Search Results (67)

Corn smut, caused by Ustilago maydis, significantly threatens maize production. This study evaluated 199 maize inbred lines at the seedling stage under greenhouse conditions for resistance to U. maydis, identifying 39 highly resistant lines. A genome-wide association study (GWAS) using the mrMLM model detected 19 significant single-nucleotide polymorphism (SNP) loci. Based on a linkage disequilibrium (LD) decay distance of 260 kb, 226 candidate genes were identified. Utilizing the significant loci chr1_244281660 and chr5_220156746, two kompetitive allele-specific PCR (KASP) markers were successfully developed. A PCR-based sequence-specific oligonucleotide probe hybridization technique applied to the 199 experimental lines and 60 validation lines confirmed polymorphism for both markers, with selection efficiencies of 48.12% and 43.33%, respectively. The tested materials were derived from foundational inbred lines of domestic and foreign origin. Analysis of 39 highly resistant lines showed that the advantageous alleles carrying thymine/cytosine (T/C) predominated at frequencies of 94.87% and 53.84%, respectively. The genotype TTCC conferred high resistance, while CCTT was highly susceptible. The resistance exhibited high heritability and significant gene-by-environment interaction. This work systematically dissects the genetic basis of common smut resistance in maize, identifies favorable alleles, and provides a novel KASP marker-based strategy for developing disease-resistant germplasm. Full article

Mannosyl erythritol lipids (MELs) are glycolipid biosurfactants produced by Ustilaginomycete yeasts. The MEL biosynthetic pathway has been characterized in Ustilago maydis where a putative transporter encoded by MMF1 is required for the secretion of the glycolipid surfactant to the extracellular space. The anamorphic yeast Moesziomyces antarcticus is a prolific producer of MELs, but the mechanism of MEL secretion is less well characterized than in U. maydis. Homologous recombination was employed to generate a disruption of the MMF1 gene in M. antarcticus JCM10317. This mutation did not prevent the intracellular accumulation of MEL species but did result in significantly reduced secretion of the conventional MEL-A, MEL-B and MEL-C species detectable by thin-layer chromatography. However, the mutant strain did secrete a glycolipid species that is distinct from conventional MEL-A/B/C and similar to a glycolipid secreted by MMF1 mutant strains of U. maydis and Pseudozyma tsukubaensis. Despite the defect in MEL secretion displayed by the M. antarcticus strain harbouring a disrupted MMF1 gene, these cells did not display a significant defect in growth or cell morphology. The findings of this investigation provide evidence that M. antarcticus MMF1 encodes a transporter required for the secretion of MELs but not required for MEL synthesis or cell growth. Full article

Fungi produce dormant structures that are responsible for protection during adverse environmental conditions and dispersal (disease spread). Ustilago maydis, a basidiomycete plant pathogen, is a model for understanding the molecular mechanisms of teliospore dormancy and germination. Dormant teliospores store components required for germination including mRNAs which may be stored as dsRNAs. RNA helicases are conserved enzymes that function to modulate, bind, and unwind RNA duplexes, and can displace other proteins. We hypothesize that RNA helicases function during teliospore dormancy to stabilize and/or modulate stored mRNAs. We identified the U. maydis udbp3 and uded1 as encoding RNA helicases of interest as they are upregulated in the dormant teliospore and decrease during germination. Experimental results suggest that udbp3 may function as a negative regulator of osmotic stress-responsive genes and that uded1 modulates stress response by repressing translation. The altered expression of uded1 also results in slow growth, polarized growth, and the formation of dsRNA. Together, the data support a role for both helicases modulating gene expression, in response to stress, leading to teliospore dormancy and also modulating responses for teliospore germination. Increasing our molecular understanding of these processes will aid in developing novel strategies to mitigate disease spread. Full article

The biotrophic fungus Ustilago maydis, which causes smut disease in maize, secretes numerous proteins upon plant colonization. Some of them, termed effectors, help to evade plant defenses and manipulate cellular processes within the host. The function of many proteins specifically secreted during infection remains elusive. In this study, we biochemically characterized one such protein, UMAG_00027, that is highly expressed during plant infection. We show that UMAG_00027 is a secreted protein with a lectin-like fold and therefore term it Llp1 (lectin-like-protein 1). Llp1 decorated the fungal cell wall of cells grown in axenic culture or proliferating in planta, which is in agreement with its potential sugar-binding ability. We were unable to identify the precise sugar moieties that are bound by Llp1. CRISPR/Cas9-mediated deletion of llp1 reveals that the gene is not essential for fungal virulence. A structural search shows the presence of several other lectin-like proteins in U. maydis that might compensate for the function of Llp1 in ∆llp1 mutants. We therefore speculate that Llp1 is part of a family of lectin-like proteins with redundant functions. Full article

Food security has been an essential priority for decades due to its direct impact on human development. Despite significant advances in meeting basic food needs, food security remains a global challenge. In Mexico, the sustainable traditional polyculture system known as milpa allows synergy among three or more food sources, increasing the economic income for peasants. We integrated the controlled production of huitlacoche (Ustilago maydis + Zea mays) galls, traditional food consumed since pre-Hispanic times, in a milpa from Las Ánimas community, Tecoanapa Municipality, Guerrero, Mexico, in order to improve food diversity, productivity, income, and food security. Two contiguous plots were evaluated: the traditional milpa system (TMS) and the milpa integrating controlled production of huitlacoche galls (TMS + Huitlacoche). Different variables were assessed including weight and size of maize ears and huitlacoche galls; degree of huitlacoche infection; production and income of maize (Zea mays), beans (Phaseolus spp.), squash (Cucurbita spp.), and huitlacoche; and sustainability indicators, maize grain sales, type of maize seed, huitlacoche sales, beans sales, and squash sales during the dry season. The integration of the controlled production of huitlacoche improved productivity and net incomes from the traditional milpa. The traditional milpa system (TMS) had an income of USD $111.00 from maize grains, while the milpa integrating controlled production of huitlacoche galls (TMS + Huitlacoche) had an income of USD $409.00 from galls. This is reflected in the cost/benefit ratio, in which milpa integrating controlled production of huitlacoche galls (TMS + Huitlacoche) was 1.48, while the traditional milpa system (TMS) was not profitable (0.91). Sustainability indicators show that the controlled production of huitlacoche galls contribute to food diversity, productivity, and income in the traditional milpa system, based on the association of maize, beans, squash, and huitlacoche, showing potential for improving food security in rural communities. Full article

In the future, the cultivation of maize will become more and more prominent. As the world’s demand for food and animal feeding increases, remote sensing technologies (RS technologies), especially unmanned aerial vehicles (UAVs), are developing more and more, and the usability of the cameras (Multispectral-MS) installed on them is increasing, especially for plant disease detection and severity observations. In the present research, two different maize hybrids, P9025 and sweet corn Dessert R78 (CS hybrid), were employed. Four different treatments were performed with three different doses (low, medium, and high dosage) of infection with corn smut fungus (Ustilago maydis [DC] Corda). The fields were monitored two times after the inoculation—20 DAI (days after inoculation) and 27 DAI. The orthomosaics were created in WebODM 2.5.2 software and the study included five vegetation indices (NDVI [Normalized Difference Vegetation Index], GNDVI [Green Normalized Difference Vegetation Index], NDRE [Normalized Difference Red Edge], LCI [Leaf Chlorophyll Index] and ENDVI [Enhanced Normalized Difference Vegetation Index]) with further analysis in QGIS. The gathered data were analyzed using R-based Jamovi 2.6.13 software with different statistical methods. In the case of the sweet maize hybrid, we obtained promising results, as follows: the NDVI values of CS 0 were significantly higher than the high-dosed infection CS 10.000 with a mean difference of 0.05422 *** and a p value of 4.43 × 10⁻⁵ value, suggesting differences in all of the levels of infection. Furthermore, we investigated the correlations of the vegetation indices (VI) for the Dessert R78, where NDVI and GNDVI showed high correlations. NDVI had a strong correlation with GNDVI (r = 0.83), a medium correlation with LCI (r = 0.56) and a weak correlation with NDRE (r = 0.419). There was also a strong correlation between LCI and GNDVI, with r = 0.836. NDRE and GNDVI indices had the correlation coefficients with a CCoeff. of r = 0.716. For hybrid separation analyses, useful results were obtained for NDVI and ENDVI as well. Full article

Maize Ustilago maydis is a disease that severely affects maize yield and quality. In this paper, we employed transcriptome sequencing and GWAS analysis to identify candidate genes and reveal disease-resistant germplasm resources, thereby laying the foundation for further analysis of the molecular mechanism of maize Ustilago maydis resistance and genetic improvement. The results of transcriptome sequencing revealed that a considerable number of receptor kinase genes, signal-transduction-related protein genes, redox-response-related genes, WRKYs, and P450s genes were significantly upregulated. There was a wide range of mutations of Ustilago maydis in maize inbred lines. Thirty-two high-resistance maize inbred lines were selected, and 16 SNPs were significantly associated with the disease index. By integrating the results of GWAS and RNA-seq, five genes related to disease resistance were identified, encoding the chitinase 1 protein, fatty acid elongase (FAE), IAA9, GATA TF8, and EREB94, respectively. It provides a certain reference for the cloning of maize anti-tumor smut genes and the breeding of new varieties. Full article

Oxidative stress represents a critical facet of the array of abiotic stresses affecting crop growth and yield. In this paper, we investigated the potential differences in the functions of two highly homologous Arabidopsis DSS1 proteins in terms of maintaining genome integrity and response to oxidative stress. In the context of homologous recombination (HR), it was shown that overexpressing AtDSS1(I) using a functional complementation test increases the resistance of the Δdss1 mutant of Ustilago maydis to genotoxic agents. This indicates its conserved role in DNA repair via HR. To investigate the global transcriptome changes occurring in dss1 plant mutant lines, gene expression analysis was conducted using Illumina RNA sequencing technology. Individual RNA libraries were constructed from three total RNA samples isolated from dss1(I), dss1(V), and wild-type (WT) plants under hydrogen peroxide-induced stress. RNA-Seq data analysis and real-time PCR identification revealed major changes in gene expression between mutant lines and WT, while the dss1(I) and dss1(V) mutant lines exhibited analogous transcription profiles. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed significantly enriched metabolic pathways. Notably, genes associated with HR were upregulated in dss1 mutants compared to the WT. Otherwise, genes of the metabolic pathway responsible for the synthesis of secondary metabolites were downregulated in both dss1 mutant lines. These findings highlight the importance of understanding the molecular mechanisms of plant responses to oxidative stress. Full article

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. Full article

The corn smut fungus, Ustilago maydis, is an excellent model for studying biotrophic plant-pathogen interactions, including nutritional adaptation to the host environment. Iron acquisition during host colonization is a key aspect of microbial pathogenesis yet less is known about this process for fungal pathogens of plants. Monothiol glutaredoxins are central regulators of key cellular functions in fungi, including iron homeostasis, cell wall integrity, and redox status via interactions with transcription factors, iron-sulfur clusters, and glutathione. In this study, the roles of the monothiol glutaredoxin Grx4 in the biology of U. maydis were investigated by constructing strains expressing a conditional allele of grx4 under the control of the arabinose-inducible, glucose-repressible promoter P_crg1. The use of conditional expression was necessary because Grx4 appeared to be essential for U. maydis. Transcriptome and genetic analyses with strains depleted in Grx4 revealed that the protein participates in the regulation of iron acquisition functions and is necessary for the ability of U. maydis to cause disease on maize seedlings. Taken together, this study supports the growing appreciation of monothiol glutaredoxins as key regulators of virulence-related phenotypes in pathogenic fungi. Full article

Ustilago maydis is a biotrophic basidiomycete fungus that infects corn plants and works as an excellent phytopathogen model, facilitating numerous genetic transformations for studying the mechanisms of plant infection. A random mutation event in the mutant strains designed to investigate the physiological significance of two plasma membrane proton-ATPases in this model resulted in a pigmented phenotype strain. For this study, the FB2 strain and the ΔPMA1 mutant were chosen to assess the pigment, which was confirmed as melanin through thin-layer chromatography, UV, and IR spectrophotometry. The melanin was observed to accumulate in the cytosol, as evident from scanning and transmission electron microscopy, and did not interfere with normal cell growth in yeast extract peptone dextrose media or minimal media. Notably, the mutant exhibited a 25% higher melanin yield compared to wild-type cells. To analyze the melanin synthesis, the tyrosinase activity was measured in a phosphate buffer at pH 6.5. The enzyme demonstrated greater activity with tyrosine as a substrate than with L-3,4 dihydroxyphenylalanine, maintaining the same trend in ion preference. Both FB2 and ΔPMA1 mutant cells were subjected to biosorption experiments, revealing that the mutants with an excess of cytosolic melanin were capable of removing at least 50 ppm of methylene blue. In conclusion, U. maydis can accumulate melanin in the cytosol without adverse physiological effects and this presents biotechnological potential for dye removal. Full article

Members of the WRKY transcription factor (TF) family are unique to plants and serve as important regulators of diverse physiological processes, including the ability of plants to manage biotic and abiotic stressors. However, the functions of specific WRKY family members in the context of maize responses to fungal pathogens remain poorly understood, particularly in response to Ustilago maydis (DC.) Corda (U. maydis), which is responsible for the devastating disease known as corn smut. A systematic bioinformatic approach was herein employed for the characterization of the maize WRKY TF family, leading to the identification of 120 ZmWRKY genes encoded on 10 chromosomes. Further structural and phylogenetic analyses of these TFs enabled their classification into seven different subgroups. Segmental duplication was established as a major driver of ZmWRKY family expansion in gene duplication analyses, while the Ka/Ks ratio suggested that these ZmWRKY genes had experienced strong purifying selection. When the transcriptional responses of these genes to pathogen inoculation were evaluated, seven U. maydis-inducible ZmWRKY genes were identified, as validated using a quantitative real-time PCR approach. All seven of these WKRY proteins were subsequently tested using a yeast one-hybrid assay approach, which revealed their ability to directly bind the ZmSWEET4b W-box element, thereby controlling the U. maydis-inducible upregulation of ZmSWEET4b. These results suggest that these WRKY TFs can control sugar transport in the context of fungal infection. Overall, these data offer novel insight into the evolution, transcriptional regulation, and functional characteristics of the maize WRKY family, providing a basis for future research aimed at exploring the mechanisms through which these TFs control host plant responses to common smut and other fungal pathogens. Full article

Common smut caused by Ustilago maydis is one of the dominant fungal diseases in plants. The resistance mechanism to U. maydis infection involving alterations in the cell wall is poorly studied. In this study, the resistant single segment substitution line (SSSL) R445 and its susceptible recurrent parent line Ye478 of maize were infected with U. maydis, and the changes in cell wall components and structure were studied at 0, 2, 4, 8, and 12 days postinfection. In R445 and Ye478, the contents of cellulose, hemicellulose, pectin, and lignin increased by varying degrees, and pectin methylesterase (PME) activity increased. The changes in hemicellulose and pectin in the cell wall after U. maydis infection were analyzed via immunolabeling using monoclonal antibodies against hemicellulsic xylans and high/low-methylated pectin. U. maydis infection altered methyl esterification of pectin, and the degree of methyl esterification was correlated with the resistance of maize to U. maydis. Furthermore, the relationship between methyl esterification of pectin and host resistance was validated using 15 maize inbred lines with different resistance levels. The results revealed that cell wall components, particularly pectin, were important factors affecting the colonization and propagation of U. maydis in maize, and methyl esterification of pectin played a role in the resistance of maize to U. maydis infection. Full article

We analyzed the global expression patterns of telomerase-negative mutants from haploid cells of Ustilago maydis to identify the gene network required for cell survival in the absence of telomerase. Mutations in either of the telomerase core subunits (trt1 and ter1) of the dimorphic fungus U. maydis cause deficiencies in teliospore formation. We report the global transcriptome analysis of two ter1Δ survivor strains of U. maydis, revealing the deregulation of telomerase-deleted responses (TDR) genes, such as DNA-damage response, stress response, cell cycle, subtelomeric, and proximal telomere genes. Other differentially expressed genes (DEGs) found in the ter1Δ survivor strains were related to pathogenic lifestyle factors, plant–pathogen crosstalk, iron uptake, meiosis, and melanin synthesis. The two ter1Δ survivors were phenotypically comparable, yet DEGs were identified when comparing these strains. Our findings suggest that teliospore formation in U. maydis is controlled by key pathogenic lifestyle and meiosis genes. Full article

The aim of this study was to investigate whether, in the context of a higher incidence of Ustilago maydis and Fusarium spp. at optimal and delayed harvest times, a higher incidence of mycotoxin contamination in maize grains could be expected. The field experiment was carried out at the Lithuanian Research Centre for Agriculture and Forestry experimental fields over three consecutive years (2020–2022). Two maize hybrids (Duxxbury and Lapriora) with different FAO numbers were used. The experimental design in the field was a randomized complete block design. Harvesting took place at three different times: first at physiological maturity, and then 10 (±2) and 20 (±2) days after the first harvest. Each hybrid had four repetitions at different harvest times. The U. maydis infection was only detected in 2021 and after the first harvest cobs were further divided into four different groups with four repetitions: healthy cobs, cobs visually infected with Fusarium spp., cobs visually infected with common smut, and cobs visually infected with both pathogens. No U. maydis-damaged maize cobs were found in 2020 and 2022. The levels of Fusarium microscopic fungi in maize grains were also from 4 to 16 times higher in 2021 than in 2020 and 2022. Harvest delays in 2020 led to a significant deoxynivalenol concentration increase in the Duxxbury hybrid and an HT-2 concentration increase in the Lapriora hybrid. In 2021, deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, and HT-2 concentrations significantly rose in both hybrids, but the T-2 concentration significantly increased only in the Lapriora hybrid. Deoxynivalenol concentrations were, respectively, 110 and 14.6 times higher than in cobs only infected with Fusarium spp. or U. maydis. Concentrations of 15-acetyl-deoxynivalenol were, respectively, 60, 67, and 43 times higher than in asymptomatic cobs and cobs only infected with Fusarium spp. or U. maydis. Cobs contaminated with both pathogens also had higher concentrations of 3-acetyl-deoxynivalenol. T-2 and HT-2 were detected in maize grains harvested from cobs infected only with Fusarium spp. The presence of U. maydis and Fusarium fungi in maize cobs, along with harvest delays, led to significant increases in mycotoxin concentrations, highlighting the importance of timely harvesting and pathogen management to mitigate mycotoxin contamination in maize grains. Full article