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1994 - 19972

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Subject: search.filters.subject.Acremonium

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    Development of the [beta]-glucuronidase reporter gene system to study Acremonium endophyte interactions with perennial ryegrass : a thesis presented in fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University
    (Massey University, 1997) Saunders, Karyn
    A transformant of the fungal endophyte Acremonium lolii, strain Lp19, containing the gusA gene under the control of the constitutive Pgpd promoter was generated, and assigned the name KS1. Analytical digests and Southern hybridisation showed that this transformant contained a single chromosomally integrated copy of the gusA gene. The transformation frequency of Lp19 was found to be very low, and attempts to increase the transformation frequency were unsuccessful. KS1 was used to artificially infect seedlings of several different genotypes of Lolium perenne, all of a single cultivar, 'Nui'. These seedlings were grown into mature plants, and the endophytically produced GUS enzyme was extracted from individual plant tissues. Assays were performed on the enzyme extracts, and the levels determined were used as a measure of endophyte metabolic activity. Alterations of the gusA gene in some plants was detected by Southern hybridisation. One alteration was found to result in loss of GUS activity, the other did not appear to alter gusA expression. Levels of transformed endophyte GUS activity were initially compared between clonal plant material of a single genotype. Statistical analysis revealed that no significant differences were detectable for a particular tissue between the different plants. This showed that plant material of identical genotype could be pooled for analysis without the pooling of the individual plants having an affect on the outcome of the analysis. Next, levels of the transformed endophyte GUS activity were compared between genetically diverse perennial ryegrass plants of cultivar 'Nui'. Significant differences in GUS activity were detected in most tissues tested between the different genotypes, with only the most mature tissue displaying no detectable differences. Finally, a single plant of each of two individual genotypes was divided into several clonal plants, and the resulting mature plants were pooled in their genotypes for analysis of GUS, peramine, ergovaline and lolitrem B levels. The F test was not particularly sensitive in this experiment, and only one major difference between genotypes could be detected. Despite this, some trends emerged which were found to be consistent with those found in other studies. Metabolic activity and peramine levels were shown to be highest in the leaf sheath tissue, with levels generally decreasing with increasing tissue age. Lolitrem B was found to be highest in leaf sheath tissue also, but with levels increasing in general with tissue age. Ergovaline levels were very low in all tissues. The results presented show the potential of the use of the GUS reporter gene system to study endophyte gene expression in planta, and pooling of plants can be carried out to allow simultaneous study of toxin expression.
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    Cloning, characterisation and evolutionary relationships of two pyr4 genes from an Acremonium endophyte of perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 1994) Collett, Michael Anthony
    A fragment of the Claviceps purpurea pyr4 gene, encoding the enzyme orotidine-5'-monophosphate decarboxylase (OMPdecarboxylase) was used to screen a genomic library to an isolate (designated Lp1) of an Acremonium sp. which grows as an endophyte in perennial ryegrass (Lolium perenne). Four positive clones, λMC11, λMC12, λMC14 and λMC20 were isolated. Three of these clones, λMC12, λMC14 and λMC20 were overlapping clones from the same locus, while λMC11 was from a different locus. Fragments of these clones which hybridised with C. purpurea pyr4 were sequenced and found to have similarity with pyr4 from other fungi of the Pyrenomycetes and related Deuteromycetes, suggesting that Lp1 has evolved from a sexual Pyrenomycetes species. The pyr4 from λMC12, λMC14 and λMC20 was designated pyr4-1 and that from λMC11 was designated pyr4-2. The predicted ORFs of the two genes were highly conserved and the 5' non-coding nucleotide sequences were the least conserved regions. RT-PCR and northern analysis of total RNA from Lp1 demonstrated that transcripts approximately 1.4 kb in length were produced from the two genes and present at similar levels. Genomic fragments containing pyr4-1 or pyr4-2 were transformed into a strain of Aspergillus nidulans which has a mutation in the pyrG gene (encoding OMPdecarboxylase). Both of the Lp1 pyr4 complemented a pyrG mutation in Aspergillus nidulans, confirming that both pyr4-1 and pyr4-2 encode functional OMPdecarboxylases. Comparisons of pyr4 restriction fragment length polymorphisms (RFLPs) from Lp1 and isolates of Epichloë typhina, E. festucae, A. lolii, A. uncinatum, and three endophyte taxonomic groupings from Festuca arundinacea: FaTG-1 (=A. coenophialum), FaTG-2 and FaTG-3 suggested that pyr4-1 originated from E. typhina, the ryegrass choke pathogen, and pyr4-2 originated from A. lolii, another endophyte from perennial ryegrass. This suggested that Lp1 is an interspecific hybrid, between E. typhina and A. lolii. Comparisons of the variable 5' non-coding nucleotide sequences from pyr4 of Lp1 and other isolates demonstrated that E. typhina, and A. lolii or E. festucae were the most likely ancestors of the two pyr4 found in Lp1. The A. lolii and E. festucae sequences were very similar, suggesting they are closely related. A. lolii has most probably evolved from an E. festucae, and in the process lost the sexual cycle. Analysis of single spore purified isolates of Lp1 revealed that Lp1 was a homokaryon for pyr4. A Southern blot of a CHEF gel of Lp1 and these single spored isolates was hybridised to a pyr4 probe and demonstrated that pyr4-1 and pyr4-2 were present on either two chromosomes of similar size, or one chromosome. The hybridisation that gave rise to Lp1 was concluded to have been a relatively recent event, given the similarity of pyr4-1 and pyr4-2 nucleotide sequences to those of their probable ancestors, and the fact that both genes are expressed and functional. Interspecific hybridisation is probably widespread in the asexual endophytes, and may be an important event in their evolution, and the evolution of other fungal species.