1. Spirodela oligorrhiza on sterile glucose-mineral medium ceased to grow three days after transfer into darkness. 2. Cytokinins, supplied in the medium, allowed continuous growth of Spirodela after transfer into darkness. Other plant growth substances, or adenine analogues, were ineffective. 3. Kinetin stimulated production of new fronds after a 24 hour lag period when added to dormant cultures although it was rapidly taken up. Kinetin reached a constant concentration in the plantlets within 30 to 60 minutes of addition to the medium. 4. In the absence of cytokinin, dormancy continued for three or four weeks after which growth spontaneously resumed in darkness. The growth rate then reached almost half that achieved with optimum kinetin concentrations in darkness. Growth continued in darkness for at least eight weeks without cytokinin. 5. Pretreatment in the light with either metabolic inhibitors, kinetin, abscisic acid, or high or low temperature, essentially eliminated the period of dormancy of Spirodela transferred to darkness in the absence of cytokinin. Growth was reduced 50% to 90% during the pretreatments. 6. The pretreatments designed to affect plastid RNA, protein or ATP production were the most effective. The fastest growth rate achieved in darkness without cytokinin after pre-treatment was 10% less than that promoted by optimum kinetin. 7. Dormant Spirodela in darkness continued to incorporate precursors into RNA, DNA and protein at a rate 50% that in growing (plus kinetin) cultures. Net rates of macromolecule accumulation were extremely slow, indicating extensive degradation. 8. Addition of kinetine to non-growing Spirodela in darkness stimulated the synthesis of RNA, DNA and protein simultaneously after a lag of approximately one hour. The rates of precursor incorporation increased to equal those in continuously growing cultures. 9. Non-growing Spriodela in darkness rapidly accumulated starch. Kinetin had little or no effect on accumulation and mobilisation of starch. 10. 14C-glucose was taken up by growing (plus kinetin) and non-growing (minus kinetin) Spirodela in darkness, and was metabolized equally in each. Three times as much 14C-glucose entered starch in the non-growing cultures. 11. A model scheme for control of dormancy in Spirodela is proposed based on an inhibitory mediator. The mediator may be similar to the hypothetical mediator of the pleiotypic response shown by animal cells.

The GTP hydrolases (GTPases) are a ubiquitous superfamily of proteins with diverse cellular roles. Members of the recently-discovered DRG subfamily have been implicated in human disease and may play a role in cell division, differentiation or death. While recent work in our laboratory has focused on the expression and subcellular localisation of DRG1 and DRG2 in the model plant Arabidopsis thaliana (Etheridge et al., 1999; Etheridge, 2002), our understanding of the role of these proteins in planta remains unclear. A yeast two-hybrid (Y2H) library screen using Arabidopsis DRG2 as bait identified 16 interacting proteins, 75% of which are predicted to reside in cellular compartments other than the cytoplasm. Since DRG2 has previously been localised to cytoplasmic vesicles, this observation suggests that DRG2 could be involved in the trafficking of these proteins to their correct cellular locations. A single DRG2 interactor, DRGIP4, was selected for detailed study and its interaction with DRG2 was confirmed in vivo and in vitro. Based on sequence similarity to Pseudomonas PCD/DCoH, DRGIP4 was renamed PDL1 (PCD/DCoH-like protein 1). Members of the widespread PCD/DCoH family are bifunctional proteins which possess catalytic and transcriptional coactivation functions in different cellular compartments and in different oligomeric states. PDL1 is encoded by a single-copy gene in Arabidopsis and shares remarkable secondary and tertiary structural conservation with members of the PCD/DCoH family and is also capable of homomeric associations. PDL1 contains an N-terminal chloroplast transit peptide (cTP) which is functional in planta and is important for interaction with DRG2, suggesting that these proteins interact in the cytoplasm prior to the import of PDL1 into the chloroplast. PDL1 lacks most of the residues important for PCD/DCoH enzymatic activity but may retain a transcriptional coactivation function in the chloroplast. PDL1 homologs in other plants also contain an N-terminal cTP and a PCD/DCoH domain. pdl1 mRNA is expressed at moderate to high levels in the aerial tissues but not in the roots. Accumulation of pdl1 transcripts is light-inducible, and the pdl1 promoter contains several cis-elements which may be responsible for light-responsive transcription. Expression of the intron-GUS reporter gene under the control of the pdl1 promoter generally correlates with mRNA accumulation but reveals tight spatial control of gene expression. Overexpression of pdl1 in transgenic plants does not result in an obvious phenotype. However, downregulation of pdl1 in transgenic plants results in a pale green leaf phenotype associated with a reduction in photosynthetic pigment content and chloroplast numbers per cell. Leaf internal architecture and chloroplast ultrastructure are unaffected in these plants. This phenotype is similar to the arc (accumulation and replication of chloroplasts) mutants and mutants in the chloroplast division and protein import machinery. PDL1 may therefore be involved in the process or regulation of chloroplast division in Arabidopsis. The pdl1 downregulation phenotype is also associated with pleiotropic effects on plant growth. A second PCD/DCoH-like protein, PDL2, was identified in the Arabidopsis genome sequence. Like PDL1, PDL2 shares limited primary sequence similarity to members of the PCD/DCoH family but retains characteristic secondary and tertiary structural features. PDL2 contains an N-terminal mitochondrial transit peptide (mTP) and is expressed at low levels in all plant tissues. PDL2 homologs in other plants also contain an N-terminal mTP and a PCD/DCoH domain. The significance of PDL2 for the function of PDL1 and DRG2 is not yet clear. Overexpression of drg1 in transgenic plants does not result in an obvious phenotype, while downregulation of drg1 in transgenic plants affects general aspects of plant growth which may be unrelated to DRG1 function. To investigate the role of DRGs in a different model system, homologs of drg1 and drg2 were isolated from Saccharomyces cerevisiae (baker’s yeast) and Schizosaccharomyces pombe (fission yeast). Knockout of either or both genes in transgenic yeast is non-lethal and results in sensitivity to agents which disrupt intra- and inter-molecular protein interactions. This phenotype is consistent with a role in protein trafficking. Overexpression of drg1 or drg2 in S. cerevisiae does not result in an obvious phenotype, while overexpression of either gene in S. pombe results in a slow growth phenotype. DRG1 and DRG2 are localised in the cytoplasm in S. pombe. The results presented here suggest that DRGs may be involved in the trafficking of proteins to different subcellular compartments. This research has provided a foundation for the detailed functional characterisation of plant and yeast DRGs and of the novel PDL family in Arabidopsis.

The GTP hydrolases (GTPases) are a ubiquitous superfamily of proteins with diverse cellular roles. Members of the recently-discovered DRG subfamily have been implicated in human disease and may play a role in cell division, differentiation or death. While recent work in our laboratory has focused on the expression and subcellular localisation of DRG1 and DRG2 in the model plant Arabidopsis thaliana (Etheridge et al., 1999; Etheridge, 2002), our understanding of the role of these proteins in planta remains unclear. A yeast two-hybrid (Y2H) library screen using Arabidopsis DRG2 as bait identified 16 interacting proteins, 75% of which are predicted to reside in cellular compartments other than the cytoplasm. Since DRG2 has previously been localised to cytoplasmic vesicles, this observation suggests that DRG2 could be involved in the trafficking of these proteins to their correct cellular locations. A single DRG2 interactor, DRGIP4, was selected for detailed study and its interaction with DRG2 was confirmed in vivo and in vitro. Based on sequence similarity to Pseudomonas PCD/DCoH, DRGIP4 was renamed PDL1 (PCD/DCoH-like protein 1). Members of the widespread PCD/DCoH family are bifunctional proteins which possess catalytic and transcriptional coactivation functions in different cellular compartments and in different oligomeric states. PDL1 is encoded by a single-copy gene in Arabidopsis and shares remarkable secondary and tertiary structural conservation with members of the PCD/DCoH family and is also capable of homomeric associations. PDL1 contains an N-terminal chloroplast transit peptide (cTP) which is functional in planta and is important for interaction with DRG2, suggesting that these proteins interact in the cytoplasm prior to the import of PDL1 into the chloroplast. PDL1 lacks most of the residues important for PCD/DCoH enzymatic activity but may retain a transcriptional coactivation function in the chloroplast. PDL1 homologs in other plants also contain an N-terminal cTP and a PCD/DCoH domain. pdl1 mRNA is expressed at moderate to high levels in the aerial tissues but not in the roots. Accumulation of pdl1 transcripts is light-inducible, and the pdl1 promoter contains several cis-elements which may be responsible for light-responsive transcription. Expression of the intron-GUS reporter gene under the control of the pdl1 promoter generally correlates with mRNA accumulation but reveals tight spatial control of gene expression. Overexpression of pdl1 in transgenic plants does not result in an obvious phenotype. However, downregulation of pdl1 in transgenic plants results in a pale green leaf phenotype associated with a reduction in photosynthetic pigment content and chloroplast numbers per cell. Leaf internal architecture and chloroplast ultrastructure are unaffected in these plants. This phenotype is similar to the arc (accumulation and replication of chloroplasts) mutants and mutants in the chloroplast division and protein import machinery. PDL1 may therefore be involved in the process or regulation of chloroplast division in Arabidopsis. The pdl1 downregulation phenotype is also associated with pleiotropic effects on plant growth. A second PCD/DCoH-like protein, PDL2, was identified in the Arabidopsis genome sequence. Like PDL1, PDL2 shares limited primary sequence similarity to members of the PCD/DCoH family but retains characteristic secondary and tertiary structural features. PDL2 contains an N-terminal mitochondrial transit peptide (mTP) and is expressed at low levels in all plant tissues. PDL2 homologs in other plants also contain an N-terminal mTP and a PCD/DCoH domain. The significance of PDL2 for the function of PDL1 and DRG2 is not yet clear. Overexpression of drg1 in transgenic plants does not result in an obvious phenotype, while downregulation of drg1 in transgenic plants affects general aspects of plant growth which may be unrelated to DRG1 function. To investigate the role of DRGs in a different model system, homologs of drg1 and drg2 were isolated from Saccharomyces cerevisiae (baker’s yeast) and Schizosaccharomyces pombe (fission yeast). Knockout of either or both genes in transgenic yeast is non-lethal and results in sensitivity to agents which disrupt intra- and inter-molecular protein interactions. This phenotype is consistent with a role in protein trafficking. Overexpression of drg1 or drg2 in S. cerevisiae does not result in an obvious phenotype, while overexpression of either gene in S. pombe results in a slow growth phenotype. DRG1 and DRG2 are localised in the cytoplasm in S. pombe. The results presented here suggest that DRGs may be involved in the trafficking of proteins to different subcellular compartments. This research has provided a foundation for the detailed functional characterisation of plant and yeast DRGs and of the novel PDL family in Arabidopsis.

1. Sixteen New Zealand and 10 overseas isolates of Phytophthora cinnamomi Rands have been compared for oospore production in paired and single cultures. Considerable variability existed among the 26 isolates in respect of mating ability and responses to physical and chemical factors. Addition of steroids and did not influence oospore production. However, hypoxanthine increased oospore production in two isolates. 2. Discovery of the role of steroids in relation to growth and asexual sporulation of Phytophthora ssp. makes it possible to replace natural media by a synthetic medium. For this purpose an investigation was made into the carbon, nitrogen, vitamin and calcium requirements of P. cinnamomi. Twenty carbon compounds were tested as major sources of carbon with Ca(NO3)2 and glutamic acid as nitrogen sources. Dextrin, starch, and sucrose were the best sources of carbon for growth and asexual reproduction. Xylose also supported abundant sporulation, although growth was slow. Organic acids and alcohols were poor sources of carbon. The best sources of organic nitrogen tested for growth were glutamine and glutamic acid. Tryptophan, phenyl-alanine, and cystine appeared to be inhibitory. Of the vitamins tested, only thiamine was required by the fungus. Five calcium compounds added singly to the basal medium gave no increase in the growth of the fungus. 3. & 4. The effect of soil extracts on sporulation of P. cinnamomi has been investigated. Non-sterile soil extracts from different soil types and from soils under different plan species all supported sporulation, stimulatory activity being markedly reduced only after 1000-fold dilution. Autumn soil was most active in stimulating sporulation, wheras summer soil was inactive. When peptone was added to summer soil, the stimulatory activity of the extract was restored. A wide variety of materials added to distilled water failed to stimulate sporulation. Soil extract lost its stimulatory properties after sterilisation by autoclaving, steaming, Seitz or sintered glass filtration, dialysis, ultrasonic disintegration, ultracentrifugation or treatment with propylene oxide or alcohol. Root exudates and microorganisms other than bacteria had no effect on sporulation. Fifty-nine bacterial cultures classified in 15 genera stimulated sporulation on one or more occasions when transferred from nutrient broth cultures but not from nutrient agar slopes. Of 16 morphologically distinct bacteria isolated from wet autumn soil, two Pseudomonas species stimulated abundant sporulation when transferred from agar plates to sterile soil extract. The activity of these isolates was lost after six weeks in culture, but was partially restored by growing them in nutrient broth. Extracts from sterilised soil inoculated with attenuated cultures of these Pseudomonas species were stimulatory but subcultures from these extracts were only stimulatory if grown in broth. The difference in ability to stimulate sporulation between cultures from broth and from agar was not due to bacterial numbers, flagellation, or fimbriation, but was associated with the additional nutrient transferred with the bacterial inoculum from a broth culture. Streptomycin was the only one of several antibiotics tested which inhibited sporulation. Irrigation of P. cinnamomi mycelium with continuously-produced sterile filtrate from soil extracts produced sporangia in 48 hours.

1. Spirodela oligorrhiza on sterile glucose-mineral medium ceased to grow three days after transfer into darkness. 2. Cytokinins, supplied in the medium, allowed continuous growth of Spirodela after transfer into darkness. Other plant growth substances, or adenine analogues, were ineffective. 3. Kinetin stimulated production of new fronds after a 24 hour lag period when added to dormant cultures although it was rapidly taken up. Kinetin reached a constant concentration in the plantlets within 30 to 60 minutes of addition to the medium. 4. In the absence of cytokinin, dormancy continued for three or four weeks after which growth spontaneously resumed in darkness. The growth rate then reached almost half that achieved with optimum kinetin concentrations in darkness. Growth continued in darkness for at least eight weeks without cytokinin. 5. Pretreatment in the light with either metabolic inhibitors, kinetin, abscisic acid, or high or low temperature, essentially eliminated the period of dormancy of Spirodela transferred to darkness in the absence of cytokinin. Growth was reduced 50% to 90% during the pretreatments. 6. The pretreatments designed to affect plastid RNA, protein or ATP production were the most effective. The fastest growth rate achieved in darkness without cytokinin after pre-treatment was 10% less than that promoted by optimum kinetin. 7. Dormant Spirodela in darkness continued to incorporate precursors into RNA, DNA and protein at a rate 50% that in growing (plus kinetin) cultures. Net rates of macromolecule accumulation were extremely slow, indicating extensive degradation. 8. Addition of kinetine to non-growing Spirodela in darkness stimulated the synthesis of RNA, DNA and protein simultaneously after a lag of approximately one hour. The rates of precursor incorporation increased to equal those in continuously growing cultures. 9. Non-growing Spriodela in darkness rapidly accumulated starch. Kinetin had little or no effect on accumulation and mobilisation of starch. 10. 14C-glucose was taken up by growing (plus kinetin) and non-growing (minus kinetin) Spirodela in darkness, and was metabolized equally in each. Three times as much 14C-glucose entered starch in the non-growing cultures. 11. A model scheme for control of dormancy in Spirodela is proposed based on an inhibitory mediator. The mediator may be similar to the hypothetical mediator of the pleiotypic response shown by animal cells.

1. Sixteen New Zealand and 10 overseas isolates of Phytophthora cinnamomi Rands have been compared for oospore production in paired and single cultures. Considerable variability existed among the 26 isolates in respect of mating ability and responses to physical and chemical factors. Addition of steroids and did not influence oospore production. However, hypoxanthine increased oospore production in two isolates. 2. Discovery of the role of steroids in relation to growth and asexual sporulation of Phytophthora ssp. makes it possible to replace natural media by a synthetic medium. For this purpose an investigation was made into the carbon, nitrogen, vitamin and calcium requirements of P. cinnamomi. Twenty carbon compounds were tested as major sources of carbon with Ca(NO3)2 and glutamic acid as nitrogen sources. Dextrin, starch, and sucrose were the best sources of carbon for growth and asexual reproduction. Xylose also supported abundant sporulation, although growth was slow. Organic acids and alcohols were poor sources of carbon. The best sources of organic nitrogen tested for growth were glutamine and glutamic acid. Tryptophan, phenyl-alanine, and cystine appeared to be inhibitory. Of the vitamins tested, only thiamine was required by the fungus. Five calcium compounds added singly to the basal medium gave no increase in the growth of the fungus. 3. & 4. The effect of soil extracts on sporulation of P. cinnamomi has been investigated. Non-sterile soil extracts from different soil types and from soils under different plan species all supported sporulation, stimulatory activity being markedly reduced only after 1000-fold dilution. Autumn soil was most active in stimulating sporulation, wheras summer soil was inactive. When peptone was added to summer soil, the stimulatory activity of the extract was restored. A wide variety of materials added to distilled water failed to stimulate sporulation. Soil extract lost its stimulatory properties after sterilisation by autoclaving, steaming, Seitz or sintered glass filtration, dialysis, ultrasonic disintegration, ultracentrifugation or treatment with propylene oxide or alcohol. Root exudates and microorganisms other than bacteria had no effect on sporulation. Fifty-nine bacterial cultures classified in 15 genera stimulated sporulation on one or more occasions when transferred from nutrient broth cultures but not from nutrient agar slopes. Of 16 morphologically distinct bacteria isolated from wet autumn soil, two Pseudomonas species stimulated abundant sporulation when transferred from agar plates to sterile soil extract. The activity of these isolates was lost after six weeks in culture, but was partially restored by growing them in nutrient broth. Extracts from sterilised soil inoculated with attenuated cultures of these Pseudomonas species were stimulatory but subcultures from these extracts were only stimulatory if grown in broth. The difference in ability to stimulate sporulation between cultures from broth and from agar was not due to bacterial numbers, flagellation, or fimbriation, but was associated with the additional nutrient transferred with the bacterial inoculum from a broth culture. Streptomycin was the only one of several antibiotics tested which inhibited sporulation. Irrigation of P. cinnamomi mycelium with continuously-produced sterile filtrate from soil extracts produced sporangia in 48 hours.

1. Spirodela oligorrhiza on sterile glucose-mineral medium ceased to grow three days after transfer into darkness. 2. Cytokinins, supplied in the medium, allowed continuous growth of Spirodela after transfer into darkness. Other plant growth substances, or adenine analogues, were ineffective. 3. Kinetin stimulated production of new fronds after a 24 hour lag period when added to dormant cultures although it was rapidly taken up. Kinetin reached a constant concentration in the plantlets within 30 to 60 minutes of addition to the medium. 4. In the absence of cytokinin, dormancy continued for three or four weeks after which growth spontaneously resumed in darkness. The growth rate then reached almost half that achieved with optimum kinetin concentrations in darkness. Growth continued in darkness for at least eight weeks without cytokinin. 5. Pretreatment in the light with either metabolic inhibitors, kinetin, abscisic acid, or high or low temperature, essentially eliminated the period of dormancy of Spirodela transferred to darkness in the absence of cytokinin. Growth was reduced 50% to 90% during the pretreatments. 6. The pretreatments designed to affect plastid RNA, protein or ATP production were the most effective. The fastest growth rate achieved in darkness without cytokinin after pre-treatment was 10% less than that promoted by optimum kinetin. 7. Dormant Spirodela in darkness continued to incorporate precursors into RNA, DNA and protein at a rate 50% that in growing (plus kinetin) cultures. Net rates of macromolecule accumulation were extremely slow, indicating extensive degradation. 8. Addition of kinetine to non-growing Spirodela in darkness stimulated the synthesis of RNA, DNA and protein simultaneously after a lag of approximately one hour. The rates of precursor incorporation increased to equal those in continuously growing cultures. 9. Non-growing Spriodela in darkness rapidly accumulated starch. Kinetin had little or no effect on accumulation and mobilisation of starch. 10. 14C-glucose was taken up by growing (plus kinetin) and non-growing (minus kinetin) Spirodela in darkness, and was metabolized equally in each. Three times as much 14C-glucose entered starch in the non-growing cultures. 11. A model scheme for control of dormancy in Spirodela is proposed based on an inhibitory mediator. The mediator may be similar to the hypothetical mediator of the pleiotypic response shown by animal cells.

The GTP hydrolases (GTPases) are a ubiquitous superfamily of proteins with diverse cellular roles. Members of the recently-discovered DRG subfamily have been implicated in human disease and may play a role in cell division, differentiation or death. While recent work in our laboratory has focused on the expression and subcellular localisation of DRG1 and DRG2 in the model plant Arabidopsis thaliana (Etheridge et al., 1999; Etheridge, 2002), our understanding of the role of these proteins in planta remains unclear. A yeast two-hybrid (Y2H) library screen using Arabidopsis DRG2 as bait identified 16 interacting proteins, 75% of which are predicted to reside in cellular compartments other than the cytoplasm. Since DRG2 has previously been localised to cytoplasmic vesicles, this observation suggests that DRG2 could be involved in the trafficking of these proteins to their correct cellular locations. A single DRG2 interactor, DRGIP4, was selected for detailed study and its interaction with DRG2 was confirmed in vivo and in vitro. Based on sequence similarity to Pseudomonas PCD/DCoH, DRGIP4 was renamed PDL1 (PCD/DCoH-like protein 1). Members of the widespread PCD/DCoH family are bifunctional proteins which possess catalytic and transcriptional coactivation functions in different cellular compartments and in different oligomeric states. PDL1 is encoded by a single-copy gene in Arabidopsis and shares remarkable secondary and tertiary structural conservation with members of the PCD/DCoH family and is also capable of homomeric associations. PDL1 contains an N-terminal chloroplast transit peptide (cTP) which is functional in planta and is important for interaction with DRG2, suggesting that these proteins interact in the cytoplasm prior to the import of PDL1 into the chloroplast. PDL1 lacks most of the residues important for PCD/DCoH enzymatic activity but may retain a transcriptional coactivation function in the chloroplast. PDL1 homologs in other plants also contain an N-terminal cTP and a PCD/DCoH domain. pdl1 mRNA is expressed at moderate to high levels in the aerial tissues but not in the roots. Accumulation of pdl1 transcripts is light-inducible, and the pdl1 promoter contains several cis-elements which may be responsible for light-responsive transcription. Expression of the intron-GUS reporter gene under the control of the pdl1 promoter generally correlates with mRNA accumulation but reveals tight spatial control of gene expression. Overexpression of pdl1 in transgenic plants does not result in an obvious phenotype. However, downregulation of pdl1 in transgenic plants results in a pale green leaf phenotype associated with a reduction in photosynthetic pigment content and chloroplast numbers per cell. Leaf internal architecture and chloroplast ultrastructure are unaffected in these plants. This phenotype is similar to the arc (accumulation and replication of chloroplasts) mutants and mutants in the chloroplast division and protein import machinery. PDL1 may therefore be involved in the process or regulation of chloroplast division in Arabidopsis. The pdl1 downregulation phenotype is also associated with pleiotropic effects on plant growth. A second PCD/DCoH-like protein, PDL2, was identified in the Arabidopsis genome sequence. Like PDL1, PDL2 shares limited primary sequence similarity to members of the PCD/DCoH family but retains characteristic secondary and tertiary structural features. PDL2 contains an N-terminal mitochondrial transit peptide (mTP) and is expressed at low levels in all plant tissues. PDL2 homologs in other plants also contain an N-terminal mTP and a PCD/DCoH domain. The significance of PDL2 for the function of PDL1 and DRG2 is not yet clear. Overexpression of drg1 in transgenic plants does not result in an obvious phenotype, while downregulation of drg1 in transgenic plants affects general aspects of plant growth which may be unrelated to DRG1 function. To investigate the role of DRGs in a different model system, homologs of drg1 and drg2 were isolated from Saccharomyces cerevisiae (baker’s yeast) and Schizosaccharomyces pombe (fission yeast). Knockout of either or both genes in transgenic yeast is non-lethal and results in sensitivity to agents which disrupt intra- and inter-molecular protein interactions. This phenotype is consistent with a role in protein trafficking. Overexpression of drg1 or drg2 in S. cerevisiae does not result in an obvious phenotype, while overexpression of either gene in S. pombe results in a slow growth phenotype. DRG1 and DRG2 are localised in the cytoplasm in S. pombe. The results presented here suggest that DRGs may be involved in the trafficking of proteins to different subcellular compartments. This research has provided a foundation for the detailed functional characterisation of plant and yeast DRGs and of the novel PDL family in Arabidopsis.

Fungal endophytes of the genus Neotyphodium form stable symbiotic associations, with grasses, that are symptomless and generally considered to be mutualistic. The benefits that these fungi confer to their grass hosts are exploited in pastoral agriculture systems. The production of a range of secondary metabolites, specifically alkaloids including peramine and ergovaline can give their host plants an ecological advantage in certain environments. Neotyphodium endophytes are asexual and have lost the ability to transfer horizontally between hosts making seed transmission a vital feature of the association. This thesis reports the occurrence of phenotypically different perennial ryegrass plants (Lolium perenne) in a population infected with Neotyphodium lolii. Here we show that the change in the plants is directly attributable to a variant endophyte that they host. Isolation of the variant endophyte reveals a change in colony growth compared to the wild-type resident endophyte in the population, which has a white and cottony phenotype. Colonies of the variant endophyte are smaller than wild-type colonies and mucoid, with hyphal filaments forming aggregates. Evidence shows that the switch between colony morphologies occurs at a very high frequency, is reversible, and appears to be environmentally induced. This suggests that the switching phenomenon involves gene regulation rather than mutation. When endophyte-free plants are infected, with either white and cottony (wild-type) or mucoid (variant) fungal colonies, they assume a morphology consistent with the state of the fungus at the time of inoculation, that is normal or dwarfed, respectively. In addition, re-isolation of endophyte from either normal or dwarfed plants always yields white and cottony or mucoid colonies, respectively, suggesting that the host environment stabilizes the state of the fungus. Proteomic profiling revealed differences in protein expression between plants infected with either the wild-type or mucoid fungus. Furthermore, host plants containing the mucoid fungus have never flowered or produced seed. Thus, if this change in the fungal symbiont occurs in a competitive natural environment the mucoid fungus and its host plant may not persist beyond the first generation. This thesis provides insights into the plastic nature of fungal endophyte/grass symbiota and discusses possible mechanisms for the observed morphological switching in culture and host dwarfing.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / 1. The most appropriate methods were investigated for producing Rous sarcoma virus of suitable quantity and quality for use in the study of the viral RNA by electron microscopy. The roller bottle method of Smith and Bernstein (1973) which was adopted, produced virus yields of up to 5mg per litre of transformed cell culture supernatant after 24 hour incubations, and 0.2mg per litre of culture supernatant after 4 hour incubations. 2. The method of purifying RNA tumour viruses which resulted in the least damage to the virions was found to be isopyncic and velocity sedimentation in Ficoll density gradients containing 5mM tris-HCl and 1mM EDTA pH 8.5. The use of solutions of sucrose or >0.1M salt resulted in both osmotic changes in the virus and viral aggregation. 3. The lipoprotein coat of the Rous sarcoma virus was shown by freeze-fracturing and electron microscopy to have properties similar to those of plasma membranes, except that the number of intramembranous particles was smaller. The hydrated diameter of Rous sarcoma virus was estimated from freeze-fracture replicas of purified virions to be 140nm. 4. Vesicular contaminants, derived from serum, were present in Rous sarcoma virus preparations that had been purified from transformed cell culture supernatants. The isolated contaminants resembled virus when examined by both freeze-fracturing and negative-staining, but were readily distinguished from virus in thin sections. The virus-like serum vesicles were present in sera from several different sources. When treated with detergent and subjected to polyacylamide gel electrophoresis, the vesicles were found to contain polypeptides that possessed similar electrophoretic mobilities to those of Rous sarcoma virus polypeptides. It is probable that extraneous nucleic acid molecules, observed in preparations of Rous sarcoma virus RNA were the result of VLSV contamination of virus suspensions. 5. Contamination of purified virus suspensions by virus-like material derived from serum was reduced by centrifugation of the serum prior to its addition to cell culture medium. Virus suspensions, purified from cell supernatants from which the contaminating vesicles had been removed, were resolved in sharp bands at p = 1.07 g/ml in Ficoll density gradients; in the analytical ultracentrifuge they sedimented as homogenous populations with a sedimentation value of 740s20,w and were observed by electron microscopy to be relatively free of contaminants. 6. The maximum length of molecules from preparations of both 60-70s and 30-40s viral RNA prepared in 80% and 50% formamide respectively was 2.5μm, but both preparations were not homogeneous since they contained other, smaller molecules. 7. A model is proposed in which the difference in physical properties between the native (60-70S) form and the denatured (30-40S) form of the viral RNA is suggested to be the result of two possible conformations of a single RNA molecule. This model is an alternative to the prevailing model in which the RNA tumour virus genome is proposed to contain a number of RNA molecules of equivalent size.