Identification of genes regulating the plant-specific expression of the ItmM gene in Epichloe festucae : this thesis is presented as a partial fulfillment of the requirements for the degree of Master of Science (Msc) in Genetics at Massey University, Palmerston North, New Zealand
The fungal endophyte Epichloë festucae forms a largely mutualistic association with the
ryegrass species Lolium perenne. E. festucae produces a range of bio-protective
alkaloids that protect the host grass from herbivory by both mammals and insects. One
such alkaloid, Lolitrem B, is a potent mycotoxin and the causative agent of ryegrass
staggers in livestock.
Ten genes required for biosynthesis of lolitrem B are encoded in the ltm gene cluster.
The ltm genes are expressed in a plant-specific manner, with high levels of expression
in planta and very low levels of expression in culture. The mechanism regulating ltm
gene expression is unknown but it is predicted to involve signalling from the host plant.
The ltmM gene was chosen for use in the investigation of ltm gene regulation because
the flanking regions do not contain retrotransposon sequence, which surrounds much of
the ltm gene cluster. To identify fungal genes involved in the plant-induced expression
of ltmM, a mutagenesis and screening system was developed using a PltmM-gusA
‘knock-in’ construct to detect expression from the ltmM promoter. Agrobacterium
tumefaciens-mediated T-DNA mutagenesis was used to create a set of mutants with
random insertions in the genome. Mutants were then screened for altered PltmM-gusA
expression, both in culture and in planta. Three mutants were identified with increased
PltmM-gusA expression in culture, however, no mutants were identified with loss of
PltmM-gusA expression in planta. This indicates that a mechanism of repression is
involved in the plant-induced expression of ltmM, either directly or indirectly.
TM mutants of interest were also observed for altered symbiosis phenotypes. Mutants
were identified with reduced colonisation rates and altered hyphal growth in planta.
Integration sites were identified for two colonisation mutants and the disrupted genes
are predicted to be the CTP:cholinephosphate cytidylyltransferase (CCT) gene PCT1
and the mitogen-activated protein kinsase kinase (MAPKK) gene mkk2.