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    Cell Wall Carbohydrate Dynamics during the Differentiation of Infection Structures by the Apple Scab Fungus, Venturia inaequalis.
    (American Society for Microbiology, 2023-06-15) Rocafort M; Srivastava V; Bowen JK; Díaz-Moreno SM; Guo Y; Bulone V; Plummer KM; Sutherland PW; Anderson MA; Bradshaw RE; Mesarich CH; Wang Y
    Scab, caused by the biotrophic fungal pathogen Venturia inaequalis, is the most economically important disease of apples. During infection, V. inaequalis colonizes the subcuticular host environment, where it develops specialized infection structures called runner hyphae and stromata. These structures are thought to be involved in nutrient acquisition and effector (virulence factor) delivery, but also give rise to conidia that further the infection cycle. Despite their importance, very little is known about how these structures are differentiated. Likewise, nothing is known about how these structures are protected from host defenses or recognition by the host immune system. To better understand these processes, we first performed a glycosidic linkage analysis of sporulating tubular hyphae from V. inaequalis developed in culture. This analysis revealed that the V. inaequalis cell wall is mostly composed of glucans (44%) and mannans (37%), whereas chitin represents a much smaller proportion (4%). Next, we used transcriptomics and confocal laser scanning microscopy to provide insights into the cell wall carbohydrate composition of runner hyphae and stromata. These analyses revealed that, during subcuticular host colonization, genes of V. inaequalis putatively associated with the biosynthesis of immunogenic carbohydrates, such as chitin and β-1,6-glucan, are downregulated relative to growth in culture, while on the surface of runner hyphae and stromata, chitin is deacetylated to the less-immunogenic carbohydrate chitosan. These changes are anticipated to enable the subcuticular differentiation of runner hyphae and stromata by V. inaequalis, as well as to protect these structures from host defenses and recognition by the host immune system. IMPORTANCE Plant-pathogenic fungi are a major threat to food security. Among these are subcuticular pathogens, which often cause latent asymptomatic infections, making them difficult to control. A key feature of these pathogens is their ability to differentiate specialized subcuticular infection structures that, to date, remain largely understudied. This is typified by Venturia inaequalis, which causes scab, the most economically important disease of apples. In this study, we show that, during subcuticular host colonization, V. inaequalis downregulates genes associated with the biosynthesis of two immunogenic cell wall carbohydrates, chitin and β-1,6-glucan, and coats its subcuticular infection structures with a less-immunogenic carbohydrate, chitosan. These changes are anticipated to enable host colonization by V. inaequalis and provide a foundation for understanding subcuticular host colonization by other plant-pathogenic fungi. Such an understanding is important, as it may inform the development of novel control strategies against subcuticular plant-pathogenic fungi.
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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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    Cell Wall Carbohydrate Dynamics during the Differentiation of Infection Structures by the Apple Scab Fungus, Venturia inaequalis
    (American Society for Microbiology, 2023-06-15) Rocafort M; Srivastava V; Bowen J; Díaz-Moreno SM; Guo Y; Bulone V; Plummer KM; Sutherland PW; Anderson MA; Bradshaw RE; Mesarich CH; Wang Y
    Scab, caused by the biotrophic fungal pathogen Venturia inaequalis, is the most economically important disease of apples. During infection, V. inaequalis colonizes the subcuticular host environment, where it develops specialized infection structures called runner hyphae and stromata. These structures are thought to be involved in nutrient acquisition and effector (virulence factor) delivery, but also give rise to conidia that further the infection cycle. Despite their importance, very little is known about how these structures are differentiated. Likewise, nothing is known about how these structures are protected from host defenses or recognition by the host immune system. To better understand these processes, we first performed a glycosidic linkage analysis of sporulating tubular hyphae from V. inaequalis developed in culture. This analysis revealed that the V. inaequalis cell wall is mostly composed of glucans (44%) and mannans (37%), whereas chitin represents a much smaller proportion (4%). Next, we used transcriptomics and confocal laser scanning microscopy to provide insights into the cell wall carbohydrate composition of runner hyphae and stromata. These analyses revealed that, during subcuticular host colonization, genes of V. inaequalis putatively associated with the biosynthesis of immunogenic carbohydrates, such as chitin and b-1,6-glucan, are downregulated relative to growth in culture, while on the surface of runner hyphae and stromata, chitin is deacetylated to the less-immunogenic carbohydrate chitosan. These changes are anticipated to enable the subcuticular differentiation of runner hyphae and stromata by V. inaequalis, as well as to protect these structures from host defenses and recognition by the host immune system. IMPORTANCE Plant-pathogenic fungi are a major threat to food security. Among these are subcuticular pathogens, which often cause latent asymptomatic infections, making them difficult to control. A key feature of these pathogens is their ability to differentiate specialized subcuticular infection structures that, to date, remain largely understudied. This is typified by Venturia inaequalis, which causes scab, the most economically important disease of apples. In this study, we show that, during subcuticular host colonization, V. inaequalis downregulates genes associated with the biosynthesis of two immunogenic cell wall carbohydrates, chitin and b-1,6-glucan, and coats its subcuticular infection structures with a less-immunogenic carbohydrate, chitosan. These changes are anticipated to enable host colonization by V. inaequalis and provide a foundation for understanding subcuticular host colonization by other plant-pathogenic fungi. Such an understanding is important, as it may inform the development of novel control strategies against subcuticular plant-pathogenic fungi.
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    The Venturia inaequalis effector repertoire is dominated by expanded families with predicted structural similarity, but unrelated sequence, to avirulence proteins from other plant-pathogenic fungi
    (BioMed Central Ltd, 2022-12) Rocafort M; Bowen JK; Hassing B; Cox MP; McGreal B; de la Rosa S; Plummer KM; Bradshaw RE; Mesarich CH
    BACKGROUND: Scab, caused by the biotrophic fungus Venturia inaequalis, is the most economically important disease of apples worldwide. During infection, V. inaequalis occupies the subcuticular environment, where it secretes virulence factors, termed effectors, to promote host colonization. Consistent with other plant-pathogenic fungi, many of these effectors are expected to be non-enzymatic proteins, some of which can be recognized by corresponding host resistance proteins to activate plant defences, thus acting as avirulence determinants. To develop durable control strategies against scab, a better understanding of the roles that these effector proteins play in promoting subcuticular growth by V. inaequalis, as well as in activating, suppressing, or circumventing resistance protein-mediated defences in apple, is required. RESULTS: We generated the first comprehensive RNA-seq transcriptome of V. inaequalis during colonization of apple. Analysis of this transcriptome revealed five temporal waves of gene expression that peaked during early, mid, or mid-late infection. While the number of genes encoding secreted, non-enzymatic proteinaceous effector candidates (ECs) varied in each wave, most belonged to waves that peaked in expression during mid-late infection. Spectral clustering based on sequence similarity determined that the majority of ECs belonged to expanded protein families. To gain insights into function, the tertiary structures of ECs were predicted using AlphaFold2. Strikingly, despite an absence of sequence similarity, many ECs were predicted to have structural similarity to avirulence proteins from other plant-pathogenic fungi, including members of the MAX, LARS, ToxA and FOLD effector families. In addition, several other ECs, including an EC family with sequence similarity to the AvrLm6 avirulence effector from Leptosphaeria maculans, were predicted to adopt a KP6-like fold. Thus, proteins with a KP6-like fold represent another structural family of effectors shared among plant-pathogenic fungi. CONCLUSIONS: Our study reveals the transcriptomic profile underpinning subcuticular growth by V. inaequalis and provides an enriched list of ECs that can be investigated for roles in virulence and avirulence. Furthermore, our study supports the idea that numerous sequence-unrelated effectors across plant-pathogenic fungi share common structural folds. In doing so, our study gives weight to the hypothesis that many fungal effectors evolved from ancestral genes through duplication, followed by sequence diversification, to produce sequence-unrelated but structurally similar proteins.
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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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    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.
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    Molecular analysis of plant innate immunity triggered by secreted effectors from bacterial and fungal pathogens of apple : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Science, Institute of Agriculture and Environment, Massey University, New Zealand
    (Massey University, 2017) Prokchorchik, Maxim
    In comparison to animals, plants do not have a dedicated immune system with mobile immune cells to protect themselves. Instead they rely on the innate immunity of each cell. Plant immunity branches into two classical layers: PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity). PTI detects the conserved molecular patterns (PAMPs) associated with pathogens and often can be overcome by pathogens translocating effector molecules into plant cells to inhibit the PTI. ETI, in turn, relies on intracellular receptors that can specifically recognize effectors or their activity and activate a rapid and robust response. The research presented in this thesis is focused on two pathogens of apple plants: the bacterial pathogen Erwinia amylovora (the causal agent of fire blight) and fungal pathogen Venturia inaequalis (the causal agent of apple scab disease). As both bacterial and fungal pathogens deliver effector molecules in order to promote their virulence, ETI engineering is a promising universal strategy to control these pathogens. In Chapter 3, the main aim was to elucidate the requirements and precise mechanism of how an important effector of E. amylovora, AvrRpt2, is recognized by the MR5 disease resistance (R) protein, derived from a hybrid apple Malus x robusta 5. I identified that a fragment of the guardee apple protein RIN4 was required and sufficient and required for MR5 activation. I further identified crucial amino acid residues responsible for this activation. Interestingly, cognate residues in RIN4 guardee homolog from Arabidopsis thaliana are responsible for suppression of the autoactivity of R protein RPS2. These findings led to the proposal of a novel hypothesis for evolutionary guardee adaption to the pool of R proteins present in plants. In Chapter 4, the main focus was to apply newly acquired whole-genome sequencing data of V. inaequalis for identifying the previously mapped AvrRvi8 effector, as well as several novel effectors predicted in silico. The sequences of these effectors were validated by amplification and resequencing of candidate genes from V. inaequalis cDNA. Further functional analysis of the selected gene candidates was performed. In addition, a library of constructs for generating V. inaequalis knock-out strains was prepared for future work. The findings from this thesis expected to be useful for breeders of apple to battle two economically important pathogens devastating the industry. Deployment of the MR5 system in apples should facilitate fire blight resistance in pipfruit and offers the opportunity for further engineering of MR5 to detect other pathogens. Furthermore, the effector library developed for V. inaequalis offers a novel tool for studying both virulence and avirulence mechanisms present in the applescab pathosystem. It is envisaged that further effector research will elucidate authentic targets critical for resistance development in apple.
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    Factors affecting spray deposits and their biological effects on New Zealand apple canopies : a thesis presented in fulfillment of the requirements for the degree of Doctor of Philosophy in Agricultural Engineering at Massey University
    (Massey University, 1998) Manktelow, David William Lewis
    A series of apple tree spraying experiments was conducted to identify factors affecting agrichemical deposits from airblast sprayers and to relate deposit observations to biological responses in selected pest, disease and physiological systems. Factors addressed included tree canopy form, application volume, travel speed and sprayer type. Several tracers were evaluated and deposits quantified by wash-off removal from bulked leaf or fruit samples drawn from 10-15 spatially consistent 1.5 m3 zones per tree. Deposit data were expressed on a tissue area basis and/or as a proportion of the spray emitted (retention). Spray deposits were compared across 11 canopy forms to identify interactions with tree size, leaf area and canopy density and volume. A two-fold difference in deposits between canopies occurred when sprays were applied at a constant chemical rate per hectare. This variability was approximately halved when chemical rates per hectare were adjusted on the basis of the canopy Tree-Row-Volume (TRV). The best TRV measurement system identified used across-row canopy spread measurements at half metre height intervals, rather than just a single measurement of canopy spread. Deposits were better correlated with TRV data than with any of the other canopy descriptors used. Canopy density was identified as an important covariate, but light penetration proved an unsuitable indicator of canopy density as it was strongly correlated with TRV. Deposit variations between zones within trees were consistent between all but the smallest canopy sprayed. Increasing the distance from the sprayer and/or increasing canopy penetration requirements reduced spray deposits. Spray retention across these canopies in full leaf ranged from 25-90%, but tended to increase with decreased application volume. There was a ca. 10-15% increase in deposits when spray volumes were reduced 4-5 times below those used in typical dilute spray volumes (ca. 2,000 1 ha-1). At high volumes with significant run-off, retention could ca. 50% of that at lower volumes. Run-off losses could be related to TRV, with significant run-off occurring once application volumes exceeded one litre per 7.5-11 m3 of TRV. Surprisingly, average deposits on 5m tail slender pyramid trees increased with increased travel speed over the range 1.9-8.8 km h-1. Within-tree spray deposit distributions were not markedly affected by the travel speeds tested with air assistance volumes of ca. 30,000 or 44,000 m3 h-1. High, but relatively consistent within-tree deposit variability was a feature of deposits from axial fan, airblast sprayers, especially when used in intensive 4-6 m tall, single leader tree plantings. Within-tree deposit variability decreased with increased application volumes. Tower sprayers provided a more even vertical distribution of spray emission points and achieved different, but not necessarily more even, within-tree deposit distributions than airblast machines. Experiments on chemical thinning, mealybug (Pseudococcus viburni) and black spot (Venturia inaequalis) control, showed the biological responses could not have been predicted from the spray deposit measurements. However, combined assessment of spray deposits and biological effects greatly facilitated interpretation of both sets of data..