Biology subjects

Bradshaw, R. E.

Publications and source records attributed to Bradshaw, R. E..

9 recordsLinked to original sources

bioRxiv2025

From Nicotiana to apple: effector screening reveals new avenues for durable scab resistance

Scab or black spot disease, caused by the fungus Venturia inaequalis, is an ongoing threat to commercial apple production. Current control methods involve extensive fungicide use and the deployment of disease-resistant apple cultivars. However, fungicide-resistant strains of V. inaequalis are becoming more prevalent, as are strains that can overcome one or more qualitative disease resistance genes in apple. To increase the durability of disease resistance, and thus decrease our reliance on fungicides, one promising approach could involve stacking endogenous and exogenous resistance genes in apple cultivars using genetic modification. As a starting point for the identification of exogenous resistance genes that are effective against V. inaequalis, 137 candidate effector (CE) proteins from this fungus, fused to a signal peptide for extracellular targeting to the apoplast, were screened for recognition by extracellular leucine-rich repeat (LRR)-receptor-like protein and LRR-receptor-like kinase immune receptors in the model angiosperm species Nicotiana benthamiana and Nicotiana tabacum using Agrobacterium tumefaciens-mediated transient transformation assays. Here, a cell death response in wild-type plants, together with a loss of this response in plants lacking the extracellular immune co-receptor NbSOBIR1 or NbBAK1, was used as an indicator of recognition. In total, six CE proteins triggered cell death in one or both Nicotiana species, but only one, a homolog of the VmE02 effector protein from the apple pathogen Valsa mali, did so in an NbSOBIR1- and NbBAK1-dependent manner. The five remaining CE proteins are homologs of other known cell death elicitors from filamentous plant pathogens for which there is evidence that they trigger non-canonical extracellular immunity in plants. One of these is an Alt a 1-like protein that also triggered cell death in apple. Collectively, these findings provide a foundation for the use of a combined set of exogenous and endogenous resistance genes in apple to provide durable protection against scab disease.

microbiology↗

bioRxiv2023

Sequential breakdown of the complex Cf-9 leaf mould resistance locus in tomato by Fulvia fulva

O_LILeaf 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 comprises five paralogous genes (Cf-9A-9E) that encode receptor-like proteins. Two of these proteins contribute to resistance: Cf-9C recognizes 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 have 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. C_LIO_LIComparative genomics, in planta transient expression assays and gene complementation experiments were used to identify Avr9B, while gene sequencing was used to assess Avr9B allelic variation across a worldwide strain collection. C_LIO_LIA 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. C_LIO_LIThis research showcases how F. fulva has evolved to sequentially break down the two functional resistance genes of the complex Cf-9 locus and highlights that this locus now has limited value for controlling leaf mould disease in worldwide commercial tomato production. C_LI

microbiology↗

bioRxiv2023

Genomic, effector protein and culture-based analysis of Cyclaneusma minus in New Zealand provides evidence for multiple morphotypes

Cyclaneusma needle cast, caused by Cyclaneusma minus, affects Pinus species around the world. Previous studies suggested the presence of two distinct morphotypes in New Zealand, verum and simile. Traditional mycological analyses revealed a third morphotype with clear differences in colony morphology and cardinal growth rates at varying temperatures. Genome sequencing of eight C. minus isolates provided further evidence of the existence of a third morphotype, named novus in this study. To further analyse these morphotypes, we predicted candidate effector proteins for all eight isolates, and also characterized a cell-death eliciting effector family, Ecp32, which is present in other pine phytopathogens. In concordance with their distinct classification into three different morphotypes, the number of Ecp32 family members differed, with patterns of pseudogenization and some family members being found exclusively in some morphotypes. We also showed that proteins belonging to the Ecp32 family trigger cell death responses in non-host Nicotiana species, and, as previously demonstrated for other plant pathogens, the C. minus proteins belonging to the Ecp32 family adopt a {beta}-trefoil fold. Understanding the geographical range and variations in virulence and pathogenicity of these morphotypes will provide a better understanding of pine needle diseases as well as enable the development of more durable methods to control this disease.

molecular biology↗

bioRxiv2022

Cell wall carbohydrate dynamics during the differentiation of infection structures by the apple scab fungus, Venturia inaequalis

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 defences 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 {beta}-1,6-glucan, are down-regulated 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 defences and recognition by the host immune system. ImportancePlant-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 down-regulates genes associated with the biosynthesis of two immunogenic cell wall carbohydrates, chitin and {beta}-1,6-glucan, and coats its infection structures with a less-immunogenic carbohydrate, chitosan. These changes are anticipated to enable subcuticular 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.

microbiology↗

bioRxiv2022

Characterization of two conserved cell death elicitor families from the Dothideomycete fungal pathogens Dothistroma septosporum and Fulvia fulva (syn. Cladosporium fulvum)

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 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 {beta}-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 {beta}-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.

molecular biology↗

bioRxiv2022

The Venturia inaequalis effector repertoire is expressed in waves, and is dominated by expanded families with predicted structural similarity to avirulence proteins from other fungi

BackgroundScab, 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. ResultsWe 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. ConclusionsOur 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.

microbiology↗

bioRxiv2021

Apoplastic effector candidates of a foliar forest pathogen trigger cell death in host and non-host plants

Forests are under threat from pests, pathogens, and changing climate. One of the major forest pathogens worldwide is Dothistroma septosporum, which causes dothistroma needle blight (DNB) of pines. D. septosporum is a hemibiotrophic fungus related to well-studied Dothideomycete pathogens, such as Cladosporium fulvum. These pathogens use small secreted proteins, termed effectors, to facilitate the infection of their hosts. The same effectors, however, can be recognised by plants carrying corresponding immune receptors, resulting in resistance responses. Hence, effectors are increasingly being exploited to identify and select disease resistance in crop species. In gymnosperms, however, such research is scarce. We predicted and investigated apoplastic D. septosporum candidate effectors (DsCEs) using bioinformatics and plant-based experiments. We discovered secreted proteins that trigger cell death in the angiosperm Nicotiana spp., suggesting their recognition by immune receptors in non-host plants. In a first for foliar forest pathogens, we also developed a novel protein infiltration method to show that tissue-cultured pine shoots can respond with a cell death response to one of our DsCEs, as well as to a reference cell death-inducing protein. These results contribute to our understanding of forest pathogens and may ultimately provide clues to disease immunity in both commercial and natural forests.

plant biology↗

bioRxiv2021

CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis

BackgroundScab, or black spot, caused by the filamentous 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. Recently, CRISPR-Cas9 has emerged as an efficient tool for gene editing in filamentous fungi. With this in mind, we set out to establish CRISPR-Cas9 as a gene editing tool in V. inaequalis. ResultsWe showed that CRISPR-Cas9 can be used for gene inactivation in the apple scab fungus. As a proof of concept, we targeted the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN), which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference. Using one of two CRISPR-Cas9 single guide RNAs (sgRNAs) 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 these 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. ConclusionsIn establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher the function of genes associated with the growth, reproduction, virulence and pathogenicity of 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.

microbiology↗

bioRxiv2017

Specific hypersensitive response-associated recognition of new apoplastic effectors from Cladosporium fulvum in wild tomato

Tomato leaf mould disease is caused by the biotrophic fungus Cladosporium fulvum. During infection, C. fulvum produces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed by Cf immune receptor genes that encode receptor-like proteins (RLPs). These RLPs recognize specific SSP effectors to initiate a hypersensitive response (HR) that renders the pathogen avirulent. C. fulvum strains capable of overcoming one or more of all cloned Cf genes have now emerged. To combat these strains, new Cf genes are required. An effectoromics approach was employed to identify wild tomato accessions carrying new Cf genes. Proteomics and transcriptome sequencing were first used to identify 70 apoplastic in planta-induced C. fulvum SSPs. Based on sequence homology, 61 of these SSPs were novel or lacked known functional domains. Seven, however, had predicted structural homology to antimicrobial proteins, suggesting a possible role in mediating antagonistic microbe-microbe interactions in planta. Wild tomato accessions were then screened for HR-associated recognition of 41 SSPs using the Potato virus X-based transient expression system. Nine SSPs were recognized by one or more accessions, suggesting that these plants carry new Cf genes available for incorporation into cultivated tomato.

microbiology↗