AbstractSome of the worlds most devastating plant pathogens are those with broad host ranges (Kamoun et al., 2015, Dean et al., 2012). Yet there has been little investigation into the molecular mechanisms that enable broad host range (Mbengue et al., 2016). Phytophthora capsici is an oomycete plant pathogen with a broad host range and is well placed to become a model organism for the investigation of the mechanisms of host range and dynamic adaption in oomycete plant pathogens (Lamour et al., 2012a).
Thus, this projects aims were two fold, 1) to develop tools for the examination of P. capsici biology and 2) to investigate the mechanisms of host specific dynamic adaption during infection. It was decided that this project would take an unbiased, “omics” based approach in defining the dynamic adaption to multiple host plants.
Initial efforts were focused on developing a technology for the isolation of P. capsici translating mRNA from infected plant tissue, to avoid excessive plant material contaminating omics analysis (Chapter 2). However, while the isolation of mRNA in ribonucleic complexes was successfully established in vitro, implementation of this technology was not feasible in samples from the infection cycle. Based on these results, a simpler methodology was developed. To investigate early changes in gene expression, induced by host-derived signals, a method that relies on the incubation of germinating cysts with plant extracts was developed and validated. The new system was then used to carry out two separate omics experiments. First RNA-sequencing of total RNA comparing tomato extract and cucumber extract at 2, 4 and 8 hours post inoculation was conducted (Chapter 3). Followed by an identical proteomics experiment (Chapter 4). Results of these experiments point to key elements and patterns of the host specific dynamic adaptation of P. capsici. We have found that host extracts were able to induce differential expression of genes, and proteins, amongst these oxidoredctase activity, and transporter proteins were found in high abundance, suggesting a necessary host-specific detoxification element to dynamic host adaptation. Proteins with phosphorylation activity, and other potential signalling molecules were also found in abundance in our suite of differentially regulated elements. However, what mechanisms induce this differential expression event, be they direct host perception or perhaps nutrient sensing, or some other indirect mechanism, is still unclear. In addition, how biologically relevant the dynamic adaptation event to host extract is to infection and colonisation of a host plant is still an open question. It was then the aim of this project to develop tools for the characterisation of the key genes identified in these two experiments. Two transformation methodologies for P. capsici were optimised, mainly for the use in the CRISPR/Cas9-system (Chapter 5). We were unable in this study to show the utility of the CRISPR/Cas9-system in P. capsici, and indeed it is still unclear how functional CRISPR/Cas will be in Phytophthora species in general. Overall this project was able to shed some light mechanism of host-specific dynamic adaption and did develop some tools for the future study of P. capsici.
|Date of Award
|Edgar Huitema (Supervisor) & Paul Birch (Supervisor)