Meiotic behaviour of the minichromosome in the phytopathogenic ascomycete Leptosphaeria maculans

All Leptosphaeria maculans field isolates displayed a minichromosome (MC) clearly separated from the overall electrokaryotype following pulsed-field gel electrophoresis. MCs exhibited a length polymorphism ranging from 650 to 950 kb. Tetrad analyses revealed the parental inheritance of MC length polymorphism (50% of the tetrads) or else the generation of novel-sized MCs (27%), which suggested that recombination occurred between MCs. Nineteen percent of the tetrads displayed a lack of the MC band in the electrokaryotype for one or two of the four resulting genotypes. Crosses between isolates carrying or lacking MCs revealed non-Mendelian segregation and suggested that some isolates could display at least two copies of the MC. Only repeated sequences hybridising to all chromosomes were isolated from the MC. Finally, saprophytic or parasitic fitness was not modified when isolates apparently lacked the MC. All these data suggested that the L. maculans MC behaves like a `B' chromosome. Received: 26 February 1996 / 2 August 1996     

Identification and characterization of polymorphic minisatellites in the phytopathogenic ascomycete Leptosphaeria maculans

Leptosphaeria maculans causes phoma stem canker, the most serious disease of oilseed rape world-wide. Sexual recombination is important in the pathogen life cycle and increases the risk of plant resistance genes being overcome rapidly. Thus, there is a need to develop easy-to-use molecular markers suitable for large-scale population genetic studies. The minisatellite MinLm1, showing six alleles in natural populations, has previously been used as a marker to survey populations. Here, we report the characterization of five new minisatellites (MinLm2–MinLm6), of which four were identified by a systematic search for tandemly repeated polymorphic regions in BAC-end sequencing data from L. maculans. Of 782 BAC-end sequences analysed, 43 possessed putative minisatellite-type repeats and four of these (MinLm3–MinLm6) displayed both consistent PCR amplification and size polymorphism in a collection of L. maculans isolates of diverse origins. Cloning and sequencing of each allele confirmed that polymorphism was due to variation in the repeat number of a core motif ranging from 11 bp (MinLm3) to 51 bp (MinLm4). The number of alleles found for each minisatellite ranged from three (MinLm4) to nine (MinLm2), with eight, five and six for MinLm3, MinLm5 and MinLm6, respectively. MinLm2–MinLm6 are all single locus markers specific to L. maculans and share some common features, such as conservation of core motifs and incomplete direct repeats in the flanking regions. To our knowledge, L. maculans is the first fungal species for which six polymorphic single locus minisatellite markers have been reported.Electronic Supplementary Material Supplementary material is available for this article at     

Major chromosomal length polymorphisms are evident after meiosis in the phytopathogenic fungus Leptosphaeria maculans

Chromosomal DNA of Australian field-isolates of the phytopathogenic ascomycete Leptosphaeria maculans was resolved by pulsed-field gel electrophoresis. All isolates examined had highly variable karyotypes. Ascospores (sexual spores) derived from single pseudothecia (sexual fruiting bodies) isolated from Brassica napus (oilseed rape) stubble were analyzed. In two tetrads four distinct karyotypes were observed, with only one chromosomal DNA band in common to all the members of each tetrad. Although isolates had highly variable karyotypes, two overall patterns were present. In one pattern there were at least 12 chromosomal DNA bands, the largest being greater than 2.2 Mb in size; in the other there were more than 15 chromosomal DNA bands, the largest being about 2.0 Mb. The chromosomal DNA preparations included mitochondrial DNA which migrated as a diffuse band between 0.10 and 0.15 Mb in size, and DNA molecules of 8 and 9 kb in size.     

Electrophoretic karyotyping of Leptosphaeria maculans differentiates highly virulent from weakly virulent isolates

Summary Whole chromosomes from the fungal phytopathogen Leptosphaeria maculans were separated by transverse alternating field electrophoresis. The chromosome complements from several isolates of both highly and weakly virulent strains were compared. Small variations in chromosome size and apparent number were detected among isolates of the same strain. However, dramatic differences in both chromosome number and size were found when isolates of the highly virulen strain were compared to those of the weakly virulent. Highly virulent isolates had 6–8 distinct bands whereas weakly virulent isolates had 12–14. The genome sizes were estimated to be at least 8.6x10⁶ base pairs for the virulent strain and 1.6x10⁷ base pairs for the weakly virulent strain. The major differences found in the chromosome complements of the two strains, in combination with results of other biochemical, morphological, and genetic studies, indicate that they are distinct species.     

Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic ascomycete Calonectria morganii

Conidia of the phytopathogenic fungus Calonectria morganii were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, governed by a heterologous fungal promoter and the Aspergillus nidulans trpC terminator. Agrobacterium tumefaciens-mediated transformation yielded stable hygromycin B-resistant clones (average number (106 per 10(7)) [corrected] conidia). Putative transformants appeared to be mitotically and meiotically stable. The presence of the hph gene was checked by PCR. In four randomly chosen transformants, single-copy integrations of the marker gene at different chromosomal sites were proven by Southern analysis.     

Genetic structure of a population of the fungus Leptosphaeria maculans in a disease nursery of Brassica napus in Australia

Microsatellite, minisatellite and mating type markers were used to determine the genetic structure of the fungus Leptosphaeria maculans within a disease nursery, where Brassica napus lines were screened for resistance to blackleg disease under high inoculum pressure. Fungal isolates were collected from pseudothecia in infected stubble and pycnidia within cotyledon lesions on seedlings within the nursery. Genetic diversity was high with gene diversity at H=0.700 across four polymorphic loci, and genotypic diversity at D=0.993. Among the 159 isolates analysed, 102 multilocus genotypes were identified. The even distribution of mating type idiomorphs MAT1-1 and MAT1-2 and gametic equilibrium within the population provided further evidence of random mating. Genetic diversity was distributed on a very fine scale in the disease nursery. The majority of genetic diversity (67%) was distributed among conidia within a lesion or among ascospores from a piece of stubble, while the remainder (33%) was distributed within lesions on seedlings or different stubble pieces. There were no among-group differences between samples from stubble and seedlings. This is consistent with the low level of genetic differentiation between the ascospore and conidia samples (F _ST=0.017) indicating that all isolates of L. maculans from the disease nursery most likely belong to one population, and that ascospores form the primary inoculum in the disease nursery.     

Comparison of the 5.8s rDNA and internal transcribed spacer sequences of isolates of Leptosphaeria maculans from different pathogenicity groups

The regions coding for the 5.8s rRNA and the flanking internal transcribed spacers (ITS1 and ITS2) from nine isolates of the blackleg pathogen Leptosphaeria maculans and one isolate of Sclerotinia sclerotiorum were amplified by the polymerase chain reaction and sequenced. Five of the L. maculans isolates were highly virulent to Brassica plants, two were weakly virulent and two were isolated from the cruciferous weed Thlaspi arvense. The 5.8s DNA sequences of all L. maculans isolates were identical. However, there were major differences in both ITS1 and ITS2 sequences that correlated with the pathogenicity grouping. Phylogenetic analysis of the ITS sequences by both parsimony and maximum-likelihood methods indicated that each pathogenicity group was statistically different from each other with the weaklyvirulent isolates being more closely related to the Thlaspi than to the highly-virulent isolates. The relationships of L. maculans to other fungi, based on a comparison of the 5.8s rDNA sequences, are discussed.     

Molecular characterization of a new hypovirus infecting a phytopathogenic fungus, Valsa ceratosperma

A double-stranded (ds) RNA, approximately 9.5kb in size; was identified in the MVC86 isolate of Valsa ceratosperma. Complete sequence of the dsRNA revealed a 9543-bp segment (excluding the 3' poly-A tail) that is predicted to encode a single large protein (P330). P330 has 63%, 49%, and 55% amino acid sequence identities to the proteins encoded by hypoviruses Cryphonectria hypovirus 3 (CHV3), CHV4, and Sclerotinia sclerotiorum hypovirus 1 (SsHV1), respectively. Like polyproteins encoded by CHV3, CHV4, and SsHV1, P330 comprises four conserved domains, including a papain-like protease, a UDP glucose/sterol glucosyltransferase (UGT), an RNA-dependent RNA polymerase (RdRp), and an RNA helicase. These molecular characteristics suggest that this dsRNA represents a new hypovirus that we tentatively designate Valsa ceratosperma hypovirus 1 (VcHV1). Phylogenetic analysis of the RdRp and RNA helicase domains of VcHV1 revealed that VcHV1, CHV3, CHV4, and SsHV1 clustered together into one clade distinct from that of CHV1 and CHV2, indicating the existence of two lineages in the family Hypoviridae. Comparison of biological properties between VcHV1-infected and VcHV1-free isogenic strains did not reveal differences in colony morphology or fungal virulence under laboratory conditions.     

Molecular characterisation of Lac s 1, the major allergen from lettuce (Lactuca sativa)

BACKGROUND: IgE sensitisation to non-specific lipid transfer proteins (nsLTP), e.g., Pru p 3 the major allergen from peach and most important allergenic LTP, is strongly associated with severe symptoms in food allergic patients. Lac s 1, a member of the nsLTP protein family, was recently identified as major allergen in lettuce (Lactuca sativa), but has not yet been investigated on the molecular basis. OBJECTIVE: Molecular characterisation and immunological comparison of Lac s 1 to peach allergen Pru p 3. METHODS: Lac s 1 cDNA was cloned by RT-PCR and natural (n) Lac s 1 was purified by a two-step chromatography. Protein structure was verified by N-terminal sequencing, mass spectrometry, and circular dichroism spectroscopy. Immunoblotting, ImmunoCAP, and competitive IgE binding experiments were performed to study the IgE sensitisation pattern and cross-reactivity with Pru p 3. Allergenic potency was analysed by histamine release assay. RESULTS: Twenty-nine lettuce allergic patients, with or without concomitant peach allergy, and 19 peach allergic patients without lettuce allergy were included in this study. IgE reactivity to lettuce was due to mono-sensitisation to Lac s 1 or cross-reactive glycan structures. Two Lac s 1 isoforms were identified which showed amino acid identity (aa-id) of 62% to each other, up to 66% to Pru p 3, and 72% to the N-terminal peptide of plane pollen LTP Pla a 3. The prevalence of IgE binding to nLac s 1 was 90% using lettuce extract in immunoblotting experiments. Enhanced sensitivity was observed in ImmunoCAP using purified nLac s 1 in comparison to extracts (93% versus 76%). Although IgE sensitisation to Lac s 1 and Pru p 3 was strongly associated, the two LTPs showed different IgE binding properties. Sensitisation to LTPs does not necessarily reflect the clinical disease, but Lac s 1 was capable of triggering histamine release as shown by positive skin test results in Lac s 1 mono-sensitised patients and by in vitro mediator release assays. CONCLUSION: Purified nLac s 1 will enhance the sensitivity in component resolved diagnosis of lettuce allergy. Similar to other cross-reactive food allergies, exclusive testing of IgE reactivities to LTP cannot be used as biomarker for clinical relevance. Our data provide indirect evidence that Pru p 3 might act as the primary sensitising agent in patients allergic to both lettuce and peach.     

Molecular karyotype of the phytopathogenic fungusSclerotinia sclerotiorum

Molecular techniques have been used to characterize different field isolates ofSclerotinia sclerotiorum, an ubiquitous phytopathogen. Chromosomal DNA resolved by pulsed-field gel electrophoresis (PFGE) revealed thatS. sclerotiorum contains at least 16 chromosomes ranging from 1.5 Mb to 4.0 Mb. The size of the haploid genome was estimated to be 43.5 Mb. Six field isolates with different levels of virulence on sunflower germlings or green beans were differentiated by random amplification of polymorphic DNA (RAPD), and analysed by clamped homogeneous electric field electrophoresis. This analysis revealed few chromosome-length polymorphisms among these strains. Chromosomal DNA hybridization indicated that the endopolygalacturonase-encodingpgl gene is localized on the smallest chromosome of all the strains, whereas the ribosomal DNA mapped to different-sized chromosomes. The less-aggressive strain was characterized by the presence of a supernumary small band, presumably consisting of dsRNA. In contrast to numerous other phytopathogenic fungi, this study reveals a strong karyotypic stability among the strains ofS. sclerotiorum which may be preserved by the sexual mode of reproduction of this species     

Molecular characterisation of GTP1, a Saccharomyces cerevisiae gene encoding a small GTP-binding protein

DNA sequence analysis upstream of the yeast DNA repair gene SNM1 revealed gene GTP1 with an ORF of 573 bp on chromosome XIII. The putative amino-acid sequence of the encoded protein shows homology to proteins of the ARF-class of small GTP-binding proteins. Homology within GTP-binding motifs is highly conserved. Gene disruption showed that GTP1 is not an essential gene and that it has no influence on the expression of the DNA repair gene SNM1 with which it shares a 191-bp promoter region.     

Genome analysis of filamentous fungi: identification and characterization of an unusual GT-rich minisatellite in the ascomycete Podospora anserina

A genomic DNA fragment, PaGT7-5, of Podospora anserina was cloned that contains three different repetitive sequence motifs including a minisatellite with an unusual structure. This element, PaMin1, consists of ten copies of a 16 bp repeat unit with five GT dinucleotide repeats. Adjacent to PaMin1, a short poly(GT) stretch and four repeats of a 12 bp sequence were identified. Six alleles which differ in the number of the minisatellite unit were demonstrated in 18 different P. anserina strains. The flanking sequences of PaMin1 did not display any sequence alterations. A PCR analysis of total DNA from cultures of different age revealed no sequence changes, indicating that this repetitive DNA remains stable during aging. Southern-blot hybridization experiments of DNA from different strains detected only minor fragment length polymorphisms in the immediate vicinity of PaMin1. In contrast, major polymorphisms were observed at a greater distance from the locus indicating that this locus can be used as an informative probe to discriminate between different P. anserina strains. Received: 12 March / 24 May 1998     

Molecular analysis of HLA-DRB1, -DQA1 and -DQB1 polymorphism in Turkey

We report the evaluation of MHC class II polymorphism in the population of Turkey. HLA-DRB1, -DQA1 and -DQB1 have been investigated by polymerase chain reaction and sequence-specific oligonucleotide probe hybridisations (PCR/SSO) and sequence-specific priming (SSP) in 250 randomly selected healthy individuals. We also report the allelic distribution of these genes. The most frequent alleles detected were DRB1*1101 (0.104), *0301 (0.092), *0701 (0.090), DQA1*0501 (0.334), *0102 (0.164) and *03 (0.148) and DQB1*0301 (0.256), *02 (0.164), *0302 (0.128). The frequent 'putative' three-locus haplotypes carry the most frequent alleles at these loci. The most frequently detected class II "haplotypes" are DRB1*1101 DQA1*0501 DQB1*0301 (0.100), DRB1*0301 DQA1*0501 DQB1*02 (0.092) and DRB1*0701 DQA1*0201 DQB1*02 (0.072). The distribution of alleles and 'putative' haplotypes has shown common features with other Mediterranean populations. The results extend the HLA map to another Mediterranean country and provide a database for further HLA-disease association studies and transplantation applications.     

Molecular characterisation of dicot-infecting mastreviruses from Australia

Monocotyledonous and dicotyledonous plant infecting mastreviruses threaten various agricultural systems throughout Africa, Eurasia and Australasia. In Australia three distinct mastrevirus species are known to infect dicotyledonous hosts such as chickpea, bean and tobacco. Amongst 34 new "dicot-infecting" mastrevirus full genome sequences obtained from these hosts we discovered one new species, four new strains, and various variants of previously described mastrevirus species. Besides providing additional support for the hypothesis that evolutionary processes operating during dicot-infecting mastrevirus evolution (such as patterns of pervasive homologous and non-homologous recombination, and strong purifying selection acting on all genes) have mostly mirrored those found in their monocot-infecting counterparts, we find that the Australian dicot-infecting viruses display patterns of phylogeographic clustering reminiscent of those displayed by monocot infecting mastrevirus species such as Panicum streak virus and Maize streak virus.     

Molecular cloning and characterisation of the genes for a non-fimbrial adhesin from Escherichia coli

A non-fimbrial adhesin (NFA-1) from the uropathogenic Escherichia coli strain 827 responsible for agglutination of human erythrocytes was cloned using the cos 4 cosmid vector. A clone was isolated which promoted haemagglutination and showed the same biological properties as the adhesin produced by the wild type strain. Both express adhesin at 37 degrees C, but not 18 degrees C nor in the presence of 1% glucose. Adhesin purified from the clone formed high molecular weight aggregates which were resolved to the 21 K dalton subunit protein seen in the wild type strain on denaturation. Binding to human kidney cells by the clone and the wild type E. coli, from which the genes were cloned, were compared in an ELISA assay and shown to be the same. The genes for the adhesin were isolated on a 15.5 kilobase BamHI-EcoRI fragment which was subjected to gamma delta mutagenesis. The NFA-1 operon was localised to a 6.5kb region of this fragment.     

Molecular analysis of DLA-DRBB1 polymorphism

The polymorphism of the canine major histocompatibility complex class II DRB gene, DRBB1, was analyzed. Exon 2 that encodes the polymorphic 81 domain was amplified by the polymerase chain reaction (PCR). The PCR product from 250 dogs was cloned and sequenced. Eighteen alleles were identified. Most of the variation in amino acid composition occurred at positions in the peptide binding site. Inheritance of these sequences showed Mendelian segregation with one or two alleles per dog. Cluster analysis of the nucleotide and predicted amino acid sequences subdivided the canine DRBB1 alleles into three major allelic groups. The number of nonsynonymous changes was higher than the number of synonymous changes in the putative antigen recognition sites indicative of positive selection. The data generated can serve as a basis for developing a typing assay for the canine DRBB1 gene.