Gerrit Haatje Jan Kema

Pseudocercospora fijiensis is the causal agent of Black Leaf Streak Disease (BLSD), also known as black Sigatoka, in banana. The disease affects many banana varieties, including the highly susceptible Cavendish banana that dominates global production and the export trade, and several cooking bananas that are a staple food for hundreds of millions of people worldwide. Currently, the disease is controlled using preventative fungicide treatments with up to 70 applications per year in Cavendish plantations, which accounts for approximately 30% of the production costs. Resistant cultivars are required for more sustainable production, but no resistance gene to BLSD has been identified. This is partly due to the poor genetic amenability of P. fijiensis and the lack of methods for functional gene analysis. To address these limitations, we developed a CRISPR/Cas9-mediated transformation system specifically optimized for P. fijiensis . We established a protocol to produce protoplasts, evaluated their capacity to regenerate into new colonies, and assessed antibiotic sensitivity. Subsequently, we confirmed the integration of foreign DNA, including resistance markers, using PEG-mediated transformation. We demonstrated targeted transformation using CRISPR-Cas9 to knockout the polyketide synthase gene PKS10-1, which is responsible for the production of the pigment melanin, and the mitogen-activated protein kinase (MAPK) gene Fus3 . Following the successful generation of knockout mutants for these genes, achieving gene targeting efficiencies of respectively 96% and 58%, we subsequently generated knockout mutants of the renowned effector Avr4 in P. fijiensis . The resulting mutants exhibited no reduction in virulence on the susceptible banana cultivar Cavendish. In addition, we used the wild-type isolate and Avr4 knockout strains to test the resistant banana accession Calcutta 4. Contrary to a previous study, we demonstrate that Avr4 does not explain the resistance of Calcutta 4, suggesting that resistance is instead triggered by the recognition of other hitherto unknown effectors. The established CRISPR/Cas9-mediated disruption system is highly efficient and enables routine functional gene characterization, which will help to elucidate genes involved in banana- P. fijiensis interaction, thereby supporting the discovery of resistance genes against BLSD.

Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB), one of the most destructive diseases of wheat worldwide. Although the Z. tritici genome encodes hundreds of predicted effector proteins, functional characterization through the use of genome-editing techniques has been limited due to low homologous recombination efficiency and extensive effector redundancy. In this study, we established and evaluated a CRISPR/Cas9-based genome editing procedure for targeted effector gene disruption in Z. tritici using in vitro-assembled Cas9-sgRNA ribonucleoprotein (RNP) complexes combined with short (60 bp) homologous donor DNA flanks. Using this approach, we successfully generated knockout mutants for a selected candidate effector gene, the Hce2 domain-containing effector Mycgr3107904. Virulence assays on the susceptible wheat cultivar Taichung 29 revealed that two independent ΔMycgr3107904 mutants exhibited a pronounced delay in symptom development compared to the wild-type strain IPO323, with disease onset and progression delayed by approximately 4-5 days. While mutant strains ultimately followed a similar disease trajectory, wild-type-infected leaves displayed extensive necrosis and pycnidia formation at earlier time points, indicating a significant reduction in virulence upon loss of Mycgr3107904. Together, our results demonstrate the feasibility of CRISPR/Cas9-mediated effector gene knockout in Z. tritici and provide functional evidence that Mycgr3107904 contributes to timely disease progression. This work advances genome editing tools for Z. tritici and facilitates systematic dissection of effector functions underlying fungal virulence.

Transposable elements (TEs) are significant drivers of genome evolution, influencing the genome dynamics of clonal fungal pathogens such as those in the Fusarium oxysporum species complex (FOSC) that cause Fusarium wilt in over 100 plant hosts. Among these, Tropical Race 4 (TR4), a clonal lineage within the FOSC, poses a severe threat to global banana production. However, the contribution of TEs to genome variation and functional traits in TR4 remains poorly understood. Here, we investigated Helitron-associated structural variations in a TR4 strain from Mozambique (M1). This revealed two large deletions in core chromosomes associated with an active FoHeli1 Helitron transposon. One of these (464 kb) disrupted 151 genes, including the entire fusaric acid (FA) biosynthetic gene cluster, consequently abolishing FA production, altering secondary metabolite profiles, and increasing sensitivity to exogenous FA. Despite these metabolic changes, infection assays using wild-type, mutant, knock-out, and complemented strains demonstrated that FA production is dispensable for TR4 virulence in banana. Our study highlights the role of FoHeli1 in modulating the genetic and metabolic landscape of TR4, underscoring the broader impact of TEs on fungal genome evolution and functional diversification, especially in clonal lineages.

The islands of Indonesia are a major part of the centre of origin of bananas. Here, we evaluated the diversity at 164 locations of Sumatra and added 20 wild banana accessions originating from other islands of Indonesia. We analysed morphological characteristics in a principal components analysis (PCA) and genotyped accessions at four nuclear gene regions, i.e. ADH3, CAT2, GBSS1 and IDH1. By combining the geographical coordinates with the morphological variation, we developed a spatial multivariate analysis. The PCA of the morphological traits clearly separated three major clusters. Based on the collected information, we propose to merge the previously described var. halabanensis and var. alasensis Nasution into the subsp. halabanesis . Moreover, we argue that the var. sumatrana should be considered as a subspecies taking into account its genetic and morphological unique characters in addition to its geographical distribution. The four nuclear genes genotyping showed a high genetic diversity of M. acuminata often in admixed stands of different haplotypes. Based on the allele frequencies, there was a significant deviation from a Hardy–Weinberg equilibrium with a high level of homozygosity. Multi-locus genetic population structure analysis confirmed the existence of three major clusters with further possible subdivisions. The discovered genetic diversity in the wild M. acuminata population across Sumatra expands the current knowledge and awaits its deployment in ongoing breeding efforts for this important crop.

Filamentous fungi have evolved compartmentalised genomes comprising conserved core regions and dynamic accessory regions, which are thought to drive adaptation to changing environments, including interactions with host organisms. Tropical Race 4 (TR4) is a lineage of banana‐infecting Fusarium spp. and causes a devastating Fusarium wilt epidemic in the industrial banana cultivar Cavendish. A recent study showed that TR4 contains a single accessory chromosome (chromosome 12), which in some strains has undergone extensive internal duplications, tripling its size compared to other closely related strains. However, the contribution of this accessory chromosome to virulence is currently unknown. Here we show that the induced loss of accessory chromosome 12 in the TR4 reference strain II5 leads to reduced virulence on banana plants. Moreover, loss of chromosome 12 co‐occurs with structural rearrangements of conserved core chromosomes. Together, our results provide new insights into the chromosome dynamics of the banana‐infecting Fusarium TR4 lineage and highlight the importance of its unique accessory chromosome in virulence.

Summary: Transposable elements can significantly influence the genome dynamics of clonal fungal plant pathogens. Tropical Race 4 (TR4) is a clonal lineage within the Fusarium oxysporum species complex that poses a substantial threat to global banana production. However, how transposable elements shape TR4’s genome and adaptation remains underexplored. We investigated the activity and impact of FoHeli1, a Helitron transposable element in the TR4 lineage, focusing on a Mozambican TR4 strain (M1) through a combination of genome analyses, metabolite profiling, and infection assays. FoHeli1 activity has been very recent within the TR4 lineage and is likely still ongoing. This has resulted in large structural variations in M1, including the loss of the conserved biosynthetic gene cluster required for the production of fusaric acid. We demonstrate that this deletion abolishes fusaric acid production and alters secondary metabolite profiles, but does not affect pathogenicity. Our results emphasize the significance of transposable elements, particularly FoHeli1, in reshaping the genetic and metabolic landscape of TR4 and challenge existing assumptions about the role of fusaric acid in pathogenicity.

Despite large omics datasets, the prediction of eukaryotic genes is still challenging. We have developed a new method to improve the prediction of eukaryotic genes and demonstrate its utility using the genome of the fungal wheat pathogen Zymoseptoria tritici. From 10,933 to 13,260 genes were predicted by four previous annotations, but only one third were identical. A novel bioinformatics suite, InGenAnnot, was developed to improve Z. tritici gene annotation using Iso-Seq full-length transcript sequences. The best gene models were selected among different ab initio gene predictions, according to transcript and protein evidence. Overall, 13,414 reannotated gene models (RGMs) were predicted, improving previous annotations. Iso-Seq transcripts outlined 5′ and 3′ untranslated regions for 73% of the RGMs and alternative transcripts mainly due to intron retention. Our results showed that the combination of different ab initio gene predictions and evidence-driven curation improved gene annotation of a eukaryotic genome. It also provided new insights into the transcriptional landscape of this fungus. [ ] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Zymoseptoria tritici is a highly damaging pathogen that causes high wheat yield losses in temperate climates. Z. tritici emerged during the domestication of wheat in the Fertile Crescent and has been extensively used as a model system for population genetic and genomic studies. New genetic tools and resources have provided a better understanding of the molecular components involved in the wheat- Z. tritici interaction, which is highlighted by the cloning of three wheat resistance genes and four Z. tritici avirulence genes. Despite the considerable progress made in the last few years, the mechanisms that mediate Z. tritici colonization remain largely unknown. In this review, we summarize the latest advances in understanding the molecular components mediating wheat- Z. tritici interactions, and we discuss future research lines to close current knowledge gaps. [ ] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

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.

Mitochondria are present in almost all eukaryotic lineages. The mitochondrial genomes (mitogenomes) evolve separately from nuclear genomes, and they can therefore provide relevant insights into the evolution of their host species. Fusarium oxysporum is a major fungal plant pathogen that is assumed to reproduce clonally. However, horizontal chromosome transfer between strains can occur through heterokaryon formation, and recently, signs of sexual recombination have been observed. Similarly, signs of recombination in F. oxysporum mitogenomes challenged the prevailing assumption of clonal reproduction in this species. Here, we construct, to our knowledge, the first fungal pan-mitogenome graph of nearly 500 F . oxysporum mitogenome assemblies to uncover the variation and evolution. In general, the gene order of fungal mitogenomes is not well conserved, yet the mitogenome of F. oxysporum and related species are highly colinear. We observed two strikingly contrasting regions in the F. oxysporum pan-mitogenome, comprising a highly conserved core mitogenome and a long variable region (6–16 kb in size), of which we identified three distinct types. The pan-mitogenome graph reveals that only five intron insertions occurred in the core mitogenome and that the long variable regions drive the difference between mitogenomes. Moreover, we observed that their evolution is neither concurrent with the core mitogenome nor with the nuclear genome. Our large-scale analysis of long variable regions uncovers frequent recombination between mitogenomes, even between strains that belong to different taxonomic clades. This challenges the common assumption of incompatibility between genetically diverse F. oxysporum strains and provides new insights into the evolution of this fungal species. IMPORTANCE: Insights into plant pathogen evolution is essential for the understanding and management of disease. Fusarium oxysporum is a major fungal pathogen that can infect many economically important crops. Pathogenicity can be transferred between strains by the horizontal transfer of pathogenicity chromosomes. The fungus has been thought to evolve clonally, yet recent evidence suggests active sexual recombination between related isolates, which could at least partially explain the horizontal transfer of pathogenicity chromosomes. By constructing a pan-genome graph of nearly 500 mitochondrial genomes, we describe the genetic variation of mitochondria in unprecedented detail and demonstrate frequent mitochondrial recombination. Importantly, recombination can occur between genetically diverse isolates from distinct taxonomic clades and thus can shed light on genetic exchange between fungal strains.