Differential gene expression during teliospore germination in <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Ustilago maydis is a model fungal pathogen that induces the formation of tumors in maize. The tumor provides an environment for hyphal differentiation, leading to the formation of thick-walled, diploid teliospores. Such spores serve as a dispersal agent for smut and rust fungi, and their germination leads to new rounds of infection. The morphological changes that occur during teliospore germination in U. maydis have been described in detail. However, the specific molecular events that facilitate this process have not been identified. Through the construction and hybridization of microarrays containing a set of 3918 non-redundant cDNAs, we have identified genes that are differentially regulated during teliospore germination. Teliospores induced to germinate for 4 and 11 h were selected for comparison with dormant teliospores. Genes identified as differentially expressed included many that are presumably involved in as yet undescribed molecular events during teliospore germination, as well as characterized genes previously shown to be required for the process. This study represents the first large-scale investigation of changes in gene expression during teliospore germination.Electronic Supplementary Material Supplementary material is available for this article at     

A comparative genomic analysis of ESTs from <Emphasis Type="Italic">Ustilago maydis</Emphasis>

A large-scale comparative genomic analysis of unisequence sets obtained from an Ustilago maydis EST collection was performed against publicly available EST and genomic sequence datasets from 21 species. We annotated 70% of the collection based on similarity to known sequences and recognized protein signatures. Distinct grouping of the ESTs, defined by the presence or absence of similar sequences in the species examined, allowed the identification of U. maydis sequences present only (1) in fungal species, (2) in plants but not animals, (3) in animals but not plants, or (4) in all three eukaryotic lineages assessed. We also identified 215 U. maydis genes that are found in the ascomycete but not in the basidiomycete genome sequences searched. Candidate genes were identified for further functional characterization. These include 167 basidiomycete-specific sequences, 58 fungal pathogen-specific sequences (including 37 basidiomycete pathogen-specific sequences), and 18 plant pathogen-specific sequences, as well as two sequences present only in other plant pathogen and plant species.Supplemental Excel Table 1 used for analysis and the derivation of Fig. 3 as well as supplemental Tables 2 and 3 are available at All ESTs used in this analysis have been submitted to GenBank. The accession numbers are CF638289–CF645747, CF663122–CF663127, and CD487847–CD490309 (Supplemental Table 3)     

Response to osmotic stress and temperature of the fungus <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Ustilago maydis is a fungal pathogen which is exposed during its life cycle to both abiotic and biotic stresses before and after the infection of maize. To cope with extreme environmental changes, microorganisms usually accumulate the disaccharide trehalose. We have investigated both the accumulation of trehalose and the activity of trehalase during the adaptation of U. maydis haploid cells to thermal, sorbitol, and NaCl stresses. Sorbitol and sodium chloride induced sustained accumulation of trehalose, while a transient increase was observed under heat stress. Sorbitol stressed cells showed higher trehalase activity compared with control cells and to those stressed by NaCl and high temperature. Addition of cycloheximide, a protein synthesis inhibitor, did not affect the trehalose accumulation during the first 15 min, but basal levels of trehalose were reached after the second period of 15 min. The proteomic analysis of the response of U. maydis to temperature, sorbitol, and salt stresses indicated a complex pattern which highlights the change of 18 proteins involved in carbohydrate and amino acid metabolism, protein folding, redox regulation, ion homeostasis, and stress response. We hypothesize that trehalose accumulation during sorbitol stress in U. maydis might be related to the adaptation of this organism during plant infection.     

Identification and characterization of secreted and pathogenesis-related proteins in <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Interactions between plants and fungal pathogens require a complex interplay at the plant–fungus interface. Extracellular effector proteins are thought to play a crucial role in establishing a successful infection. To identify pathogenesis-related proteins in Ustilago maydis we combined the isolation of secreted proteins using a signal sequence trap approach with bioinformatic analyses and the subsequent characterization of knock-out mutants. We identified 29 secreted proteins including hydrophobins and proteins with a repetitive structure similar to the repellent protein Rep1. Hum3, a protein containing both, a hydrophobin domain and a repetitive Rep1-like region, is shown to be processed during passage through the secretory pathway. While single knock-outs of hydrophobin or repellent-like genes did not affect pathogenicity, we found a strong effect of a double knock-out of hum3 and the repetitive rsp1. Yeast-like growth, mating, aerial hyphae formation and surface hydrophobicity were unaffected in this double mutant. However, pathogenic development in planta stops early after penetration leading to a complete loss of pathogenicity. This indicates that Hum3 and Rsp1 are pathogenicity proteins that share an essential function in early stages of the infection. Our results demonstrate that focusing on secreted proteins is a promising way to discover novel pathogenicity proteins that might be broadly applied to a variety of fungal pathogens.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Identification of plant-regulated genes in<Emphasis Type="Italic"> Ustilago maydis</Emphasis> by enhancer-trapping mutagenesis

To identify plant-induced genes in the maize pathogenic fungus Ustilago maydis we have developed a genetic screen that combines REMI (restriction enzyme mediated integration) mutagenesis with enhancer trapping using the gene for Green Fluorescent Protein (GFP) as vital reporter. Of 2350 insertion mutants isolated, three were shown to express GFP only after the fungus had come into contact with the host maize plant. One of the genes tagged was mfa1, which encodes the pheromone precursor, while the second gene, pig2 , codes for a product that showed similarity to protein disulfide isomerase. The third integration event had occurred in a locus which we designated the p -locus. This locus contains 11 genes in a 24-kb stretch. Of these, pig3 , 4 , 5 , 6 and 7 show a plant-regulated expression pattern, while the other genes found at the locus (designated npi) do not. Of the plant-regulated genes only two were found to be similar to database entries: the pig4 product is related to membrane transporters of the major facilitator family, while the pig6 protein shows similarity to multidrug transporters. Detailed expression studies revealed that the five plant-regulated genes at the p -locus differ in their expression profiles. Mutants deleted for each of them showed no apparent phenotype, while the npi1 gene appeared to be essential. A viable deletion encompassing the entire p -locus could be generated when npi1 function was provided ectopically. This deletion mutant also showed no obvious alteration in virulence.Communicated by C. P. HollenbergThe first three authors contributed equally to this work     

The short form of the recombinant CAL-A-type lipase UM03410 from the smut fungus <Emphasis Type="Italic">Ustilago maydis</Emphasis> exhibits an inherent <Emphasis Type="Italic">trans</Emphasis>-fatty acid selectivity

The Ustilago maydis lipase UM03410 belongs to the mostly unexplored Candida antarctica lipase (CAL-A) subfamily. The two lipases with […] the highest identity are a lipase from Sporisorium reilianum and the prototypic CAL-A. In contrast to the other CAL-A-type lipases, this hypothetical U. maydis lipase is annotated to possess a prolonged N-terminus of unknown function. Here, we show for the first time the recombinant expression of two versions of lipase UM03410: the full-length form (lipUMf) and an N-terminally truncated form (lipUMs). For comparison to the prototype, the expression of recombinant CAL-A in E. coli was investigated. Although both forms of lipase UM03410 could be expressed functionally in E. coli, the N-terminally truncated form (lipUMs) demonstrated significantly higher activities towards p-nitrophenyl esters. The functional expression of the N-terminally truncated lipase was further optimized by the appropriate choice of the E. coli strain, lowering the cultivation temperature to 20 °C and enrichment of the cultivation medium with glucose. Primary characteristics of the recombinant lipase are its pH optimum in the range of 6.5–7.0 and its temperature optimum at 55 °C. As is typical for lipases, lipUM03410 shows preference for long chain fatty acid esters with myristic acid ester (C14:0 ester) being the most preferred one. More importantly, lipUMs exhibits an inherent preference for C18:1Δ9 trans and C18:1Δ11 trans-fatty acid esters similar to CAL-A. Therefore, the short form of this U. maydis lipase is the only other currently known lipase with a distinct trans-fatty acid selectivity.     

Absence of repellents in <Emphasis Type="Italic">Ustilago maydis</Emphasis> induces genes encoding small secreted proteins

The rep1 gene of the maize pathogen Ustilago maydis encodes a pre-pro-protein that is processed in the secretory pathway into 11 peptides. These so-called repellents form amphipathic amyloid fibrils at the surface of aerial hyphae. A SG200 strain in which the rep1 gene is inactivated (∆rep1 strain) is affected in aerial hyphae formation. We here assessed changes in global gene expression as a consequence of the inactivation of the rep1 gene. Microarray analysis revealed that only 31 genes in the ∆rep1 SG200 strain had a fold change in expression of ≥2. Twenty-two of these genes were up-regulated and half of them encode small secreted proteins (SSPs) with unknown functions. Seven of the SSP genes and two other genes that are over-expressed in the ∆rep1 SG200 strain encode proteins that can be classified as secreted cysteine-rich proteins (SCRPs). Interestingly, most of the SCRPs are predicted to form amyloids. The SCRP gene um00792 showed the highest up-regulation in the ∆rep1 strain. Using GFP as a reporter, it was shown that this gene is over-expressed in the layer of hyphae at the medium-air interface. Taken together, it is concluded that inactivation of rep1 hardly affects the expression profile of U. maydis, despite the fact that the mutant strain has a strong reduced ability to form aerial hyphae.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Purification and Characterization of an Extracellular Non-Aspartyl Acid Protease (pumAe) from <Emphasis Type="Italic">Ustilago maydis</Emphasis>

The proteinase pumAe was purified to homogeneity from haploid U. maydis FB1 growing on acid mineral medium. The purification procedure consisted of ammonium sulfate fractionation and gel filtration chromatography, resulting in a 7.7% recovery and a 15.1-fold increase in specific activity. The molecular weight of the enzyme was estimated to be 72 kDa and 74 kDa by gel filtration chromatography and SDS-PAGE, respectively. Enzymatic activity was optimal at pH 4.0 and at 45°C toward hemoglobin, and the pI was determined to be 5.5. The effects of six protease inhibitors on pumAe were tested, and no inhibitory effect was observed. The pure enzyme degraded gelatin and albumin, but casein and collagen were not degraded. The K_m value was 3.5 M, and the V_max value was 11430 mol h^–1 mg^–1 for Suc-R-P-F-H-L-L-V-Y-MCA.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Pedogamy in <Emphasis Type="Italic">Neidium</Emphasis> (<Emphasis Type="Italic">Bacillariophyceae</Emphasis>)

Single (unpaired) vegetative cells of freshwater pennate diatom Neidium cf. ampliatum differentiated into gametangia and produced a single zygote (auxospore) via a pedogamic process. The gametic nuclei fused after auxospore expansion had begun. The auxospore expanded in parallel to the apical axis of the gametangium.     

Replication timing of large <Emphasis Type="Italic">Sorex granarius</Emphasis> (<Emphasis Type="Italic">Soricidae</Emphasis>, <Emphasis Type="Italic">Eulipotyphla</Emphasis>) telomeres

Previously, we described the unique feature of telomeric regions in Iberian shrew Sorex granarius: its telomeres have two ranges of size, very small (3.8 kb of telomeric repeats on average) and very large discontinuous telomeres (213 kb) interrupted with 18S rDNA. In this study, we have demonstrated extraordinary replication pattern of S. granarius large telomeres that have not been shown before in other studied mammal. Using the ReD-FISH procedure, we observed prolonged, through S period, large telomere replication. Furthermore, revealed ReD-FISH asymmetric signals were probably caused by partial replication of telomeres within an hour of 5-bromodeoxyuridine treatment due to the large size and special organization. We also found that in contrast to the telomeric halo from primary fibroblasts of bovine, mink, and common shrew, telomere halo of S. granarius consists of multiple loops bundled together, some of which contain rDNA. Here, we suggested several replicons firing possibly stochastic in each large telomere. Finally, we performed the TIF assay to reveal DNA damage responses at the telomeres, and along with TIF in nuclei, we found large bodies of telomeric DNA and ?-H2AX in the cytoplasm and on the surface of fibroblasts. We discuss the possibility of additional origin activation together with recombination-dependent replication pathways, mainly homologous recombination including BIR for replication fork stagnation overcoming and further S. granarius large telomere replication.     

Three new species of <Emphasis Type="Italic">Stigmidium</Emphasis> and <Emphasis Type="Italic">Sphaerellothecium</Emphasis> (lichenicolous ascomycetes) on <Emphasis Type="Italic">Stereocaulon</Emphasis>

Sphaerellothecium stereocaulorum sp. nov., Stigmidium beringicum sp. nov., Stigmidium stereocaulorum sp. nov. and goniocysts are described on Stereocaulon species from the northern Holarctic. Endococcus nanellus is reported new to Alaska and Mongolia. Taxonomical novelties Sphaerellothecium stereocaulorum Zhurb. & Triebel, Stigmidium beringicum Zhurb. & Triebel, Stigmidium stereocaulorum Zhurb. & Triebel.     

Tissue culture of <Emphasis Type="Italic">Sinningia speciosa</Emphasis> and analysis of the <Emphasis Type="Italic">in vitro</Emphasis>-generated <Emphasis Type="Italic">tricussate whorled phyllotaxis</Emphasis> (<Emphasis Type="Italic">twp</Emphasis>) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l⁻¹ NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.