Identification and characterisation of novel virulence factors from Dothideomycete pathogens : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatū, New Zealand
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Date
2023
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Massey University
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Abstract
The Dothideomycetes class of fungi contains some of the most important plant pathogens, including Dothistroma septosporum and Fulvia fulva. Dothistroma needle blight, caused by D. septosporum, is a devasting disease of pines that has been increasing in severity and incidence worldwide. Tomato leaf mould, caused by F. fulva, has recently risen in importance after overcoming resistance breeding efforts. There is now an urgent need to identify novel genes from these pathogens that encode virulence factors, such as effector proteins. These proteins contribute to pathogen virulence, so the study of virulence factors not only enables a deeper understanding of how pathogens and their hosts interact at the molecular level, but also provides information that may lead to the development of new methods for disease control. As such, the aim of this thesis was to identify and characterise new candidate virulence factors from D. septosporum and F. fulva. Candidate virulence factor CfEcp11-1 from F. fulva, previously identified to trigger plant defences in wild tomato cultivars, was a particular focus in Chapter 2. Two new homologues of CfEcp11-1 were identified from Fusarium oxysporum and suggested to be part of the same Leptosphaeria AviRulence and Suppressing (LARS) effector family. Recognition of CfEcp11-1 by the tomato receptor was also examined through the design of chimeric and mutant protein sequences. CRISPR/Cas9 was used for the first time in F. fulva to disrupt CfEcp11-1 and generate single- and double-copy mutants; to the best of our knowledge this was the first report of multiple gene copy disruption by CRISPR/Cas9 in a fungal pathogen. In Chapter 3, candidate virulence factors from D. septosporum and F. fulva were identified through a prediction pipeline, which selected proteins with predicted roles as effectors that manipulate plant defences, or as transcription factors which regulate the expression of other genes. Because D. septosporum and F. fulva are hemibiotrophic pathogens, they transition from colonising living plant tissue to killing and feeding on dead tissue, a transition termed the necrotrophic switch. It is currently unknown what mechanisms govern this important disease process, so a key focus was on candidate virulence factors with a possible role in the necrotrophic switch. In Chapter 4, some of these candidates were further characterised, and new candidates identified, from proteomic analysis of the culture filtrates of D. septosporum and F. fulva grown in different conditions. Existing transcriptomic data were used to assess which of these proteins were likely to be functional in planta. Among those identified were several characterised effectors, such as Cf/DsEcp2-1, Cf/DsEcp20-1, Cf/DsEcp20-3, and CfAvr4E, which were secreted in culture despite having known functions in planta. Novel candidate virulence factors from D. septosporum and F. fulva were also identified in this analysis, including a Nis1 domain-containing protein from D. septosporum with a possible role in the necrotrophic switch. This analysis illustrates that in culture proteomic analysis can be a useful tool for the identification of candidate effector proteins. In Chapter 5, two candidate virulence factor genes of D. septosporum with predicted roles in the necrotrophic switch were disrupted through CRISPR/Cas9 gene editing; this was the first use of this method for gene disruption in D. septosporum. The disruption mutants were tested for virulence on the P. radiata host. One of the mutants was disrupted in DsCE3, which was suggested to be a virulence factor, with a possible role in the necrotrophic switch. Overall, the results presented in this thesis have provided new research methodologies as well as valuable knowledge about the molecular tools these two pathogens use to invade their hosts. Whilst further work is required, these developments will ultimately aid future disease control strategies in pine and tomato.
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Keywords
Dothideomycetes, Virulence (Microbiology), Genetic aspects, Pathogenic microorganisms