Fig 1.
Transcription factor genes on the pathogenicity chromosome have homologs on the core genome and on other accessory chromosomes.
Schematic representation of the chromosomes of Fol and the position of transcription factor genes on the pathogenicity chromosome (chromosome 14) and their homologs on other chromosomes. All accessory regions are depicted in light grey and core chromosomes in dark grey. Note that small accessory regions are attached to core chromosomes 1 and 2. Grey ribbons indicate duplicated genomic regions. Transcription factor genes are indicated with colored bars, each transcription factor gene family is represented in a separate circos plot.
Fig 2.
Transcription factors encoded on accessory chromosomes of F. oxysporum have diverged more than core-encoded transcription factors between Fusarium species.
Phylogenetic tree of the Tf1 (Ftf1) family based on protein sequence alignments, including homologs in Fol, other F. oxysporum ff. spp. and other Fusarium species. The aTf1 homologs encoded on the pathogenicity chromosome in Fol are boxed, and the core homolog (cTf1) is written in italics. Cloned genes are indicated with aTF1 or cTF1. aTF1*: short version of aTF1, aTF1**: long version of aTF1. A grey bar indicates the clade with Fol, Fv, Fg and Fs core-encoded homologs. Phylogenetic trees of the other transcription factor families are presented in S1 Fig.
Fig 3.
DNA binding motifs are not diversified within transcription factor families, but some motifs are enriched on accessory chromosomes.
A) DNA binding motifs determined by DNA binding arrays for several accessory and core transcription factor pairs. Accessory transcription factors were cloned from the pathogenicity chromosome in all cases except aTF1; this transcription factor was cloned from another accessory chromosome. Of transcription factors with a reliable DNA binding site on one or two arrays, the top 8-mers for both arrays are given (in color: significant enriched binding [E-score > 0.45], in grey, E-score < 0.45). For each transcription factor: upper 8-mer: HK array, lower 8-mer: ME array. For transcription factors with significantly enriched binding to ten 8-mers or more, a motif was constructed of all 8-mers with significant binding (right hand side of the figure). B) Sge1 can transcriptionally activate an UAS-less CYC1 promoter via the presence of the Wor1 DNA binding site. Activation is lost when the Wor1 DNA binding site is absent or mutated (lower panel). Activation assays were performed in duplicate, error bars represent standard deviation. C) The number of genes with one or more transcription factor DNA binding site (TF DBS) in the promoter, for the complete genome, for a subgenome (core, accessory or pathogenicity chromosome), and for a subset of genes (up- or down-regulated during infection). Boxes indicate a significant enrichment (P value < 0.01 after Bonferroni correction). The motif found in effector promoters earlier (aacTGCCGa) and overlapping with the Tf1 DNA binding site has been included.
Fig 4.
Expression of aTF1 (FTF1), cTF1 (FTF2) or SGE1 from a constitutive promoter activates the SIX1 promoter.
Quantitative measurement of GFP fluorescence in strains expressing transcription factor genes from the FEM1 promoter. For each transcription factor fluorescence of 2*10^6 spores was measured in several independent transformants, as indicated with letters or numbers. Psix1 is the background strain (Fol007 with the Psix1GFP reporter construct).
Fig 5.
aTf1 and cTf1 (Ftf1 and Ftf2) are transcriptional activators.
aTf1 and cTf1 can transcriptionally activate an UAS-less CYC1 promoter via the presence of two Tf1 DNA binding sites. Activation is lost when the Tf1 DNA binding site is absent or mutated (lower panel). Activation assays were performed in duplicate, error bars represent the standard deviation.
Fig 6.
Fold induction of aTF1 (FTF1), cTF1 (FTF2) and SGE1 expression in overexpressors and during infection.
Fold induction of SGE1, aTF1 and cTF1 transcript abundance during Fol4287-infection (compared to axenic growth of Fol4287) and in their respective overexpressors compared to the background strain (Fol007 Psix1GFP). Red hued bars: aTF1 overexpressors, orange hued bars: cTF1 overexpressors, purple hued bars: SGE1 overexpressors. Blue bars: comparison between Fol4287 in planta and Fol4287 axenic cultures. The wild type strains Fol4287 and Fol007 are very similar [7,50]. One star (*): p<0.05, two stars (**): p<0.01. Note that to calculate fold change, all values of zero were replaced by 0.1 (half of the lowest value in the dataset). This includes the aTF1 levels in cTF1 and SGE1 overexpressors.
Fig 7.
Upstream regions of genes up-regulated in strains overexpressing aTF1 (FTF1), cTF1 (FTF2) or SGE1 are enriched for the respective DNA binding sites.
The number of genes with ≥1 or ≥3 transcription factor DNA binding sites (TF DBS) in the promoter region, for the complete genome, or for a subset of genes. DNA binding sites are i) the Tf1 DNA binding site (≥3 occurrences), ii) the motif found in the promoters of effector genes, overlapping with the Tf1 DNA binding site (≥1 occurrences) and iii) the Sge1 DNA binding site (≥1 occurrences). Subsets of genes are: up- or down-regulated during infection (in planta UP and in planta DOWN), up- or down-regulated in the aTF1, cTF1 or SGE1 overexpressor (aTF1 UP, cTF1 UP, SGE1 UP, aTF1 DOWN, cTF1 DOWN and SGE1 DOWN), genes that code for small secreted proteins, genes with a MIMP (miniature impala) in the promoter and genes that encode proteins that have been found in xylem sap of infected plants [34]. Boxes indicate a significant enrichment (P <0.01 after Bonferroni correction).
Fig 8.
Expression of aTF1 (FTF1), cTF1 (FTF2) or SGE1 from the FEM1 promoter induces many plant-responsive genes on the pathogenicity chromosome.
A) Heatmap of differentially expressed genes during infection or in aTF1, cTF1 or SGE1 overexpressors for the pathogenicity chromosome, other accessory regions and core genome. Displayed is the log2 fold change for each differentially expressed gene (rows), with yellow indicating up-regulation compared to the control (hues cover the log2 fold range from 0 to 5). Blue indicates down-regulation compared to the control (hues cover the log2 fold range from 0 to -5). The log2 fold change values of genes that are not significantly different from the control were set to zero. Each condition is a separate column, with two independent transformants (thus 2 columns) per transcription factor. The order of the rows reflects clustering of similar expression patterns. B) Enlarged heatmap of the pathogenicity chromosome, adorned with gene names: (putative) effector genes are shown in pink, transcription factor genes in blue (aTF1 17458 = FOXG_17458, aTF1 14275 = FOXG_14257, both the longer version of aTF1, aTF1 16414 = FOXG_16414, shorter version of aTF1), other genes are shown in black. SM-47 to SM-56: putative secondary metabolite gene cluster, FOXG_17447 to FOXG_17456.
Fig 9.
Induction of effector gene expression mediated by aTF1 (FTF1) overexpression is Sge1 dependent.
Relative expression as determined by Q-RT-PCR of SIX1 (A), SIX3 (B), SGE1 (C) and aTF1 (D) in WT, aTF1 overexpressor (aTF1 OE), aTF1 overexpressor transformed with a SGE1 deletion construct, integrated ectopically (aTF1 ectopic, two independent transformants), and aTF1 overexpressor with SGE1 deleted (aTF1 OE Δsge1, two independent transformants). Relative expression is calculated as gene expression/expression of EF1α. Error bars indicate standard deviation.