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1.
J Appl Microbiol ; 113(5): 1145-53, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22830361

ABSTRACT

AIM: To develop a pyrosequencing assay to monitor the frequency of alleles of an avirulence gene, AvrLm4, in populations of sexual spores of Leptosphaeria maculans, a fungal pathogen of canola (Brassica napus). METHODS AND RESULTS: The predominant mutation in AvrLm4 responsible for virulence to the corresponding resistance gene, Rlm4, is a single nucleotide polymorphism (SNP) at base 358. Pyrosequencing primers were designed to amplify a 90-bp region that included this SNP. The assay was developed and validated by analysing the frequency of AvrLm4 in isolate mixtures of different proportions. Furthermore, the frequency of avrLm4 (virulence allele) determined by pyrosequencing of populations of sexual spores was consistent with the frequency of avrLm4 determined by Sanger sequencing of the entire AvrLm4 gene from single isolates cultured from the same stubble. CONCLUSION: This high-throughput assay can play an important role in predicting the risk of resistance breakdown in crops. SIGNIFICANCE AND IMPACT OF THE STUDY: Similar assays can be applied to monitor frequencies of fungicide resistance in pathogens of crops and to assay diversity in microbial soil communities such as in soil samples from bat caves where white-nose syndrome has been detected.


Subject(s)
Ascomycota/genetics , Brassica napus/microbiology , Gene Frequency , Plant Diseases/microbiology , Alleles , Ascomycota/pathogenicity , DNA Primers/genetics , DNA, Fungal/genetics , High-Throughput Nucleotide Sequencing , Mutation , Polymorphism, Single Nucleotide , Reproducibility of Results , Sequence Analysis, DNA , Spores, Fungal/genetics , Virulence/genetics
2.
Plant Dis ; 90(2): 190-198, 2006 Feb.
Article in English | MEDLINE | ID: mdl-30786411

ABSTRACT

The infection by Leptosphaeria maculans of Brassica napus cultivars with major gene resistance derived from Brassica rapa subsp. sylvestris was studied in southeastern Australia. Following the commercial release of these cultivars in Australia in 2000, plants with stem cankers were first reported in 2002 at two geographically isolated regions in South Australia and New South Wales. In 2003, this study showed that the major gene resistance had been overcome in an area of approximately 50,000 ha in South Australia and in two fields in New South Wales (0.5 and 30 ha). There was no relationship between disease severity and incidence in 2003 and the proximity to the sites where resistance breakdown occurred in 2002. At some locations, the frequency of isolates able to overcome the B. rapa subsp. sylvestris-derived resistance had increased between 2002 and 2003. Isolates cultured from canola cultivars with either B. rapa subsp. sylvestris-derived resistance or polygenic resistance showed host specificity when inoculated onto cultivars with B. rapa subsp. sylvestris-derived or polygenic resistance, respectively. The most likely cause of the resistance breakdown was the rapid increase in frequency of L. maculans isolates virulent on this particular resistance source. The selection pressure leading to this increased frequency was probably mediated by the planting of cultivars harboring the major resistance gene in the same locations for a 3-year period, and the ability of the pathogen to produce large numbers of asexual and sexual spores.

3.
Phytochemistry ; 62(2): 147-53, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12482449

ABSTRACT

Analysis of sterols in mycelia of the ascomycete, Leptosphaeria maculans by gas chromatography-mass spectrometry revealed that ergosterol comprised 95% of the total sterols, with eight other sterols comprising the remaining 5%. Six of these latter sterols were putative precursors of ergosterol and their presence suggested a pathway for ergosterol biosynthesis in this fungus. Ergosterol biosynthesis in fungi is inhibited by the triazole antifungal agent flutriafol. When L. maculans was grown in the presence of flutriafol, ergosterol content decreased while two 14 alpha-methylated sterols, 24-methylene dihydrolanosterol and obtusifoliol, accumulated.


Subject(s)
Ascomycota/chemistry , Mycelium/chemistry , Plant Diseases/microbiology , Sterols/biosynthesis , Sterols/isolation & purification , Ascomycota/metabolism , Gas Chromatography-Mass Spectrometry , Molecular Structure , Mycelium/metabolism , Sterols/chemistry , Time Factors
4.
Fungal Genet Biol ; 33(1): 1-14, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11407881

ABSTRACT

The loculoascomycete Leptosphaeria maculans (anamorph: Phoma lingam) causes blackleg of Brassicas, including Brassica napus (canola or rapeseed). This fungus probably comprises several morphologically similar species; taxonomic relationships between them are being clarified and nomenclature is being revised. The pathotype ("A" group) responsible for major economic losses to canola has been studied in more detail than other members of this species complex and is the focus of this review. L. maculans is haploid, outcrossing, can be transformed, and has a genome size of about 34 Mb. Preliminary genetic and physical maps have been developed and three genes involved in host specificity have been mapped. As yet, few genes have been characterized. Chemical analysis of fungal secondary metabolites has aided understanding of taxonomic relationships and of the host-fungal interaction by the unraveling of pathways for detoxification of antimicrobial phytoalexins. Several phytotoxins (host and nonhost specific) have been identified and a complex pattern of regulation of their synthesis by fungal and host metabolites has been discovered.


Subject(s)
Ascomycota/physiology , Brassica/microbiology , Plant Diseases/microbiology , Ascomycota/genetics , Ascomycota/ultrastructure , Brassica napus/microbiology , Chromosome Mapping , Mycotoxins/chemistry , Mycotoxins/metabolism , Piperazines/chemistry , Piperazines/metabolism
5.
Genome ; 44(2): 167-71, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11341726

ABSTRACT

An opsin gene (ops) has been characterized from Leptosphaeria maculans, the ascomycete that causes black-leg disease of Brassica species. This is the second opsin identified outside the archaeal and animal kingdoms. The gene encodes a predicted protein with high similarity (70.3%) and identity (53.3%) to the nop-1 opsin of another ascomycete Neurospora crassa. The L. maculans opsin also has identical amino acid residues in 20 of the 22 residues in the retinal-binding pocket of archaeal opsins. Opsin, on the fourth largest chromosome of L. maculans and 22 cM from the mating type locus, is the first cloned gene to be mapped in L. maculans. Opsin is transcribed at high levels in mycelia grown in the presence and absence of light; this pattern is in contrast with that of the N. crassa opsin, which is transcribed only in the light.


Subject(s)
Ascomycota/genetics , Fungal Proteins/genetics , Genes, Fungal , Genes, Mating Type, Fungal , Rod Opsins/genetics , Amino Acid Sequence , Animals , Archaea/genetics , Ascomycota/pathogenicity , Base Sequence , Brassica/microbiology , Chromosome Mapping , DNA Primers/genetics , Fungal Proteins/chemistry , Light , Molecular Sequence Data , Neurospora crassa/genetics , Plant Diseases/microbiology , Protein Structure, Secondary , Rod Opsins/chemistry , Species Specificity , Transcription, Genetic/radiation effects
6.
Mol Plant Pathol ; 2(4): 241-55, 2001 Jul 01.
Article in English | MEDLINE | ID: mdl-20573012

ABSTRACT

Summary Recently many fungal genes have been identified that, when disrupted, result in strains with a reduction or total loss of disease symptoms. Such pathogenicity genes are the subject of this review. The large number of pathogenicity genes identified is due to the application of tagged mutagenesis techniques (random or targeted). Genes have been identified with roles in the formation of infection structures, cell wall degradation, overcoming or avoiding plant defences, responding to the host environment, production of toxins, and in signal cascades. Additionally, genes with no database matches and with 'novel' functions have also been found. Improved technologies for mutation analysis and for sequencing and analysing fungal genomes hold promise for identifying many more pathogenicity genes.

7.
Mol Gen Genet ; 263(3): 463-70, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10821180

ABSTRACT

A gene encoding a cyanide hydratase was cloned from an aggressive isolate of Leptosphaeria maculans, the fungus which causes blackleg disease of oilseed Brassica spp. This enzyme catalyses the breakdown of hydrogen cyanide to a less toxic compound, formamide. The predicted amino acid sequence of cyanide hydratase in L. maculans is 77% and 82% identical to cyanide hydratases from two other ascomycetes, Gloeocercospora sorghi and Fusarium lateritium, respectively. The gene is present as a single copy in the L. maculans genome, in both aggressive and non-aggressive isolates, although there is a restriction fragment length polymorphism between these two isolate groups for this gene. The cyanide hydratase promoter contains four putative target sites for GATA transcription factors, proteins that regulate nitrogen metabolism and other processes. Transcription of cyanide hydratase in an aggressive L. maculans isolate is induced strongly by potassium cyanide. Transcription of the gene is detectable in cotyledons of Brassica juncea and B. napus during infection. L. maculans can utilise the reaction product, formamide, as a sole source of nitrogen.


Subject(s)
Ascomycota/enzymology , Ascomycota/genetics , Genes, Fungal , Hydro-Lyases/genetics , Amino Acid Sequence , Brassica/microbiology , Chromosomes, Fungal , Cloning, Molecular , DNA, Complementary/metabolism , Gene Library , Glucosinolates/metabolism , Hydrogen Cyanide/metabolism , Hydrolysis , Molecular Sequence Data , Sequence Homology, Amino Acid , Transcription, Genetic
8.
Gene ; 248(1-2): 89-97, 2000 May 02.
Article in English | MEDLINE | ID: mdl-10806354

ABSTRACT

The ascomycete, Leptosphaeria maculans, causes blackleg disease of oilseed Brassica spp. such as canola (Brassica napus). We have cloned a gene encoding endopolygalacturonase, pg1, and two genes encoding cellulases, cel1 and cel2, in L. maculans. These genes are not clustered in the genome, as they are located on different chromosomes. The deduced amino acid sequences of all three genes predict an N-terminal signal sequence, as is common for secreted fungal enzymes that degrade plant cell walls. The endopolygalacturonase encoded by pg1 shows the highest similarity (54% amino acid identity) to endopolygalacturonase 4 from Botrytis cinerea. Both cel1 and cel2 appear to encode cellobiohydrolase, and neither gene encodes a recognizable cellulose-binding domain or linker region. Transcription of pg1 is induced in cultures containing 1% polygalacturonic acid or pectin, and cel1 is induced in 1% cellulose or carboxymethylcellulose, as shown by Northern analysis. Glucose represses the induction of cel1 caused by cellulose and carboxymethylcellulose, but does affect transcription of pg1. Transcription of cel2 (but not cel1 or pg1) is detectable during infection of B. napus and B. juncea cotyledons and leaves using reverse transcription-PCR.


Subject(s)
Ascomycota/genetics , Brassica/microbiology , Cell Wall/metabolism , Cellulase/genetics , Polygalacturonase/genetics , Amino Acid Sequence , Ascomycota/enzymology , Brassica/metabolism , Cellulase/metabolism , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , DNA, Plant/chemistry , DNA, Plant/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Fungal , Genes, Plant/genetics , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Plant Diseases/microbiology , Polygalacturonase/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transcription, Genetic
9.
Mol Plant Pathol ; 1(5): 293-302, 2000 Sep 01.
Article in English | MEDLINE | ID: mdl-20572976

ABSTRACT

Abstract A genetic and physical map has been developed for the loculoascomycete Leptosphaeria maculans, a pathogen of oilseed Brassicas. The genetic map was constructed from 58 F(1) progeny and comprises 155 amplified fragment length polymorphic (AFLP) markers, three random amplified polymorphic DNA (RAPD) markers, the mating type locus and a host specificity locus conferring the ability to form lesions on Brassica juncea. Twenty-one linkage groups, 5 pairs, and 18 unlinked markers were assigned, and the genome size was 1520 cM. Pulsed field gel electrophoresis experiments showed that the parental isolates each had 16 chromosomes and a genome size of about 33.5 Mb. Attempts to anchor a large number of markers to chromosomes were hampered by difficulties in converting AFLPs into RFLP markers, and because many markers bound to every chromosome, indicating that L. maculans has a high level of dispersed repetitive sequences. This fungus displays chromosomal length polymorphisms, but in the cross examined, the linkage and physical maps were essentially congruent and there was no evidence of translocations. The host specificity locus is 18 cM from the nearest AFLP marker and is located on a chromosome sized 1.85 Mb in the virulent parent. The mating type locus is on a chromosome sized 2.6 Mb and coincident on an AFLP marker amplified from the virulent parent. The derived amino acid sequence of part of this marker has some conserved amino acids present in the High Mobility Group DNA binding domain of MAT-2 mating type genes of other ascomycetes.

10.
Microbiol Res ; 152(3): 261-7, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9352662

ABSTRACT

In the ascomycete Leptosphaeria maculans tandem repeats of ribosomal DNA (rDNA) are restricted to one or two particular chromosomes of the 15 chromosomes of 19 field isolates examined. Ribosomal DNA can account for size differences of 35% between homologous chromosomes in a particular tetrad. During crossing, no detectable recombination between blocks of tandem repeats, nor changes in their size occur. The organisation of rDNA in L. maculans differs from many other haploid fungi. Firstly, sequence heterogeneity occurs within tandem repeats of rDNA; regularly spaced Sal 1 sites (0.25 Mb apart) are present within a 1.4 Mb block of tandem repeats. Secondly, individual isolates have different-sized rDNA repeats; this variation occurs in the non-transcribed intergenic spacer region. Thirdly, there is a wide range in the copy number of the rDNA repeat (from 56 to 225) amongst only four field isolates examined.


Subject(s)
Ascomycota/genetics , DNA, Fungal/chemistry , DNA, Ribosomal/chemistry , Chromosomes, Fungal , Repetitive Sequences, Nucleic Acid
11.
Electrophoresis ; 18(9): 1544-7, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9378119

ABSTRACT

Pulsed field gel electrophoresis (PFGE), or electrophoretic karyotyping, separates chromosomal-sized pieces of DNA in agarose gels where the orientation of the electric field is periodically altered. This technique has revealed that many fungi have a high degree of chromosomal length polymorphisms. Often the only isolates with identical karyotypes are derived from a single clone, thus PFGE provides a 'genetic fingerprint' for them. The size range and number of chromosomes within isolates of a particular species are usually constant, hence PFGE can distinguish between morphologically similar fungi. This technique can also be used to follow inheritance of chromosomal length polymorphisms and shows that in some fungi novel-sized chromosomes are produced during meiosis. As well as resolving the nuclear (A-type) chromosomes, it can also resolve dispensable (B-type) chromosomes and cytoplasmic genomes including mitochondrial DNA and linear plasmids. The application of this technique to Australian isolates of Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus), is discussed.


Subject(s)
Ascomycota/genetics , DNA, Fungal/analysis , Electrophoresis, Gel, Pulsed-Field , Genes, Fungal , Ascomycota/ultrastructure , Brassica/microbiology , Chromosomes, Fungal/ultrastructure , Meiosis , Mitochondria/chemistry , Plasmids
12.
Gene ; 158(1): 153-4, 1995 May 26.
Article in English | MEDLINE | ID: mdl-7789806

ABSTRACT

The 5' flanking region and the first 704 bp of the nitrite reductase-encoding gene (niiA) of Leptosphaeria maculans has been sequenced. The niiA start codon is 1438 bp downstream from the stop codon of the nitrate reductase-encoding gene (niaD) and the two genes are transcribed in the same direction.


Subject(s)
Ascomycota/genetics , Genetic Linkage , Nitrate Reductases/genetics , Nitrite Reductases/genetics , Transcription, Genetic , Amino Acid Sequence , Ascomycota/enzymology , Base Sequence , DNA, Recombinant , Molecular Sequence Data , Nitrate Reductase , Sequence Homology, Amino Acid
13.
Mol Gen Genet ; 247(4): 416-22, 1995 May 20.
Article in English | MEDLINE | ID: mdl-7770048

ABSTRACT

Pulsed field gel electrophoresis experiments show that chromosomal length polymorphisms are produced during meiosis in the ascomycetous plant pathogen Leptosphaeria maculans. Homologues in tetrads of L. maculans were identified on the basis of their binding to chromosome-specific probes that included beta-tubulin, nitrate reductase, 18S ribosomal DNA and two Random Amplified Polymorphic DNA (RAPD) markers. Changes in size of homologues were followed during meiosis. Significant karyotype variation was evident due to the random assortment of parental homologues of different sizes. In most cases, the progeny had the same-sized homologues as the parents; however, in some instances novel-sized homologues were detected that varied in size from those of the parents by up to 50 kb. Our results are consistent with the hypothesis that these novel chromosomal length polymorphisms are produced by reciprocal recombination between parental homologous chromosomes of unequal sizes.


Subject(s)
Ascomycota/genetics , Base Sequence , Chromosomes/ultrastructure , DNA Primers/chemistry , DNA, Fungal/genetics , Meiosis , Molecular Sequence Data , Polymorphism, Genetic , RNA, Ribosomal, 18S/genetics , Tubulin/genetics
14.
Curr Genet ; 27(3): 239-42, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7736608

ABSTRACT

Southern hybridisation, polymerase chain reaction (PCR), and nucleotide sequence, data indicate that the 5s ribosomal RNA (rRNA) gene is linked to the main rRNA gene repeat in the nuclear genome of four cryptomonad algae (Rhinomonas pauca, Storeatula major, Komma caudata, and isolate Cs 134). The 5s gene is apparently transcribed in the same direction as the large and small subunit rRNA genes. The intergenic spacer between the 5s gene and the large subunit rRNA gene exhibits length and sequence polymorphism among the different species. Cryptomonads contain two different eukaryotic genomes: the host nucleus and the nucleus of a eukaryotic endosymbiont. Mapping experiments with isolated chromosomes of the host and endosymbiont genomes showed that the intergenic spacer between the large subunit and the 5s rRNA gene, which was amplified from total DNA by PCR, was derived from the host nuclear genome.


Subject(s)
DNA, Ribosomal/genetics , Eukaryota/genetics , RNA, Ribosomal, 5S/genetics , Base Sequence , Molecular Sequence Data , Polymerase Chain Reaction , Polymorphism, Genetic , RNA, Ribosomal, 5S/chemistry , Sequence Homology, Nucleic Acid
15.
Curr Genet ; 26(3): 276-80, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7859312

ABSTRACT

Two linear DNA plasmids (pLm9 and pLm10, sized 9 and 10 kb respectively) were isolated from the phytopathogenic ascomycete Leptosphaeria maculans, using pulsed-field gel electrophoresis. pLm9 and pLm10 are found only in aggressive isolates of L. maculans but, because aggressive and non-aggressive strains appear to be different species, these plasmids are probably not involved in pathogenicity. pLm9 and pLm10 copurify with a mitochondrially-enriched cell fraction, and do not hybridise to chromosomal or mitochondrial DNA, or to each other. Exonuclease digestions suggest that both these molecules contain covalently-bound proteins at their 5' termini. pLm9 hybridises to the RNA polymerase of a linear plasmid from the ascomycete Podospora anserina, and pLm10 hybridises to the DNA polymerase from the same P. anserina plasmid, suggesting that pLm9 and pLm10 encode their own replication and transcription enzymes.


Subject(s)
Ascomycota/genetics , Plasmids/isolation & purification , Ascomycota/physiology , Cell Nucleus , DNA Probes , DNA, Mitochondrial , DNA-Directed DNA Polymerase/genetics , DNA-Directed RNA Polymerases/genetics , Electrophoresis, Gel, Pulsed-Field , Fungal Proteins , Genome, Fungal , Mitochondria
16.
Mol Gen Genet ; 244(1): 1-8, 1994 Jul 08.
Article in English | MEDLINE | ID: mdl-8041355

ABSTRACT

The nitrate reductase (niaD) gene was isolated from the phytopathogenic loculoascomycete Leptosphaeria maculans by screening a genomic DNA library with the Aspergillus nidulans niaD gene. The L. maculans niaD gene is the first protein-encoding gene characterised from this fungus. It encodes a predicted protein of 893 amino acids and contains four putative introns at positions in the gene equivalent to those of four of the six introns in the A. nidulans niaD gene. Mutants defective in niaD and molybdenum cofactor gene(s) of L. maculans have been isolated. Transformation of a L. maculans niaD mutant with a 3.8 kb SacII fragment containing the L. maculans niaD gene restored wild-type growth on nitrate as a sole nitrogen source. The niaD gene is present as a single copy on a chromosome which ranges in size from 2.6 to 2.8 Mb between the different L. maculans isolates examined.


Subject(s)
Ascomycota/enzymology , Chromosomes, Fungal , Nitrate Reductases/genetics , Amino Acid Sequence , Base Sequence , Chlorates , Chromosome Mapping , Cloning, Molecular , DNA, Fungal , Molecular Sequence Data , Mutation , Nitrate Reductase , Nitrate Reductases/metabolism , Sequence Homology, Amino Acid , Transformation, Genetic
17.
Curr Genet ; 24(1-2): 107-13, 1993.
Article in English | MEDLINE | ID: mdl-8358816

ABSTRACT

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.


Subject(s)
Ascomycota/genetics , Polymorphism, Genetic , Ascomycota/growth & development , Ascomycota/isolation & purification , Australia , Base Sequence , Chromosomes, Fungal , DNA, Fungal , Electrophoresis, Gel, Pulsed-Field , Karyotyping , Meiosis , Molecular Sequence Data
18.
Curr Genet ; 22(6): 455-61, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1473177

ABSTRACT

Southern hybridization and polymerase chain reaction data indicate that the 5S ribosomal RNA gene is linked to the ribosomal RNA gene repeat unit in the oomycetes, Phytophthora vignae, P. cinnamomi, P. megasperma f.sp. glycinea and Saprolegnia ferax, and is apparently transcribed in the same direction as the large and small subunit ribosomal RNA genes. The polymerase chain reaction has been used to amplify all components of the entire ribosomal RNA gene repeat unit for each of these oomycetes. The total size of all amplified products is identical to the size of the ribosomal RNA gene repeat unit, as determined by Southern analysis.


Subject(s)
Genes, Fungal , Oomycetes/genetics , Phytophthora/genetics , RNA, Ribosomal, 5S/genetics , RNA, Ribosomal/genetics , Base Sequence , Blotting, Southern , DNA, Ribosomal/genetics , Molecular Sequence Data , Oligonucleotides , Polymerase Chain Reaction , RNA, Fungal/genetics
19.
Histochem J ; 14(6): 949-66, 1982 Nov.
Article in English | MEDLINE | ID: mdl-7174355

ABSTRACT

The cellular sites of the glycoproteins Group 1 allergen (glycoprotein 1) and Antigen A (glycoprotein 2) in mature ryegrass pollen have been investigated by immunoelectron microscopy. Radioimmunoassays confirm previous findings of cross-reactivity between the purified glycoprotein antigens at the high immunoglobulin G (IgG) concentrations used for localization. Freeze-drying of anthers followed by anhydrous processing has been employed because of the water solubility and mobility of the glycoproteins. A double-embedding technique has been developed. This involves, first, embedding anthers in the water-soluble plastic resin JB-4, sectioning and incubating in ferritin-labelled antisera by the indirect method. The sections are then embedded in Spurr's resin for ultra-thin sectioning. Both glycoproteins are found in the following sites: (1) exine and intine wall layers; (2) pollen cytoplasm; (3) the orbicules and anther loculus; and (4) the anther cuticle. In the exine arcades and surface and in the anther loculus, the ferritin label is bound to pollenkitt. The finding that the glycoproteins are in similar sites is predictable in view of the cross-specificity of the antisera. The extent of antibody penetration of the plastic sections has been examined; labelling is confined to cut grains and absent from intact grains.


Subject(s)
Allergens/analysis , Ferritins , Glycoproteins/analysis , Pollen/analysis , Cross Reactions , Histocytochemistry , Immunoglobulin G/immunology , Microscopy, Electron , Radioimmunoassay
20.
Clin Allergy ; 12(3): 259-68, 1982 May.
Article in English | MEDLINE | ID: mdl-7105390

ABSTRACT

Immunoglobulin E (IgE), directed against components of Acacia (wattle) pollen, has been detected by radioallergosorbent tests (RAST) in the sera of some children and adults who develop allergic symptoms in the presence of flowering Acacia trees in Australia. All these subjects also had high levels of IgE directed against Lolium perenne (rye grass) pollen. Inhibition by RAST showed that most of the IgE molecules which bound to Acacia pollen components also bound to L. perenne pollen extracts, and to Glycoprotein 1, the major allergen of L. perenne pollen. In these assays, the allergens have been immobilized on polyvinyl chloride microtitre trays: the sensitivity of this approach is compared to that of commercial RAST kits.


Subject(s)
Acacia/immunology , Cross Reactions , Edible Grain/immunology , Pollen/immunology , Secale/immunology , Adult , Binding Sites, Antibody , Binding, Competitive , Child , Conjunctivitis/diagnosis , Conjunctivitis/immunology , Humans , Immunoglobulin E/analysis , Immunoglobulin E/biosynthesis , Immunoglobulin G/analysis , Immunoglobulin G/biosynthesis , Plant Extracts/immunology , Pollen/analysis , Radioallergosorbent Test , Rhinitis, Allergic, Seasonal/diagnosis , Rhinitis, Allergic, Seasonal/immunology , Skin Tests
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