Biology subjects

Bowen, J. K.

Publications and source records attributed to Bowen, J. K..

8 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↗

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

The Bcvic1 and Bcvic2 vegetative incompatibility genes in Botrytis cinerea encode proteins with domain architectures involved in allorecognition in other filamentous fungi

Vegetative incompatibility is a fungal allorecognition system characterised by the inability of genetically distinct conspecific fungal strains to form a viable heterokaryon, and is controlled by multiple polymorphic loci termed vic (vegetative incompatibility) or het (heterokaryon incompatibility). We have genetically identified and characterised the first vic locus in the economically important, plant-pathogenic, necrotrophic fungus Botrytis cinerea. A bulked segregant approach coupled with whole genome Illumina sequencing in near-isogenic lines of cinerea was used to map a 60-kb genomic region for a vic locus. Within that locus, we identified two adjacent, highly polymorphic open reading frames, Bcvic1 and Bcvic2, which encode predicted proteins that contain domain architectures implicated in vegetative incompatibility in other filamentous fungi. Bcvic1 encodes a predicted protein containing a putative serine esterase domain, a NACHT family of NTPases domain, and several Ankyrin repeats. Bcvic2 encodes a putative syntaxin protein containing a SNARE domain; such proteins typically function in vesicular transport. Deletion of Bcvic1 and Bcvic2 individually had no effect on vegetative incompatibility. However, deletion of the region containing both Bcvic1 and Bcvic2 resulted in mutant lines that were severely restricted in growth and showed loss of vegetative incompatibility. Complementation of these mutants by ectopic expression restored the growth and vegetative incompatibility phenotype, indicating that Bcvic1 and Bcvic2 are controlling vegetative incompatibility at this vic locus. Author SummaryFungal colonies are characterised by radiating filaments, termed hyphae, which often fuse to form a highly interconnected individual. This is advantageous since it enables efficient water and nutrient utilisation across a colony network. However, hyphal fusion is not necessarily restricted to within an individual colony, with potential for hyphal fusion between individuals belonging to the same species. There are, however, drawbacks to this. For instance, viruses that detrimentally affect a colony may be transmitted, with their infection leading to a reduction in the virulence of a pathogenic species. Fungi have therefore developed complex systems to prevent fusion between genetically distinct individuals of the same species. This phenomenon is termed vegetative incompatibility and results in the death of fused cells and cessation of transfer of cellular contents from one individual to another. We have identified the first genes in the fungal plant pathogen Botrytis cinerea that control this phenomenon. They resemble genes that control vegetative incompatibility in other fungi, and genes involved in immunity in plants and animals. Uncovering further genes involved in vegetative incompatibility in B. cinerea may pave the way for the development of a super donor strain capable of overriding vegetative incompatibility to transmit viruses, thus enabling their exploitation as potent control agents against this damaging plant pathogen.

genetics↗

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

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↗

bioRxiv2020

Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

European canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Real-time quantitative reverse transcription PCR (qRT-PCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate qRT-PCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

plant biology↗

bioRxiv2018

Draft Genome Sequence of the Asian Pear Scab Pathogen, Venturia nashicola

Venturia nashicola, which causes scab disease of Asian pear, is a host-specific, biotrophic fungus, with a sexual stage that occurs during saprobic growth. V. nashicola is endemic to Asia and is regarded as a quarantine threat to Asian pear production outside of this continent. Currently, fungicide applications are routinely used to control scab disease. However, fungicide resistance in V. nashicola, as in other fungal pathogens, is an ongoing challenge and alternative control or prevention measures that include, for example, the deployment of durable host resistance, are required. A close relative of V. nashicola, V. pirina, causes scab disease of European pear. European pear displays non-host resistance (NHR) to V. nashicola and Asian pears are non-hosts of V. pirina. It is anticipated that the host specificity of these two fungi is governed by differences in their effector arsenals, with a subset responsible for activating NHR. The Pyrus-Venturia pathosystems provide a unique opportunity to dissect the underlying genetics of non-host interactions and to understand coevolution in relation to this potentially more durable form of resistance. Here, we present the first V. nashicola draft whole genome sequence (WGS), which is made up of 40,800 scaffolds (totalling 45 Mb) and 11,094 predicted genes. Of these genes, 1,232 are predicted to encode a secreted protein by SignalP, with 273 of these predicted to be effectors by EffectorP. The V. nashicola WGS will enable comparison to the WGSs of other Venturia spp. to identify effectors that potentially activate NHR in the pear scab pathosystems.

genomics↗