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Population differentiation and recombination in wheat scab populations of Gibberella zeae from the United States.

In limited previous studies of the Ascomycete fungus Gibberella zeae in North America, the populations examined were genetically and phenotypically diverse and could be viewed as subsamples of a larger population. Our objective in this study was to test the hypothesis that a homogeneous, randomly mating population of G. zeae is contiguous throughout the central and eastern United States across a span of several years. We analysed presence/absence alleles based on amplified fragment length polymorphisms (AFLPs) at 30 loci, 24 of which are defined genetically on a linkage map of G. zeae, from > 500 isolates in eight field populations from seven states collected during the 1998, 1999 and 2000 cropping seasons. All these strains had AFLP profiles similar to those of standard isolates of G. zeae phylogenetic lineage 7. All the populations are genetically similar, have high genotypic diversity and little or no detectable genetic disequilibrium, and show evidence of extensive interpopulation genetic exchange. Allele frequencies in some of the populations examined are not statistically different from one another, but others are. Thus, the populations examined are not mere subsamples from a single, large, randomly mating population. Geographic distance and genetic distance between populations are correlated significantly. The observed differences are relatively small, however, indicating that while genetic isolation by distance may occur, genetic exchange has occurred at a relatively high frequency among US populations of G. zeae. We think that these differences reflect the time required for the alleles to diffuse across the distances that separate them, because relatively little linkage disequilibrium is detected either in the population as a whole or in any of the individual subpopulations.

Gene Frequency↗

Type IV pili are involved in plant-microbe and fungus-microbe interactions.

Adherence of bacteria to eukaryotic cells is essential for the initiation of infection in many animal and human pathogens, e.g. Neisseria gonorrhoeae and Pseudomonas aeruginosa. Adhesion-mediating type IV pili, filamentous surface appendages formed by pilin subunits, are crucial virulence factors. Here, we report that type IV pilus-dependent adhesion is also involved in plant-bacteria and fungus-bacteria interactions. Nitrogen-fixing, endophytic bacteria, Azoarcus sp., can infect the roots of rice and spread systemically into the shoot without causing symptoms of plant disease. Formation of pili on solid media was dependent on the pilAB locus. PilA encodes an unusually short (6.4 kDa) putative pilin precursor showing 100% homology to the conserved N-terminus of the Pseudomonas aeruginosa type IV pilin. PilB encodes for a 14.2 kDa polypeptide showing similarity to FimF, a component of type I fimbriae of Escherichia coli. It was found to be extruded beyond the cell surface by immunofluorescence studies, and it may, therefore, be part of a pilus assembly complex or the pilus itself. Both genes are involved in the establishment of bacteria on the root surface of rice seedlings, as detected by fluorescence microscopy. Moreover, both genes are necessary for bacterial adhesion to the mycelium of an ascomycete, which was isolated from the same rhizosphere as the bacteria. In co-culture with the fungus, Azoarcus sp. forms complex intracytoplasmic membranes, diazosomes, which are related to efficient nitrogen fixation. Adhesion to the mycelium appears to be crucial for this process, as diazosomes were absent and nitrogen fixation rates were decreased in pilAB mutants in co-culture.

Amino Acid Sequence↗

Blue light adaptation and desensitization of light signal transduction in Neurospora crassa.

The ascomycete Neurospora crassa has the capacity of adapting to a given light quantity, leading to transient blue light responses under continuous light conditions. Here, we present an investigation of this photoadaptation phenomenon. We demonstrated previously that two proteins of the Neurospora blue light signal transduction chain, WC1 and WC2, are subject to light-dependent phosphorylation. WC1 was phosphorylated in parallel with the transient increase in transcript levels of light-regulated genes. Using the light-dependent phosphorylation of WC1 as a marker for an active signalling state of WC1, we show that the transiency of Neurospora blue light responses results from desensitization of the photoreceptor and/or the signalling cascade. Furthermore, a Neurospora mutant was characterized that revealed a specific defect in photoadaptation. In this mutant, the transient expression of light-regulated genes under continuous light, the temporary insensitivity after a light pulse and the capability of differentiating between and adapting to low and high light intensities were abolished. The corresponding protein seems to represent a central component of a negative feedback desensitization mechanism. This negative feedback regulation requires continuous and light-dependent protein de novo biosynthesis.

Adaptation, Physiological↗

The nsdD gene encodes a putative GATA-type transcription factor necessary for sexual development of Aspergillus nidulans.

The ability to reproduce both sexually and asexually is one of the characteristics of the homothalic ascomycete Aspergillus nidulans. Unlike the other Aspergillus species, A. nidulans undergoes sexual development that seems to be regulated by internal and external stimuli. To begin to understand the sexual reproduction of A. nidulans we previously isolated and characterized several NSD (never in sexual development) mutants that failed to produce any sexual reproductive organs, and identified four complementation groups, nsdA, nsdB, nsdC, and nsdD. The nsdD gene has been isolated, and it is predicted to encode a GATA-type transcription factor with the type IVb zinc finger DNA-binding domain. The mRNA of the nsdD gene started to accumulate in the early phase of vegetative growth, and the level increased as sexual development proceeded. However, it decreased during asexual sporulation and no nsdD mRNA was detected in conidia. Deletion of nsdD resulted in no cleistothecia (fruiting bodies) formation, even under the conditions that preferentially promoted sexual development, indicating that nsdD is necessary for sexual development. In contrast, when the nsdD gene was over-expressed, sexual-specific organ (Hülle cell) was formed even in submerged culture, which normally completely blocked sexual development, and the number of cleistothecia was also dramatically increased on solid medium. These results lead us to propose that the nsdD gene functions in activating sexual development of A. nidulans. Multiple copies of the nsdD gene could suppress nsdB5 and veA1, indicating that either nsdD acts downstream of these genes or possibly functions in overlapping pathway(s).

Aspergillus nidulans↗

CPMK2, an SLT2-homologous mitogen-activated protein (MAP) kinase, is essential for pathogenesis of Claviceps purpurea on rye: evidence for a second conserved pathogenesis-related MAP kinase cascade in phytopathogenic fungi.

Cpmk2, encoding a mitogen-activated protein (MAP) kinase from the ascomycete Claviceps purpurea, is an orthologue of SLT2 from Saccharomyces cerevisiae, the first isolated from a biotrophic, non-appressorium-forming pathogen. Deletion mutants obtained by a gene replacement approach show impaired vegetative properties (no conidiation) and a significantly reduced virulence, although they retain a limited ability to colonize the host tissue. Increased sensitivity to protoplasting enzymes indicates that the cell wall structure of the mutants may be altered. As the phenotypes of these mutants are similar to those observed in strains of the rice pathogen, Magnaporthe grisea, that have been deprived of their MAP kinase gene mps1, the ability of cpmk2 to complement the defects of delta mps1 was investigated. Interestingly, the C. purpurea gene, under the control of its own promoter, was able to complement the M. grisea mutant phenotype: transformants were able to sporulate and form infection hyphae on onion epidermis and were fully pathogenic on barley leaves. This indicates that, despite the differences in infection strategies, which include host and organ specificity, mode of penetration and colonization of host tissue, CPMK2/MPS1 defines a second MAP kinase cascade (after the Fus3p/PMK1 cascade) essential for fungal pathogenicity.

Claviceps↗

The siderophore system is essential for viability of Aspergillus nidulans: functional analysis of two genes encoding l-ornithine N 5-monooxygenase (sidA) and a non-ribosomal peptide synthetase (sidC).

The filamentous ascomycete A. nidulans produces two major siderophores: it excretes triacetylfusarinine C to capture iron and contains ferricrocin intracellularly. In this study we report the characterization of two siderophore biosynthetic genes, sidA encoding l-ornithine N(5)-monooxygenase and sidC encoding a non-ribosomal peptide synthetase respectively. Disruption of sidC eliminated synthesis of ferricrocin and deletion of sidA completely blocked siderophore biosynthesis. Siderophore-deficient strains were unable to grow, unless the growth medium was supplemented with siderophores, suggesting that the siderophore system is the major iron assimilatory system of A. nidulans during both iron depleted and iron-replete conditions. Partial restoration of the growth of siderophore-deficient mutants by high concentrations of Fe(2+) (but not Fe(3+)) indicates the presence of an additional ferrous transport system and the absence of an efficient reductive iron assmilatory system. Uptake studies demonstrated that TAFC-bound iron is transferred to cellular ferricrocin whereas ferricrocin is stored after uptake. The siderophore-deficient mutant was able to synthesize ferricrocin from triacetylfusarinine C. Ferricrocin-deficiency caused an increased intracellular labile iron pool, upregulation of antioxidative enzymes and elevated sensitivity to the redox cycler paraquat. This indicates that the lack of this cellular iron storage compound causes oxidative stress. Moreover, ferricrocin biosynthesis was found to be crucial for efficient conidiation.

Amino Acid Sequence↗

Purification and properties of hydroquinone hydroxylase, a FAD-dependent monooxygenase involved in the catabolism of 4-hydroxybenzoate in Candida parapsilosis CBS604.

The ascomycetous yeast Candida parapsilosis CBS604 catabolizes 4-hydroxybenzoate through the initial formation of hydroquinone (1, 4-dihydroxybenzene). High levels of hydroquinone hydroxylase activity are induced when the yeast is grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, 1,3-dihydroxybenzene or 1, 4-dihydroxybenzene as the sole carbon source. The monooxygenase constitutes up to 5% of the total amount of protein and is purified to apparent homogeneity in three chromatographic steps. Hydroquinone hydroxylase from C. parapsilosis is a homodimer of about 150 kDa with each 76-kDa subunit containing a tightly noncovalently bound FAD. The flavin prosthetic group is quantitatively resolved from the protein at neutral pH in the presence of chaotropic salts. The apoenzyme is dimeric and readily reconstituted with FAD. Hydroquinone hydroxylase from C. parapsilosis catalyzes the ortho-hydroxylation of a wide range of monocyclic phenols with the stoichiometric consumption of NADPH and oxygen. With most aromatic substrates, no uncoupling of hydroxylation occurs. Hydroxylation of monofluorinated phenols is highly regiospecific with a preference for C6 hydroxylation. Binding of phenol highly stimulates the rate of flavin reduction by NADPH. At pH 7.6, 25 degrees C, this step does not limit the rate of overall catalysis. During purification, hydroquinone hydroxylase is susceptible towards limited proteolysis. Proteolytic cleavage does not influence the enzyme dimeric nature but results in relatively stable protein fragments of 55, 43, 35 and 22 kDa. N-Terminal peptide sequence analysis revealed the presence of two nick sites and showed that hydroquinone hydroxylase from C. parapsilosis is structurally related to phenol hydroxylase from Trichosporon cutaneum. The implications of these findings for the catalytic mechanism of hydroquinone hydroxylase are discussed.

Amino Acid Sequence↗

The determination of the partial 18 S ribosomal DNA sequences of Cordyceps species.

Cordyceps species, which are used in Chinese traditional medicines, are fungal parasites of insects. In this study the partial nucleotide sequences of 18 S ribosomal DNA from four Cordyceps species were determined and compared with the sequences of published ascomycetes. The sequence data support the concept that Cordyceps species belong to the pyrenomycetes. Based on sequence data the phylogenetic tree was constructed using the neighbor-joining (NJ) method. Diversity in the phylogenetic tree was found for Cordyceps species. A new classification of Cordyceps species can be constructed based on the phylogenetic information obtained from such rDNA sequences.

Animals↗

Heteroduplex mobility assay of the D1/D2 region of the 26S rDNA for differentiation of Saccharomyces species.

AIMS: We present the HMA method for Saccharomyces differentiation using the PCR amplified D1/D2 26S rDNA. METHODS AND RESULTS: This methodology is based on heteroduplex formation when two different DNAs are hybridized. We tested 11 type cultures of Saccharomyces, 27 different cultures of S. cerevisiae and four other ascomycetic genera. CONCLUSION: The method was capable of differentiating Saccharomyces species and was mainly very efficient for S. cerevisiae identification. HMA can probably be applied in other genera, where identification is sometimes difficult only by conventional traits, which are based on physiology and morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: HMA provides a rapid and relatively simple molecular tool, contributing for yeast taxonomy.

DNA, Fungal↗

Effect of aeration and agitation on the production of mycelial biomass and exopolysaccharides in an enthomopathogenic fungus Paecilomyces sinclairii.

AIMS: The objective of the present study was to investigate the influence of aeration rate and agitation intensity on the production of mycelial biomass and exopolysaccharide (EPS) in Paecilomyces sinclairii. METHODS AND RESULTS: The P. sinclairii was cultivated under various aeration and agitation conditions in a 5 l stirred-tank bioreactor. The highest mycelial biomass (30.5 g l-1) and EPS production (11.5 g l-1) were obtained at a high aeration rate (3.5 v.v.m.) and at a high agitation speed (250 rev min-1). The apparent viscosities (6000-8000 cP) of fermentation broth increased rapidly towards the end of fermentations at high aeration and agitation conditions. CONCLUSIONS: The high level of dissolved oxygen achieved at a high aeration rate (3.5 v.v.m.) associated with higher hyphal density eventually resulted in enhanced EPS production. Agitation intensity was also proved to be a critical factor influencing on both the mycelial biomass and EPS production: high agitation speeds up to 250 rev min-1 were preferred to the yields of biomass and EPS production. SIGNIFICANCE AND IMPACT OF THE STUDY: The critical effects of aeration and agitation in the culture process of P. sinclairii were found, which is widely applicable to other kinds of basidiomycetes or ascomycetes in their submerged culture processes.

Aerobiosis↗

Antimicrobial azaphilones from the fungus Hypoxylon multiforme.

Four new azaphilones named multiformins A - D together with a known compound 4 : 5:4':5'-tetrahydroxy-1 : 1'-binaphthyl were isolated from the methanolic stromatal extract of the xylariaceous ascomycete Hypoxylon multiforme. Their absolute structures were characterised by 2D-NMR, UV, IR, mass and CD spectroscopy. Multiformins A - D showed strong and apparently non-selective antimicrobial activity.

Anti-Infective Agents↗

The fungal teratogen secalonic acid D is an inhibitor of protein kinase C and of cyclic AMP-dependent protein kinase.

The teratogenic metabolite secalonic acid D deriving from the ergot-producing, rye-infecting ascomycete fungus Claviceps purpurea and from Penicillum oxalicum is an inhibitor of Ca2+- and phospholipid-dependent protein kinase C (PKC) and of the catalytic subunit of cyclic AMP-dependent protein kinase (cAK) (C50 values 15 microM and 12 microM, respectively). Secalonic acid D also inhibits Ca2+-calmodulin-dependent myosin light chain kinase (MLCK) and plant Ca2+-dependent protein kinase (CDPK). The inhibition of cAK by secalonic acid D is competitive with respect to both peptide substrate and ATP. However, secalonic acid D does not inhibit a high-affinity nucleotide-binding phosphatase from potato. A variety of other naturally-occurring teratogenic agents are not inhibitors of the protein kinases examined.

Amino Acid Sequence↗

Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain.

Many fungal pathogens must enter plant cells for successful colonization. Barley mildew resistance locus o (Mlo) is required for host cell invasion upon attack by the ascomycete powdery mildew fungus, Blumeria graminis f.sp. hordei, and encodes the founder of a family of heptahelical integral membrane proteins unique to plants. Recessively inherited loss-of-function mutant alleles (mlo) result in effective penetration resistance to all isolates of the biotrophic parasite. We used noninvasive fluorescence-based imaging to show that fluorescently tagged MLO protein becomes redistributed in the plasma membrane (PM) and accumulates beneath fungal appressoria coincident with the initiation of pathogen entry into host cells. Polarized MLO accumulation occurs once upon attack and appears to be independent of actin cytoskeleton function. Likewise, barley ROR2 syntaxin, a genetically defined component of penetration resistance to B. graminis f.sp. hordei, and a subset of predicted PM-resident proteins become redistributed to fungal entry sites. We previously identified calmodulin, a cytoplasmic calcium sensor, as an interactor and positive regulator of MLO activity and demonstrate here by FRET microscopy an increase in MLO/calmodulin FRET around penetration sites coincident with successful host cell entry. Our data provide evidence for the formation of a pathogen-triggered PM microdomain that is reminiscent of membrane microdomains (lipid rafts) induced upon attempted entry of pathogenic bacteria in animal cells.

Calmodulin↗

Lateral transfer of mating system in Stemphylium.

The fungal genus Stemphylium (Ascomycota) contains selfing species that evolved from outcrossing ancestors. To find out how selfing originated, we analyzed the Stemphylium MAT loci that regulate sexual reproduction in ascomycetes and compared MAT structures and phylogeny with a multigene Stemphylium species phylogeny. We found that some Stemphylium species' MAT loci contained a single gene, either MAT1-1 or MAT1-2, whereas others contained a unique fusion of the MAT1-1 and MAT1-2 regions. In all fused MAT regions, MAT1-1 was inverted and joined to a forward-oriented MAT1-2 region. As in the closely related Cochliobolus, Stemphylium species with fused MAT regions were able to self. Structural and phylogenetic analyses of the MAT loci showed that the selfing-conferring fused MAT regions were monophyletic with strong support. However, in an organismal phylogeny of Stemphylium species based on 106 isolates and four loci unrelated to mating, selfing arose in two clades, each time with strong support. Isolates with identical fused MAT regions were present in both clades. We showed that a one-time origin of the fused MAT loci, followed by a horizontal transfer across lineages, was compatible with the data. Another group of selfers in Stemphylium only had forward-oriented MAT1-1 at their MAT loci, constituting an additional and third origin of selfing in Stemphylium.

Ascomycota↗

Evolution of genomic diversity and sex at extreme environments: fungal life under hypersaline Dead Sea stress.

We have found that genomic diversity is generally positively correlated with abiotic and biotic stress levels (1-3). However, beyond a high-threshold level of stress, the diversity declines to a few adapted genotypes. The Dead Sea is the harshest planetary hypersaline environment (340 g.liter-1 total dissolved salts, approximately 10 times sea water). Hence, the Dead Sea is an excellent natural laboratory for testing the "rise and fall" pattern of genetic diversity with stress proposed in this article. Here, we examined genomic diversity of the ascomycete fungus Aspergillus versicolor from saline, nonsaline, and hypersaline Dead Sea environments. We screened the coding and noncoding genomes of A. versicolor isolates by using >600 AFLP (amplified fragment length polymorphism) markers (equal to loci). Genomic diversity was positively correlated with stress, culminating in the Dead Sea surface but dropped drastically in 50- to 280-m-deep seawater. The genomic diversity pattern paralleled the pattern of sexual reproduction of fungal species across the same southward gradient of increasing stress in Israel. This parallel may suggest that diversity and sex are intertwined intimately according to the rise and fall pattern and adaptively selected by natural selection in fungal genome evolution. Future large-scale verification in micromycetes will define further the trajectories of diversity and sex in the rise and fall pattern.

Biological Evolution↗

On the similarity between the tRNAs of organelles and prokaryotes.

Fluorescence studies with organelle transfer RNAs separated from their cytoplasmic counterparts revealed that phenylalanine tRNA from Euglena chloroplasts or Neurospora mitochondria does not contain a fluorescent "base Y." In contrast, cytoplasmic phenylalanine tRNA from Euglena and cytoplasmic tRNA from Neurospora were found to contain fluorescent bases. The fluorescence-emission spectra of Neurospora cytoplasmic tRNAs and those of the related ascomycete Saccharomyces cerevisiue were observed to be quite different. The results support the generalization that eukaryotic tRNAs contain a fluorescent base, but indicate that their respective organelle tRNAs do not. They also indicate a striking parallelism between organelle and prokaryotic tRNAs.

Acylation↗

Clustering of spore-specific genes in Aspergillus nidulans.

We have investigated the chromosomal organization of genes that are expressed specifically in the asexual spores (conidia) of the Ascomycete fungus Aspergillus nidulans, using two experimental approaches. In the first, 30 different recombinant clones, containing long nuclear DNA inserts and at least one spore-specific gene, were selected randomly. The total number of spore-specific genes present in each clone was then determined by RNA blot analysis. In the second approach, several chromosomal recombinant DNA libraries, having average insert lengths ranging from 1 to 15 kilobases, were constructed. The fraction of clones in each library having one or more spore-specific poly(A)+RNA-coding regions was then determined by colony or plaque filter hybridization with radiolabeled, spore-specific, complementary DNA. The results from these experiments were compared to statistical predictions based on the assumption that the spore-specific genes are randomly distributed in the Aspergillus genome. In both cases, the experimental values deviated significantly from the predicted values, demonstrating that the spore-specific genes are nonrandomly arranged in the genome. Rather, they appear frequently to occur in tightly linked clusters.

Aspergillus nidulans↗

Transformation of Aspergillus nidulans by using a trpC plasmid.

We constructed a chimeric plasmid carrying a complete copy of the trifunctional trpC gene from the Ascomycete fungus Aspergillus nidulans. This plasmid, designated pHY201, replicates in Escherichia coli, where it confers resistance to ampicillin and chloramphenicol and complements trpC mutants lacking phosphoribosylanthranilate isomerase activity. We used pHY201 to transform an A. nidulans trpC- strain to trpC+ at frequencies of greater than 20 stable transformants per microgram of DNA. Southern blot analysis of DNA from transformants showed that pHY201 DNA had integrated into the A. nidulans chromosomes in a majority of cases. Most of the integration events appeared to occur at the site of the trpC- allele of the recipient strain. In several instances, we succeeded in recovering pHY201, or derivatives thereof, from A. nidulans transformants by restriction endonuclease digestion of chromosomal DNA, ligation, and transformation of E. coli.

Ampicillin↗