Fusarium wilt disease of lettuce caused by the soilborne fungus Fusarium oxysporum f. sp. lactucae (Fola) causes substantial crop losses worldwide. Four races of Fola have been identified with race 1 (Fola1) and the more recently emerged race 4 (Fola4) predominant in Europe. Recent analysis of Fola1 and Fola4 genomes alongside PCR-based isolate profiling determined that while both Fola1 and Fola4 isolates harbour homologues of the putative effector genes Secreted in Xylem (SIX) 9 and SIX14, Fola4 has additionally gained SIX8, which is divergently transcribed with Pair with Six Eight 1 (PSE1) or Pair with Six Eight 1- Like (PSL1) genes. Transcriptomic analyses at a single timepoint also demonstrated high expression levels for SIX8, SIX9, SIX14 and PSL1 during Fola4 lettuce infection. In this study we demonstrate that SIX8, SIX9 and SIX14 expression levels increase up to 96 h following Fola4 lettuce infection, hence confirming their potential importance in wilt disease. The role of SIX8 in virulence on lettuce was then investigated through phenotyping of knockout mutants generated by CRISPR-Cas9 gene editing. ΔSIX8 mutants resulted in reduced disease in both in vitro lettuce seedling and pot-based lettuce plant glasshouse bioassays compared to wild-type Fola4. Complementation of SIX8 back into one of the knockout mutants using Agrobacterium-mediated transformation restored full virulence on lettuce. We conclude that SIX8 plays a role in Fola4 virulence and discuss the hypothesis that SIX8 may allow Fola4 to overcome some sources of Fola1 resistance and hence infect a different range of lettuce cultivars.

Abstract Defoliating (D) strains of the vascular wilt fungus Verticillium dahliae cause severe yield losses in cotton and olive, but the genetic basis of this pathotype remained unknown. Using comparative genomics, functional genetics, structural analysis, and phylogenomics, we identify a D-pathotype–specific genomic region encoding two duplicated secreted effector genes. Simultaneous deletion of both copies abolishes pathogenicity and defoliation in cotton and olive, and affects virulence in Nicotiana benthamiana and Arabidopsis thaliana . Expression of the effector in non-defoliating strains induces cotton defoliation, and purified protein causes wilting and leaf drop. Structural analyses reveal a previously uncharacterized protein fold conserved across Verticillium and Fusarium species, with evidence of functional diversification and host specificity. Phylogenomic and genomic context analyses indicate repeated horizontal transfer mediated by giant transposable elements known as Starships . Together, these findings identify the D effector as a central determinant of defoliation and virulence and show how Starship -mediated gene transfer drives emergence of an agriculturally important fungal trait.

In many fungal phytopathogens, infection is regulated by accessory genomic regions enriched in facultative heterochromatin, but the precise role of this chromatin state in virulence is poorly understood. Here we examined the two facultative heterochromatic marks H3K27me and H3K36me in the tomato-pathogenic Fusarium oxysporum isolate Fol 4287, by genetically analyzing the corresponding histone methyltransferases Kmt6 and Ash1. Fol has three KMT6 homologs, two of which, KMTB/C, are encoded on an accessory chromosome. Loss of the core gene KMT6A resulted in strongly reduced but not abolished H3K27me, revealing that methylation of subtelomeres can be taken over by the accessory copy Kmt6b. To the best of our knowledge, this demonstrates for the first time that accessory fungal histone modifiers can contribute to the overall epigenetic profile. Deletion of KMT6A or ASH1 resulted in derepression of in planta-induced effector genes under axenic conditions. KMT6A deletion mutants were blocked at early stages of root infection and were non-pathogenic on tomato plants, but were still able to kill the animal host Galleria mellonella. Constitutive overexpression of the pathogenicity-related transcription factor FTF1 in the ∆kmt6a background restored effector gene activation in planta but not pathogenicity, suggesting that additional facultative heterochromatin-regulated virulence factors are essential for plant infection.

A major question in infectious disease research is how bacteria have evolved into highly niche-adapted pathogens with efficient host infection strategies. The plant pathogenic bacterium Xanthomonas campestris is subdivided into pathovars that occupy two distinct niches of the same plant leaf: the vasculature and the mesophyll tissue. Using a pangenome comparison of 94 X. campestris isolates, we discovered that the vasculature-infecting pathovar emerged in one monophyletic clade, has lost its CRISPR-Cas system, and showed an increase in both genomic plasticity and acquisition of virulence factors, such as type III effector proteins, compared with the ancestral pathovar. In addition, we show that the CRISPR spacers of isolates belonging to the ancestral pathovar map to plasmids that circulate in Xanthomonas populations and encode high numbers of transposons and virulence factors, suggesting that CRISPR-Cas restricts gene flow toward this pathovar. Indeed, we demonstrate experimentally reduced plasmid uptake in a CRISPR-Cas-encoding isolate. Based on our data, we propose that the loss of the CRISPR-Cas system was a pivotal step in X. campestris evolution by facilitating increased genome dynamics and the emergence of the vasculature-adapted X. campestris pathovar campestris, a major pathogen of Brassica crops.

Fusarium oxysporum (Fo) is a soil-borne fungal pathogen that causes wilt disease in over one hundred plant species, with host-specific strains classified into formae speciales (ff. spp.). For example, Fo f. sp. melonis (Fom) only causes disease in melon while Fo f. sp. radicis-cucumerinum (Forc) can infect multiple cucurbit species. The virulence factors underlying host specificity in these cucurbit-infecting formae speciales have largely remained elusive, limiting our understanding of Fo-host interactions. A previous study identified Effector for Cucurbit Compatibility 1a (ECC1a), an avirulence protein from Fom that restricts cucumber infection when introduced into Forc. Here, we show that ECC1a is part of a previously unrecognized effector gene family, ECC1, abundantly present in strains that infect one or more cucurbit species. However, the role of this family in host compatibility is still poorly understood. Using gene knockout- and replacement strategies, we show that the ECC1 gene family contributes to virulence of both Forc and Fom on cucumber and melon. Specifically, ECC1a contributes to Fom virulence on melon and Forc virulence on cucumber. ECC1b appears to be primarily involved in Fom virulence on melon.Expression profiling reveals a potential role of ECC1 during early stages of infection, suggesting involvement in initial host colonization. Together, these findings highlight the host- and forma specialis-specific functions of ECC1 homologs in Fo pathogenicity.

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 these chromosomes are missing in the reference genome. Pangenome variation graphs offer a reference-free alternative for studying these chromosomes. Here, we constructed a pangenome variation graph for 73 diverse Fusarium oxysporum genomes, a major fungal plant pathogen with a compartmentalized genome that includes conserved core as well as variable accessory chromosomes. To obtain insights into accessory chromosome dynamics, we first constructed a chromosome similarity network using all-vs-all similarity mapping. We identified eleven core chromosomes conserved across all strains and a substantial number of highly variable accessory chromosomes. Some of these accessory chromosomes are host-specific and likely play a role in determining host range. Using a k-mer based approach, we further identified the presence of these accessory chromosomes in all available (581) F. oxysporum assemblies and corroborated the occurrence of host-specific accessory chromosomes. To further analyze the evolution of chromosomes in F. oxysporum, we constructed a pangenome variation graph per group of homologous chromosomes. This reveals that accessory chromosomes are composed of different stretches of accessory regions, and possibly rearrangements between accessory regions gave rise to these mosaic accessory chromosomes. Furthermore, we show that accessory chromosomes are likely horizontally transferred in natural populations. Our findings demonstrate that a pangenome variation graph is a powerful approach to elucidate the evolutionary dynamics of accessory chromosomes in F. oxysporum, which is not only a useful resource for Fusarium but also provides a framework for similar analyses in other species containing accessory chromosomes.

Vascular wilt diseases, caused by the soilborne pathogen Fusarium oxysporum (Fo), significantly impact agriculture and horticulture. Traditional management strategies are often environmentally harmful and not effective once the pathogen has entered the host. Consequently, understanding the molecular mechanisms underlying resistance to pathogenic Fo strains is crucial for developing novel control strategies. In tomato, resistance to F. oxysporum f. sp. lycopersici (Fol) can be mediated by R genes, such as I-1, I-2 or I-3, or by Fo endophytes. Fol inoculation of resistant lines results in high accumulation of pathogenesis-related 5 (PR-5) proteins. However, the role of xylem-localised PR-5 proteins in Fol resistance in tomato plants remains unknown. Here, we demonstrate that apoplastic fluid enriched with recombinant PR-5x or NP24 exhibits antifungal activity against Fol. Using CRISPR/Cas9-mediated gene editing and overexpression studies, the role of PR-5x and NP24 in disease resistance was studied. Loss-of-function mutations in either PR-5 isoform did not compromise Fol resistance. However, overexpression of NP24 in tomato plants resulted in decreased susceptibility to Fol and surprisingly enhanced susceptibility to Ralstonia solanacearum. The role of PR-5x in Fol resistance remains undetermined as lines overexpressing the transgene could not be identified. Our findings suggest that PR-5 proteins are involved in restricting Fol proliferation in the xylem vasculature and thereby contribute to disease resistance.

Vascular wilt disease, caused by the soil-borne fungus Fusarium oxysporum (Fo), poses a threat to many crop species. Four different tomato resistance (R) genes (I-1, I-2, I-3, and I-7) have been identified to confer protection against Fo f.sp. lycopersici (Fol). These I genes are root-expressed and mount an immune response upon perception of the invading fungus. Despite immune activation, Fol is still able to colonize the xylem vessels of resistant tomato lines. Yet, the fungus remains localized in the vessels and does not colonize adjacent tissues or cause disease. The molecular mechanism constraining Fol in the vascular system of the stem remains unclear. We here demonstrate that an I-2-resistant rootstock protects a susceptible scion from Fusarium wilt, notwithstanding fungal colonization of the susceptible scion. Proteomic analyses revealed the presence of fungal effectors in the xylem sap of infected plants, showing that the lack of fungal pathogenicity is not due to its inability to express its virulence genes. To identify mobile root-derived proteins, potentially involved in controlling fungal proliferation, comparative xylem sap proteomics was performed. We identified distinct pathogenesis-related (PR) protein profiles in xylem sap from Fol-inoculated I-1, I-2, I-3, and I-7 resistant lines. Despite structural diversity, all four immune receptors trigger the accumulation of a common set of four PR proteins: PR-5x, PR-P2, and two glucan endo-1,3-β-D-glucosidases. This research provides insights into Fusarium resistance mechanisms and identifies a core set of proteins whose abundance correlates with defense against Fusarium wilt.

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Fungal effectors (small-secreted proteins) have long been considered as species or even subpopulation-specific. The increasing availability of high-quality fungal genomes and annotations has allowed the identification of trans-species or trans-genera families of effectors. Two avirulence effectors, AvrLm10A and AvrLm10B, of Leptosphaeria maculans, the fungus causing stem canker of oilseed rape, are members of such a large family of effectors. AvrLm10A and AvrLm10B are neighbouring genes, organized in divergent transcriptional orientation. Sequence searches within the L. maculans genome showed 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, AvrLm10A_like1/AvrLm10B_like1 and AvrLm10A_like2/AvrLm10B_like2, the second one had the ability to physically interact, similarly to what was previously described for the AvrLm10A/AvrLm10B pair, and cross-interactions were also detected for two pairs. AvrLm10A homologues were identified in more than 30 Dothideomycete and Sordariomycete plant-pathogenic fungi. One of them, SIX5, is an effector from Fusarium oxysporum f. sp. lycopersici physically interacting with the avirulence effector Avr2. We found that AvrLm10A/SIX5 homologues were associated with at least eight distinct putative effector families, suggesting that AvrLm10A/SIX5 is able to cooperate with different effectors. These results point to a general role of the AvrLm10A/SIX5 proteins as "cooperating proteins", able to interact with diverse families of effectors whose encoding gene is co-regulated with the neighbouring AvrLm10A homologue.

Vascular wilt diseases caused by Fusarium oxysporum are a major threat to many agriculturally important crops. Genetic resistance is rare and inevitably overcome by the emergence of new races. To identify potentially durable and non-race-specific genetic resistance against Fusarium wilt diseases, we set out to identify effector targets in tomato that mediate susceptibility to the fungus. For this purpose, we used the SIX8 effector protein, an important and conserved virulence factor present in many pathogenic F. oxysporum isolates. Using protein pull-downs and yeast two-hybrid assays, SIX8 was found to interact specifically with two members of the tomato TOPLESS family: TPL1 and TPL2. Loss-of-function mutations in TPL1 strongly reduced disease susceptibility to Fusarium wilt and a tpl1;tpl2 double mutant exerted an even higher level of resistance. Similarly, Arabidopsis tpl;tpr1 mutants became significantly less diseased upon F. oxysporum inoculation as compared to wildtype plants. We conclude that TPLs encode susceptibility genes whose mutation can confer resistance to F. oxysporum.

The staggering biodiversity of angiosperms has been difficult to reconcile with the gradual Darwinian process thought to create it. Changes in climate through the Earth's history could have instigated this diversification, but perceived ...Although climate change has been implicated as a major catalyst of diversification, its effects are thought to be inconsistent and much less pervasive than localized climate or the accumulation of species with time. Focused analyses of highly speciose ...

Species of the Fusarium solani species complex (FSSC) are responsible for the Fusarium wilt disease of melon (Cucumis melo), a major disease of this crop in Iran. According to a recent taxonomic revision of Fusarium based primarily on multilocus phylogenetic analysis, Neocosmospora, a genus distinct from Fusarium sensu stricto, has been proposed to accommodate the FSSC. This study characterized 25 representative FSSC isolates from melon collected in 2009-2011 during a field survey carried out in five provinces of Iran. Pathogenicity assays showed the isolates were pathogenic on different varieties of melon and other cucurbits, including cucumber, watermelon, zucchini, pumpkin, and bottle gourd. Based on morphological characteristics and phylogenetic analysis of three genetic regions, including nrDNA internal transcribed spacer (ITS), 28S nrDNA large subunit (LSU) and translation elongation factor 1-alpha (tef1), Neocosmospora falciformis (syn. F. falciforme), N. keratoplastica (syn. F. keratoplasticum), N. pisi (syn. F. vanettenii), and Neocosmospora sp. were identified among the Iranian FSSC isolates. The N. falciformis isolates were the most numerous. This is the first report of N. pisi causing wilt and root rot disease in melon. Iranian FSSC isolates from different regions in the country shared the same multilocus haplotypes suggesting a long-distance dispersal of FSSC, probably through seeds.

Objective Wilt caused by Fusarium oxysporum f. sp. melonis (Fom) is one of the most widespread and destructive melon diseases worldwide. Whole-genome sequencing data of a diverse set of Fom strains, as well as several non-pathogenic strains isolated from melon from different parts of the world are described here. These data shed light on the genetic diversity, population structure and the potential evolutionary trajectories which have led to the emergence of different Fom races, and will facilitate identification of avirulence genes which will be helpful to develop resistant melon cultivars. Data description Genomic DNA was extracted from mycelium of 38 Fusarium oxysporum (Fo) strains collected from different parts of the world including Belgium, China, France, Iran, Israel, Japan, Mexico, New Zealand, Spain, the Netherlands, and the United States. The genomes were sequenced to ≈ 20 × coverage using the Illumina Hiseq Xten system, resulting in paired-end reads of 151 bp and assemblies of 1675 (Fom-18L) to 4472 (Fom-R12-13) scaffolds. The genome sequences are available in the National Center for Biotechnology Information (NCBI) and the Sequence Read Archive (SRA) under Project number PRJNA596396 and PRJNA596396, respectively. The presented data set can be useful to identify the genes associated with pathogenic strategies.

Understanding biotic changes that occur alongside climate change constitute a research priority of global significance. Here, we address a plant pathogen that poses a serious threat to life on natural oases, where climate change is already taking a toll and severely impacting human subsistence. Fusarium oxysporum f. sp. albedinis is a pathogen that causes dieback disease on date palms, a tree that provides several critical ecosystem services in natural oases; and consequently, of major importance in this vulnerable habitat. Here, we assess the current state of global pathogen spread, we annotate the genome of a sequenced pathogen strain isolated from the native range and we analyse its in silico secretome. The palm dieback pathogen secretes a large arsenal of effector candidates including a variety of toxins, a distinguished profile of secreted in xylem proteins (SIX) as well as an expanded protein family with an N-terminal conserved motif [SG]PC[KR]P that could be involved in interactions with host membranes. Using agrobiodiversity as a strategy to decrease pathogen infectivity, while providing short term resilient solutions, seems to be widely overcome by the pathogen. Hence, the urgent need for future mechanistic research on the palm dieback disease and a better understanding of pathogen genetic diversity.

1 Abstract The 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 .

The 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.

Many plant pathogenic fungi contain conditionally dispensable (CD) chromosomes that are associated with virulence, but not growth in vitro. Virulence-associated CD chromosomes carry genes encoding effectors and/or host-specific toxin biosynthesis enzymes that may contribute to determining host specificity. Fusarium oxysporum causes devastating diseases of more than 100 plant species. Among a large number of host-specific forms, F. oxysporum f. sp. conglutinans ( Focn ) can infect Brassicaceae plants including Arabidopsis ( Arabidopsis thaliana ) and cabbage. Here we show that Focn has multiple CD chromosomes. We identified specific CD chromosomes that are required for virulence on Arabidopsis, cabbage, or both, and describe a pair of effectors encoded on one of the CD chromosomes that is required for suppression of Arabidopsis-specific phytoalexin-based immunity. The effector pair is highly conserved in F. oxysporum isolates capable of infecting Arabidopsis, but not of other plants. This study provides insight into how host specificity of F. oxysporum may be determined by a pair of effector genes on a transmissible CD chromosome.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.