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    CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis
    (Elsevier BV on behalf of the British Mycological Society, 2022-01) Rocafort M; Arshed S; Hudson D; Sidhu JS; Bowen JK; Plummer KM; Bradshaw RE; Johnson RD; Johnson LJ; Mesarich CH; Brown NA
    Apple scab, caused by the fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Using the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN) as a target for inactivation, which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference, we show, for the first time, that the CRISPR-Cas9 gene editing system can be successfully applied to the apple scab fungus. More specifically, using a CRISPR-Cas9 single guide RNA (sgRNA) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ∼16.7% gene inactivation efficiency. Notably, of the six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. In establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher gene function in this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.
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    Unravelling the molecular basis of subcuticular host-colonization by the apple scab fungus, Venturia inaequalis : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Science, School of Agriculture and Environment, Massey University
    (Massey University, 2023) Rocafort Ferrer, Mercedes
    Scab, caused by the fungal pathogen V. inaequalis (Vi), is the most economically important apple disease. During infection, Vi occupies the subcuticular environment, where it develops specialized infection structures, called stromata and runner hyphae. These structures are thought to be important for fungal nutrition and the delivery of proteins, with many of these anticipated to function as virulence factors (effectors) in promoting host infection or avirulence factors (Avr effectors) in triggering host resistance. To date, nothing is known about how these structures are differentiated and protected from recognition by the host immune system. Likewise, little is known about the identity and function of Vi effector proteins. To better control scab, a greater understanding of the molecular mechanisms underpinning infection structure differentiation and protection, as well as Vi virulence and avirulence, is first needed. In Chapter 2, a comprehensive review of apoplastic effector proteins from plant-associated fungi (and oomycetes) was provided. Given that Vi is an extracellular pathogen, this review provided insights into the potential types of effector proteins secreted by Vi into the subcuticular environment. Then, in Chapter 3, a multidisciplinary approach based on bioinformatics, transcriptomics, and structural biology was used to identify and characterize Vi effector candidates (ECs). This revealed that ECs were predominantly expressed in two temporal waves, and that many belonged to expanded protein families with predicted structural similarity to virulence and avirulence effectors from other plant-pathogenic fungi. This analysis helped to generate a list of ECs for further study and contributed to a better understanding of effector biology and evolution. Next, in Chapter 4, a multidisciplinary approach based on transcriptomics, proteomics, glycomics, and confocal microscopy was used to study Vi cell wall carbohydrate composition during the differentiation of infection structures. This iii revealed that Vi down-regulates genes putatively associated with the biosynthesis of immunogenic carbohydrates, and deacetylates surface-exposed chitin to the less immunogenic carbohydrate, chitosan. Finally, in Chapter 5, CRISPR-Cas9 technology was applied to Vi for the first time, which will enable genes identified in this study to be functionally characterized. Altogether, this thesis has furthered our understanding of the Vi –apple pathosystem and has provided novel data that can be used to inform the development of new scab control strategies against Vi.
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    The influence of lime sulphur on the quality and sulphur content of organic 'Royal Gala' and 'Braeburn' apples : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Agribusiness at Massey University
    (Massey University, 2007) Ihringer, Diana
    Black spot or apple scab is a major disease in apple (Malus domestica) production. Its control is especially difficult in organic production systems that rely on copper- and sulphur-based fungicides which are not very effective and demand a high number of applications throughout the season. The most commonly used fungicide in organic apple production is lime sulphur, which is known to be phytotoxic, especially towards the cultivar 'Braeburn'. The influence of different application rates of lime sulphur (1% anti 2%) was evaluated when applied 11 times throughout the growing season from October to February. As varieties differ in their susceptibility to lime sulphur, the two cultivars 'Royal Gala' and 'Braeburn' were compared in this study. Black spot incidence and severity, russet development and postharvest quality parameters were evaluated. At harvest, residues of sulphur on and in the apple were determined as total sulphur, total water-soluble non-protein thiol compounds and cysteine content. Both cultivars behaved similarly to the application of lime sulphur, but 'Braeburn' was affected to a greater extent. Lime sulphur decreased background colour, blush, firmness, soluble solids content and dry matter content in both cultivars; fruit size in 'Braeburn' and titratable acidity in 'Royal Gala'. The changes observed can possibly be attributed at least in part to the decrease in the photosynthetic rate, which was especially drastic in 'Braeburn'. Lime sulphur caused increased russet on 'Royal Gala', but not on 'Braeburn'. Significant sulphur residues were found in the skin and flesh of both cultivars and part of the lime sulphur applied was metabolized into water-soluble non-protein thiols and cysteine. These results are of significant interest to the organic industry as the use of lime sulphur may compromise the residue-free status of organic apples and could have an influence on consumer acceptance and flavour.