Article Figures & Data
Figures
- FIG 1
Phylogenetic relationship of ISS-F3/F4 among other F. oxysporum isolates. (A) The phylogenetic tree constructed from EF-1α sequences of 62 F. oxysporum isolates places ISS-F3/F4 in clade 3, belonging to a clonal lineage shared by lycopersici_016, lycopersici_072, lycopersici_075, and Fo47. Other Fusarium species (bottom) were included to root the tree. The bootstrap confidence (in percent; 1,000 replicates performed) is shown in red numbers in the tree. (B) Presence/absence profiles of ISS-F3/F4 with 62 other strains based on 104 candidate effector proteins. Colored boxes indicate the presence of a particular effector gene, with gray indicating its absence in the genome. The list of effector genes is shown in Data Set S4. Formae speciales that cause disease in the same host cluster together, as seen by the groupings of green (cucurbit-infecting strains), red (tomato-infecting strains), and yellow (banana-infecting strains). Dark gray represents non-plant-pathogenic isolates, and pink represents strains isolated from or flown to the ISS. Blue represents other plant pathogens. ISS-F3/F4 contained relatively few effector genes compared to the number found in the plant pathogens (green, red, yellow, and blue) and showed the most similar pattern (number and type) to Fo47, a non-plant-pathogenic soil isolate with a biocontrol function.
- FIG 2
Single nucleotide polymorphism (SNP) analysis comparing ISS-F3/F4 to the reference genome Fo47. (A) Maximum likelihood tree constructed from SNPs found in the coding regions of the genomes of ISS-F3, ISS-F4, and 8 other F. oxysporum strains (see Table S3 in Data Set S5 for reasons for inclusion in analysis). ISS-F3/F4 form their own clonal lineage distinct from Fo47 and the other strains. A similar tree was inferred when all SNPs across the genome were analyzed. (B) The frequency of SNPs across the entire genome (black), the noncoding region of the genome (purple), and the coding region of the genome (yellow) is summarized in the bar graph. Fol016 and 26381, well-defined strains, known to be different than Fo47, had a lower frequency of SNPs than ISS-F3/F4.
- FIG 3
Summary of gene ontology (GO) annotation of the predicted genes in ISS-F3/F4. Predicted genes in ISS-F3/F4 were predicted using AUGUSTUS and then annotated using Blast2GO. Functions could be assigned to 10,789 of the 16,648 genes in ISS-F3 and 11,305 of the 16,729 genes in ISS-F4. (A) Bar charts summarizing the most abundant GO terms for ISS-F3. BP, biological processes; MF, metabolic function; CC, cellular components. (B) Bar charts summarizing the abundance of enzyme classes in ISS-F3. NB, ISS-F3/F4 had the same distributions of GO and enzyme class abundances, and thus, only ISS-F3 is shown for simplicity. The distributions in ISS-F3/F4 were similar to Fo47 (non-plant-pathogenic soil isolate) and FOSC-3a (clinical isolate) (data not shown).
- FIG 4
Annotation of protein domains and protein families with InterProScan. Protein families were classified and domains were predicted using InterProScan. The heat map shows the relative abundances of the top 100 most abundant domains (A) and families (B) in ISS-F3/F4, in addition to a non-plant-pathogenic soil isolate (Fo47) and a clinical isolate, isolated from a patient with fusariosis (FOSC-3a). Red represents high relative abundances, and green represents low relative abundances.
- FIG 5
Presence/absence profile of polyketide synthases (PKSs) detected in the genomes of ISS-F3/F4. The sequences of predicted genes that had a ketoacyl synthase domain (one of 3 essential domains in polyketide synthases [PKSs]) were compared with BLAST against a PKS database (35) to determine which PKSs ISS-F3/F4 had the ability to produce. Eleven PKSs found in ISS-F3/F4 are also present in 12 other F. oxysporum species that have been previously studied (35). However, there were two additional PKSs, unkA and unkB, in ISS-F3/F4 that did not have any matches in the database. The first column represents the PKS, the last column shows the polyketide that the PKS makes (if known), and the middle columns indicate the F. oxysporum strains. Colored boxes indicate the presence of a specific PKS in the genome. FoZG, Fo47; FoYG, FOSC-3a; FoXG, lycopersici 4287; FoXB, Fo5176; FoQG, raphani NRRL 54005; FoTG, vasinfectum NRRL 25433; FoMG, melonis NRRL 26406; FoIG, cubense tropical race 4; FoCG, radicis-lycopersici 26381; FoWG, MN25; FoVG, pisi (HDV 247); FoPG, conglutinans race 2 NRRL 54008. Amino acid sequences for the unknown PKSs in ISS-F3 can be found in Data Set S1 (g14942 and g11694), and those for ISS-F4 can be found in Data Set S2 (g15849 and g12099). NB, the sequences are identical between ISS-F3/F4.
- FIG 6
Virulence of Fusarium oxysporum strains in C. elegans AU37 model. (A) Kaplan-Meier survival curves to determine total death of worms caused by F. oxysporum strains (n = 90 worms per fungal strain, results accumulated from 3 different experiments, 3 biological replicates per experiment, 10 worms per replicate). P < 0.0001 for F4 and F4antib compared to all other strains. (B) Kaplan-Meier survival curves to determine the hypha-related death of worms (n = 90 worms per fungal strain, results accumulated from 3 different experiments, 3 biological replicates per experiment, 10 worms per replicate). P = 0.0129 for F4 and F4antib compared to all other strains. (C and D) Comparison of hypha-related and total deaths of C. elegans caused by ISS-F4 (C) and ISS-F3 (D). Total deaths of C. elegans caused by ISS-F4 were significantly higher than the deaths associated with hyphae piercing through the body (P = 0.0102, n = 90, log rank [Mantel-Cox] test). There is no statistical difference between total and hypha-related deaths caused by ISS-F3 (P = 0.2465, n = 90). NB, 293sp is the IMV00293 strain that was isolated from Chernobyl and grown on the ISS for 12 days, and 293gr is IMV00293 that was concomitantly grown for 12 days on Earth.
- FIG 7
Images of C. elegans AU37 with hyphae of F. oxysporum ISS-F4 piercing through worm body. Microscopic images taken at 22 h (A) and 46 h (B) after coincubation of C. elegans with ISS-F4 conidia. Solid arrows point out the hyphae piercing through the worm body. Dotted arrows show growing extended hyphae that initially penetrated from the intestinal tube. The images were taken with the Olympus SZX16 microscope (×5 magnification).
Tables
- TABLE 1
General assembly statistics for the draft genomes of Fusarium oxysporum ISS-F3/F4 strains
Isolate Read count
(million)k-mer size N50 (bp) No. of scaffolds
(total)No. of scaffolds
over 1 kbMax scaffold
length (Mb)Genome
size (Mb)GC content (%) Coverage (×) ISS-F3 48 86 923,684 6,166 736 3.24 53.6 47.05 90 ISS-F4 42 86 940,233 7,066 948 2.23 53.8 46.88 80
TABLE S2
F. oxysporum k-mer database. The sequences shown represent the 50-bp k-mer sequences that are unique to each strain and not present in the other 64 strains examined. Download Table S2, TXT file, 0.05 MB.
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FIG S1
GO annotation of F. oxysporum ISS-F3/F4 strains. Bar graphs showing the 50 most abundant GO terms for ISS-F3 (A) and ISS-F4 (B) for each of the three categories: biological processes (BP), top; metabolic function (MF), middle; and cellular components (CC), bottom. Download FIG S1, PDF file, 0.1 MB.
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DATA SET S1
Summary of BLAST results, gene ontology (GO), and InterProScan (IPS) annotations for ISS-F3. Download Data Set S1, XLSX file, 9.5 MB.
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DATA SET S2
Summary of BLAST results, gene ontology (GO), and InterProScan (IPS) annotations for ISS-F4. Download Data Set S2, XLSX file, 9.6 MB.
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FIG S2
Annotation of protein domains and protein families with InterProScan. Protein families were classified and domains predicted using InterProScan. Heat maps in panels A and B capture the presence/absence profiles of all 2,468 domains and 3,061 families detected in the 4 strains. Gray bars indicate that a specific domain (A) or family (B) was absent in a specific genome. Of the family and domains that are present, the color gradient shows the relative abundances within the genome, with light red being the most relatively abundant and light green being the least relatively abundant. Download FIG S2, PDF file, 0.2 MB.
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DATA SET S3
Summary of relative abundances of protein families (A) and protein domains (B) in the genomes of four F. oxysporum strains. Download Data Set S3, XLSX file, 0.2 MB.
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DATA SET S4
List of effector genes used in Fig. 1B. Download Data Set S4, XLSX file, 0.01 MB.
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DATA SET S5
Table S1 is a summary of 65 genomes downloaded from GenBank that were used to generate F. oxysporum strain-specific k-mers (ftp://ftp.ncbi.nlm.nih.gov/genomes/genbank/fungi/Fusarium_oxysporum/). Table S3 is a list of F. oxysporum strains in addition to ISS-F3 and ISS-F4 that were used in SNP analysis. The “Reason” column lists the reasons for including these strains for comparison. Download Data Set S5, XLSX file, 0.01 MB.
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FIG S3
A comparison of biosynthetic gene clusters in F. oxysporum strains. Biosynthetic gene clusters, and thus the ability to produce secondary metabolites, were analyzed in ISS-F3/F4 using antiSMASH. Included for comparison were the biosynthetic gene clusters in Fo47 (a non-plant-pathogenic soil isolate) and FOSC-3a (a clinical isolate). The boxes with numbers listed to the right of the strain name indicate the number of clusters in the genome for that specific type (i.e., for T1pks there were 7 clusters in strain ISS-F3). The letters indicate the name of the compound produced or the name of the polyketide synthase (PKS) or nonribosomal peptide synthase (NRPS). Download FIG S3, PDF file, 0.2 MB.
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FIG S4
Sequence comparison of radiation resistance genes among 69 F. oxysporum strains. An inferred maximum likelihood tree was constructed from concatenated sequences of rad54, rad53, rad51, rad2, and phr1 (a photolyase), genes that have been shown to play a role in radiation resistance in fungi. Fusarium proliferatum and Fusarium fujikuroi were used to root the tree. Since ISS-F3, ISS-F4, IMV00293, VEG-01C1, and VEG-01C2 were all cultured from radiation-rich environments, it was hypothesized that they would share similar sequences in these genes, distinct from the other 65 F. oxysporum strains. However, as the tree shows, this was not the case. Download FIG S4, PDF file, 0.05 MB.
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