Fig 1.
Phenotypes and phylogeny of three isolates of Fusarium oxysporum f. sp. niveum (Fon) sequenced in this study.
(A) Potato dextrose agar (PDA) cultures of FonR1, FonR2, and FonR3 (NXWFW008) and disease phenotypes on differential hosts. PDA cultures were photographed 5 days post inoculation (dpi), while the plant infections were photographed 28 dpi on 11-day-old seedlings. Mock was inoculated with water. Different watermelon cultivars used to identify the physiological races of Fon were PKR6 (resistant to R1, R2, but susceptible to R3), Sugarlee and Calhoun Gray (resistant to R1, but susceptible to R2 and R3), and G42 (susceptible to all three races). For each treatment, 15 plants were inoculated, and the experiment was repeated three times. (B) Phylogenetic tree of 20 F. oxysporum strains including FonR1, FonR2, and FonR3. The tree was rooted by F. verticillioides strain BRIP53590. All branches are supported by 100% bootstrap values. Clades were annotated as described in O’Donnell et al. [20].
Table 1.
Genome assembly and annotation statistics for isolates belonging to three races of Fusarium oxysporum f. sp. niveum.
Fig 2.
Gap-free genome assembly of FonR3 and accessory genome identification of FonR1, FonR2, FonR3.
(A) An ideogram of the most virulent race, FonR3, compared to the Fo47 genome was visualized using GenomeSyn [80]. Genomic features, including gene density, GC content, TE content, telomeres, centromeres, and genome synteny with Fo47 chromosomes, are shown on the ideograms. The yellow background indicates the accessory regions. (B) Dotplots of FonR1, FonR2, and FonR3 genome alignments with Fo47. The color scale shows the sequence identity of the aligned sequences. (C) Comparison of three isolates in terms of total length, the number of annotated genes, and predicted effectors in core (light colors) and accessory (dark colors) genomic regions.
Fig 3.
Comparative genomics of FonR1, FonR2, and FonR3 gap-free genome assemblies reveals core and lineage-specific accessory regions/chromosomes.
(a) Chromosomes of FonR1, FonR2, FonR3. Light colors represent core chromosomes (CC), while dark colors represent accessory chromosomes/regions (AC/AR). (b) Predicted effectors (black) and unique effectors (red) are shown. (c) Links showing synteny between three genome sequences. Gray links are between CCs while colored links are between AC/ARs.
Fig 4.
Comparative transcriptomics of three Fon race isolates during watermelon infection.
(A) Kernel density plots of gene expression log2FC(Fold Change) values between control and 6 dpi of the G42 cultivar. The median log2FC values are shown as bold numbers and dashed vertical lines. (B) k-means clustering of the expression of the single-copy orthologous genes of FonR1, FonR2, and FonR3. The figure was generated using ClusterGVis [72] with k = 20. Only the clusters with a difference among the three isolates are shown. The full image, including all clusters, can be found in S5 Fig.
Fig 5.
Features of FonR3SE1, a critical effector for FonR3 infection.
(A) A structure diagram for the FonR3SE1 gene with domains and cysteine residues. (B) Predicted 3D protein structure of FonR3SE1 without signal peptide using Alphafold2 and ColabFold software [40,81]. (C) The expression of FonR3SE1 in the vegetative growth stage (0 dpi) and in planta (1, 3, 6 dpi). (D) Greenhouse bioassays for the functional study of FonR3SE1 were repeated independently three times, with eight to ten plants for each treatment. One representative experiment was shown here. Left lane: colony of WT FonR3, the FonR3SE1 mutant, and its complemented strain on PDA plates at 5 dpi; Right lane: Infected PKR6 plants with mock (water) and the corresponding strains from the left at 28 dpi using 11-day-old seedlings. (E) Disease index quantification of infected PKR6 seedlings from 7 to 28 dpi. Disease index was evaluated based on a 7-scale rating: 0 = asymptomatic, 1 = slight stunted growth and yellowing, 3 = stunted growth and yellowing, 5 = wilting, 7 = dead. Wilcoxon rank-sum test between 28 dpi disease indices of WT and mutants was applied. p-value < 10-4 is denoted as ****. (F) Fresh weights of the above-ground infected plants at 28 dpi. Different letters above the bars represented significant differences between treatments using ANOVA analysis followed by Duncan’s multiple range test (p = 0.05).
Fig 6.
A schematic diagram summarizing the evolution of virulence in F. oxysporum f. sp. niveum (Fon).
Core (gray) and accessory (yellow) chromosomes/regions of FonR1, FonR2, and FonR3, and the accessory genome expression patterns with the enriched GO terms in infection on the susceptible G42 cultivar are shown. While FonR1 and FonR2 cannot infect the resistant cultivar PKR6, FonR3 can infect it and cause wilt symptoms through the FonR3SE1 effector. This image was created by Biorender under an Academic Individual License.