Browsing by Author "Mesarich CH"

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    Apoplastic effector candidates of a foliar forest pathogen trigger cell death in host and non-host plants
    (2021-08-09) Hunziker L; Tarallo M; Gough K; Guo M; Hargreaves C; Loo TS; McDougal RL; Mesarich CH; Bradshaw RE
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    Apoplastic effector candidates of a foliar forest pathogen trigger cell death in host and non-host plants.
    (7/10/2021) Hunziker L; Tarallo M; Gough K; Guo M; Hargreaves C; Loo TS; McDougal RL; Mesarich CH; Bradshaw RE
    Forests are under threat from pests, pathogens, and changing climate. A major forest pathogen worldwide is the hemibiotroph Dothistroma septosporum, which causes dothistroma needle blight (DNB) of pines. While D. septosporum uses effector proteins to facilitate host infection, it is currently unclear whether any of these effectors are recognised by immune receptors to activate the host immune system. Such information is needed to identify and select disease resistance against D. septosporum in pines. We predicted and investigated apoplastic D. septosporum candidate effectors (DsCEs) using bioinformatics and plant-based experiments. We discovered DsCEs that trigger cell death in the angiosperm Nicotiana spp., indicative of a hypersensitive defence response and suggesting their recognition by immune receptors in non-host plants. In a first for foliar forest pathogens, we developed a novel protein infiltration method to show that tissue-cultured pine shoots can respond with a cell death response to a DsCE, as well as to a reference cell death-inducing protein. The conservation of responses across plant taxa suggests that knowledge of pathogen-angiosperm interactions may also be relevant to pathogen-gymnosperm interactions. These results contribute to our understanding of forest pathogens and may ultimately provide clues to disease immunity in both commercial and natural forests.
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    Beyond the genomes of Fulvia fulva (syn. Cladosporium fulvum) and Dothistroma septosporum: New insights into how these fungal pathogens interact with their host plants.
    (BSPP and John Wiley and Sons, Inc., 2023-05-01) Mesarich CH; Barnes I; Bradley EL; de la Rosa S; de Wit PJGM; Guo Y; Griffiths SA; Hamelin RC; Joosten MHAJ; Lu M; McCarthy HM; Schol CR; Stergiopoulos I; Tarallo M; Zaccaron AZ; Bradshaw RE
    Fulvia fulva and Dothistroma septosporum are closely related apoplastic pathogens with similar lifestyles but different hosts: F. fulva is a pathogen of tomato, whilst D. septosporum is a pathogen of pine trees. In 2012, the first genome sequences of these pathogens were published, with F. fulva and D. septosporum having highly fragmented and near-complete assemblies, respectively. Since then, significant advances have been made in unravelling their genome architectures. For instance, the genome of F. fulva has now been assembled into 14 chromosomes, 13 of which have synteny with the 14 chromosomes of D. septosporum, suggesting these pathogens are even more closely related than originally thought. Considerable advances have also been made in the identification and functional characterization of virulence factors (e.g., effector proteins and secondary metabolites) from these pathogens, thereby providing new insights into how they promote host colonization or activate plant defence responses. For example, it has now been established that effector proteins from both F. fulva and D. septosporum interact with cell-surface immune receptors and co-receptors to activate the plant immune system. Progress has also been made in understanding how F. fulva and D. septosporum have evolved with their host plants, whilst intensive research into pandemics of Dothistroma needle blight in the Northern Hemisphere has shed light on the origins, migration, and genetic diversity of the global D. septosporum population. In this review, we specifically summarize advances made in our understanding of the F. fulva-tomato and D. septosporum-pine pathosystems over the last 10 years.
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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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    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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    Characterization of two conserved cell death elicitor families from the Dothideomycete fungal pathogens Dothistroma septosporum and Fulvia fulva (syn. Cladosporium fulvum)
    (Frontiers Media S.A., 2022-09-08) Tarallo M; McDougal RL; Chen Z; Wang Y; Bradshaw RE; Mesarich CH; Wang Y
    Dothistroma septosporum (Ds) and Fulvia fulva (Ff; previously called Cladosporium fulvum) are two closely related Dothideomycete fungal species that cause Dothistroma needle blight in pine and leaf mold in tomato, respectively. During host colonization, these pathogens secrete virulence factors termed effectors to promote infection. In the presence of corresponding host immune receptors, however, these effectors activate plant defenses, including a localized cell death response that halts pathogen growth. We identified two apoplastic effector protein families, Ecp20 and Ecp32, which are conserved between the two pathogens. The Ecp20 family has four paralogues in both species, while the Ecp32 family has four paralogues in D. septosporum and five in F. fulva. Both families have members that are highly expressed during host infection. Members of the Ecp20 family have predicted structural similarity to proteins with a β-barrel fold, including the Alt a 1 allergen from Alternaria alternata, while members of the Ecp32 family have predicted structural similarity to proteins with a β-trefoil fold, such as trypsin inhibitors and lectins. Using Agrobacterium tumefaciens-mediated transient transformation assays, each family member was assessed for its ability to trigger cell death in leaves of the non-host species Nicotiana benthamiana and N. tabacum. Using this approach, FfEcp20-2, DsEcp20-3, and FfEcp20-3 from the Ecp20 family, and all members from the Ecp32 family, except for the Ds/FfEcp32-4 pair, triggered cell death in both species. This cell death was dependent on secretion of the effectors to the apoplast. In line with recognition by an extracellular immune receptor, cell death triggered by Ds/FfEcp20-3 and FfEcp32-3 was compromised in N. benthamiana silenced for BAK1 or SOBIR1, which encode extracellular co-receptors involved in transducing defense response signals following apoplastic effector recognition. We then investigated whether DsEcp20-3 and DsEcp20-4 triggered cell death in the host species Pinus radiata by directly infiltrating purified protein into pine needles. Strikingly, as in the non-host species, DsEcp20-3 triggered cell death, while DsEcp20-4 did not. Collectively, our study describes two new candidate effector families with cell death-eliciting activity from D. septosporum and F. fulva and provides evidence that members of these families are recognized by plant immune receptors.
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    Characterization of two conserved cell death elicitor families from the Dothideomycete fungal pathogens Dothistroma septosporum and Fulvia fulva (syn. Cladosporium fulvum)
    (Frontiers Media S.A., 2022-09-08) Tarallo M; McDougal RL; Chen Z; Wang Y; Bradshaw RE; Mesarich CH; Wang Y
    Dothistroma septosporum (Ds) and Fulvia fulva (Ff; previously called Cladosporium fulvum) are two closely related Dothideomycete fungal species that cause Dothistroma needle blight in pine and leaf mold in tomato, respectively. During host colonization, these pathogens secrete virulence factors termed effectors to promote infection. In the presence of corresponding host immune receptors, however, these effectors activate plant defenses, including a localized cell death response that halts pathogen growth. We identified two apoplastic effector protein families, Ecp20 and Ecp32, which are conserved between the two pathogens. The Ecp20 family has four paralogues in both species, while the Ecp32 family has four paralogues in D. septosporum and five in F. fulva. Both families have members that are highly expressed during host infection. Members of the Ecp20 family have predicted structural similarity to proteins with a β-barrel fold, including the Alt a 1 allergen from Alternaria alternata, while members of the Ecp32 family have predicted structural similarity to proteins with a β-trefoil fold, such as trypsin inhibitors and lectins. Using Agrobacterium tumefaciens-mediated transient transformation assays, each family member was assessed for its ability to trigger cell death in leaves of the non-host species Nicotiana benthamiana and N. tabacum. Using this approach, FfEcp20-2, DsEcp20-3, and FfEcp20-3 from the Ecp20 family, and all members from the Ecp32 family, except for the Ds/FfEcp32-4 pair, triggered cell death in both species. This cell death was dependent on secretion of the effectors to the apoplast. In line with recognition by an extracellular immune receptor, cell death triggered by Ds/FfEcp20-3 and FfEcp32-3 was compromised in N. benthamiana silenced for BAK1 or SOBIR1, which encode extracellular co-receptors involved in transducing defense response signals following apoplastic effector recognition. We then investigated whether DsEcp20-3 and DsEcp20-4 triggered cell death in the host species Pinus radiata by directly infiltrating purified protein into pine needles. Strikingly, as in the non-host species, DsEcp20-3 triggered cell death, while DsEcp20-4 did not. Collectively, our study describes two new candidate effector families with cell death-eliciting activity from D. septosporum and F. fulva and provides evidence that members of these families are recognized by plant immune receptors.
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    Chromosome-level assembly of the Phytophthora agathidicida genome reveals adaptation in effector gene families
    (Frontiers Media S.A., 2022-11-02) Cox MP; Guo Y; Winter DJ; Sen D; Cauldron NC; Shiller J; Bradley EL; Ganley AR; Gerth ML; Lacey RF; McDougal RL; Panda P; Williams NM; Grunwald NJ; Mesarich CH; Bradshaw RE; Hane J
    Phytophthora species are notorious plant pathogens, with some causing devastating tree diseases that threaten the survival of their host species. One such example is Phytophthora agathidicida, the causal agent of kauri dieback - a root and trunk rot disease that kills the ancient, iconic and culturally significant tree species, Agathis australis (New Zealand kauri). A deeper understanding of how Phytophthora pathogens infect their hosts and cause disease is critical for the development of effective treatments. Such an understanding can be gained by interrogating pathogen genomes for effector genes, which are involved in virulence or pathogenicity. Although genome sequencing has become more affordable, the complete assembly of Phytophthora genomes has been problematic, particularly for those with a high abundance of repetitive sequences. Therefore, effector genes located in repetitive regions could be truncated or missed in a fragmented genome assembly. Using a combination of long-read PacBio sequences, chromatin conformation capture (Hi-C) and Illumina short reads, we assembled the P. agathidicida genome into ten complete chromosomes, with a genome size of 57 Mb including 34% repeats. This is the first Phytophthora genome assembled to chromosome level and it reveals a high level of syntenic conservation with the complete genome of Peronospora effusa, the only other completely assembled genome sequence of an oomycete. All P. agathidicida chromosomes have clearly defined centromeres and contain candidate effector genes such as RXLRs and CRNs, but in different proportions, reflecting the presence of gene family clusters. Candidate effector genes are predominantly found in gene-poor, repeat-rich regions of the genome, and in some cases showed a high degree of duplication. Analysis of candidate RXLR effector genes that occur in multicopy gene families indicated half of them were not expressed in planta. Candidate CRN effector gene families showed evidence of transposon-mediated recombination leading to new combinations of protein domains, both within and between chromosomes. Further analysis of this complete genome assembly will help inform new methods of disease control against P. agathidicida and other Phytophthora species, ultimately helping decipher how Phytophthora pathogens have evolved to shape their effector repertoires and how they might adapt in the future.
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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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    Draft Genome Sequence of the Asian Pear Scab Pathogen, Venturia nashicola
    (2018-06-22) Johnson S; Jones D; Thrimawithana AH; Deng CH; Bowen JK; Mesarich CH; Ishii H; Won K; Bus VGM; Plummer KM
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    Pucciniastrum minimum is the causal agent of blueberry leaf rust on different Vaccinium species in Hawke’s Bay, New Zealand
    (Springer Nature on behalf of the Australasian Plant Pathology Society Inc, 2023-03-01) Chen X; Mesarich CH; Kerckhoffs H; Hutchins D; Sofkova-Bobcheva S
    Blueberry leaf rust has become a prevalent disease in New Zealand blueberry production. To identify the pathogen responsible for this disease in the Hawke’s Bay region of New Zealand, leaves showing signs or symptoms of rust infection were collected from three blueberry cultivars (‘Centra Blue’ [Rabbiteye], ‘Georgia Dawn’ [Southern Highbush] and ‘Nui’ [Northern Highbush]) and the pathogen subjected to morphological characterization using both scanning electron and bright-field microscopy. Meanwhile, genomic DNA was extracted from urediniospores of infected leaves collected from cultivar ‘Rahi’ (Rabbiteye) and the Internal Transcribed Spacer (ITS) region was sequenced and compared to the corresponding nucleotide sequence of known rust pathogens. Results from both experiments indicated that Pucciniastrum minimum (syn. Thekopsora minima) was the causal agent of blueberry leaf rust disease in Hawke’s Bay. Next, the level of disease caused by P. minimum was quantified on 23 blueberry cultivars in this region during the 2019 blueberry production season. Here, a total of 20 leaves selected from each cultivar were continually monitored, and the lesion area was calculated using ImageJ based on images taken in the field. Based on this analysis, all leaves were found to be infected by the rust pathogen. However, disease intensity, as a function of the ‘area under the disease progress curve’ (AUDPC) value, was found to be different. This suggests that certain cultivars display a lower disease intensity during the harvest season. Further field assessment covering a whole growing cycle will give a better understanding about blueberry leaf rust infection on these cultivars.
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    Secreted Glycoside Hydrolase Proteins as Effectors and Invasion Patterns of Plant-Associated Fungi and Oomycetes.
    (2022) Bradley EL; Ökmen B; Doehlemann G; Henrissat B; Bradshaw RE; Mesarich CH
    During host colonization, plant-associated microbes, including fungi and oomycetes, deliver a collection of glycoside hydrolases (GHs) to their cell surfaces and surrounding extracellular environments. The number and type of GHs secreted by each organism is typically associated with their lifestyle or mode of nutrient acquisition. Secreted GHs of plant-associated fungi and oomycetes serve a number of different functions, with many of them acting as virulence factors (effectors) to promote microbial host colonization. Specific functions involve, for example, nutrient acquisition, the detoxification of antimicrobial compounds, the manipulation of plant microbiota, and the suppression or prevention of plant immune responses. In contrast, secreted GHs of plant-associated fungi and oomycetes can also activate the plant immune system, either by acting as microbe-associated molecular patterns (MAMPs), or through the release of damage-associated molecular patterns (DAMPs) as a consequence of their enzymatic activity. In this review, we highlight the critical roles that secreted GHs from plant-associated fungi and oomycetes play in plant-microbe interactions, provide an overview of existing knowledge gaps and summarize future directions.
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    Sequential breakdown of the Cf-9 leaf mould resistance locus in tomato by Fulvia fulva.
    (John Wiley and Sons Ltd on behalf of New Phytologist Foundation, 2024-06-24) de la Rosa S; Schol CR; Ramos Peregrina Á; Winter DJ; Hilgers AM; Maeda K; Iida Y; Tarallo M; Jia R; Beenen HG; Rocafort M; de Wit PJGM; Bowen JK; Bradshaw RE; Joosten MHAJ; Bai Y; Mesarich CH
    Leaf mould, caused by Fulvia fulva, is a devastating disease of tomato plants. In many commercial tomato cultivars, resistance to this disease is governed by the Cf-9 locus, which encodes five paralogous receptor-like proteins. Two of these proteins confer resistance: Cf-9C recognises the previously identified F. fulva effector Avr9 and provides resistance during all plant growth stages, while Cf-9B recognises the yet-unidentified F. fulva effector Avr9B and provides mature plant resistance only. In recent years, F. fulva strains have emerged that can overcome the Cf-9 locus, with Cf-9C circumvented through Avr9 deletion. To understand how Cf-9B is circumvented, we set out to identify Avr9B. Comparative genomics, transient expression assays and gene complementation experiments were used to identify Avr9B, while gene sequencing was used to assess Avr9B allelic variation across a world-wide strain collection. A strict correlation between Avr9 deletion and resistance-breaking mutations in Avr9B was observed in strains recently collected from Cf-9 cultivars, whereas Avr9 deletion but no mutations in Avr9B were observed in older strains. This research showcases how F. fulva has evolved to sequentially break down the Cf-9 locus and stresses the urgent need for commercial tomato cultivars that carry novel, stacked resistance genes active against this pathogen.
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    Targeted Gene Mutations in the Forest Pathogen Dothistroma septosporum Using CRISPR/Cas9.
    (8/04/2022) McCarthy HM; Tarallo M; Mesarich CH; McDougal RL; Bradshaw RE
    Dothistroma needle blight, caused by Dothistroma septosporum, has increased in incidence and severity over the last few decades and is now one of the most important global diseases of pines. Disease resistance breeding could be accelerated by knowledge of pathogen virulence factors and their host targets. However, this is hindered due to inefficient targeted gene disruption in D. septosporum, which is required for virulence gene characterisation. Here we report the first successful application of CRISPR/Cas9 gene editing to a Dothideomycete forest pathogen, D. septosporum. Disruption of the dothistromin pathway regulator gene AflR, with a known phenotype, was performed using nonhomologous end-joining repair with an efficiency of > 90%. Transformants with a range of disruption mutations in AflR were produced. Disruption of Ds74283, a D. septosporum gene encoding a secreted cell death elicitor, was also achieved using CRISPR/Cas9, by using a specific donor DNA repair template to aid selection where the phenotype was unknown. In this case, 100% of screened transformants were identified as disruptants. In establishing CRISPR/Cas9 as a tool for gene editing in D. septosporum, our research could fast track the functional characterisation of candidate virulence factors in D. septosporum and helps set the foundation for development of this technology in other forest pathogens.
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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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    Why a strategic shift in action is needed to recognise and empower Indigenous plant pathology knowledge and research
    (Springer Nature on behalf of the Australasian Plant Pathology Society Inc, 2024-05-01) Ehau-Taumaunu H; Williams NM; Marsh A; Waipara NW; Higgins CM; Geering ADW; Mesarich CH; Rigano LA; Summerell BA; Johnson GI; Williamson P; MacDiarmid RM
    Plant pathology researchers play a pivotal role in thought leadership and its translation to action regarding the recognition and demonstration of the value of Indigenous knowledge and science. For many scientists, navigating the space of Indigenous rights and perspectives is challenging. In pursuit of a cultural shift in research and development within the field of plant pathology, the 2019–2021 Management Committee of the Australasian Plant Pathology Society (APPS) undertook a review and modernization of the Society’s Constitution. The aim was to ensure its alignment with principles that foster inclusivity of Indigenous peoples in the development and implementation of relevant research projects impacting their communities. Additionally, a dynamic repository of guidelines and resources was compiled. These resources are designed to assist plant pathologists, while respecting and not superseding the guidance provided by local Indigenous researchers, practitioners, and advisors. The collective efforts of plant pathologists hold immense potential in championing Indigenous Peoples and their rights, steering the field toward a more inclusive and equitable future. This paper builds upon the thesis presented in the APPS Presidential Address at the Biennial APPS Conference in 2021, held virtually in lutruwita (Tasmania) on the unceded lands of the Palawa people. It underscores the potential impact when plant pathologists unite in advocating for Indigenous Peoples and their rightful place within the field.