The two paralogous kiwellin proteins KWL1 and KWL1-b from maize are structurally related and have overlapping functions in plant defense.

Many plant-pathogenic bacteria and fungi deploy effector proteins that down-regulate plant defense responses and reprogram plant metabolism for colonization and survival in planta Kiwellin (KWL) proteins are a widespread family of plant-defense proteins that target these microbial effectors. The KWL1 protein from maize (corn, Zea mays) specifically inhibits the enzymatic activity of the secreted chorismate mutase Cmu1, a virulence-promoting effector of the smut fungus Ustilago maydis. In addition to KWL1, 19 additional KWL paralogs have been identified in maize. Here, we investigated the structure and mechanism of the closest KWL1 homolog, KWL1-b (ZEAMA_GRMZM2G305329). We solved the Cmu1-KWL1-b complex to 2.75 Å resolution, revealing a highly symmetric Cmu1-KWL1-b heterotetramer in which each KWL1-b monomer interacts with a monomer of the Cmu1 homodimer. The structure also revealed that the overall architecture of the heterotetramer is highly similar to that of the previously reported Cmu1-KWL1 complex. We found that upon U. maydis infection of Z. mays, KWL1-b is expressed at significantly lower levels than KWL1 and exhibits differential tissue-specific expression patterns. We also show that KWL1-b inhibits Cmu1 activity similarly to KWL1. We conclude that KWL1 and KWL1-b are part of a redundant defense system that tissue-specifically targets Cmu1. This notion was supported by the observation that both KWL proteins are carbohydrate-binding proteins with distinct and likely tissue-related specificities. Moreover, binding by Cmu1 modulated the carbohydrate-binding properties of both KWLs. These findings indicate that KWL proteins are part of a spatiotemporally coordinated, plant-wide defense response comprising proteins with overlapping activities.

Microbial Diversity in the Edible Gall on White Bamboo Formed by the Interaction between Ustilago esculenta and Zizania latifolia.

An edible gall is formed between the third and fourth nodes beneath the apical meristem near the base of Zizania latifolia shoots. This gall is harbored by and interacts with the smut fungus Ustilago esculenta. The gall is also a valuable vegetable called "white bamboo," jiaobai or gausun in China and makomotake in Japan. Five samples of the galls harvested at different stages of swelling were used to isolate microorganisms by culturing. Isolated fungal and bacterial colonies were identified by DNA sequencing and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, respectively. Several strains of U. esculenta as well as 6 other species of fungi and 10 species of bacteria were isolated. The microbiome was also evaluated by simple and outlined DNA profiling with automated rRNA intergenic spacer analysis (ARISA), and the amount of DNA of U. esculenta was determined by qPCR. At least 16 species of fungi and 40 species of bacteria were confirmed by ARISA of the overall sample. Interestingly, the greatest bacterial diversity, i.e., 18 species, was observed in the most mature sample, whereas the fungal diversity observed in this sample, i.e., 4 species, was rather poor. Based on qPCR, U. esculenta occurred in samples from all stages; however, the abundance of U. esculenta exhibited unique U-shaped relationships with growth. These results may explain why the interaction between U. esculenta and Z. latifolia also influences the unique microbial diversity observed throughout the growth stages of the swollen shoot, although the limited sample size does not allow conclusive findings.

Investigation on the differentiation of two Ustilago esculenta strains - implications of a relationship with the host phenotypes appearing in the fields.

BACKGROUND: Ustilago esculenta, a pathogenic basidiomycete fungus, infects Zizania latifolia to form edible galls named Jiaobai in China. The distinct growth conditions of U. esculenta induced Z. latifolia to form three different phenotypes, named male Jiaobai, grey Jiaobai and white Jiaobai. The aim of this study is to characterize the genetic and morphological differences that distinguish the two U. esculenta strains. RESULTS: In this study, sexually compatible haploid sporidia UeT14/UeT55 from grey Jiaobai (T strains) and UeMT10/UeMT46 from white Jiaobai (MT strains) were isolated. Meanwhile, we successfully established mating and inoculation assays. Great differences were observed between the T and MT strains. First, the MT strains had a defect in development, including lower teliospore formation frequency and germination rate, a slower growth rate and a lower growth mass. Second, they differed in the assimilation of nitrogen sources in that the T strains preferred urea and the MT strains preferred arginine. In addition, the MT strains were more sensitive to external signals, including pH and oxidative stress. Third, the MT strains showed an infection defect, resulting in an endophytic life in the host. This was in accordance with multiple mutated pathogenic genes discovered in the MT strains by the non-synonymous mutation analysis of the genome re-sequencing data between the MT and T strains (GenBank accession numbers of the genome re-sequencing data: JTLW00000000 for MT strains and SRR5889164 for T strains). CONCLUSION: The MT strains appeared to have defects in growth and infection and were more sensitive to external signals compared to the T strains. They displayed an absolutely stable endophytic life in the host without an infection cycle. Accordingly, they had multiple gene mutations occurring, especially in pathogenicity. In contrast, the T strains, as phytopathogens, had a complete survival life cycle, in which the formation of teliospores is important for adaption and infection, leading to the appearance of the grey phenotype. Further studies elucidating the molecular differences between the U. esculenta strains causing differential host phenotypes will help to improve the production and formation of edible white galls.

Investigation on the biotrophic interaction of Ustilago esculenta on Zizania latifolia found in the Indo-Burma biodiversity hotspot.

Ustilago esculenta is a uniquely flavored biotrophic smut fungus that forms a smut gall on the top internodal region of Zizania latifolia, a perennial wild rice found in the Indo-Burma biodiversity hotspot. The smut gall is an edible vegetable locally called "kambong" in Manipur, India. The life cycle of the fungus was studied in vitro and its biotrophism was observed during different stages of the plant growth starting from the bud stage to decaying stage using light, fluorescent and electron microscopy. The size of the smut gall and the number of internodes below the apical smut gall varied significantly (P < 0.05). Examination of various parts of infected plants using culture methods, microscopy and polymerase chain reaction revealed that Ustilago esculenta colonized Zizania latifolia in a non-systemic manner. Spores and fragmented hyphae of U. esculenta were present in the rhizome of infected plant throughout the year, but shoot interiors were without any fungal structures from April until September. The smut region of infected plants in early September to December were heavily sporulated with fragmented hyphae, while the nodal regions of infected plants had no spores and fragmented hyphae. Hyphae and spores were also absent in the internodes and membranes aboveground up to smut region of infected plants but were present in the old rhizomes.

Ustilago echinata: Infection in a Mixed Martial Artist Following an Open Fracture.

Ustilago, a common fungal parasite of grains, is infrequently isolated as a pathogen in humans. We describe a case of Ustilago echinata infection following an open distal tibia fracture, review the current literature of this genus as a cause of invasive fungal infection in humans, and discuss management issues.

Biodegradation of cefdinir by a novel yeast strain, Ustilago sp. SMN03 isolated from pharmaceutical wastewater.

Cefdinir, a semi-synthetic third generation cephalosporin antibiotic being considered as an emerging pollutant, demands removal from aquatic ecosystems. A yeast strain isolated from pharmaceutical wastewater which was identified as Ustilago sp. SMN03 by molecular techniques and was found to be capable of utilizing cefdinir as a sole carbon source. The isolate was found to degrade 81 % of cefdinir within 6 days under optimized conditions viz. pH 6.0, temperature 30 °C, a shaking speed of 120 rpm, an inoculum dosage of 4 % (w/v) and an initial cefdinir concentration of 200 mg L(-1). Kinetic studies revealed that cefdinir degradation followed the pseudo-first order model, a rate constant of 0.222 per day and a half-life period of 3.26 days. Using LC-MS analysis, six novel intermediates formed during the cefdinir degradation were identified and characterized. FT-IR analysis showed that the functional groups ranging from 1,766 to 1,519 cm(-1), characteristic for lactam ring were completely removed during the cefdinir degradation. The opening of the ß-lactam ring was one of the major steps in the cefdinir degradation process. Based on the results from the present study, a possible pathway of cefdinir degradation by Ustilago sp. SMN03 was proposed. To the best of our knowledge, this is the first report on microbial degradation of cefdinir by yeast.

Isolation and screening of glycolipid biosurfactant producers from sugarcane.

Forty-three fungal producers for glycolipid biosurfactants, mannosylerythritol lipids (MELs), were isolated from leaves and smuts of sugarcane plants. These isolates produced MELs with sugarcane juice as nutrient source. The strains were taxonomically categorized into the genera Pseudozyma and Ustilago on the basis of partial sequences of the ribosomal RNA gene.

Morphological and molecular differences in two strains of Ustilago esculenta.

Ustilago esculenta is a fungal endophyte of Zizania latifolia that plays an important agricultural role in this vegetable crop. The purpose of this study was to characterize sporidial (T) and mycelial (M-T) strains of U. esculenta isolated from sporulating and non-sporulating galls on plants growing in Zhejiang province, China. Morphological comparisons of the T strain and M-T strain were made by optical and scanning electron microscope observation. Genetic differences were examined by sequencing the ITS region of the fungus and examining differential protein expression by two-dimensional gel electrophoresis and MALDI-TOF-MS/MS. The sporidial (T) and mycelial (M-T) strains differed in morphological characteristics of their in vitro single colony formations and in cell shape. Alignment of ITS sequences of the T strain and M-T strain revealed a single mutation between the T strain and M-T strain, but the sequences were the same within strains. A total of 146 proteins were only expressed in the M-T strain, and 242 proteins were only expressed in the T strain isolated from infected plants. A total of 222 proteins were up-regulated or down-regulated in the T strain when compared with the M-T strain. Of these, 18 proteins were identified and eight were associated with processes involving energy metabolism and the cytoskeleton. Two morphology-related proteins, MAP kinase kinase and actin, were differentially expressed. The differences noted in the T strain and M-T strain may lead to a better understanding of the life cycle and morphogenesis in U. esculenta.

Isolation of a natural solopathogenic strain of Sporisorium reilianum f.sp. zeae (Ustilaginaceae, Basidiomycetes).

Sporisorium reilianum f.sp. zeae (Kühn) Langdon and Fullerton (Basidiomycota, Ustilaginaceae) is the causal agent of head smut of maize and sorghum. The parasitism is initiated by the fusion of two compatible sporidia which give rise to the formation of dikaryotic pathogen hyphae. However, in Ustilaginaceae, some fuzzy diploid strains could also be formed. These strains are solopathogen as they can infect a host in absence of crossing with a compatible haploid sporidia. A solopathogenic strain of S. refilianum was obtained using an original protocol. Sporidia were isolated from germinated teliospores and spread on solid medium to identify stable fuzzy solopathogenic strain. Confocal observations of the solopathogenic strain (SRZS1) after nucleus staining with propidium iodide indicates that they are formed by rounded shape cells which are monokaryotic. A CAPS approach was used to analysis the matb gene of S. reilianum. The presence of two matb loci in SRZS1 showed that this monocaryotic strain is diploid. The pathogenicity of SRZS1 was investigated by maize infection. Our results confirmed that SRZS1 is infectious, induces some typical symptoms in maize but could not sporulate and form sori.

Toxic effects of Ustilago maydis and fumonisin B1 in rats.

The toxicity of Ustilago maydis and the possible synergism with fumonisin B1 (FB1) were studied in Fischer rats by evaluating pathological changes and biochemical parameters in blood serum (LDH, ALT, GGT, ChE) and tissue homogenate of brain and liver (AChE, ChE, GGT, ALP). One experimental group (US) consumed diet with 70% of U. maydis galls and the other group (US+FB1) was fed pellets containing 70% of U. maydis galls and 1 mg of FB1 per kg of diet for 17 days. Control group (C) consumed standard pellets. During the trial, experimental animals were more excited, showing hyperactivity. Body mass gains slightly increased in both groups compared to the control. Gross pathological changes in liver, lungs, uterus and ovaries were more pronounced in the US+FB1 than in the US group. Specific catalytic activities of AChE decreased by 61% and by 63% in the liver and brain homogenate of the US group (p<0.05) compared to the control, indicating neurotoxic activity of U. maydis. Also, specific catalytic concentration of AChE and ALP was significantly decreased in the liver of the US+FB(1) group (p<0.05). Activity of LDH in the blood serum was increased up to 166% and 165% in the US+FB1 group (p<0.05) compared to the control and US group values, respectively, which indicates that FB1 was responsible for the disruption of cell membrane integrity. These findings suggest that Ustilago maydis and FB1 showed neurotoxicity in Fischer rats, which could be related to the alkaloids of U. maydis and disruption of sphingolipid metabolism by FB1 activity.

Use of PCR to detect infection of differentially susceptible maize cultivars using Ustilago maydis strains of variable virulence.

Ustilago maydis was specifically detected in infected maize plants by means of the polymerase chain reaction (PCR) using oligonucleotides corresponding to a specific region downstream of the homeodomain of the bE genes of the pathogen. The reaction gave rise to amplification of a ca. 500-bp product when tested with U. maydis DNA, but no amplification was detected with DNA from fungi not related to U. maydis. Using these primers, U. maydis was detected in infected maize plants from differentially susceptible cultivars as early as 4 days after inoculation with strains of variable degrees of virulence. Detection of U. maydis at early stages of infection, or in asymptomatic infected plants should assist in studies on plant-pathogen interactions.

The effect of meteorological factors on the daily variation of airborne fungal spores in Granada (southern Spain).

A study was made of the link between climatic factors and the daily content of certain fungal spores in the atmosphere of the city of Granada in 1994. Sampling was carried out with a Burkard 7-day-recording spore trap. The spores analysed corresponded to the taxa Alternaria, Ustilago and Cladosporium, with two morphologically different spore types in the latter genus, cladosporioides and herbarum. These spores were selected both for their allergenic capacity and for the high level of their presence in the atmosphere, particularly during the spring and autumn. The spores of Cladosporium were the most abundant (93.82% of the total spores identified). The Spearman correlation coefficients between the spore concentrations studied and the meteorological parameters show different indices depending on the taxon being analysed. Alternaria and Cladosporium are significantly correlated with temperature and hours of sunlight, while Ustilago shows positive correlation indices with relative humidity and negative indices with wind speed.

The Ustilago maydis regulatory subunit of a cAMP-dependent protein kinase is required for gall formation in maize.

In the plant, filamentous growth is required for pathogenicity of the corn smut pathogen Ustilago maydis. Earlier, we identified a role for the cAMP signal transduction pathway in the switch between budding and filamentous growth for this fungus. A gene designated ubc1 (for Ustilago bypass of cyclase) was found to be required for filamentous growth and to encode the regulatory subunit of a cAMP-dependent protein kinase (PKA). Here, we show that ubc1 is important for the virulence of the pathogen. Specifically, ubc1 mutants are able to colonize maize plants and, like the wild-type pathogen, cause localized symptoms in association with the presence of hyphae. However, in contrast to plants infected with wild-type cells that often developed galls from initially chlorotic tissue, plants infected with the ubc1 mutant did not produce galls. These data suggest that PKA regulation is critical for the transition from saprophytic to pathogenic growth and from vegetative to reproductive development. Plate mating assays in which exogenous cAMP was applied suggested that the cAMP and b mating-type morphogenetic pathways may be coordinated.

Nonsterol related resistance in Ustilago maydis to the polyene antifungals, amphotericin B and nystatin.

Two amphotericin B resistant mutants of Ustilago maydis were isolated following direct selection from a wild-type population. Each mutant was demonstrated to be cross-resistant to nystatin yet remained sensitive to azoles. Sterol analysis indicated a sterol profile similar to the parent strain, precluding the involvement of an alteration in ergosterol biosynthesis as the cause of polyene resistance.

A study of the aeromycoflora of Cádiz: relationship to anthropogenic activity.

We describe a quantitative and qualitative study of the fungal spores found in the air of Cádiz during 1989 using a Cour-type trap. The results of this study can be extrapolated to other coastal cities of southern Europe with a Mediterranean climate. The spores identified have been classified into 25 taxonomic categories. The most abundant were Cladosporium, Chaetomium and Ustilago, and the most frequent, in addition to those mentioned, were Alternaria, Ascophyta and Venturia. The great abundance of Cladosporium is in accordance with the coastal situation of the city. Cladosporium, Alternaria, Curvularia and Stemphylium reached maximum concentrations jointly in October, 1989. They showed mutual cross-reactions. Ustilago and Nigrospora appeared during the period of cereal harvesting and storage.

Induced mutagenesis in Ustilago maydis. I. Isolation and characterization of a radiation-revertible allele of the structural gene for nitrate reductase.

A UV-revertible mutant of the nar1 structural gene for nitrate reductase was isolated in wild-type (nar+ nir+) Ustilago maydis. It proved to be vigorously revertible by gamma rays as well. Genetic analysis revealed that the strain carried a single, nonleaky, recessive allele (nar1-m) with an unusually high spontaneous reversion rate (approximately 3 X 10(-5)/div.). Reliable reversion frequencies were determined with a special agar medium that reduced the normally high level of residual growth observed on nitrate minimal agar. Radiation-induced reversion frequencies in the homozygous diploid were approximately twice those in the haploid. Following crosses to wild type, two revertants (one spontaneous and one UV-induced) were found to map at nar1. Although the molecular basis of nar1-m reversion is not known, available data suggest that some form of point mutation is involved.

Induced mutagenesis in Ustilago maydis. II. An in vivo biochemical assay.

UV gamma radiation-induced reversion to nar+ in a nar1-m nir1-1 strain of Ustilago maydis was found to occur under nongrowth conditions by performing the in vivo assay for functional nitrate reductase described by Resnick and Holliday (1971) who previously demonstrated that nonviable cells may still synthesize normal or near-normal levels of activity. Reversion frequencies of a signle gamma-irradiated culture were estimated in two cell populations by different methods: (A) among surviving clones after plating, and (B) among all cells (viable and nonviable) in suspension in the absence of postirradiation cell division. At gamma doses (300, 500 krad) corresponding to considerable cell killing (35%, 2% survival), reversion frequency by either method was the same. This supports the conclusion that mutation induction by gamma rays and its expression occur in nonviable cells with the same frequency as among survivors. If an error-prone repair system is assumed to be responsible for the observed gamma revertibility, then it is argued that this process is constituitive rather than inducible and that it is recombination-independent.

Formation of protoplasts from Ustilago maydis.

Protoplasts of Ustilago maydis were obtained by incubating sporidia of the fungus with a combination of Helicase and a commercial "Onozuka" R-10 enzyme preparation of Trichoderma harzianum in the presence of 0.6 M (NH4)2 SO4 as an osmotic stabilizer. In the presence of the organic stabilizers sorbitol and sucrose, however, the release of protoplasts was inhibited. Combinations of Helicase with other lytic enzymes such as cellulase from Aspergillus niger, cellulase and hemicellulase from Rhizopus, and Driselase or Nagarse were inactive.