Treena Burgess

Ectomycorrhizas are formed as the outcome of a symbiotic association between the fine roots of the majority of temperate forest tree species and numerous species of higher order fungi. The development of an ectomycorrhizal structure involves the attraction of the fungal symbiont to the root surface, its recognition and attachment, the subsequent proliferation of the hyphre on the root surface and the formation of the Hartig net between root cells. Ectomycorrhizal formation is a complex and dynamic process during which the morphology, physiology and biochemistry of the individual symbionts alter, culminating in the formation of the symbiotic structure. These phenotypic changes are pre-empted by an alteration in gene expression and protein biosynthesis of both symbionts. However, whilst this is a recognised principle, the critical stages of control, at which compatibility, aggressiveness and specificity are determined, are unknown. These issue scan be approached by exploiting the natural variation that exists within a fungal population. This thesis focuses on Pisolithus-Eucalyptus ectomycorrhizas as a model system and exploits intraspecific variation in Pisolithus to investigate changes in protein biosynthesis during ectomycorrhizal formation. The Pisolithus-Eucalyptus symbiosis is well suited to such a study as both symbionts grow rapidly in vitro and ectomycorrhizas are produced within days. The taxonomy of the genus Pisolithus is currently unclear. Thus, before selecting isolates for developmental studies it was necessary to classify available isolates. One hundred isolates of Pisolithus, predominantly collected within Australia, were assessed and found to vary greatly in basidiocarp and basidiospore morphology and culture characteristics. These isolates were also classified according to separation of soluble polypeptides using one dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (lD-SDS-PAGE). This technique resulted in groups that corresponded to host species and geographic location. Subsequently, 20 Pisolithus isolates, covering a range of hosts, basidiocarp types and geographic locations were compared on their ability to form mycorrhiza in vitro with Eucalyptus grandis and to stimulate seedling growth in vivo. There was a large variation between isolates in the rate of mycorrhizal development ranging from incompatible to very aggressive. In vivo observations of growth stimulation of E. grandis, under conditions of limiting phosphorus, were positively correlated to the extent of mycorrhizal development in vitro. Isolates of differing aggressiveness were then selected to examine early changes in protein biosynthesis during ectomycorrhizal development. This study was facilitated by the development of a simple and reproducible in vitro system to rapidly produce eucalypt ectomycorrhizas. The seedlings germinated and grew in the presence of fungal exudates resulting in a two fold increase in the emergence of lateral root tips compared to seedlings germinated separately and challenged later. In addition, the lateral root tips of inoculated seedlings emerged closer to the primary root apex. The differentiation of ectomycorrhizas was followed by examining lateral tips basipetally along a single primary root. Protein biosynthesis was examined over a time sequence (pre-contact to 8 days after contact) using 2D-PAGE of proteins labelled by in vivo incorporation of [35S] radiolabelled amino acids. For compatible isolates, ectomycorrhizal development was accompanied by an apparent loss of plant polypeptides, differential accumulation of some fungal polypeptides and the emergence of symbiosis-specific polypeptides. The accumulation of fungal polypeptides was more rapid than expected from the fungal biomass. Protein biosynthesis with the incompatible isolate was unaltered and roots sampled at 8 days had the same 2D-PAGE profile as an artificial mix of protein from non-inoculated seedlings and fungal hyphre. The rate of accumulation of fungal proteins was dependent upon the aggressiveness of the isolate. In order to relate the changes in protein biosynthesis more closely to the developmental stage of the ectomycorrhizas, protein biosynthesis was related to primary root age by dividing the primary root into 10 mm segments. As the age of the primary root segment increased, so did the age of ectomycorrhizallateral tips which had emerged on that segment. The youngest segment of the primary root, which included the primary root apex, produced a biosynthesis profile very similar to that of non-inoculated roots. By contrast, the older segments of the primary root, produced a biosynthesis profile very similar to that of the free-living hyphre. Thus, the changes in protein biosynthesis along the primary root were similar to those observed during the time sequence except that abundant plant polypeptides were restricted to the youngest segment. It was concluded that, the domination of the fungal partner in the biosynthesis of developing ectomycorrhizas is probably a consequence of stimulated fungal growth and a corresponding decrease in plant meristematic activity. In both experiments, the major changes in protein biosynthesis were observed in a group of acid polypeptides, of fungal origin, found between 29 and 37 kDa which constituted about 50% of protein biosynthesis in fully developed ectornycorrhizas. These polypeptides f1rst appeared at contact and increased during ectomycorrhizal formation. Fractionation of total soluble protein into cell wall, membrane, soluble cytoplasmic and secreted fractions, revealed that some of these acid polypeptides were bound to the cell membrane whilst others are associated with the cell wall or secreted into the external medium of free-living cultures. Some isomers which were stimulated in ectomycorrhizas were also present in free-living hyphre grown at high glucose levels, suggesting that the expression of these polypeptides could be constitutive in response to increased carbohydrate activity at the root surface. However, other isomers were more highly expressed in the ectomycorrhizas. These polypeptides are potentially involved in recognition, attachment and the formation of the extracellular matrix of the mantle. The role of this polypeptide group was less significant in mature ectomycorrhizas, further supporting their role in the formation of the symbiosis rather than in its functioning. In conclusion, fungal protein biosynthesis is dominant during the early stages of ectomycorrhizal development. Secreted proteins, either bound to the cell wall or free, play an important role in determining success of symbiosis, and the rapid stimulation of these fungal proteins is indicative of a compatible association. To pinpoint the exact stage of induction of these proteins, individual inoculated tips ranging in development from pre-contact to maturity must be examined. This can be facilitated by the purification of specific proteins and the raising of antibodies to be used in immunolocalization or to probe for corresponding eDNA's.