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    Identification and characterization of effector proteins from pine needle 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
    (Massey University, 2022) Massoco Tarallo, Mariana
    Collectively, Dothistroma septosporum, Cyclaneusma minus and Phytophthora pluvialis cause serious foliar diseases on Pinus radiata in New Zealand and on many other pine species worldwide. Considering the ecological and economic importance of forest trees, understanding how these pathogens interact with their hosts on a molecular level is critical as it could lead to new and durable approaches to control the diseases they cause. Pathogens have the ability to deliver proteinaceous virulence factors, termed effectors, into the apoplast and cell cytoplasm of their host plants. Effectors typically promote host colonization through suppression of the plant immune system. However, in resistant host plants, one or more of these effectors can be recognized by corresponding immune receptors to activate the plant immune system. Often, one of the main outputs of this immune system is a localised cell death reaction, termed the hypersensitive response (HR), which renders the pathogen unable to cause disease (avirulent). The general goal of this thesis was to identify shared candidate effector (CE) proteins between the three foliar pine pathogens and to characterise their virulence (or avirulence) functions. This is important because disease resistance based on core effectors that are vital for a pathogen’s ability to cause disease is more likely to be durable. Using a combination of “omics” information and bioinformatic tools, two sets of orthologous CE proteins were identified between D. septosporum, C. minus and P. pluvialis, while several other sets were identified between the two fungal pathogens. Some of these CEs had the ability to trigger cell death responses in non-host Nicotiana plants, and some were shown to activate Nicotiana benthamiana genes involved in pathogen-associated molecular pattern-triggered immunity and HR. CEs were also screened in the host, Pn. radiata, using a method developed in this thesis, where it was determined that some of these CEs also trigger cell death. Two conserved cell death elicitor families, Ecp20 and Ecp32, were identified from D. septosporum and its close relative Fulvia fulvum, and the cell death triggered by some family members in N. benthamiana was shown to require membrane-localized receptor-like proteins. Tertiary structure predictions of CEs provided insights into the possible roles and host targets of these proteins during pine infection. Moreover, a shared β-trefoil fold was found between sequence-unrelated CE proteins from the three pine pathogens, along with evidence that they are also present in many other fungal species. A CRISPR/Cas9 gene editing methodology was applied to D. septosporum for the first time, which allowed for the functional characterization of three D. septosporum CE genes, two of which are also present in C. minus and P. pluvialis. Collectively, this thesis provides a significant advance in our understanding of pine-pathogen interactions at the molecular level and provides a blueprint for similar studies in other forest pathosystems.
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    Identification of novel avirulence effectors in the Dothideomycete plant pathogens, Venturia inaequalis and Cladosporium fulvum : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Sciences at Massey University, Manawatū, New Zealand
    (Massey University, 2022) de la Rosa, Silvia
    Venturia inaequalis and Cladosporium fulvum are important fungal pathogens of crop species, causing scab and leaf mould disease of apple and tomato, respectively. Resistance to these pathogens is governed by Rvi (apple) and Cf (tomato) resistance (R) genes. These R genes encode immune receptors that recognize specific pathogen virulence factors, termed avirulence (Avr) effectors, to activate plant defenses. Notably, isolates or strains of V. inaequalis and C. fulvum have emerged that can overcome resistance mediated by specific R genes in their respective hosts. To better understand how these pathogens cause disease or overcome resistance, and to monitor the occurrence of resistance-breaking isolates or strains in the field, Avr effectors from V. inaequalis and C. fulvum must be identified and functionally characterized. Using a combined comparative genomics and phenotyping approach based on progeny from a sexual cross between V. inaequalis isolates that differ in their ability to overcome Rvi4 resistance in apple, a strong candidate for the corresponding AvrRvi4 effector gene was identified (Chapter 2). Similarly, using a comparative genomics approach based on in planta-expressed effector candidates from C. fulvum strains that differ in their ability to overcome Cf-9B resistance in tomato, combined with functional assays, the corresponding Avr9B effector gene was identified (Chapter 4). In the resistance-breaking isolates or strains studied, the candidate AvrRvi4 gene was disrupted, while the Avr9B gene had been deleted. Consistent with most fungal Avr effectors and their genes, both the AvrRvi4 candidate and Avr9B are highly expressed in planta, and encode small, secreted cysteine-rich proteins. The AvrRvi4 candidate forms part of an expanded protein family in V. inaequalis, with members predicted to adopt a β sandwich fold similar to structurally characterized fungal effectors. Avr9B, however, is predicted to adopt a novel protein fold. Finally, using a heterologous expression approach, three in planta-expressed candidate effectors from V. inaequalis were found to trigger defense responses in non-host plants (Nicotiana spp.), suggesting they are recognized by R proteins in these species (Chapter 3). Taken together, this thesis has increased our understanding of the molecular mechanisms responsible for the activation and circumvention of resistance by V. inaequalis and C. fulvum, which will in turn direct host cultivar deployment and disease control strategies in the field.