Biology subjects

Fokkens, L.

Publications and source records attributed to Fokkens, L..

7 recordsLinked to original sources

bioRxiv2025

Mutation or deletion of the Avr6 avirulence effector gene of Fulvia fulva leads to breakdown of the Cf-6 leaf mold resistance locus in tomato

Genetic resistance mediated by Cf cell-surface receptors is a cornerstone of tomato breeding against leaf mold disease caused by the fungal pathogen Fulvia fulva. After widespread deployment of the Cf-9 resistance locus, breeders increasingly relied on Cf-6, yet Cf-6-mediated resistance has already been overcome in multiple regions. The molecular basis of this breakdown, and the identity of the matching avirulence (Avr) effector, have remained unresolved. Here, we use comparative genomics and functional genetics to identify the F. fulva effector Avr6 as the previously described apoplastic protein Ecp5. Sequencing of Cf-6-breaking strains revealed either deletion of the Ecp5 locus or non-synonymous mutations in its coding sequence. Using transient expression, targeted gene knockout, and complementation assays, we show that Ecp5 is both necessary and sufficient for Cf-6-mediated recognition and defence activation. We then use Avr6-triggered cell death as a phenotypic marker in a bulked segregant analysis combined with Comparative Subsequence Sets analysis (CoSSa) to map Cf-6 to a [~]2 Mb interval on the short arm of chromosome 12 that overlaps the previously described Cf-Ecp5.12 locus. Sequencing of Avr6 in a broad strain collection reveals a predominantly conserved allele under apparent purifying selection, with recent independent loss-of-function variants in Europe and South America that confer virulence on Cf-6 plants. Together, these findings establish the Avr6-Cf-6 gene-for-gene pair, explain the erosion of Cf-6-mediated resistance, and illustrate how effector loss or mutation provides a rapid route for F. fulva adaptation, with important implications for designing more durable resistance strategies against tomato leaf mold.

microbiology↗

bioRxiv2024

Reference-free identification and pangenome analysis of accessory chromosomes in a major fungal plant pathogen

Accessory chromosomes, found in some but not all individuals of a species, play an important role in pathogenicity and host specificity in fungal plant pathogens. However, their variability complicates reference-based analysis, especially when chromosomes are missing from reference genomes. Pangenome variation graphs offer a reference-free alternative for studying these chromosomes. Here, we constructed a pangenome variation graph for Fusarium oxysporum, a major fungal plant pathogen with a compartmentalized genome. To study accessory chromosomes, we constructed a chromosome similarity network and identified eleven conserved core chromosomes and many highly variable accessory chromosomes. Some of these are host-specific and are likely involved in determining host range, which we corroborate by analyzing nearly 600 F. oxysporum assemblies. By a reconstruction of pangenome variation graph per homologous chromosomes, we show that these evolve due to extensive structural variation as well as the exchange of genetic material between accessory chromosomes giving rise to these mosaic accessory chromosomes. Furthermore, we show that accessory chromosomes are horizontally transferred in natural populations. We demonstrate that pangenome variation graphs are a powerful approach to elucidate the evolutionary dynamics of accessory chromosomes in F. oxysporum and provides a computational framework for similar analyses in other species that encode accessory chromosomes.

genomics↗

bioRxiv2022

Fusarium oxysporum Effector Clustering version 2 (FoEC2): an updated pipeline to infer host range

1The fungus Fusarium oxysporum is infamous for its devastating effects on economically important crops worldwide. F. oxysporum isolates are grouped into formae speciales based on their ability to cause disease on different hosts. Assigning F. oxysporum strains to formae speciales using non-experimental procedures has proven to be challenging due to their genetic heterogeneity and polyphyletic nature. However, genetically diverse isolates of the same forma specialis encode similar repertoires of effectors, proteins that are secreted by the fungus and contribute to the establishment of compatibility with the host. Based on this observation, we previously designed the F. oxysporum Effector Clustering (FoEC) pipeline which is able to classify F. oxysporum strains by forma specialis based on hierarchical clustering of the presence of predicted putative effector sequences, solely using genome assemblies as input. Here we present the updated FoEC2 pipeline which is more user friendly, customizable and, due to multithreading, has improved scalability. It is designed as a Snakemake pipeline and incorporates a new interactive visualization app. We showcase FoEC2 by clustering 537 publicly available F. oxysporum genomes and further analysis of putative effector families as multiple sequence alignments. We confirm classification of isolates into formae speciales and are able to further identify their subtypes. The pipeline is available on github: https://github.com/pvdam3/FoEC2.

bioinformatics↗

bioRxiv2022

The neighboring genes AvrLm10A and AvrLm10B are part of a large multigene family of cooperating effector genes conserved in Dothideomycetes and Sordariomycetes

With only a few exceptions, fungal effectors (small secreted proteins) have long been considered as species- or even isolate-specific. With the increasing availability of high-quality fungal genomes and annotations, trans-species or trans-genera families of effectors are being uncovered. Two avirulence effectors, AvrLm10A and AvrLm10B, of Leptosphaeria maculans, the fungus responsible for stem canker of oilseed rape, are members of such a large family of effectors. AvrLm10A and AvrLm10B are neighboring genes, organized in divergent transcriptional orientation. Sequence searches within the L. maculans genome show that AvrLm10A/AvrLm10B belong to a multigene family comprising five pairs of genes with a similar tail-to-tail organization. The two genes in a pair always had the same expression pattern and two expression profiles were distinguished, associated with the biotrophic colonization of cotyledons and / or petioles and stems. Of the two protein pairs further investigated Lmb_jn3_08094/Lmb_jn3_08095 and Lmb_jn3_09745 / Lmb_jn3_09746, one (Lmb_jn3_09745 / Lmb_jn3_09746) had the ability to physically interact, similarly to what was previously described for the AvrLm10A/AvrLm10B pair. AvrLm10A homologues are present in more than 30 Dothideomycete and Sordariomycete plant-pathogenic fungi whereas fewer AvrLm10B homologues were identified. One of the AvrLm10A homologues, SIX5, is an effector from Fusarium oxysporum f.sp. lycopersici physically interacting with the avirulence effector Avr2. We found that AvrLm10A homologues were associated with at least eight distinct putative effector families, suggesting an ability of AvrLm10A/SIX5 to cooperate with diverse effectors. These results point to a general role of the AvrLm10A/SIX5 protein as a cooperator protein, able to interact with diverse families of effectors whose encoding gene is co-regulated with the neighboring AvrLm10A homologue.

plant biology↗

bioRxiv2020

A chromosome-scale genome assembly for the Fusarium oxysporum strain Fo5176 to establish a model Arabidopsis-fungal pathosystem

Plant pathogens cause widespread yield losses in agriculture. Understanding the drivers of plant-pathogen interactions requires decoding the molecular dialog leading to either resistance or disease. However, progress in deciphering pathogenicity genes has been severely hampered by suitable model systems and incomplete fungal genome assemblies. Here, we report a significant improvement of the assembly and annotation of the genome of the Fusarium oxysporum (Fo) strain Fo5176. Fo comprises a large number of serious plant pathogens on dozens of plant species with largely unresolved pathogenicity factors. The strain Fo5176 infects Arabidopsis thaliana and, hence, constitutes a highly promising model system. We use high-coverage Pacific Biosciences Sequel long-read and Hi-C sequencing data to assemble the genome into 19 chromosomes and a total genome size of 67.98 Mb. The genome has a N50 of 4 Mb and a 99.1% complete BUSCO score. Phylogenomic analyses based on single-copy orthologs clearly place the Fo5176 strain in the Fo f sp. conglutinans clade as expected. We generated RNAseq data from culture medium and plant infections to train gene predictions and identified [~]18,000 genes including ten effector genes known from other Fo clades. We show that Fo5176 is able to infect cabbage and Brussel sprouts of the Brassica oleracea, expanding the usefulness of the Fo5176 model pathosystem. Finally, we performed large-scale comparative genomics analyses comparing the Fo5176 to 103 additional Fo genomes to define core and accessory genomic regions. In conjunction with the molecular tool sets available for A. thaliana, the Fo5176 genome and annotation provides a crucial step towards the establishment of a highly promising pathosystem.

genomics↗

bioRxiv2020

A partial pathogenicity chromosome in Fusarium oxysporum is sufficient to cause disease and can be horizontally transferred

During host colonization, plant pathogenic fungi secrete proteins, called effectors, to facilitate infection. Collectively, effectors may defeat the plant immune system, but usually not all effectors are equally important for infecting a particular host plant. In Fusarium oxysporum f.sp. lycopersici, all known effector genes - also called SIX genes - are located on a single accessory chromosome which is required for pathogenicity and can also be horizontally transferred to another strain. To narrow down the minimal region required for virulence, we selected partial pathogenicity chromosome deletion strains by fluorescence-assisted cell sorting of a strain in which the two arms of the pathogenicity chromosome were labelled with GFP and RFP, respectively. By testing the virulence of these deletion mutants, we show that the complete long arm and part of the short arm of the pathogenicity chromosome are not required for virulence. In addition, we demonstrate that smaller versions of the pathogenicity chromosome can also be transferred to a non-pathogenic strain and they are sufficient to turn the non-pathogen into a pathogen. Surprisingly, originally non-pathogenic strains that had received a smaller version of the pathogenicity chromosome were much more aggressive than recipients with a complete pathogenicity chromosome. Whole genome sequencing analysis revealed that partial deletions of the pathogenicity chromosome occurred mainly close to repeats, and that spontaneous duplication of sequences in accessory regions is frequent both in chromosome deletion strains and in horizontal transfer (recipient) strains. Author SummaryFungal genomes can often be divided into a core genome, which is essential for growth, and an accessory genome which is dispensable. The accessory genome in fungi can be beneficial under some conditions. For example, in some plant-pathogenic fungi, virulence genes are present in the accessory genome, which enable these fungi to cause disease on certain hosts. In Fusarium oxysporum f.sp. lycopersici, which infects tomato, all host-specific virulence genes are located on a single accessory chromosome. This pathogenicity chromosome can be horizontally transferred between strains. Here, we found that many suspected virulence genes are in fact not required for virulence because strains without a large part of the pathogenicity chromosome, including these genes, showed no reduced virulence. In addition, we demonstrate that partial pathogenicity chromosomes can be horizontally transferred to a non-pathogen. Surprisingly, originally non-pathogenic strains that had received a partial pathogenicity chromosome were more virulent than strains that had received the complete pathogenicity chromosome.

microbiology↗

bioRxiv2018

The multi-speed genome of Fusarium oxysporum reveals association of histone modifications with sequence divergence and footprints of past horizontal chromosome transfer events

Fusarium oxysporum is an economically important pathogen causing wilting or rotting disease symptoms in a large number of crops. It is proposed to have a structured, \"two-speed\" genome: i.e. regions containing genes involved in pathogenicity cluster with transposons on separate accessory chromosomes. This is hypothesized to enhance evolvability. Given the continuum of adaptation of all the genes encoded in a genome, however, one would expect a more complex genome structure. By comparing the genome of reference strain Fol4287 to those of 58 other Fusarium oxysporum strains, we found that some Fol4287 accessory chromosomes are lineage-specific, while others occur in multiple lineages with very high sequence similarity - but only in strains that infect the same host as Fol4287. This indicates that horizontal chromosome transfer has been instrumental in past host-switches. Unexpectedly, we found that the sequence of the three smallest core chromosomes (Chr. 11, 12 and 13) is more divergent than that of the other core chromosomes. Moreover, these chromosomes are enriched in genes involved in metabolism and transport and genes that are differentially regulated during infection. Interestingly, these chromosomes are -like the accessory chromosomes- marked by histone H3 lysine 27 trimethylation (H3K27me3) and depleted in histone H3 lysine 4 dimethylation (H3K4me2). Detailed genomic analyses revealed a complex, \"multi-speed genome\" structure in Fusarium oxysporum. We found a strong association of H3K27me3 with elevated levels of sequence divergence that is independent of the presence of repetitive elements. This provides new leads into how clustering of genes evolving at similar rates could increase evolvability.\n\nAuthor summaryFungi that cause disease on plants are an increasingly important threat to food security. New fungal diseases emerge regularly. The agricultural industry makes large investments to breed crops that are resistant to fungal infections, yet rapid adaptation enables fungal pathogens to overcome this resistance within a few years or decades. It has been proposed that genome compartmentalization of plant pathogenic fungi, in which infection-related genes are clustered with transposable elements (or jumping genes) into separate, fast-evolving regions, enhances their adaptivity. Here, we aimed to shed light on the possible interplay between genome organization and adaptation. We measured differences in sequence divergence and dispensability between and within individual chromosomes of the important plant pathogen Fusarium oxysporum. Based on these differences we defined four distinct chromosomal categories. We then mapped histone modifications and gene expression levels under different conditions for these four categories. We found a division of labor between chromosomes, where some are pathogenicity chromosomes - specialized towards infection of a specific host, while others are enriched in genes involved in more generic infection-related processes. Moreover, we confirmed that horizontal transfer of pathogenicity chromosomes likely plays an important role in gain of pathogenicity. Finally, we found that a specific histone modification is associated with increased sequence divergence.

genomics↗