Development of a genetic transformation system in the family Ceratocystidaceae with specific reference to Ceratocystis albifundus

Abstract

The usefulness of transformation systems in fungal pathogens has been well described. Although functional studies in the Ceratocystidaceae is lagging behind model fungi from Aspergillus and Neurospora, the economic impact of many species in this family on both the agricultural and forestry industries speaks to the need to address this. Developing reliable transformation systems is the first step towards functionally studying various aspects related to the biology of these important fungi, including the systems underlying pathogenicity. This dissertation aimed to address this need for transformation systems by using a two-pronged approach. The first explored the viability of transferring an Agrobacterium-mediated transformation system, that has been established in Ceratocystis albifundus, across species and generic boundaries. During the course of this study, the existing protocol was successfully optimised to improve the yield of transformants from C. albifundus. When this protocol was applied as is to eight other species representing eight Ceratocystidaceae genera, successful transformation was possible in six of the species representing six genera. This finding indicates that the technology is highly transferable, even among distant relatives from the same family. It should be noted that the transformation efficiency was markedly lower in these species when compared to C. albifundus. Several suggestions were made for adjustments that should improve these efficiencies for future studies. The current study produced a small collection of random transformants expressing green fluorescent protein, making them potentially useful for studies looking at the infection pathway of these pathogens. Although the Agrobacterium system is effective for transformations in C. albifundus, this approach requires the use of the Ti plasmid which necessitates multiple cloning steps when doing targeted mutagenesis. As a means of addressing these expensive and time-consuming cloning steps, the second approach was an attempt to establish a PEG/CaCl2-protoplast transformation protocol in this species. This system can make use of linear DNA, such as PCR products, making it ideal for high-throughput gene editing. The use of a PEG/CaCl2-protoplast transformation system relies on the successful production of viable protoplasts, and a protocol was created to produce these from C. albifundus conidia. When these protoplasts were used for transformation with PEG/CaCl2-protoplast protocols, no transformants were obtained in spite of the many alterations that were made to various parts of the protocol. However, the study still provides a tangible starting point for future attempts at optimizing the system. Additionally, the ability to produce protoplasts from C. albifundus will allow other transformation systems to be tested that also depend on the use of these specialized cells. Protoplasts are also useful beyond transformation and could be used to determine the chromosome number in this important pathogen, something that has previously not been possible. The work presented in this dissertation provides a benchmark for the transformation of other Ceratocystidaceae species that has not yet been investigated. It is envisioned that the findings and technologies presented here may serve to initiate future studies characterising gene function in these globally important plant pathogens.