Patterns of expression of a lolitrem biosynthetic gene in the Epichloë festucae-perennial ryegrass symbiosis

Lolitrem B is synthesized by Epichlo? festucae in associations with Pooid grasses. A complex cluster of at least 10 genes (ltm genes) is required for its synthesis. An early step in this pathway is catalyzed by ltmM, a symbiosis-expressed gene. PltmM-gusA reporter gene analysis was used to monitor ltmM gene expression patterns in planta. The minimum promoter length required for high-level gusA expression in infected seedlings is in the range of 480 to 782 bp. gusA was expressed by the endophyte in all infected vegetative plant tissues and in epiphyllous hyphae. Spikelets from reproductive tillers were analyzed at different developmental stages. During pre-anthesis, gusA expression was observed in all infected floral organs except the immature gynoecium. In post-anthesis florets, gene expression occurred almost exclusively in the gynoecium. Expression of gusA by the endophyte was observed in germinating seeds 24 h postimbibition and seedlings older than 6 days postimbibition in hyphae from the mesocotyl to the tip of the emerging first leaf. This work provides a detailed analysis of the spatial and temporal expression patterns of a symbiosis-expressed gene in planta.     

Functional analysis of a beta-1,6-glucanase gene from the grass endophytic fungus Epichloë festucae

β-1,6-glucanases degrade the polysaccharide β-1,6-glucan, a cell wall component in some filamentous fungi. A single copy of a β-1,6-glucanase gene, designated gcnA, was identified in each of the grass endophytic fungi Neotyphodium lolii and Epichloë festucae. Phylogenetic analysis indicates that the GcnA protein is a member of glycosyl hydrolase family 5, and is closely related to fungal β-1,6-glucanases implicated in mycoparasitism. The E. festucae gcnA gene was expressed in mycelium grown in culture and in both vegetative and reproductive tissues of perennial ryegrass. A gcnA replacement mutant had reduced β-1,6-glucanase activity when grown in media containing pustulan as the major carbon source. β-1,6-glucanase activity was restored in the replacement mutant by introducing multiple copies of the gcnA gene. Growth of ΔgcnA and gcnA-overexpressing strains in vegetative grass tissues was indistinguishable from wild type strains.     

Detection of a restriction site polymorphism within the human α-globin gene complex

Summary A mutant RsaI restriction endonuclease site of high frequency has been identified in individuals of German, Greek, Italian, and Turkish origin. The mutation was found within the -globin gene complex and is located 0.7 kb 5 to the -globin gene. In individuals of central European origin 34 out of 58 chromosomes exhibited the -gene linkage to the presence of the polymorphic site, and thus a preliminary estimate of the gene frequency for this allele would be 0.59. DNA analysis data of individuals derived from Mediterranean populations indicate a distribution of this polymorphic marker in similar frequencies.     

Epichloë endophytes: fungal symbionts of grasses

Recent developments have increased our understanding of the evolution of mutualistic associations between Epichlo? endophytes and their grass hosts. Most of the asexual species appear to be interspecific hybrids. Although endophytes form compatible associations with their natural hosts, transfers to other hosts elicit a range of incompatible reactions. Recently, the genes involved in the synthesis of the secondary metabolites ergot alkaloids and indole-diterpenes, which confer protective benefits on the association, have been cloned.     

Functional analysis of an indole-diterpene gene cluster for lolitrem B biosynthesis in the grass endosymbiont Epichloë festucae

Epichloë festucae Fl1 in association with Lolium perenne synthesizes a diverse range of indole-diterpene bioprotective metabolites, including lolitrem B, a potent tremorgen. The ltm genes responsible for the synthesis of these metabolites are organized in three clusters at a single sub-telomeric locus in the genome of E. festucae. Here we resolve the genetic basis for the remarkable indole-diterpene diversity observed in planta by analyzing products that accumulate in associations containing ltm deletion mutants of E. festucae and in cells of Penicillium paxilli containing copies of these genes under the control of a P. paxilli biosynthetic gene promoter. We propose a biosynthetic scheme to account for this metabolic diversity.     

An ‘open’ structure of the RecOR complex supports ssDNA binding within the core of the complex

Efficient DNA repair is critical for cell survival and the maintenance of genome integrity. The homologous recombination pathway is responsible for the repair of DNA double-strand breaks within cells. Initiation of this pathway in bacteria can be carried out by either the RecBCD or the RecFOR proteins. An important regulatory player within the RecFOR pathway is the RecOR complex that facilitates RecA loading onto DNA. Here we report new data regarding the assembly of Deinococcus radiodurans RecOR and its interaction with DNA, providing novel mechanistic insight into the mode of action of RecOR in homologous recombination. We present a higher resolution crystal structure of RecOR in an ‘open’ conformation in which the tetrameric RecR ring flanked by two RecO molecules is accessible for DNA binding. We show using small-angle neutron scattering and mutagenesis studies that DNA binding does indeed occur within the RecR ring. Binding of single-stranded DNA occurs without any major conformational changes of the RecOR complex while structural rearrangements are observed on double-stranded DNA binding. Finally, our molecular dynamics simulations, supported by our biochemical data, provide a detailed picture of the DNA binding motif of RecOR and reveal that single-stranded DNA is sandwiched between the two facing oligonucleotide binding domains of RecO within the RecR ring.     

Role of odour compounds in the attraction of gamete vectors in endophytic Epichloë fungi

Grass-infecting Epichlo? endophytes (Ascomycota, Calvicipitaceae) depend on Botanophila flies for gamete transfer, while fly larvae feed and develop on the fertilized fungal fruiting structures. Flies are known to be attracted by volatile signals, but the exact mechanisms of chemical communication and the degree of specialization are unknown. Headspace samples collected from five different Epichlo? species were analysed with respect to physiologically active substances using Botanophila flies. In field bioassays using synthetic compounds, their attractiveness and the specificity of the Epichlo?-Botanophila attraction were investigated. The identification of a new natural product, methyl (Z)-3-methyldodec-2-enoate, attracting Botanophila flies is reported here, and chokol K is confirmed as an attractive compound. Different blends of the two compounds attracted Botanophila flies under field conditions, but the three fly taxa present at the study site showed no preference for specific blends of volatiles. Chemical communication in the Epichlo?-Botanophila system relies on a few specific compounds, known as a communication system with 'private channels'. Although ratios of emitted compounds vary in different Epichlo? species, this seems not to lead to specialized attraction of Botanophila flies. Low selective pressure for specialization may have maintained a more generalist interaction between fungi and flies.     

Genetic structure of natural populations of the grass endophyte Epichloë festucae in semiarid grasslands

Plants of red fescue (Festuca rubra), a commercially important turf grass, are infected by the fungal endophyte Epichloë festucae in semiarid natural grasslands, known as dehesas, in western Spain. We used amplified fragment length polymorphism (AFLP) markers to analyse the genetic polymorphism existing in two natural populations of Epichloë festucae. Linkage disequilibrium and the presence of clonal lineages indicated that nonrecombinant asexual reproduction predominates in both populations. However, most genetic variation detected was found to occur within populations, with only a moderate amount of genetic differentiation between populations (F_ST: 0.197). Overall, the study suggests that dehesa grasslands are useful reservoirs of Epichloë festucae endophytes, and provides information on population structure which is relevant to design sampling strategies.     

A new species of Epichloë symbiotic with Chinese grasses

Epichlo? species are fungal symbionts (endophytes) of grasses, six European and four North American biological species in genus Epichlo? have been described in previous researches. In this study we describe a new Epichlo? species, Epichlo? yangzii Li et Wang, found in natural symbioses with Roegneria kamoji native to China. We investigated the host specificity, morphology, interfertility tests and molecular phylogenetic evidences of this new species. The results indicated that E. yangzii is host specific and seedborne. Most morphological characteristics of this new species are typical in the genus. However differences are evident in several features including size of perithecia, asci and ascospores. In mating tests E. yangzii was not interfertile with E. elymi isolates from related hosts in genera Elymus. Phylogenetic relationships based on sequences of beta-tubulin gene (tub2) introns and translation elongation factor 1-alpha gene (tef1) introns showed that members of the new species grouped into exclusive clades with high bootstrap value.     

Polarity of meiotic gene conversion is 5′ to 3′ within the niaD gene of Aspergillus nidulans

We have examined polarity of meiotic gene conversion in the niiA-niaD gene cluster of Aspergillus nidulans in two-point crosses. The type and position of the mutations represented by the niaD alleles and the correlation between the relative frequency of gene conversion and the physical position of these mutations were determined. We show that polarity of meiotic gene conversion is 5 to 3 (transcribed strand) within the niaD gene. Additional crosses involving a niiA allele and a niaD allele show little polarity of gene conversion, which suggests that the recombination events leading to restoration of the niaD gene are initiated upstream of the coding region of the niaD gene but within the niiA-niaD gene cluster, possibly within the intergenic promoter region.     

The role of host-specificity in the reproductive isolation of Epichloë endophytes revealed by reciprocal infections

Speciation in sexually reproducing organisms hinges on reproductive barriers that reduce gene flow between species or preclude the formation of hybrids. Here, we studied potential reproductive barriers in four members of the Epichloë typhina (Ascomycota, Clavicipitaceae) complex, i.e. Epichloë typhina infecting Dactylis glomerata, E. typhina subsp. clarkii infecting Holcus lanatus, E. typhina subsp. poae infecting Poa nemoralis and E. typhina infecting P. trivialis. Reciprocal inoculation tests showed that these endophytes are host-specific. This suggests that reproductive isolation among Epichloë strains may be the result of specialization to one host, on which mating between different individuals occurs. Furthermore, significantly lower infection frequencies of F1 progeny from crosses between host-strains compared to parental strains and within host-strain progeny suggest that host-dependent effects upon hybrid fitness exist, which would conform to an extrinsic postzygotic isolation barrier. Our results may explain, why members of the E. typhina complex remain genetically differentiated in natural populations.     

Alleles of the rat T-cell receptor β chain gene complex

Inbred rat strains provide a rich source of genetic diversity in immunologically relevant gense. We have characterized the alleles of one of these genes, encoding the rat T-cell receptor C1 chain, by Southern blots and nucleic acid sequencing. The Cb1 gene segments from DA and LEW rats display complex allotypic variation: both coding and noncoding regions contain multiple nucleotide substitutions. In addition, there is a polymorphic insertion of a rat repetitive LINE element 3 to the coding region. The Cb1 alleles are one part of larger Tcrb haplotypes, containing V, D, and J elements; complete Cb1 genomic nucleotide sequences, and a partial list of the strain distribution of the two alleles, are described in this report.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M65136.     

Variation in horizontal and vertical transmission of the endophyte Epichloë elymi infecting the grass Elymus hystrix

Systemic fungal endophytes (Clavicipitaceae) of grasses reproduce sexually when the fungus forms stromata and contagious ascospores, or asexually by vertical transmission of hyphae into seeds and seedlings. Vertical transmission is predicted to favor reduced virulence compared with horizontal transmission in systems with both types of transmission. Here, variation in vertical and horizontal transmission and its potential heritability in a host grass-endophyte interaction, Elymus hystrix infected with Epichlo? elymi, were examined in natural populations and two common garden experiments using field-collected host tillers and seed progeny of maternal plants with known infection phenotypes. Transmission mode exhibited year-to-year variation in field and common garden environments. In the common garden there were consistent differences among maternal plant families in stroma production and significant correlations between stroma production in the common garden and in natural populations. Transmission mode differed among maternal families, spanning a continuum from pure vertical transmission to a high proportion of stroma production and horizontal transmission potential. Vertical transmission to seeds occurred at high rates in all maternal families regardless of their stroma production. Observed patterns of variation indicate that endophyte transmission mode and correlated changes in virulence can respond to selection by biotic and abiotic factors.     

Epichloë endophyte affects the root colonization pattern of belowground symbionts in a wild grass

Plants host multiple symbionts that interact with each other affecting plant performance and regulating their establishment. Here, we analyzed how the association with Epichloë endophytes affects belowground colonization by Dark Septate Endophytes (DSE) and arbuscular mycorrhizal fungi (AMF) in the grass Bromus auleticus. Epichloë-symbiotic (E+) and Epichloë-non symbiotic (E−) plants were sampled from a long-term experimental plot and colonization structures were analyzed in the roots. We also examined the influence of Epichloë exudates on the in vitro growth of DSE Microdochium bolleyi isolated from roots. Epichloë symbiosis increased AMF colonization, although differences were not significant. Despite the lack of differences in total DSE colonization, in concordance with in vitro findings, a higher significant abundance of microsclerotia was observed in E+ plants. A negative correlation between total mycorrhizal and DSE was found. Our findings show a more uniform root colonization pattern in E+ plants, suggesting a root symbiosis modulating role.     

Distribution of the ΔF508 mutation in 194 Spanish cystic fibrosis families

Summary Spanish cystic fibrosis (CF) families (n = 194) have been analysed for the ΔF508 mutation, and for closely linked DNA markers. The ΔF508 mutation accounts for 50% of CF chromosomes. Four haplotypes are associated with the deletion, and at least seven haplotypes carry other mutations. The second major CF mutation is associated with pancreatic insufficiency and occurred in the same haplotype in which the ΔF508 arose. Only 31% of Spanish CF patients with no family history of the disease can be accurately diagnosed; about 50% of CF carriers can be detected in the Spanish population.     

A high proportion of mutations in the BRCA1 gene in German breast/ovarian cancer families with clustering of mutations in the 3′ third of the gene

We have analyzed 61 German breast and breast/ovarian cancer families for BRCA1 mutations using single-strand conformation polymorphism analysis (SSCP) followed by sequencing. Forty-seven of the families had at least three cases (at least two under 60 years) and 14 families had only two cases of breast/ovarian cancer (at least one under 50 years). Twenty-eight families were breast/ovarian and 33 were breast cancer-only families. Eighteen mutations in BRCA1 were detected in 11/28 breast/ovarian cancer families and 7/33 breast cancer families and none in the families with only two cases. We identified 17 truncation mutations (8 frameshift, 7 nonsense and 2 splice variants) and one missense mutation. Seven of these are novel and two, the 5382insC and 5622C→T mutations, occurred in two apparently unrelated families. The genotype of the two families with the 5382insC mutation is compatible with the rare haplotype segregating with the 5382insC mutation in different populations, further supporting its European origin. One unclassified missense alteration, R841W, was found in one family but did not segregate with the disease, suggesting that it is more likely a polymorphism. We also report and discuss the sequence of several new unclassified single-nucleotide changes first identified by SSCP. Of the 18 mutations, 13 occurred in the 3′ third of the gene (end of exon 11–24) and ovarian cancers were found in eight of these families. Received: 5 February 1998 / Accepted: 7 April 1998