Anther-smut infection of Silene alba caused by Ustilago violacea: factors determining fungal reproduction

Summary The anther-smut fungus Ustilago violacea sporulates in flowers of the dioecious host plant Silene alba. Growth chamber comparisons of healthy and diseased plants, with the genetic background of host and pathogen controlled, revealed that fungal infection increases the number of flowers produced per plant and decreases the size of individual flowers. There were few consistent effects of plant genotype or fungal isolate on diseased flower traits, but differences between the plant sexes were apparent. Stimulation of flower production is proportionally greater in females than males: thus, although healthy male plants produce many more flowers than healthy females, sexual differences in diseased flower number are reduced. Sexual differences in diseased flower size also exist, with male flowers smaller than females. A field inoculation study confirmed dimorphism in diseased flower size and demonstrated that spore production per flower was greater for males than females for all flower size classes.     

Use of Internal Transcribed Spacer Primers and Fungicide Treatments to Study the Anther-Smut Disease, Microbotryum violaceum (=Ustilago violacea), of White Campion Silene alba (=Silene latifolia)

We report the construction and use of polymerase chain reaction (PCR) primers for detection of Microbotryum violaceum in Silene alba. Such primers show that the pathogen is present in the aboveground bud meristems, flower pedicels, and flower-stem internodes but not in the flowering-stem leaves and roots of flowering plants. Use of primers also showed that the pathogen was absent from plants that had been inoculated with the fungus but remained healthy. The fungicides Folicur and Bayleton cured plants of the disease, and the absence of the fungus from such cured plants was confirmed using the PCR primers.     

Acquisition of mitochondrial DNA by a transformation vector for Ustilago violacea

Plasmid pUCH1 is a 5.2-kb pUC18 construct bearing the hygB gene fused to a promoter from Cochliobolus heterostrophus. Haploid cells of the basidiomycete, Ustilago violacea, were transformed with this plasmid. In addition to multiple integrations of plasmid sequences into U. violacea nuclear DNA, vector sequences independent of the nuclear genome were indicated by Southern-blot analysis using all or part of pUCH1 as a probe. Hybridization also revealed intact pUCH1 and several larger derivatives in satellite bands from CsCl-bis-benzamide gradients of whole cellular DNA and in DNA from purified mitochondria [mitochondrial (mt) DNA preparations] of transformed U. violacea; circular DNAs consistent with the sizes of DNAs in these satellite bands were seen in electron microscope analyses of the same mt DNA preparations as well. The plasmids could be detected in mt DNA preparations even after 30 generations of transformant growth under selective pressure. Transformation of Escherichia coli by these mt DNA preparations produced bacterial transformants bearing intact pUCH1, as well as several pUCH1 derivatives, including pUCH2, an approx. 8.0-kb plasmid. A 2.5-kb EcoRI fragment from pUCH2 showed only weak hybridization with pUCH1. This unique fragment did hybridize strongly with mt DNA from untransformed U. violacea. This derivative thus appears to have acquired mt sequences from U. violacea.     

Myosin-V, Kinesin-1, and Kinesin-3 cooperate in hyphal growth of the fungus Ustilago maydis

Long-distance transport is crucial for polar-growing cells, such as neurons and fungal hyphae. Kinesins and myosins participate in this process, but their functional interplay is poorly understood. Here, we investigate the role of kinesin motors in hyphal growth of the plant pathogen Ustilago maydis. Although the microtubule plus-ends are directed to the hyphal tip, of all 10 kinesins analyzed, only conventional kinesin (Kinesin-1) and Unc104/Kif1A-like kinesin (Kinesin-3) were up-regulated in hyphae and they are essential for extended hyphal growth. deltakin1 and deltakin3 mutant hyphae grew irregular and remained short, but they were still able to grow polarized. No additional phenotype was detected in deltakin1rkin3 double mutants, but polarity was lost in deltamyo5rkin1 and deltamyo5rkin3 mutant cells, suggesting that kinesins and class V myosin cooperate in hyphal growth. Consistent with such a role in secretion, fusion proteins of green fluorescent protein and Kinesin-1, Myosin-V, and Kinesin-3 accumulate in the apex of hyphae, a region where secretory vesicles cluster to form the fungal Spitzenk?rper. Quantitative assays revealed a role of Kin3 in secretion of acid phosphatase, whereas Kin1 was not involved. Our data demonstrate that just two kinesins and at least one myosin support hyphal growth.     

Genetics of Ustilago violacea. XXXII. Genetic evidence for transposable elements

Crosses between Ustilago violacea mutant strains with different color phenotypes that were derived from the 1.A1 and 2.A2 laboratory strains yielded, as expected, bisectored teliospore colonies with the parental colors as well as the a-1 and the a-2 mating-types. Generally, wild teliospore collections usually produced sporidia of both mating-types, providing two-mating-type (TMT) strains. Occasionally, however, sporidia with only one mating-type allele, a-1 or a-2, were obtained from teliospores, providing one-mating-type (OMT) strains. Crosses between OMT and laboratory strains with different color phenotypes gave (1) bisectored teliospore colonies with the parental colors or colonies with a parental color and a nonparental color and (2) nonsectored colonies with the nonparental color or with the parental color. The frequencies for the occurrence of non-parental color ranged from 41% to 93%, depending on the strain. The yield of teliospore colonies was usually reduced for these crosses. In many of these teliospore colonies, morphologically-altered sporidia (MAS phenotype) were observed. The morphology and the size of the sporidia with the MAS phenotype differed from those of teliospore colonies of the crosses between the laboratory strains. In addition, these sporidia did not form conjugants. A cross involving the TMT strains C449 yielded the MAS phenotype as well as a high incidence of tetrad colonies with a nonparental color. The high degree of instability of the parental color phenotypes, and the high frequency of the appearance of nonparental color phenotypes as well as the appearance of the MAS phenotype, are in accord with the presence of active and inactive transposable elements in the OMT strains, TMT strains, and laboratory strains.     

A novel class of small amphipathic peptides affect aerial hyphal growth and surface hydrophobicity in Ustilago maydis.

Ustilago maydis, a fungal pathogen of corn, can alternate between yeast-like and filamentous growth. This dimorphic switch is governed by the mating-type loci. We have identified an abundant class of small SDS-insoluble cell wall proteins, designated repellents, specifically present in the filamentous form. Genetic analysis revealed that these peptides are processed from a single precursor protein, Rep1. Rep1 comprises 652 amino acids with a leader sequence for secretion. A characteristic feature of Rep1 is 12 repeats of a 37 amino acid consensus sequence; 10 of these repeats are separated by Kex2 protease cleavage sites. In (delta)rep1 mutants formation of aerial hyphae and surface hydrophobicity were reduced dramatically. This and the fact that expression of rep1 is regulated by the mating-type loci indicates that repellents play a structural role in the formation of aerial hyphae.     

Diurnal and nocturnal pollination of Silene alba (Caryophyllaceae)

Flowers that are open for >12 h may be visited by both diurnal and nocturnal pollinators. I compared the effectiveness (measured as seed production and pollen movement distance) of diurnal and nocturnal pollinators of Silene alba, a species whose flowers open in evening but close by midmorning the following day. By bagging flowers either during evening hours or during daylight hours or both day and night, I compared seed production caused by diurnal and nocturnal pollinators. Flowers exposed only to nocturnal visitors (mostly sphingid and noctuid moths) produced significantly more seeds than flowers exposed only to diurnal visitors (bees, flies, and wasps). Fluorescent dye applied to anthers moved significantly further and to more stigmas at night than during the day. In both measures of pollination effectiveness, nocturnal-visiting moths are better pollinators of S. alba than are the diurnal-visiting bees, flies, and wasps. These data support the hypothesis that floral phenology is an adaptation to expose flowers to the most effective pollinators.     

A class-V myosin required for mating, hyphal growth, and pathogenicity in the dimorphic plant pathogen Ustilago maydis

In the early stages of plant infection, yeast-like haploid sporidia of Ustilago maydis respond to pheromone secreted by compatible partners by forming conjugation tubes. These then fuse to generate a dikaryotic hypha that forms appressoria to penetrate the host plant. As a first step toward understanding the structural requirements for these transitions, we have identified myo5, which encodes a class-V myosin. Analysis of conditional and null mutants revealed that Myo5 plays nonessential roles in cytokinesis and morphogenesis in sporidia and is required for hyphal morphology. Consistent with a role in morphogenesis, a functional green fluorescent protein-Myo5 fusion protein localized to the bud tip and the hyphal apex as well as to the septa and the spore wall during later stages of infection. However, the loss of Myo5 did not affect the tip growth of hyphae and sporidia. By contrast, Myo5 was indispensable for conjugation tube formation. Furthermore, myo5 mutants were impaired in the perception of pheromones, which indicates a particular importance of Myo5 in the mating process. Consequently, few mutant hyphae were formed that penetrated the plant epidermis but did not continue invasive growth. These results indicate a crucial role of Myo5 in the morphogenesis, dimorphic switch, and pathogenicity of U. maydis.     

Somatic nuclear division in the sporidia of Ustilago violacea. III. Ultrastructural observations.

The paper provides detailed ultrastructural observations on nuclear division in the smut fungus Ustilago violacea and is based on previous light-microscopic work outlining the division in living and stained cells. The division as in many other Basidiomycetes is not intranuclear, but occurs within a partially disrupted membrane. The division takes place after migration of most of the nucleus into the bud cell, after limited breakdown of the nuclear membrane, and after the formation of a spindle between two spindle-pole bodies (SPB). The remaining part of the nucleus containing the nucleolus is left behind in the parent cell and degenerates there. The SPB, as in other Basidiomycetes, is a dome-shaped relatively structureless body, quite distinct from the flat plaques of many Ascomycetes and the elaborat centrioles of Phycomycetes. The SPB divides shortly before migration into the daughter cell and invariably is located at the apex of the migrating nucleus. Nuclear division is completed when the two masses of chromatin clustered about each of the SPB's are separated as the spindle elongates. One daughter neculeus reforms in the bud and the other is reformed in the mother cell. Cells fixed and stained by conventional light-microscopic methods were examined in the light of the electron-microscopic observations to determine whether these procedures induce artefacts. Aceto-orcein and Giemsa when used cold were found to produce relatively artefact-free preparations. However, previous results in which the cells were warmed gently in these stains are now seen to contain artefacts in the form of contracted chromatinic granules often arranged in chains. These artefacts may provide useful information but clearly they must be interpreted cautiously until the nature of the changes induced by heating are known.     

Genetics of Ustilago violacea. XXXV. Transposition in Haploid and Diploid Sporidia and Germinating Teliospores

Ustilago violacea sporidia of the white (w) MAD strain (a-2 w lys-3 ino-1 thi) incubated on minimal medium containing 100 mM KClO3 (potassium chlorate) produced only colonies with the pink phenotype. Sporidia from these colonies retained their pink color on complex medium. Sporidia of the diploid D1 strain (a-1 y nic-1 thi/a-2 w met-1 arg-f Chl70 thi) and of the diploid D2 strain (a-1 y his-1 glu-1 thi/a-2 w lys-3 ino-1 thi) produced pink colonies on complex medium. Streaks of diploid D1 sporidia from the pink colonies were stable on complex medium. In contrast, streaks of diploid D2 sporidia, which are heterozygous for the MAD strain, were unstable, initially producing pink colonies on complex medium but then, with continued incubation, producing white termini. Sporidia from the white termini with diploid morphology continued to yield white colonies. Teliospore colonies from three crosses with the MAD strain as a common parent were uniformly pink or had a pink sector instead of the expected uniformly white colonies or colonies having a white sector. Four of 20 and 13 of 20 teliospore colonies, respectively, from two of the three crosses had both a-1 and a-2 sporidia, and the remaining colonies had only a-1 or only a-2 sporidia. All 40 teliospore colonies from the third cross had only a-1 or only a-2 sporidia. All of these observations indicated that the MAD strain may have two autonomous, transactive transposable elements in different chromosomes and that insertional mutations in at least two haplolethal loci were responsible for the teliospore colonies with only a-1 or only a-2 mating type. Crossing over between a haplolethal locus and the centromere would account for teliospore colonies with both a-1 and a-2 sporidia.     

The effects of stigma age on receptivity in Silene alba (Caryophyllaceae)

Silene alba, a perennial, dioecious plant, produces flowers that open in the evening and can remain open and receptive to pollination for up to 5 d, though in hot and dry conditions the flowers will wilt during the day only to reopen night after night. In the field, it is visited by two different kinds of pollinators with differential success: moths visit the flowers at night, and their movements result in broad pollen dispersal and large seed production, whereas bees, wasps, and flies visit the flowers in the mornings and have decreased pollination effectiveness. However, this differential success may be due to a decrease in stigmatic receptivity soon after the flowers open. We performed controlled pollinations to determine the effect of stigma age on pollen germination and seed set. We pollinated flowers at 12-h intervals up to 120 h and divided these into two sets: from one set, we removed stigmas 24 h after pollination to examine percentage of pollen germination. The second set of flowers was allowed to produce fruits, and the seeds were counted and weighed. Pollen germination declined significantly with stigma age, but there was no significant effect of stigma age at pollination on the number or mass of resulting seeds. Thus, the decreased pollination success of bees is not due to a decrease in stigmatic receptivity but is most likely a result of pollinator inefficiency.     

Microtubules are dispensable for the initial pathogenic development but required for long-distance hyphal growth in the corn smut fungus Ustilago maydis

Fungal pathogenicity often involves a yeast-to-hypha transition, but the structural basis for this dimorphism is largely unknown. Here we analyze the role of the cytoskeleton in early steps of pathogenic development in the corn pathogen Ustilago maydis. On the plant yeast-like cells recognize each other, undergo a cell cycle arrest, and form long conjugation hyphae, which fuse and give rise to infectious filaments. F-actin is essential for polarized growth at all these stages and for cell-cell fusion. Furthermore, F-actin participates in pheromone secretion, but not perception. Although U. maydis contains prominent tubulin arrays, microtubules are neither required for cell-cell recognition, nor for cell-cell fusion, and have only minor roles in morphogenesis of yeast-like cells. Without microtubules hyphae are formed, albeit at 60% reduced elongation rates, but they reach only approximately 50 mum in length and the nucleus fails to migrate into the hypha. A similar phenotype is found in dynein mutants that have a nuclear migration defect and stop hyphal elongation at approximately 50 mum. These results demonstrate that microtubules are dispensable for polarized growth during morphological transition, but become essential in long-distance hyphal growth, which is probably due to their role in nuclear migration.     

Biomechanical models of hyphal growth in actinomycetes

The tip growth of filamentary actinomycetes is investigated within the framework of large deformation membrane theory in which the cell wall is represented as a growing elastic membrane with geometry-dependent elastic properties. The model exhibits realistic hyphal shapes and indicates a self-similar tip growth mechanism consistent with that observed in experiments. It also demonstrates a simple mechanism for hyphal swelling and beading that is observed in the presence of a lysing agent.