Double chitin synthetase mutants from the corn smut fungus Ustilago maydis

Using genetic crosses between single chs mutants of Ustilago maydis inoculated into maize ( Zea mays ) seedlings, two classes of double mutants affected in genes coding for chitin synthetases were isolated: chs3 / chs4 , and chs4 / chs5 . Analysis of the mutants showed almost no change in their phenotype compared with wild-type strains. Growth rate, effect of stress conditions, dimorphic transition and mating were not affected. The only salient differences were increased sensitivity to osmotics at acid pH, and decrease in chitin synthetase activity, especially when measured with CO²⁺, and in chitin content. Most significant was a decrease in virulence, although this appeared to be due a factor unrelated to CHS genes. These data can be taken as further evidence that multigenic control of chitin synthetase in fungi operates as a safety mechanism to guarantee fungal viability in changing and hostile environmental conditions.     

玉米黑粉菌致病的分子机制研究进展

玉米黑粉菌（Ustilago maydis）可在其宿主植物玉米（Zea mays L.）地上部的所有器官诱导肿瘤发生。玉米黑粉菌成功定殖宿主并诱导形成肿瘤取决于与宿主植物多方位、多层次的相互作用以及该过程中发生的复杂的细胞和分子事件。本文综述了玉米黑粉菌与玉米互作研究的最新进展,介绍了玉米黑粉菌通过分泌效应子入侵、定殖玉米植株以及植株在分子水平上对入侵的响应;阐述了活体营养建立过程中,玉米黑粉菌与玉米通过效应子、激素、糖代谢酶和转运蛋白的差异调节,协调受感染宿主组织重新编程发育成膨大的植物肿瘤的关键因素,并对今后的研究方向进行了展望。     

Disruption of two genes for chitin synthase in the phytopathogenic fungus Ustilago maydis

The phytopathogenic fungus Ustilago maydis exhibits a dimorphic transition in which non-pathogenic, yeast-like cells mate to form a pathogenic, filamentous dikaryon. Northern analysis indicated that two chitin synthase genes, chs1 and chs2, from U. maydis are expressed at similar levels in yeast-like cells and in cells undergoing the mating reaction leading to the filamentous cell type. A mutation was constructed in each of the chitin synthase genes by targeted gene disruption. Each mutant showed a reduction in the level of trypsin-activated enzyme activity, compared with a wild-type strain, but retained the wild-type morphology, the ability to mate and the ability to form the filamentous pathogenic cell type.     

Purification and characterization of aminopeptidase (pumAPE) from Ustilago maydis

The aminopeptidase pumAPE was purified from the haploid fungus Ustilago maydis FB1 strain. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included anion-exchange, hydrophobic interaction, and gel filtration chromatography, resulting in a 23% recovery. The molecular mass of the dimeric enzyme was estimated to be 110 kDa and 58 kDa by gel filtration chromatography and SDS-PAGE, respectively. Enzymatic activity was optimal at pH 7.0 and at 35 degrees C toward Lys-pNA and the pI was determined to be 5.1. The enzyme was inhibited by EDTA-Na2, 1,10- phenanthroline, bestantin, PMSF and several divalent cations (Cu2+, Hg2+ and Zn2+). The aminopeptidase showed a preference for lysine and arginine in the N-position. The K(m) value was 54.4 microM and the Vmax value was 408 micromolmin(-1)mg(-1) for Lys-pNA.     

Influence of carbon and nitrogen concentration on itaconic acid production by the smut fungus Ustilago maydis

Itaconic acid is a valuable platform compound for the production of bio‐based polymers, chemicals, and fuels. Ustilago maydis is a promising host for the production of itaconic acid from biomass‐derived substrates due to its unicellular growth pattern and its potential to utilize biomass‐derived sugar monomers and polymers. The potential of U. maydis for industrial itaconate production was assessed in pH‐controlled batch fermentations with varying medium compositions. Using 200 g/L glucose and 75 mM ammonium, 44.5 g/L of itaconate was produced at a maximum rate of 0.74 g L^?1 h^?1. By decreasing the substrate concentrations to 50 g/L glucose and 30 mM ammonium, a yield of 0.34 g/g (47 mol%) could be achieved. Itaconate production from xylose was also feasible. These results indicate that high itaconic acid titers can be achieved with U. maydis. However, further optimization of the biocatalyst itself through metabolic engineering is still needed in order to achieve an economically feasible process, which can be used to advance the development of a bio‐based economy.     

Endocytosis in the plant-pathogenic fungus Ustilago maydis

Summary. Filamentous fungi are an important group of tip-growing organisms, which include numerous plant pathogens such as Magnaporthe grisea and Ustilago maydis. Despite their ecological and economical relevance, we are just beginning to unravel the importance of endocytosis in filamentous fungi. Most evidence for endocytosis in filamentous fungi is based on the use of endocytic tracer dyes that are taken up into the cell and delivered to the vacuole. Moreover, genomewide screening for candidate genes in Neurospora crassa and U. maydis confirmed the presence of most components of the endocytic machinery, indicating that endocytosis participates in filamentous growth. Indeed, it was shown that in U. maydis early endosomes cluster at sites of growth, where they support morphogenesis and polar growth, most likely via endosome-based membrane recycling. In humans, such recycling processes to the plasma membrane involve small GTPases such as Rab4. A homologue of this protein is encoded in the genome of U. maydis but is absent from the yeast Saccharomyces cerevisiae, suggesting that Rab4-mediated recycling is important for filamentous growth. Furthermore, human Rab4 regulates traffic of early endosomes along microtubules, and a similar microtubule-based transport is described for U. maydis. These observations suggest that Rab4-like GTPases might regulate endosome- and microtubule-based recycling during tip growth of filamentous fungi. Correspondence and reprints: MPI für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, 35043 Marburg, Federal Republic of Germany.     

黄淮海地区玉蜀黍黑粉菌群体遗传结构分析

利用17个多态性ISSR引物对2017年在我国黄淮海地区采集的41株玉蜀黍黑粉菌Ustilago maydis样本进行了种群遗传结构分析。基于UPGMA法和贝叶斯模型对玉蜀黍黑粉菌群体遗传结构分析显示,采集的样本可以分为两大群,且种群的划分表现出了与来源地理纬度的一致性。因此按照各样本所属地理纬度将样本分为北纬33°-34°、北纬35°-36°、北纬37°-38° 3大种群。结果表明,分离自北纬33°-34°地区的玉蜀黍黑粉菌遗传多样性最丰富,Nei’s基因多样性（H）为0.2216,Shannon指数（I）为0.3300。分子方差分析（AMOVA）显示,变异中来自种群内部的变异为86%,种群间变异14%（φpt=0.141）。     

Mode of Action and Genetic Analysis of Resistance to Fluazinam in Ustilago maydis

Ustilago maydis strains, with low to moderate resistance to fluazinam (Rf ranging from 11.8 to 80), were isolated in a mutation frequency of 0.75 × 10⁻⁷ after chemical mutagenesis with N‐methyl‐N‐nitro‐N‐nitrosoguanidine (MNNG). Genetic analysis resulted in the identification of two chromosomal genes. A study of the effect of mutant genes in the phytopathogenic fitness of U. maydis revealed that the resistance mutations had no apparent effect on mycelia growth rate and pathogenicity on young corn plants. Cross‐resistant studies showed that the mutations for resistance to fluazinam were also responsible for resistance to oligomycin, but not to dinitrophenol. A dose‐dependent inhibition of glucose oxidation in whole cells was observed by both fluazinam and oligomycin, and a complete inhibition was found at 40 μg/ml. The results obtained provide strong evidence that the mode of action of fluazinam consists of the inhibition the fungal cell's energy production process through direct inhibition of the ATP synthetase.     

Proteinases and exopeptidases from the phytopathogenic fungus Ustilago maydis

The proteolytic system of the phytopathogenic and dimorphic fungus Ustilago maydis is not known. In this work, we report the presence of at least four proteases from two haploid strains of U. maydis. Activities of two proteinases pumA and pumB, aminopeptidase pumAPE, and dipeptidylaminopeptidase pumDAP were measured under several nutritional and morphological conditions, including the yeast-mycelium transition. The activity of pumA was found in the intracellular and extracellular fractions, pumAi and pumAe, respectively. The latter activity was detected only during the yeast-mycelium dimorphic transition induced by growth at acid pH in a medium containing ammonium as the sole nitrogen source. Activity of pumAe was partially inhibited by Pepstatin A, which also inhibited mycelium formation. Activity of pumAi was inhibited by this specific inhibitor of aspartyl-proteases. Activity of pumB was detected in intracellular and extracellular fractions, mostly bound to an endogenous inhibitor, which was removed by treatment at acid pH. This fungus contains at least two soluble pumAPE, which might be metallo-proteases, because they were inhibited by EDTA and 1-10, phenanthroline. When the fungus was grown in media containing proline or corn infusion as the nitrogen source, an intracellular pumDAP activity was detected. No carboxypeptidase activity was found with N-benzoyl-l-tyrosine-4-nitroanilide as substrate in any of the conditions tested in any of the U. maydis strains analyzed.     

Progress in pathogenesis research of Ustilago maydis,and the metabolites involved along with their biosynthesis

Ustilago maydis is a pathogenic fungus that causes corn smut. Because of its easy cultivation and genetic transformation, U. maydis has become an important model organism for plant-pathogenic basidiomycetes. U. maydis is able to infect maize by producing effectors and secreted proteins as well as surfactant-like metabolites. In addition, the production of melanin and iron carriers is also associated with its pathogenicity. Here, advances in our understanding of the pathogenicity of U. maydis, the metabolites involved in the pathogenic process, and the biosynthesis of these metabolites, are reviewed and discussed. This summary will provide new insights into the pathogenicity of U. maydis and the functions of associated metabolites, as well as new clues for deciphering the biosynthesis of metabolites.     

Pheromone-induced G2 arrest in the phytopathogenic fungus Ustilago maydis

In the corn smut fungus Ustilago maydis, pathogenic development is initiated when two compatible haploid cells fuse and form the infectious dikaryon. Mating is dependent on pheromone recognition by compatible cells. In this report, we set out to evaluate the relationship between the cell cycle and the pheromone response in U. maydis. To achieve this, we designed a haploid pheromone-responsive strain that is able to faithfully reproduce the native mating response in nutrient-rich medium. Addition of synthetic pheromone to the responsive strain induces the formation of mating structures, and this response is abolished by mutations in genes encoding components of the pheromone signal transduction cascade. After recognition of pheromone, U. maydis cells arrest the cell cycle in a postreplicative stage. Visualization of the nucleus and microtubule organization indicates that the arrest takes place at the G₂ phase. Chemical-induced cell cycle arrest and release in the presence of pheromone further support this conclusion.     

Genome‐wide assessment of tandem repeat markers for biogeographical analyses of the corn smut fungus,Ustilago maydis

It has been postulated that the fungus Ustilago maydis followed its host maize around the world. In order to understand the biogeography of this fungus relative to its host, we used a bioinformatics approach to develop 86 tandem repeat markers useful for population studies of U. maydis. We characterized repeat motifs between two and 155 nucleotides using 36 isolates from USA, Mexico and South America. Our data suggested that (i) repeat motif length does not predict the number of alleles for that locus, and (ii) that the more polymorphic markers can resolve differences in a small geographical area.     

Regulation of cell separation in the dimorphic fungus Ustilago maydis

During its haploid phase the dimorphic fungus Ustilago maydis grows vegetatively by budding. We have identified two genes, don1 and don3, which control the separation of mother and daughter cells. Mutant cells form tree-like clusters in liquid culture and grow as ring-like (donut-shaped) colonies on solid medium. In wild-type U. maydis cells, two distinct septa are formed during cytokinesis and delimit a fragmentation zone. Cells defective for either don1 or don3 display only a single septum and fail to complete cell separation. don1 encodes a guanine nucleotide exchange factor (GEF) of the Dbl family specific for Rho/Rac GTPases. Don3 belongs to the germinal-centre-kinase (GC) subfamily of Ste20-like protein kinases. We have isolated the U. maydis homologues of the small GTP binding proteins Rho2, Rho3, Rac1 and Cdc42. Out of these, only Cdc42 interacts specifically with Don1 and Don3 in the yeast two-hybrid system. We propose that Don1 and Don3 regulate the initiation of the secondary septum, which is required for proper cell separation.     

Completion of the sexual cycle and demonstration of genetic recombination in Ustilago maydis in vitro

The heterobasidiomycetes responsible for plant smuts obligatorily require their hosts for the completion of the sexual cycle. Accordingly, the sexual cycle of these fungi could so far be studied only by infecting host plants. We have now induced Ustilago maydis, the causative agent of corn smut, to traverse the whole life cycle by growing mixtures of mating-compatible strains of the fungus on a porous membrane placed on top of embryogenic cell cultures of its host Zea mays. Under these conditions, mating, karyogamy and meiosis take place, and the fungus induces differentiation of the plant cells. These results suggest that embryogenic maize cells produce diffusible compounds needed for completion of the sexual cycle of U. maydis, as the plant does for the pathogen during infection. Received: 16 February 1999 / Accepted: 30 June 1999