and its control efficacy at 300 g/hm2 reached 81.77%, which was significantly higher than that of pyraclostrobin and epoxiconazole at the same dosage. After 1:1 mixing of pydiflumetofen with the above control agents, the control efficacies of these mixtures at the same dosage increased compared to the use of a single agent. All the above results demonstrated that pydiflumetofen exhibits excellent inhibitory activity against B. maydis and good control of SCLB, suggesting that it could be used as an alternative fungicide for the control of SCLB.

Aggressive strains of the late wilt fungus of corn exist in Israel in mixed populations and can specialize in disrupting growth or plant health

Maize late wilt disease caused by the fungus Magnaporthiopsis maydis significantly damages crops in Israel and in other countries. Resistant maize cultivars are the preferred method for disease restraining. However, the pathogen populations of Spain and Egypt have varying aggressiveness, and virulent strains can overcome host resistance. In 2001 and from 2016 to -2019, 17 M. maydis strains were isolated from infected maize fields in Israel. The isolates’ effects on seed germination, plant development, and disease symptoms severity were evaluated. The isolates from Israel display a diverse degree of aggressiveness that is not linked to their geographic distribution. The virulent strains are found in mixed populations, whereas less virulent M. maydis isolates exist. Aggressive strains harmed the development of plants and ears and caused severe wilting and death. In contrast, plants inoculated with less virulent strains exhibited only mild dehydration signs, and crop yield was similar to that of the non-infected control. Interestingly, different host cultivars can evoke specific virulence of M. maydis strains. Moreover, some pathogen strains significantly repress plant development, while the impact of other strains was evidenced by wilting symptoms. The current research further increases our understanding of the pathogen and our ability to control it.

Techno-Economic Analysis as a Driver for Optimisation of Cellobiose Lipid Fermentation and Purification.

Cellobiose lipids (CL) are glycolipids synthesized by Ustilaginaceae species with potential application as detergents or in cosmetics. This study identified process optimisation potential for CL fermentation based on process modelling and techno-economic analysis. Using a stoichiometric equation based on laboratory data, we calculated the maximum possible CL yield YP/S of 0.45 gCL·gglucose −1 at the biomass yield of 0.10 gBiomass·gglucose −1 with an Ustilago maydis strain. Due to substrate inhibition that may occur at high glucose concentrations, a fed-batch process to increase biomass and CL concentrations was considered in our model. Simulation of different process scenarios showed that the choice of aeration units with high oxygen transfer rates and adaptation of power input to oxygen uptake can significantly decrease electricity consumption. We further assessed scenarios with different fermentation media and CL purification methods, suggesting additional process optimisation potential. Here the omission of vitamins from the fermentation medium proved to be a possible mean to enhance process economy, without compromising CL productivity.

Ustilago maydis Metabolic Characterization and Growth Quantification with a Genome-Scale Metabolic Model.

Ustilago maydis is an important plant pathogen that causes corn smut disease and serves as an effective biotechnological production host. The lack of a comprehensive metabolic overview hinders a full understanding of the organism’s environmental adaptation and a full use of its metabolic potential. Here, we report the first genome-scale metabolic model (GSMM) of Ustilago maydis (iUma22) for the simulation of metabolic activities. iUma22 was reconstructed from sequencing and annotation using PathwayTools, and the biomass equation was derived from literature values and from the codon composition. The final model contains over 25% annotated genes (6909) in the sequenced genome. Substrate utilization was corrected by BIOLOG phenotype arrays, and exponential batch cultivations were used to test growth predictions. The growth data revealed a decrease in glucose uptake rate with rising glucose concentration. A pangenome of four different U. maydis strains highlighted missing metabolic pathways in iUma22. The new model allows for studies of metabolic adaptations to different environmental niches as well as for biotechnological applications.

An arabinose-induced enhancement of asexual reproduction and concomitant changes in metabolic state in the filamentous fungus Bipolaris maydis.

l-Arabinose, a major constituent pentose of plant cell-wall polysaccharides, has been suggested to be a less preferred carbon source for fungi but to be a potential signalling molecule that can cause distinct genome-wide transcriptional changes in fungal cells. Here, we explore the possibility that this unique pentose influences the morphological characteristics of the phytopathogenic fungus Bipolaris maydis strain HITO7711. When grown on plate media under different sugar conditions, the mycelial dry weight of cultures on l-arabinose was as low as that with no sugar, suggesting that l-arabinose does not substantially contribute to vegetative growth. However, the intensity of conidiation on l-arabinose was comparable to or even higher than that on d-glucose and on d-xylose, in contrast to the poor conidiation under the no-sugar condition. To explore the physiological basis of the passive growth and active conidiation on l-arabinose, we next investigated cellular responses of the fungus to these sugar conditions. Transcriptional analysis of genes related to carbohydrate metabolism showed that l-arabinose stimulates carbohydrate utilization through the hexose monophosphate shunt (HMP shunt), a catabolic pathway parallel to glycolysis and which participates in the generation of the reducing agent NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate). Then, the HMP shunt was impaired by disrupting the related gene BmZwf1, which encodes glucose-6-phosphate dehydrogenase in this fungus. The resulting mutants on l-arabinose showed remarkably decreased conidiation, but a conversely increased mycelial dry weight compared with the wild-type. Our study demonstrates that l-arabinose acts to enhance resource allocation to asexual reproduction in B. maydis HITO7711 at the cost of vegetative growth, and suggests that this is mediated by the concomitant stimulation of the HMP shunt.

Effectors with Different Gears: Divergence of Ustilago maydis Effector Genes Is Associated with Their Temporal Expression Pattern during Plant Infection.

Plant pathogens secrete a variety of effector proteins that enable host colonization but are also typical pathogen detection targets for the host immune system. Consequently, effector genes encounter high selection pressures, which typically makes them fast evolving. The corn smut pathogen Ustilago maydis has an effector gene repertoire with a dynamic expression across the different disease stages. We determined the amino acid divergence of U. maydis effector candidates with Sporisorium reilianum orthologs, a close relative of U. maydis. Intriguingly, there are two distinct groups of effector candidates, ones with a respective conserved and diverged protein evolution. Conservatively evolving effector genes especially have their peak expression during the (pre-)penetration stages of the disease cycle. In contrast, expression of divergently evolving effector genes generally peaks during fungal proliferation within the host. To test if this interspecific effector diversity corresponds to intraspecific diversity, we sampled and sequenced a diverse collection of U. maydis strains from the most important maize breeding and production regions in China. Effector candidates with a diverged interspecific evolution had more intraspecific amino acid variation than candidates with a conserved evolution. In conclusion, we highlight diversity in evolution within the U. maydis effector repertoire with dynamically and conservatively evolving members.

Understanding Ustilago maydis Infection of Multiple Maize Organs.

Ustilago maydis is a smut fungus that infects all aerial maize organs, namely, seedling leaves, tassels, and ears. In all organs, tumors are formed by inducing hypertrophy and hyperplasia in actively dividing cells; however, the vast differences in cell types and developmental stages for different parts of the plant requires that U. maydis have both general and organ-specific strategies for infecting maize. In this review, we summarize how the maize–U. maydis interaction can be studied using mutant U. maydis strains to better understand how individual effectors contribute to this interaction, either through general or specific expression in a cell type, tissue, or organ. We also examine how male sterile maize mutants that do not support tumor formation can be used to explore key features of the maize anthers that are required for successful infection. Finally, we discuss key unanswered questions about the maize–U. maydis interaction and how new technologies can potentially be used to answer them.

A detached leaf assay to rapidly screen for resistance of maize to Bipolaris maydis, the causal agent of southern corn leaf blight

Southern corn leaf blight (SCLB), caused by the fungus Bipolaris maydis, is a disease that significantly affects maize productivity across the globe. A detached leaf assay (DLA) was developed to rapidly assess maize resistance to SCLB. Several experiments were conducted to: (i) identify a highly virulent B. maydis isolate; and to determine the most appropriate (ii) phytohormone to maintain viability of maize leaf tissue, (iii) leaf age for the assay, and (iv) inoculum concentration. Once optimized, the DLA was compared with screenhouse and field experiments. Use of DLA required a maximum of 28 days for resistance assessment, in contrast to screenhouse and field tests at a minimum of 33 and 72 days, respectively. DLA positively correlated with screenhouse (r = 0.48, P = 0.08) and field experiments (r = 0.68, P = 0.008). Assessments of diverse B. maydis strains and host genotypes indicated that the DLA could be used to detect both highly virulent SCLB strains and highly resistant maize genotypes. Here we report that DLA is a rapid, reliable technique to screen maize resistance to SCLB. Use of this tool in maize breeding programs can speed up the process of identification of sources of resistance to multiple variants of SCLB.

<i>Ustilago maydis </i>yeast stage found on imported sweet corn

During the inspection of imported sweet corn (Zea mays), a specimen with dry rot symptoms was detected by Ministry for Primary Industries quarantine officers. A sample was sent to the MPI Plant Health and Environment Laboratory for diagnostics, and initial examination found a layer of yeast cells on the surface of the symptomatic tissue. The fungus was cultured and identified based on DNA sequences as Ustilago maydis. While the corn-smut pathogen U. maydis is well known to cause tumour like galls on corn kernels, it is a less recognised fact that this fungus can also grow as a yeast. To determine if the yeast stage could have been associated with the dry-rot symptoms observed on the specimen, healthy material was inoculated with the isolated U. maydis strain. No symptoms developed on inoculated material, indicating that the yeast cells were likely multiplying on the surface of the cut corn ear as saprobes. To our knowledge, this is the first report of U. maydis yeast stage on corn ears and indicates a previously unconsidered pathway for the organism. For the yeast stage to cause disease, mating with a compatible mating type on the surface of a living host plant would be required.

Biomass-Derived Production of Itaconic Acid as a Building Block in Specialty Polymers.

Biomass, the only source of renewable organic carbon on Earth, offers an efficient substrate for bio-based organic acid production as an alternative to the leading petrochemical industry based on non-renewable resources. Itaconic acid (IA) is one of the most important organic acids that can be obtained from lignocellulose biomass. IA, a 5-C dicarboxylic acid, is a promising platform chemical with extensive applications; therefore, it is included in the top 12 building block chemicals by the US Department of Energy. Biotechnologically, IA production can take place through fermentation with fungi like Aspergillus terreus and Ustilago maydis strains or with metabolically engineered bacteria like Escherichia coli and Corynebacterium glutamicum. Bio-based IA represents a feasible substitute for petrochemically produced acrylic acid, paints, varnishes, biodegradable polymers, and other different organic compounds. IA and its derivatives, due to their trifunctional structure, support the synthesis of a wide range of innovative polymers through crosslinking, with applications in special hydrogels for water decontamination, targeted drug delivery (especially in cancer treatment), smart nanohydrogels in food applications, coatings, and elastomers. The present review summarizes the latest research regarding major IA production pathways, metabolic engineering procedures, and the synthesis and applications of novel polymeric materials.

Online evaluation of the metabolic activity of Ustilago maydis on (poly)galacturonic acid

BackgroundPectin is a rather complex and highly branched polysaccharide strengthening the plant cell wall. Thus, many different pectinases are required for an efficient microbial conversion of biomass waste streams with a high pectin content like citrus peel, apple pomace or sugar beet pulp. The screening and optimization of strains growing on pectic substrates requires both, quantification of the residual substrate and an accurate determination of the enzymatic activity. Galacturonic acid, the main sugar unit of pectin, is an uncommon substrate for microbial fermentations. Thus, growth and enzyme production of the applied strain has to be characterized in detail to understand the microbial system. An essential step to reach this goal is the development of online monitoring tools.ResultsIn this study, a method for the online determination of residual substrate was developed for the growth of the plant pathogenic fungus Ustilago maydis on pectic substrates such as galacturonic acid. To this end, an U. maydis strain was used that expressed a heterologous exo-polygalacturonase for growth on polygalacturonic acid. The growth behavior on galacturonic acid was analyzed by online measurement of the respiration activity. A method for the online prediction of the residual galacturonic acid concentration during the cultivation, based on the overall oxygen consumption, was developed and verified by offline sampling. This sensitive method was extended towards polygalacturonic acid, which is challenging to quantify via offline measurements. Finally, the enzymatic activity in the culture supernatant was calculated and the enzyme stability during the course of the cultivation was confirmed.ConclusionThe introduced method can reliably predict the residual (poly)galacturonic acid concentration based on the overall oxygen consumption. Based on this method, the enzymatic activity of the culture broth of an U. maydis strain expressing a heterologous exo-polygalacturonase could be calculated. It was demonstrated that the method is especially advantageous for determination of low enzymatic activities. In future, it will be applied to U. maydis strains in which the number of produced hydrolytic enzymes is increased for more efficient degradation.

The complete genomic sequence of a novel botybirnavirus isolated from a phytopathogenic Bipolaris maydis.

Bipolaris maydis is the causal agent of corn southern leaf blight. Here, we report a novel double-stranded RNA (dsRNA) mycovirus designated Bipolaris maydis botybirnavirus 1 (BmBRV1) from B. maydis strain JZ11 in Jingzhou, Hubei province of China. BmBRV1 has a genome consisting of two dsRNAs (dsRNA1 and dsRNA2) with a size of 6435 and 5987bp, respectively, each of which contains a single open reading frame (ORF). The two polyproteins encoded by dsRNA1 and dsRNA2 share the highest amino acid identities of 81.8 and 75.3%, respectively, with the RdRp and coat protein of Sclerotinia sclerotiorum botybirnavirus 1 (SsBRV1), a tentative species of the genus Botybirnavirus. Phylogenetic analysis based on the amino acid sequences of RdRp indicated that BmBRV1 belongs to a distinct species of the newly proposed family Botybirnaviridae.

Combined analysis of genome-wide expression profiling of maize (Zea mays L.) leaves infected with Ustilago maydis.

Although many gene expression profiling studies of maize leaves infected with Ustilago maydis have been published, heterogeneity of the results, caused by various data processing methods and pathogenic strains in different data sets, remains strong. Hence, we conducted a combined analysis of six genome-wide expression data sets of maize leaves infected with five different U. maydis strains by using the same pre-processing and quality control procedures. Six data sets were regrouped into five groups according to pathogenic strain used. Subsequently, each group of data set was processed by Multi-array Average for pre-processing and by pair-wise Pearson correlation for quality control. The differentially expressed genes were calculated by a standard linear mixed-effect model and then validated by various sensitivity analysis and multiple evidences. Finally, 44 unique differentially expressed genes were identified. Pathway enrichment analysis indicated that these genes related to response to fungus, oxidation-reduction, transferase activity, and several carbohydrate metabolic and catabolic processes. In addition, the hub genes within protein-protein interaction networks showed high relevance with the basic pathogenesis. We report a highly credible differentially expressed list, and the genes with multiple validations may denote a common signature of U. maydis in maize, which provides a new window for disease-resistant protection of maize plants.

The 'PhenoBox', a flexible, automated, open-source plant phenotyping solution.

Summary There is a need for flexible and affordable plant phenotyping solutions for basic research and plant breeding.We demonstrate our open source plant imaging and processing solution (‘PhenoBox’/‘PhenoPipe’) and provide construction plans, source code and documentation to rebuild the system. Use of the PhenoBox is exemplified by studying infection of the model grass Brachypodium distachyon by the head smut fungus Ustilago bromivora, comparing phenotypic responses of maize to infection with a solopathogenic Ustilago maydis (corn smut) strain and effector deletion strains, and studying salt stress response in Nicotiana benthamiana.In U. bromivora‐infected grass, phenotypic differences between infected and uninfected plants were detectable weeks before qualitative head smut symptoms. Based on this, we could predict the infection outcome for individual plants with high accuracy. Using a PhenoPipe module for calculation of multi‐dimensional distances from phenotyping data, we observe a time after infection‐dependent impact of U. maydis effector deletion strains on phenotypic response in maize. The PhenoBox/PhenoPipe system is able to detect established salt stress responses in N. benthamiana.We have developed an affordable, automated, open source imaging and data processing solution that can be adapted to various phenotyping applications in plant biology and beyond.

Itaconic Acid Production by Filamentous Fungi in Starch-Rich Industrial Residues.

Several fungi and starch-rich industrial residues were screened for itaconic acid (IA) production. Out of 15 strains, only three fungal strains were found to produce IA, which was confirmed by HPLC and GC-MS analysis. These strains were identified as Aspergillus terreus strains C1 and C2, and Ustilago maydis strain C3 by sequencing of 18S rRNA gene and internal transcribed spacer regions. Cis-aconitate decarboxylase (cad) gene, which encodes a key enzyme in IA production in A. terreus, was characterized from strains C1 and C2. C1 and C2 cad gene sequences showed about 96% similarity to the only available GenBank sequence of A. terreus cad gene. 3-D structure and cis-aconitic acid binding pocket of Cad enzyme were predicted by structural modeling. Rice, corn and potato starch wastes were screened for IA production. These materials were enzymatically hydrolyzed under experimentally optimized conditions resulting in the highest glucose production of 230mg/mL from 20% potato waste. On comparing the production potential of selected strains with different wastes, the best IA production was achieved with strain C1 (255.7mg/L) using potato waste. Elemental composition as well as batch-to-batch variation in waste substrates were analyzed. The difference in IA production from two different batches of potato waste was found to inversely correlate with their phosphorus content, which indicated that A. terreus produced IA under phosphate limiting condition. The potato waste hydrolysate was deionized to remove inhibitory ions like phosphate, resulting in improved IA production of 4.1g/L by C1 strain, which is commercially competitive.

Fungal derived cytokinins are necessary for normal Ustilago maydis infection of maize

Phytohormones derived from fungi play a key role in regulating plant–pathogen interactions; however, deciphering the separate contributions of pathogen and plant during infection has been difficult. Here, the Ustilago maydis–Zea mays pathosystem was used to investigate this chemical exchange. Ustilago maydis, the causative agent of maize smut, produces cytokinins (CK), which are a group of phytohormones responsible for directing plant development. The characteristic symptom of smut disease is the formation of tumours composed of plant and fungal tissue. Isopentenyltransferase (IPT) catalyses the rate‐limiting step in CK biosynthesis, and U. maydis strains in which the sole tRNA‐ipt gene was deleted no longer produced CKs. These deletion strains elicited fewer, smaller tumours than the pathogenic strain SG200. High performance liquid chromatography‐electrospray ionization tandem mass spectrometry (HPLC‐ESI MS/MS) was used to detect and quantify phytohormone levels in infected tissue. This revealed that key hormone changes in SG200 infections were not present in infections by deletion strains, suggesting that CK production by U. maydis is required for the altered phytohormone profile in infected tissue relative to uninfected tissue. Separate analyses indicated that U. maydis tRNA‐ipt mutants might be altered in their ability to metabolize CKs taken up from the environment. Mining the U. maydis genome identified genes encoding putative CK signalling and biosynthesis proteins.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions.

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell's DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.