Abstract
Starships form a recently discovered superfamily of giant transposons in Pezizomycotina fungi, implicated in mediating horizontal transfer of diverse cargo genes between fungal genomes. Their elusive nature has long obscured their significance, and their impact on genome evolution remains poorly understood. Here, we reveal a surprising abundance and diversity of Starships in the phytopathogenic fungus Verticillium dahliae. Remarkably, Starships dominate the plastic genomic compartments involved in host colonization, carry multiple virulence-associated genes, and exhibit genetic and epigenetic characteristics associated with adaptive genome evolution. Phylogenetic analyses suggest extensive horizontal transfer of Starships between Verticillium species and, strikingly, from distantly related Fusarium fungi. Finally, homology searches and phylogenetic analyses suggest that a Starship contributed to de novo virulence gene formation. Our findings illuminate the profound influence of Starship dynamics on fungal genome evolution and the development of virulence.
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Introduction
Transposable elements (TEs, transposons) are ubiquitous mobile genetic elements in all life forms. Originally, these have been seen as selfish elements carrying only genetic information for their proliferation, but presently are appreciated to shape genome structure and function, and drive evolutionary innovations1. Whereas most TE superfamilies have simple structures and encode few proteins2, giant TEs are tens to hundreds of kilobases (kb) in size and carry tens to hundreds of cargo genes3,4,5.
Starships are giant TEs (15–700 kb) that were recently discovered in Pezizomycotina fungi, the largest subdivision of Ascomycota, and typically contain a tyrosine recombinase (YR) “captain” gene as the first gene, required for transposition, while cargo genes are variable and functionally diverse5,6,7,8. How Starships impact global genome evolution, and the range and extent to which Starships have shaped genomes over time, remains enigmatic. Starship detection is technically challenging owing to the large diversity in cargo that can include abundant repeats7, and relies on the detection of the presence/absence of YR-containing inserts in orthologous sites among highly contiguous genome assemblies5. While 143 Starships were identified in this manner in a systematic search of 2899 fungal genomes, 10,628 “orphan” captain-like YR genes remained, suggesting many overlooked Starships6. Starships occupy up to 2.4% of the genome of the human pathogen Aspergillus fumigatus, show extensive presence/absence variation, and contain many differentially expressed cargo genes upon infection9. Moreover, in the plant pathogen Macrophomina phaseolina, 30% of chromosomal translocations, inversions, and putative chromosomal fusions occur near Starship insertions7. Interestingly, Starships can transfer horizontally between closely-related fungi of the same order, and can transfer important traits such as pathogenicity8,10,11,12,13,14,15,16.
Pathogens and their hosts typically engage in molecular arms races, with the pathogen exploiting secreted virulence factors (effectors) to mediate host colonization, while hosts employ immune receptors for pathogen interception17,18,19. To avoid recognition, pathogen effector catalogs are highly dynamic and variable, mediated by a “two-speed genome” organization in which virulence genes co-localize in highly plastic genomic regions that are enriched in repetitive elements and particular epigenetic features20,21,22,23,24,25,26. Accordingly, the fungus Verticillium dahliae that causes vascular wilt disease in hundreds of hosts27,28,29 contains plastic ‘adaptive genomic regions’ (AGRs)30 that are enriched in virulence genes31,32,33,34,35,36, transcriptionally active TEs30,37,38, and structural variations32,37,38,39, associated with a unique chromatin profile and physical interactions in the nucleus30,40,41,42. Besides V. dahliae, the Pezizomycotina Verticillium genus contains nine additional plant-associated species28. Thus far, two Starships have been identified in a single strain of V. dahliae6. Here, we queried 56 highly contiguous Verticillium genome assemblies for Starships to analyze their association with the evolution of AGRs and virulence on plant hosts.
Results
A wealth of Starships occurs in the Verticillium genus
To identify Starships across the Verticillium genus, we collected 56 high-quality Verticillium genome assemblies, comprising 36 V. dahliae strains and 20 strains of the nine remaining species (Fig. 1a and Supplementary Data 1, 2) and queried these genomes for Starships using “Starfish”6. We uncovered 54 Starships that belong to 24 haplotypes of 14 naves of seven families (Fig. 1a, b and Supplementary Data 3). Between one and three Starships were detected in 33 of the 56 strains belonging to seven of the ten Verticillium species (Fig. 1a, b). Thus, most Verticillium genomes contain a Starship, and several genomes even contain multiple. Moreover, as these Starships range from 17 to 625 kilobases (kb) (Fig. 1c), and larger ones typically contain multiple YR genes of different naves (Fig. 1c, d), these likely represent nested Starship insertions. Thus, the final number of Verticillium Starships is likely under-estimated.
a The tree in black shows the phylogeny of the 56 strains used in this study across the Verticillium genus, divided into the Flavexudans (FE) and Flavnonexudans (FNE) clades28, based on whole-genome sequence alignments. Circle colors indicate the ten Verticillium spp. while the label comprises species abbreviation followed by strain name. The tree in gray shows only the V. dahliae strains at increased resolution. Scale bars indicate phylogenetic distances expressed as nucleotide substitutions per site. b Repertoires of Starship haplotypes (hap.) per strain. Starships were classified according to previous studies5,6. Columns indicate Starship haplotypes defined by k-mer similarity and named according to captain navis and family (Supplementary Fig. S1b), whereas heatmap colors show haplotype member counts. “Id” refers to identical Starships (coverage and nucleotide sequence identity >98%) within a haplotype. c Size of the different Verticillium Starships. Points indicate individual Starships listed in Supplementary Data 3. Gray crossbars and error bars indicate the median and 95% confidence interval range of Starship lengths for each navis. d Captain/captain-like tyrosine recombinase (YR) navis repertoires in Starships with multiple YR genes. e Captain/captain-like YR gene classification per strain where “Starship (captain)” indicates YR genes located as the first gene at the 5’-terminus of a Starship for which both borders could reliably be identified with the Starfish tool6, “Starship (non-captain)” refers to YR genes located at other sites in a Starship, suggesting nested Starships with unidentified boundaries. Furthermore, “other Starship region” indicates YR gene presence in regions that could not reliably be identified as Starship with Starfish, but that are syntenic to reference Starships. Finally, “unaffiliated” refers to YR genes that cannot reliably be affiliated with a Starship region.
Verticillium genomes exhibit extensive large-scale genomic rearrangements that may have affected the integrity of prior inserted Starships32,37,43. However, Starfish cannot identify fragmented Starships, rearranged Starship insertion sites, or Starship insertions into lineage-specific regions6. To identify such Starships, we queried for reference captain and captain-like YRs previously identified in Pezizomycotina genomes6, revealing 2–18 homologs in each strain, amounting to 556 YR genes of 38 naves and seven families (Supplementary Fig. S1 and Supplementary Data 4). Only 21% of them belong to Starships identified by Starfish (Fig. 1e), while the remaining 79% could point to unidentified Starships. Additionally, to detect potential Starships and Starship-like regions that may have been overlooked due to genomic rearrangements and insertion into lineage-specific regions, we identified regions syntenic to Starships as “Starship regions”. Hereafter, “Starships” refer to those identified using Starfish, while “Starship regions” collectively refer to Starships regions plus syntenic regions. Intriguingly, such Starship regions occur in all strains (Supplementary Data 5), and half of the captain-like YR genes that did not occur in Starships appeared in such Starship regions (Fig. 1e). Thus, we reveal abundant Starships and their remnants in the Verticillium genus.
Starships are hotspots of large-scale genomic rearrangements
To detail how genomic rearrangements affected Starships, we compared telomere-to-telomere genome assemblies of V. dahliae strains JR2 and VdLs17 that comprise dozens of large-scale genomic rearrangements32,37,44. In strain JR2, we detected three large Starships of 0.50–0.54 Mb each, belonging to haplotypes Ar1h1, Ar4h1, and Se1h1, plus additional Starship regions, collectively accounting for 5.3% (1.92 Mb) of the genome (36.15 Mb) (Fig. 2a). Although no Starships were detected in VdLs17 by Starfish, Starship regions account for 2.7% (0.96 Mb) of the genome (35.97 Mb) (Fig. 2a). Intriguingly, 60% of the inter-chromosomal rearrangement breakpoints between these strains occurred in Starship regions (Fig. 2a and Supplementary Fig. S2). Accordingly, Starship regions were mainly detected in AGRs that are enriched in such rearrangements32,37 (Fig. 2a, b). In strain JR2, 92% (1.77 Mb) of the Starship regions colocalized with AGRs (Supplementary Data 6). Moreover, 53% of the total AGR complement belongs to Starships. In VdLs17, 94% (0.90 Mb) of the Starship regions colocalized with 22% of the AGR complement (Supplementary Data 6).
a Circular plots showing the locations of Starship regions, adaptive genomic regions (AGRs), and genomic rearrangements between V. dahliae strains JR2 (upper eight chromosomes) and VdLs17 (lower eight chromosomes). Tracks are filled with colors representing either core, AGR, or centromeric regions, overlaid with bold lines and arrows representing Starship regions and Starships colored by haplotype. Overlapping arrows in a single region indicate that the Starship orientation is not determined due to the presence of captains at the 5’ end of both strands. Regular triangles point to the captain and captain-like tyrosine recombinase (YR) genes, colored by family and annotated with navis identification (ID). The overlaps among these elements and rearrangement breakpoints are shown in Supplementary Fig. S2 at a higher resolution. Colored bands at the inner edge of tracks represent genetic elements grouped into protein coding sequence (CDS) and transposable element (TE). Ribbons connect syntenic regions (>80% nucleotide sequence identity over 10 kb). b Total length of genomic compartments in V. dahliae strains JR2 and VdLs17. c Violin plots depicting the sequence alignment coverage determined by comparing JR2 genomic compartments against 35 V. dahliae genomes (Supplementary Fig. S3). Points indicate the coverage, and the color represents genome-wide average nucleotide identity (ANI) for each genome alignment (n = 35), while blue crossbars indicate the median values. Different letter labels indicate significant differences (two-sided Dunn’s test, adjusted p < 0.05). d Violin plots depicting the fold-enrichment of structural variations (SVs) (insertion and deletion (INDEL) without size definition, translocation (TRA), inversion (INV), and duplication (DUP)) in the three genomic compartments compared with the genome-wide average. SVs were determined by comparing the genome of V. dahliae strain JR2 against the 35 V. dahliae genomes. Points indicate the fold-enrichment determined for each genome (n = 35), while blue crossbars indicate the median values. Different letter labels indicate significant differences for each genetic variation (two-sided Dunn’s test, adjusted p < 0.05). e Violin plots depicting length of unaligned regions accompanied by SVs in JR2 genomic compartments. Points indicate the length of every gap (n = 644 in Starship regions, n = 1163 in other AGRs, and n = 8877 in core regions) found between the JR2 genome and 35 V. dahliae genomes in a color and shape representing SV type, while blue crossbars indicate the median values. Different letter labels indicate significant differences (two-sided Dunn’s test, adjusted p < 0.05). f Starship rearrangements between the genomes of V. dahliae strains JR2 (X-axis) and GF1192 (Y-axis). Diagonal lines indicate synteny, while the color represents nucleotide sequence identity. Bars and triangles aligned to the plots indicate the positions of Starships and YR genes. The left and right plots are superimposed in Supplementary Fig. S4b to make the breakpoints of inter-chromosomal rearrangements clearer.
Since significant AGR proportions could not be assigned to Starships in JR2 (47%) and VdLs17 (78%), we determined whether Starship regions in AGRs display different characteristics than other AGRs. Pairwise genomic alignments of the JR2 genome with those of the other 35 V. dahliae strains revealed significantly lower alignment coverage in Starship regions (median 31%) than in other AGRs (median 85%) and core genomic regions (median 93%) (Fig. 2c and Supplementary Fig. S3), indicating enhanced presence/absence variation or sequence divergence in Starship regions. Moreover, we revealed an extreme enrichment in structural variation, particularly concerning translocations, inversions, and duplications, in Starships (Fig. 2d). Regions lacking alignment accompanying these rearrangements were significantly larger in Starship regions (median 14.7 kb) than in other AGRs (median 5.9 kb) and core regions (median 1.7 kb) (Fig. 2e and Supplementary Fig. S4a), underscoring the extensive presence/absence variation in Starship regions. Intriguingly, some genomic rearrangements even occurred between Starships as the Ar1h1 Starship in V. dahliae strain GF1192 is largely syntenic to the JR2 Ar1h1 Starship, but synteny lacks at four sites that are syntenic to parts of the JR2 Ar4h1 Starship (Fig. 2f and Supplementary Fig. S4b), leading to Starship diversification.
Starships display typical traits of plastic genomic regions
Like plastic genomic regions of many filamentous plant pathogens20,21,22,23,24, V. dahliae AGRs are enriched in effector genes, in planta induced genes with facultative heterochromatic histone modifications (H3K27me3)30,32,41, and repetitive elements including active TEs37,38. Intriguingly, the previously characterized effector genes Ave131,33 and Av234,45 occur in Ar1h1 and Ar4h1 Starships (Fig. 3a, b). While no difference in in planta gene induction and H3K27me3 levels could be observed between Starship regions and other AGRs (Fig. 3c, d), the average TE density and expression were higher in Starship regions than in other AGRs (Fig. 3e–g). TEs in many fungi are typically inactivated by repeat-induced point mutation (RIP) that introduces cytosine-to-thymine mutations during sexual stages46,47. The majority of TEs in other AGRs and core regions showed the signature of RIP (positive composite RIP index (CRI) values48), but the majority of TEs in Starship regions did not (Fig. 3h), consistent with their expression levels (Supplementary Fig. S5). V. dahliae AGRs have furthermore been characterized by segmental duplications37 that physically interact in the nucleus42. Interestingly, consistent with the segmental duplication pattern (Figs. 2d, 3i), such bipartite long-range chromatin interactions occur between the Ar1h1 and Ar4h1 Starships, between nested Starships within the Ar1h1 and Ar4h1 Starships, and between the Se1h1 Starship and syntenic Starship regions in the JR2 genome (Fig. 3j). Collectively, our data indicate that Starships in V. dahliae are strongly associated with traits that characterize AGRs.
a Verticillium phylogeny as described in Fig. 1a. b Presence or absence of (a)virulence-associated genes in the 56 Verticillium strains. Square colors indicate Starships in which the genes were detected, while the other orthologs found in syntenic Starship regions are not colored. Circles indicate the presence of orthologs with 100% gene coverage and the fill color representing nucleotide identity. c Violin plots depicting the in planta expression induction of genes residing in the three genomic compartments in V. dahliae strain JR2. Induction levels are represented by fold-changes (FC) of gene expression in planta (Arabidopsis thaliana) versus in vitro (potato dextrose broth, PDB). Points indicate FC values for each gene (n = 450 in Starship regions, n = 547 in other adaptive genomic regions (AGRs), and n = 10,638 in core regions), while blue crossbars indicate median values. Different letter labels indicate significant differences (two-sided Dunn’s test, adjusted p < 0.05). d Violin plots depicting histone H3K27me3 levels for the three JR2 genomic compartments expressed as ChIP-Seq read counts per million (CPM) normalized by bin length. Each point indicates the value for a bin (~10 kb) (n = 192 in Starship regions, n = 160 in other AGRs, and n = 3145 in core regions), while blue crossbars indicate median values. Different letter labels indicate significant differences (two-sided Dunn’s test, adjusted p < 0.05). e Stacked bar plots depicting the transposable element (TE) density in the JR2 genomic compartments over 10 kb windows (n = 188 in Starship regions, n = 153 in other AGRs, and n = 3125 in core regions). Different letter labels indicate significant differences (two-sided Dunn’s test, adjusted p < 0.05). f Proportion of expressed (transcripts per million (TPM) >0) and non-expressed (TPM = 0) TEs in the JR2 genomic compartments (n = 284 in Starship regions, n = 202 in other AGRs, and n = 1261 in core regions). Different letter labels indicate significant differences (two-sided Fischer’s test with Bonferroni correction, adjusted p < 0.05). g Violin plots depicting the TE expression levels in the three JR2 genomic compartments for V. dahliae cultivated in PDB. Points indicate TPM values for individual TEs excluding non-expressed ones (n = 217 in Starship regions, n = 78 in other AGRs, and n = 650 in core regions), while blue crossbars indicate median values. Different letter labels indicate significant differences (two-sided Dunn’s test, adjusted p < 0.05). h Violin plots depicting repeat-induced point mutation (RIP) signature of TEs in the JR2 genomic compartments. Points indicate composite RIP index (CRI)48 for individual TEs (n = 284 in Starship regions, n = 202 in other AGRs, and n = 1261 in core regions), while blue crossbars indicate median values. Different letter labels indicate significant differences (two-sided Dunn’s test, adjusted p < 0.05). i Locations of Starships and AGRs in the JR2 genome. See Fig. 2a legend for the details of symbols. Ribbons connect pairs of segmentally duplicated regions that share >80% nucleotide sequence identity over 10 kb, with a color representing identity. j Long-range chromatin interactions in the JR2 genome. Ribbons connect genomic regions that are separated in the genome but physically interact in nuclei by colors that represent the intensity.
Starships carry virulence cargo genes
To assess whether previously characterized virulence genes besides Ave1 and Av2 appear as cargo, we queried the Starships for Verticillium genes that are described in the Pathogen–Host Interactions Database (PHI-base)49. This revealed that the G-LSR2 region, proposed to confer cotton-specific virulence in V. dahliae50, occurs in a Ph1h1 Starship, while the Vl43-LS region that affects V. longisporum virulence51 occurs in a Se1h1 Starship (Fig. 3a, b). In addition, several Starships (Se1h1, Se1h2, Se1h3, and En3h1) contain orthologs of other genes that were shown to contribute to virulence. These genes encode transporters, β-tubulin, and regulators of infection structure morphogenesis and pH-signaling52,53,54,55,56 (Supplementary Fig. S6 and Supplementary Data 7), demonstrating that Verticillium Starships carry diverse virulence-associated cargo genes.
Starship dynamics within and between Verticillium genomes
HGT among Verticillium spp. may have contributed to the shaping of AGRs57. To detect possible Starship transfer between Verticillium spp., we utilized an implicit phylogenetic method that identifies segments with increased sequence similarity over the genome-wide average nucleotide identity (ANI)58. This analysis revealed that several Starships, such as those belonging to haplotypes Se1h1 and Ar1h1, as well as syntenic Starship regions have higher ANI levels than the genome-wide average between the Verticillium species in which they occur (Fig. 4a, b and Supplementary Data 8). The most conspicuous is the Ar1h1 Starship that carries Ave1. The Ar1h1 Starship and its syntenic regions share over 99% identity over 98% coverage across 0.5 Mb between V. dahliae and V. nonalfalfae, while the genome-wide average identity between these species is only 93% (Fig. 4c and Supplementary Data 8).
a Verticillium phylogeny as described in Fig. 1a. b Occurrence of diverse Starships across the Verticillium genus. Columns indicate different Starships with their length and number of captain and captain-like tyrosine recombinase (YR) genes. The heatmap indicates the query coverage, Average Nucleotide Identity (ANI) of Starship regions (Starship ANI), and the difference between Starship ANI and genome-wide ANI for each pairwise strain comparison to detect potential horizontal transfers. Red arrows indicate the Starship highlighted in (c). c Circular plot depicting the synteny among regions of four V. dahliae and two V. nonalfalfae strains harboring orthologous Ar1h1 Starships. See Fig. 2a legend for the details of symbols. d Synteny plots between the V. dahliae JR2 genome and the Ar1h1 Starhip regions in (c). Each plot indicates the synteny between the JR2 chromosomes (Y-axis) and each chromosome (chr) or contig (ctg) containing the Ar1h1 Starship (X-axis), with diagonal lines colored by syntenic JR2 chromosome. Red background indicates the coordinates of Ar1h1 Starship.
To further support Starship mobility, we compared synteny of orthologous Starship flanking regions, revealing that Se1h1 and Ar1h1 Starships occur in several non-homologous genomic regions in different Verticillium clades (Fig. 4c, d and Supplementary Fig. S7). For instance, the Ar1h1 Starship and its syntenic regions that occur in nine V. dahliae strains and in two V. nonalfalfae strains collectively inserted into regions that are orthologous to five different JR2 chromosomes (Fig. 4d), suggesting at least four Ar1h1 Starship movements. Se1h1 Starships in three V. dahliae strains, one V. longisporum strain, and one V. nonalfalfae strain collectively occur in regions that are orthologous to four different JR2 chromosomes (Supplementary Fig. S7c). Se1h1 Starships in some strains appear to have been generated by rearrangements between regions orthologous to two JR2 chromosomes (chr2 and chr5) but have been inserted into two other different chromosomal regions (JR2 chr1 and chr4), suggesting at least two movements (Supplementary Fig. S7c).
Comparisons among orthologous Starship regions across Verticillium strains revealed various diversification patterns (Supplementary Fig. S8). Besides nested Starship insertions (e.g., Ar3h1 Starship in Ar1h1 Starship, Supplementary Fig. S8a and Fig. 1c, d), we typically observed gain and loss of cargo elements in TE-enriched Starships (e.g., Pr1h1 Starships, Supplementary Fig. S8b, c). We furthermore observed invasion of orthologous sites by different Starships that contain orthologous captains, but otherwise lack synteny (e.g., Ar1h1 and Ar1h2 Starships, Supplementary Fig. S8d), which points towards independent insertions as orthologous captains target particular sequences as Starship insertion sites6,8,59.
Cross-order horizontal Starship transfer
To detect possible horizontal Starship transfer between Verticillium and other fungi, we queried all Pezizomycotina genomes available in the GenBank WGS database (10,113 genomes from 668 genera, Supplementary Fig. S9a and Supplementary Data 9) with Verticillium Starships. Intriguingly, regions syntenic to Ph1h1 and Ph1h2 Starships that were detected in only three V. dahliae strains (CQ2, VD991, and XJ592) (Fig. 4a, b), were found in the genomes of various Fusarium species, some of which contain regions syntenic to the G-LSR2 region (Ph1h1 Starship cargo) that was associated with V. dahliae virulence50 (Fig. 5a and Supplementary Fig. S9b). Since Fusarium belongs to Hypocreales and synteny to these Verticillium Starships lacks in non-Verticillium genomes of the Glomerellales to which Verticillium spp. belong, these results suggest horizontal transfer of Ph1h1 and Ph1h2 Starships between Verticillium and Fusarium.
a Occurrence of Verticillium Starships across 10,113 Pezizomycotina genomes. Cell colors and labels denote the percentage of genomes with hits (e-value < 0.05 and total query coverage >50% or >30 kb) in each genus. b Phylogeny of Starships that occur in the Verticillium and Fusarium genera based on k-mer similarity. Scale bar indicates the Mash distance that represents the k-mer difference140. Circles at the nodes represent bootstrap values for 1000 iterations. Red arrows indicate the Starship highlighted in (c). c Similarity of Ph1h2 Starships and surrounding regions between V. dahliae and F. keratoplasticum. d Possibility of horizontal gene transfer (HGT) between Verticillium and fungi belonging to the other orders of Pezizomycotina for genes residing in the three genomic compartments in V. dahliae strain JR2. High Alien index (AI) values (≥45) suggest HGT, while moderate values (>0 and <45) suggest a weak HGT possibility. Points in the left violin plots indicate AI values of individual genes (n = 457 (including captain and captain-like genes predicted de novo) in Starship regions, n = 547 in other adaptive genomic regions (AGRs), and n = 10638 in core regions), while blue crossbars indicate median values. Different letter labels indicate significant differences (two-sided Dunn’s test, adjusted p < 0.05). The right bar plots depict the percentage of genes with high, moderate, and low AI values. ND indicates that AI was not determined because of lacking hits in both ingroup and outgroup. e Occurrence of homologs of Verticillium Starship cargo genes and transposable elements (TEs) across 10,113 Pezizomycotina genomes. Square colors represent the percentage of genomes with hits (e-value < 0.05, over 70% nucleotide identity over 50% coverage) in each group. Circles indicate the coverage and identity of the best hits. The lower panel indicates AI values as described in (d).
To identify the directionality of horizontal transfer, we analyzed a k-mer-based phylogeny of Starships60. To this end, we identified 52 Starships in 283 high-quality Fusarium genome assemblies (Supplementary Data 10, 11) and analyzed their phylogeny together with the 54 Verticillium Starships, revealing that most Starships clustered according to genus (Fig. 5b). In contrast, the Verticillium Ph1h1 and Ph1h2 Starships phylogenetically localized in the Fusarium Starship clade (Fig. 5b), suggesting Fusarium to Verticillium transfer. Moreover, we identified a near-identical Ph1h2 Starship that shares over 99% nucleotide identity over 50 kb in three V. dahliae strains and in F. keratoplasticum (Fig. 5c). The Starship flanking regions are syntenic in three phylogenetically clustered V. dahliae strains, suggesting that the Ph1h2 Starship invaded V. dahliae from F. keratoplasticum before these strains diverged (Fig. 5c).
To further explore potential Starship-associated HGTs overlooked by the synteny search, we tested HGT signatures for each Starship cargo element using the Alien index (AI) that detects higher similarity to outgroup than ingroup orthologs61. We queried orthologs of Verticillium genes in the Pezizomycotina genomes and calculated AI scores with non-Verticillium Glomerellales genera as ingroup and the other 74 orders as outgroup. This showed that the median AI for JR2 genes is higher in Starship regions (45) than in other AGR (0) and core regions (−59), and that the proportion of genes with AI ≥45 (indicative of HGT) is higher in Starship regions (19%) and other AGRs (17%) than in core regions (4%), suggesting cross-order HGT in Starship regions and other AGRs (Fig. 5d). Among the virulence-associated cargo genes, Av2 as well as the G-LSR2 genes showed AI >45 with hits in the various Fusarium spp. of the Hypocreales while lacking ingroup hits (Fig. 5e). Horizontal Av2 transfer was further supported by phylogenetic analyses in which Verticillium orthologs were detected in a clade within a larger clade of Fusarium orthologs (Supplementary Fig. S10a, b). Fusarium Av2 orthologs were found proximal to captain-like YR genes, which suggests Starship association (Supplementary Fig. S10c). We also tested the HGT signature of transcriptionally active Verticillium TEs62 that occur in Starship regions (Supplementary Fig. S11a). Surprisingly, seven out of nine cargo TEs showed AI >45 with the best outgroup hits in five orders, including Hypocreales, while six of them lacked ingroup hits (Fig. 5e). Orthologs of the five cargo TEs also occurred in Fusarium Starships (Supplementary Fig. S11b), suggesting horizontal transfer via Starships. Collectively, our results provide evidence for horizontal transfer of various Starship cargo elements between Verticillium and fungi that even belong to other orders.
Starships mediate de novo gene birth
Starships carry diverse lineage-specific genes7, yet their origin remains enigmatic. We pursued the evolution of the NLP6 effector gene that occurs in a limited number of V. dahliae strains, including VdLs1763, and was detected near a Starship region (Supplementary Fig. S12a). NLP6 belongs to the family of necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), many members of which confer virulence63,64. As multiple NLP paralogs occur in Verticillium genomes63, we first analyzed the phylogeny of NLP homologs in the 56 Verticillium genomes. NLP6 occurs in the same clade as NLP3 orthologs which were detected in all 56 strains (Fig. 6a). Intriguingly, NLP6 is more closely related to NLP3 orthologs of V. nubilum and species of the FE clade than to those of V. dahliae or other species of the FNE clade (Fig. 6a). The similarity between NLP3 orthologs and NLP6 occurs at the C- but not at the N-terminus (Supplementary Fig. S12b). Rather, the 3’ end (115-450 nt) of NLP6 shares 90% nucleotide identity with the 3’ end (379-714 nt) of V. nubilum NLP3, which greatly exceeds the genome average (82%) (Fig. 6b, c, Supplementary Fig. S12c, and Supplementary Data 8), suggesting that the 3’ part of V. dahliae NLP6 is derived from a horizontally transferred NLP3 ortholog. To explore the origin of the 5’ part (1-114 nt) of NLP6, we queried the Verticillium genomes and detected hits with 96% nucleotide identity in non-coding regions in Se1h1 Starships in four Verticillium species, which we termed tNLP6 (for truncated NLP6) (Fig. 6d–f). Intriguingly, three tNLP6 copies were detected around NLP6 in VdLs17 (Fig. 6e). Collectively, these results suggest that NLP6 could be formed through the fusion of a non-coding Starship element with the 3’ part of an NLP3 ortholog through a genomic rearrangement (Fig. 6g). Interestingly, although a role of NLP3 in fungal virulence could not be demonstrated, deletion of NLP6 results in reduced symptom development in V. dahliae-inoculated tomato plants (Fig. 6h and Supplementary Fig. S13). Collectively, these results demonstrate that Starships mediated the emergence of a virulence-associated lineage-specific effector gene in V. dahliae.
a Phylogeny of necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) in 56 strains across the Verticillium genus with all NLP clades collapsed except for the NLP3 clade. The red arrow points to NLP6. Scale bar indicates amino acid substitutions per site. Bootstrap values (>95%) for 1000 iterations are shown at the nodes. Circle colors indicate Verticillium spp. from which each NLP homolog was derived. Flavexudans (FE) and Flavnonexudans (FNE) indicate the two clades within the Verticillium genus28. b Verticillium phylogeny as described in Fig. 1a. Circle colors indicate Verticillium spp. with the same color coding as in (a). c, d Coverage plots for alignments of NLP6 with NLP3 genes (c) and for alignments of a truncated NLP6 (tNLP6: 1-114 nt of NLP6) with genome sequences (d). The X-axes represent the nucleotide positions in NLP6 or tNLP6 of V. dahliae VdLs17. Bar colors indicate the identity of the region and its difference from genome-wide ANI between each Verticillium strain and VdLs17. e Location of tNLP6 in Starship in three Verticillium species. See Fig. 2a legends for the details of symbols. f Location of tNLP6 in non-coding regions of V. dahliae strains. g Proposed model for NLP6 evolution. h Symptoms of tomato plants inoculated with wild-type (WT) and three independent NLP6 deletion (Δ) lines of V. dahliae strain VdLs17 at 14 days post inoculation. Points indicate relative values for individual plants (n = 6), divided by the mean of WT-inoculated plants. Crossbars and error bars indicate mean ± standard deviation. Different letter labels indicate significant differences (two-sided Tukey’s test, adjusted p < 0.05).
Discussion
Here, we reveal the unprecedented impact of Starships on fungal genome evolution through four main findings. First, every Verticillium genome contains Starships or their remnants. Secondly, these Starships compose the vast majority of AGRs that govern pathogenicity on plant hosts. Thirdly, extensive horizontal Starship transfer occurred between V. dahliae and phylogenetically diverse fungi. Fourthly, Starships contributed to the de novo generation of a novel virulence gene. Thus, Starships are not only instrumental for Verticillium genome evolution, but also fundamental to evolutionary innovations that led to plant pathogenicity.
Thus far, little is known about Starship abundance in individual fungal genomes. We identified 24 Starship haplotypes in 56 Verticillium genomes. These Starships generally occur in multiple Verticillium strains, indicating invasion before strain diversification. While some remained intact, others got disrupted by genomic rearrangements. The abundance of Starships exceeds the abundance previously recorded in A. fumigatus, where 20 Starships and their remnants were identified in a survey of 519 strains, and not all strains contained Starships9. In M. phaseolina, four Starships and remnants were identified in 12 isolates7. Overall, our study reveals an unprecedented abundance of Starships across Verticillium genomes.
We could unequivocally demonstrate that Starships make up about half of AGRs in the V. dahliae JR2 genome, while the remaining AGRs could not be assigned to Starships. AGRs were initially identified as lineage-specific genomic regions by comparisons among V. dahliae strains32, while additional AGRs were subsequently identified based on their characteristic chromatin profile30. Interestingly, the Starship regions identified in this study correspond to the initially identified AGRs. However, besides their chromatin profile, like Starships, also the non-Starship AGRs are enriched in horizontally transferred genes and genomic rearrangements. Thus, we speculate that these AGRs may be derived from unidentified Starships that became disrupted by genomic rearrangements. In support of this hypothesis, Verticillium genomes likely still contain many unidentified Starship regions, given 211 captain-like YR genes unassigned to known Starship regions in the 56 genomes, the majority (127/211) of which occur in AGRs (Supplementary Data 4). Overall, we conclude that Starships are the major constituents of AGRs in V. dahliae.
Many filamentous plant pathogens carry plastic genomic regions similar to AGRs20,21,22,23,24, but how these regions evolved remains elusive26. Although several Starships have been identified in Pezizomycotina plant pathogens7,12,14,15,65,66,67,68, the association of these Starships with the typical plastic genomic regions has not been noted previously. The evolution of plastic genomic regions is known to be associated with the activity of smaller TEs20,26. Our results show that TEs in Starship regions are more highly expressed and show weaker RIP signatures than those in the other regions of the V. dahliae genome. RIP mutates TEs during sexual stages in Pezizomycotina fungi47. As V. dahliae is presumed to reproduce asexually69, it has been hypothesized that TEs showing RIP signatures could have been mutated in its sexually active ancestor in which RIP was active30. We have previously shown that TEs in lineage-specific AGRs, which correspond to Starship regions identified in this study, are enriched in evolutionarily younger TEs that proliferated after the speciation of V. dahliae, whereas other regions are enriched in older TEs that proliferated before speciation37. Therefore, the enrichment of transcriptionally active TEs in Starship regions could be due to their invasion of V. dahliae after the inactivation of TEs in other genomic regions. In support of this hypothesis, our results suggest the horizontal transfer of active TEs in Starships. These findings highlight the close relationships among plastic genomic regions, Starships, and the activity of other TEs.
HGT has driven virulence evolution in diverse fungal pathogens70,71. HGT between phylogenetically close fungi is thought to be mediated by anastomosis and horizontal chromosome transfer, while the mechanisms of HGT between phylogenetically distant fungi remain elusive70,71. Our phylogenetic analyses suggest the horizontal Starship transfer between V. dahliae and V. nonalfalfae on the one hand, and distantly related Fusarium fungi on the other hand. In addition, Starship regions in V. dahliae are enriched in cargo elements horizontally transferred to/from fungi belonging to further diverse Pezizomycotina orders. Thus, while previous studies have shown Starship-mediated HGT between fungi within the same order8,10,11,12,13,14,15,16, our study extends this observation by revealing that Starships mediated HGT between phylogenetically distant fungi of different orders. There are many reports of cross-order HGT between diverse Pezizomycotina pathogens, though the mechanism remains generally unknown72,73,74,75,76,77,78. Further identification of Starships in diverse taxa may help to understand the extent to which Starships have mediated such HGT events.
Pathogens exploit diverse effectors to colonize hosts, but it often remains unclear where, when, and how such effectors evolved, given that they are often considered lineage-specific inventions79. Since pathogen effectors often lack conserved protein domains, it has been speculated that such effectors may have evolved de novo79. De novo genes are defined to originate, at least in part, from non-coding DNA sequences, but the underlying evolutionary mechanisms remain poorly understood80,81. Our results suggest that a Starship contributed to the emergence of a virulence effector gene from a non-coding cargo sequence by the fusion with a section of a conserved effector gene. Given the strong association of Starships with plastic genomic regions enriched in (a)virulence effector genes, further elucidation of Starship-mediated gene evolution can be fundamental to understanding how novel virulence genes evolve.
Methods
Genome assemblies and annotations
Genome sequences were collected from public databases or generated in this study (Supplementary Data 1, 9, 10). Fungal genomic DNA was extracted and sequenced using Oxford Nanopore Technologies as previously described34. Sequencing reads were assembled with Canu version 2.282, and genome assembly quality was evaluated with BUSCO version 5.7.0 with the datasets eukaryota_odb10 and glomerellales_odb1083,84. The genome-wide average nucleotide identity (ANI) was calculated with FastANI version 1.3385 that maps query genomic sequences fragmented to 3 kb to a reference genome and calculates the average of the maximum identity of the mapped regions.
Genes and TEs in V. dahliae strain JR2 were identified previously38,44. In all other genomes, repetitive sequences were predicted with RepeatModeler version 2.0.586 and were soft-masked with RepeatMasker version 4.1.587. Genes were then predicted with the BRAKER version 3.0.8 pipeline C88, which trains GeneMark-EP+ version 4.72_lic89 and AUGUSTUS version 3.0.890 with information on splice sites, start, stop, and coding features using reference fungal proteins in OrthoDB version 1191. Orthologous gene groups were identified by eggNOG-mapper version 2.1.1292 using eggNOG database version 5.0.293. TEs were further characterized with the TE annotation pipeline EDTA version 2.2.194 with curated TEs of V. dahliae VdLs1762.
Identification of Starships and Starship regions
Starships and captain/captain-like tyrosine recombinase (YR) genes were identified with Starfish version 1.0.0 according to the standard workflow6. First, the captain candidate YR genes were identified de novo from the genomes using the Starfish gene finder module with MetaEuk version 6.a5d39d995 and HMMER version 3.3.296 based on sequence similarity to the Pezizomycotina captain/captain-like YR database attached to Starfish. The identified YR genes were grouped into families as defined previously6 based on a similarity search with HMMER version 3.3.296. The YR genes were then grouped into naves by clustering of Verticillium YRs using MMseqs2 version 14.7e284 easy-cluster97 with thresholds of 50% amino acid sequence identity and 25% coverage. Each Verticillium YR navis was named with two letters of the YR family followed by an identifier number. Then, Starship candidates were identified via multiple sequence alignments among the 56 Verticillium genomes or among the 283 Fusarium genomes that were selected based on a relatively low number of contig/scaffold numbers, suggesting a relatively highly contiguous genome assembly (Supplementary Data 2, 10), by the Starfish element finder module using BLAST version 2.12.0+98 and MUMmer version 4.0.0rc199. After the initial screening by Starfish, confident Starships were curated by (1) removing Starship candidates that lack flanking synteny over 20 kb against orthologous sites without Starships through manual inspection with Starfish pairViz, (2) removing Starship candidates that contain serial N stretches (N ≥ 3) in scaffolds, and (3) unifying redundantly detected Starships due to the presence of YR genes on both strands around the 5’ ends. The Starships were grouped into haplotypes based on the captain navis and nucleotide k-mer similarities detected by sourmash version 4.8.3100 with Starfish sim with default k-mer size 5106,9, followed by the clustering using mcl version 14-137101 with minimal similarity threshold 0.05. Each Verticillium Starship haplotype was named after the captain navis ID followed by a suffix. Genomic regions orthologous to Starship insertion sites across Verticillium strains (Supplementary Fig. S7) were identified by the Starfish region finder module based on the presence of low copy number (up to 5) eggNOG orthologs.
Starship regions were annotated by two approaches. First, regions downstream of YRs were annotated as Starship regions using Starfish extend6 with default settings using BLAST version 2.12.0+98 based on similarity against curated Starships (Supplementary Data 12). Second, other Starship regions were identified irrespective of the presence of YR genes based on synteny against curated Starships through the alignments with nucmer of MUMmer version 4.0.0rc1 with options maxmatch and minimal alignment length 10,000, followed by filtering using delta-filter with the threshold of >80% nucleotide identity99 (Supplementary Data 13). Starships identified in this study (Supplementary Data 3, 11) were used as reference. The Starship regions identified by the two approaches were merged with bedtools version 2.30.0102 to determine the final coordinates of Starship regions (Supplementary Data 5). The pairwise sequence alignments between each Starship and syntenic Starship regions in each genome (Fig. 4b) were also performed using nucmer with the same settings.
The genomic compartments of V. dahliae strains JR2 and VdLs17 were assigned based on the overlap of Starships and Starship regions with the previously assigned AGRs, core genomic regions30, and centromeres103. AGRs in the JR2 genome were identified by chromatin profiling30, while AGRs in the other strains refer to regions syntenic to AGRs in JR2 and regions absent in JR242. To identify AGRs in strains other than JR2, genome alignments were performed with nucmer of MUMmer version 4.0.0rc199, followed by filtering of alignments with >80% nucleotide identity over 1 kb. The regions aligned to the JR2 AGRs and not aligned to the JR2 genome were merged with bedtools version 2.30.0102 and then filtered by the length threshold of >1 kb to determine the final AGRs. The “Starship” compartment refers to Starships and additional Starship regions, “other AGR” refers to AGRs that do not belong to Starship regions, “centromere” refers to previously identified centromeres, and “core” refers to core genomic regions that do not belong to Starship regions or centromeres. Genes and TEs in the JR2 genome were assigned to genomic compartments based on the location of their midpoints. TE counts over 10 kb windows in each genomic compartment were calculated using bedtools version 2.30.0 intersect102. Compartments smaller than 10 kb were omitted from the TE density analysis. The composite RIP index (CRI) of TEs was calculated as previously described48. Briefly, CRI was determined by subtracting the RIP substrate index ((CpA + TpG)/(ApC + GpT)) from the RIP product index (TpA/ApT), as CpA dinucleotides are preferentially targeted by RIP.
Analysis of genomic syntenies and variations
Genomic syntenies and variations were identified with MUMmer version 4.0.0rc199. Specifically, segmental duplications in the JR2 genome were identified by genome self-alignment using nucmer with options maxmatch, nosimplify, and minimal alignment length 10,000, followed by filtering with delta-filter. Inter-genomic syntenies and variations were identified by pairwise genome alignments with nucmer with option minimal alignment length 10,000, followed by filtering and analysis by dnadiff. The genome-wide alignment coverage was calculated by integrating all fragmented hits. Among the structural variations (SVs) shown in this study, insertion/deletion (INDEL) corresponds to “GAP” and “BRK” in MUMmer4, while translocation (TRA) corresponds to “JMP” and “SEQ”. SV frequency was calculated by dividing the SV counts by the aligned length for each genomic compartment or genome. SV fold-enrichment was calculated by dividing the frequency in each genomic compartment by the genome-wide frequency.
Transcriptome, epigenome, and 3D genome analyses
Global gene expression in V. dahliae strain JR2 was analyzed using the previous RNA sequencing (RNA-Seq) read data104 obtained from the NCBI short read archive (SRA) under the accession numbers listed in Supplementary Data 14. RNA-Seq reads were filtered with fastp version 0.19.5105 and mapped to the unmasked JR2 genome with STAR version 2.7.10a106 with previously applied options to allow multiple mapped reads for TE expression measurement38. The mapped reads were counted for each gene and TE by TEtranscripts version 2.2.1 with mode multi for 1,000 iterations to allow fractional counting of multi-mapped reads107. Fold change (FC) of gene expression was analyzed by DESeq2 version 1.42.1108. Genes and TEs with no sequencing reads in all samples were excluded from the DESeq2 analysis. Transcripts per million (TPM) were calculated using the established formula109. Mean TPM values of three biological replicates for individual genes/TEs were used for the data visualization and statistics.
Global histone H3K27me3 modifications in V. dahliae strain JR2 were analyzed using the previous chromatin immunoprecipitation sequencing (ChIP-Seq) data30 obtained from NCBI SRA under the accession numbers listed in Supplementary Data 14. ChIP-Seq reads were filtered with fastp version 0.19.5105 and mapped to the JR2 genome masked as previously described30 using BWA version 0.7.17 with the BWA-MEM algorithm110. The mapped reads were sorted and indexed with samtools version 1.10111 and counted with featureCounts version 2.0.1112 with the option to count multi-mapped reads fractionally for each genomic compartment over 10 kb windows. The normalized counts per million (CPM) were calculated using the TPM formula109 by replacing transcripts with bins. The mean normalized CPM values of two biological replicates for respective bins were used for the data visualization and statistics.
Long-range chromatin interactions in V. dahliae strain JR2 were previously identified through chromatin conformation capture (Hi-C)42. The previously analyzed data were directly used for visualization with the Starship positions.
Local similarity searches
Local similarity searches were performed with BLAST version 2.15.0+ or 2.16.0+98. The nucleotide-to-nucleotide searches were performed with the blastn algorithm with options word size 11 and e-value 0.05. The protein-to-translated nucleotide searches were performed with the tblastn algorithm with an e-value threshold of 0.05. The query Verticillium gene and TE sequences were obtained from GenBank and FungiDB under the accession numbers listed in Supplementary Data 15, and genes including introns were used as queries. The sequences and metadata of other virulence-associated proteins were obtained from PHI-base version 4.1749. Pezizomycotina genomes were obtained from whole-genome shotgun (WGS) sequences in GenBank under the accession numbers listed in Supplementary Data 9. Fungal taxonomic data were obtained from the National Center for Biotechnology Information (NCBI) Taxonomy database. The coordinates of Av2 orthologs (Supplementary Data 16) were identified by a blastn search of the Pezizomycotina genomes with V. dahliae Av2 or the Av2 ortholog of Fusarium phyllophilum (FpAv2) under the accession numbers in Supplementary Data 15, followed by the selection of hits with >70% identity and >80% coverage.
Alien index (AI) values were calculated by the formula AI = log((best e-value for ingroup) + e-200) - log((best e-value for outgroup) + e-200)61 using hits with >50% coverage against Verticillium gene/TE queries by the blastn search. The e-value for no hits was set to 1 for queries with hits in either ingroup or outgroup, whereas AI values for queries with no hits in both ingroup and outgroup were not determined. The 332 non-Verticillium genomes of the order Glomerellales were used as ingroup, while the 9641 genomes of the remaining 74 orders of Pezizomycotina were used as outgroup (Supplementary Data 9).
Phylogenetic analyses
Verticillium phylogeny was inferred with REALPHY version 1.13113. Briefly, genome sequences were fragmented into overlapping 50 bp subsequences and mapped to the JR2 genome with Bowtie version 2.2.5114. Based on single-nucleotide polymorphisms in the aligned regions, the maximum likelihood phylogenetic tree was built with PhyML version 3.3.20220408115.
Starship phylogeny was inferred by k-mer comparisons with mashtree version 1.4.6 with default k-mer size of 21, accuracy option mindepth 0, and bootstrapping for 1,000 iterations116.
For the phylogenetic analyses of Av2, nucleotide sequences of Av2 orthologs were extracted from the Pezizomycotina genomes at the coordinates listed in Supplementary Data 16 using SeqKit version 2.3.0 subseq117. The nucleotide sequences of Av2 were aligned by MAFFT versions 7.511 or 7.526 with the L-INS-i method that iteratively refines local alignments118, followed by removal of ambiguously aligned sites with trimAl version 1.4.rev15 with option strict119. After multiple sequence alignments, maximum likelihood phylogeny was inferred by IQ-TREE version 2.0.3120 with the best substitution model TIM2e + G4 suggested by ModelFinder121 and the ultrafast bootstrap approximation for 1000 iterations122.
For the phylogenetic analysis of NLPs, putative Verticillium proteins that contain an NPP1 domain64 were identified based on the homology to reference sequences in the Pfam database123 (Pfam accession PF05630.16) using HMMER version 3.3.296. Putative secretion signals were identified with SignalP 6.0124 or SignalP 3.0125. The deduced NLP amino acid sequences were aligned and trimmed as described for Av2. The phylogeny of NLPs was also inferred as described for Av2 with the different best substitution model VT + F + I + G4. NLP orthologs were numbered by the monophyletic relationship with NLP1 to NLP9 of V. dahliae strain VdLs1739,63 in the GenBank RefSeq database under the accession numbers listed in Supplementary Data 15.
Targeted deletion of NLPs from the Verticillium dahliae genome
To generate NLP6 and NLP3 deletion constructs, flanking sequences of its coding sequence were amplified from genomic DNA of V. dahliae strains VdLs17 and JR2, respectively, using primers listed in Supplementary Data 17. The amplified products were cloned into pRF-HU2 as described previously126, and subsequent Agrobacterium tumefaciens-mediated transformation of V. dahliae was performed as described previously127. Transformants were selected on PDA supplemented with cefotaxime (Duchefa, Haarlem, The Netherlands) at 200 μg/ml and hygromycin (Duchefa) at 50 μg/ml, and homologous recombination was PCR-verified. For genetic complementation, the coding sequence of NLP6 was cloned into the pFBT005 vector as previously described128, after which the NLP6 deletion mutants were transformed using the A. tumefaciens-mediated transformation method described above.
Pathogenicity assays were performed on ten-day-old tomato seedlings (MoneyMaker) plants using root-dip inoculation as previously described129. Disease symptoms were scored up to 14 dpi, pictures were taken, and ImageJ was used to determine canopy areas while fungal colonization was determined with real-time PCR. To this end, stem sections were taken at the height of the first internode, flash-frozen in liquid nitrogen, ground to powder, and genomic DNA was isolated. Real-time PCR was performed with a quantitative PCR core kit for SYBR Green I (Eurogentec, Seraing, Belgium) on an ABI7300 PCR machine (Applied Biosystems, Foster City, CA, U.S.A.). The V. dahliae internal transcribed spacer (ITS) levels were used relative to tomato ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) levels to quantify fungal colonization of tomato plants36.
Statistics
Data were analyzed with R version 4.4.2130 and the R package “tidyverse” version 2.0.0131. The normality and homoscedasticity of data were tested by the Shapiro–Wilk test and Bartlett’s test, respectively, with the R base package “stats” version 4.3.1130. The multiple comparisons of data with non-normal distributions and unequal variances (p < 0.05) were performed by Dunn’s test with the Bonferroni correction with the R package “dunn.test” version 1.3.6132. The multiple comparisons of data with normal distributions and equal variances (p < 0.05) were performed by Tukey’s test with the R package “multcomp” version 1.4.26133. The multiple comparisons of the proportions of two categorical variables were performed by Fisher’s test with the Bonferroni correction with the R package “RVAideMemoire” version 0.9-83-11134. The exact adjusted p-values of multiple comparisons are provided in the Source Data files. The Spearman’s rank correlation coefficient was calculated with the R base package “stats” version 4.3.1130.
Data visualization
Data were visualized with the R packages. Circular and linear plots of genomic regions were generated with “circlize” version 0.4.16135 and ‘gggenomes’ version 1.0.0136 or “genoPlotR” version 0.8.11137, respectively. Phylogenetic trees were visualized with “ggtree” version 3.10.1138. Other plots were generated with “ggplot2” version 3.5.1139.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
The genomes assembled in this study and previous studies57,103 have been submitted to NCBI under the BioProject accession PRJNA1253319. Genome annotation files and genome assemblies with complex gap information103 have been deposited at Zenodo (https://doi.org/10.5281/zenodo.15450312). Other genome sequence, RNA-Seq, and ChIP-Seq data used in this study are available in the NCBI database under the accession numbers listed in Supplementary Data 1, 9, 10, 14–16. Source data are provided with this paper.
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Acknowledgements
Y.S. acknowledges funding through an Overseas Research Fellowship from the Japan Society for the Promotion of Science. B.P.H.J.T. acknowledges funding by the Alexander von Humboldt Foundation in the framework of an Alexander von Humboldt Professorship endowed by the German Federal Ministry of Education and is furthermore supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy – EXC 2048/1 – Project ID: 390686111. R.B. and M.H. acknowledge funding by the Research Foundation Flanders (FWO project ID: G004022N). We thank Dr. Edgar A. Chavarro-Carrero for providing sequencing read data of the V. dahliae genomes assembled in this study.
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Y.S. and B.P.H.J.T. conceived the project. Y.S., M.F.S., and B.P.H.J.T. designed the analyses. R.B. and M.H. contributed to the discovery of the NLP6 origin. G.C.M.v.d.B. and P.S. performed fungal gene disruption and pathogenicity assays. Y.S., G.C.M.v.d.B., P.S., and B.P.H.J.T. analyzed the data. Y.S. and B.P.H.J.T. wrote the manuscript with feedback from all authors.
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Sato, Y., Bex, R., van den Berg, G.C.M. et al. Starship giant transposons dominate plastic genomic regions in a fungal plant pathogen and drive virulence evolution. Nat Commun 16, 6806 (2025). https://doi.org/10.1038/s41467-025-61986-6
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DOI: https://doi.org/10.1038/s41467-025-61986-6
