This dissertation examines fungal genetics in two systems: Penicillium chrysogenum and Rhizopogon salebrosus. Penicillium chrysogenum holds historical and medical significance for the discovery and development of penicillin as an antibiotic. Rhizopogon salebrosus is an ecologically important mycorrhizal fungus that forms symbioses with both green plants and non-photosynthetic mycoheterotrophic plants. Mycoheterotrophs, plants that parasitize mycorrhizal fungi and cheat the classical mycorrhizal symbiosis, have been integral to the discovery and continuing study of mycorrhizal fungi.
The first chapter describes the utilization of assembly-free synteny analysis to identify rearrangements in wild P. chrysogenum strains. While traditional methods of mutagenesis and selection have been effective in improving production of compounds to commercial scale, the genetic changes behind the altered phenotypes have remained largely unclear. We utilized high-throughput Illumina short read sequencing of a wild Penicillium chrysogenum strain in order to make whole genome comparisons to a sequenced improved strain (WIS 54-1255). We developed an assembly-free method of identifying rearrangements and validated the in silico predictions with a PCR-based assay followed by Sanger sequencing. In addition to finding a previously published inversion in the penicillin biosynthesis cluster, we located several genes related to penicillin production associated with these rearrangements. By comparing the configuration of rearrangement events among several historically important strains known to be good penicillin producers to a collection of recently isolated wild strains, we suggest that wild strains with rearrangements similar to those in known good penicillin producers may be viable candidates for further improvement efforts.
The second chapter describes use of the phytohormone gibberellic acid (GA), a phytohormone which induces germination in many plants, to induce asymbiotic germination of two obligate mycoheterotrophic plants. These plants had previously only been seen to germinate in the presence of a symbiotic fungus, and such limitations prevented further study with these plants. We found that GA stimulates Pterospora andromedea germination at frequencies that can be far higher than that achieved with its fungal symbiont, R. salebrosus, alone. With constant exposure to 0.5M GA, we found nearly 80% germination, as opposed to only 15% with R. salebrosus. Even a short exposure to GA is sufficient for significantly enhanced germination. One day of exposure to 0.5mM GA induced ~55% stage 3 germination, and three days of exposure led to ~ 70% stage 3 germination. Exposing seeds to a lower dosage (0.1mM GA) for a longer time (2 weeks) produced a comparable germination rate. The closely related plant Sarcodes sanguinea was more resistant to GA stimulation. Exposure to GA is the only known method of inducing monotrope germination without the presence of a specific fungal symbiont. GA stimulation achieved workable germination rates for further studies, and GA may also be used as an improved assay for seed viability.
In the third chapter we utilize GA germination of P. andromedea seeds to examine transcriptional differences in R. salebrosus with partner and non-partner mycohterotroph seeds. The monotropes constitute a group of obligately dependent, non-photosynthetic mycoehterotrophs with extreme host specificity. Here we generate a de novo transcriptome assembly and examine differential expression when R. salebrosus interacts with seeds from partner (R. salebrosus type) and non-partner (R. arctostaphyli type) P. andromedea. Because P. andromedea seeds are very small, with few nutritional resources, germination in the presence of a suitable host fungus is a critical step in the establishment of symbiosis. We have identified putative genes in categories thought to be important for ectomycorrhizal symbiosis. These include stress response, host defense, cellular transport, secretion, carbohydrate metabolism and transport, and signal transduction. To our knowledge, this is the first study of gene expression with any mycorrhizal fungus involved with a mycoheterotrophic plant.
