Figure 1.
Characteristics of Verticillium dahliae (Vd) and V. albo-atrum (Vaa) used in the comparative genomics analyses, showing hallmark morphological features of the fungi, and aspects of plant colonization.
A) The darkly pigmented microsclerotia of Vd; B) Dark resting mycelia of Vaa; C) Confocal laser scanning microscopy of a cross section of a spinach plant stem showing colonization of the vascular bundles in a ring-like arrangement with Vd (strain VdSo316) expressing green fluorescent protein (GFP) using filters set to detect autofluorescence and GFP; D) Confocal laser scanning microscopy of the same spinach stem shown in C, without filter to detect plant autofluorescence, and thus only GFP signal from the fungus is detectable; The confocal laser scanning microscopy was performed as previously described [103]; E) Lettuce plants sliced longitudinally through the crown and taproot, showing the vascular discoloration of the root and crown in the symptomatic plant on the right (arrow), but not in the asymptomatic plant on the left; F) Microsclerotia of Vd embedded in lettuce roots. Scale bar = A = 50 µm; B, C, D = 100 µm; F = approx. 1 mm.
Figure 2.
Phylogenetic relationships of the vascular wilt pathogens Verticillium dahliae, V. albo-atrum and Fusarium oxysporum, and other fungi relevant to this study.
The vascular wilt pathogens are ascomycetes and belong to different subclasses in the Sordariomycetes as indicated by vertical bars on the right. Aspergillus niger of the Eurotiomyctes is chosen as the outgroup.
Table 1.
Comparison of genome statistics between V. dahliae and V. albo-atrum.
Table 2.
Carbohydrate-Active Enzymes of Vd and Vaa in comparison to other sequenced ascomycete fungal genomes.
Figure 3.
Pectinolytic machinery in Verticillium dahliae, illustrating the enzymes that cleave homogalacturonan, xylogalacturonan and rhamnogalacturonan.
The expanded gene families in Verticillium genomes are highlighted in orange (see text and methods for details). *Enzymes of glycoside hydrolase (GH) families GH88 and GH105 act on the reaction products of polysaccharide lyase (PL) family members PL4 and PL11. The representation of the complex polysaccharidic constituents of pectins is adapted from [41].
Figure 4.
Global view of syntenic alignments between V. dahliae (Vd) and V. albo-atrum (Vaa) and the distribution of transposable elements and EST alignments.
Vd linkage groups (black bars) are shown as the reference, and the length of the light grey background to the left of each linkage group (in the scale of Mb) is defined by the Vd optical map. For each chromosome, column a represents the Vd genomic scaffolds positioned on the optical linkage groups separated by scaffold breaks. Scaffold numbers are adjacent to the blocks; column b displays the syntenic mapping of Vaa scaffolds; column c, color red shows the density of transposable elements calculated with a 10 kb window; and color black represents the AT-rich regions; column d represents the density of ESTs calculated with a 10 kb window. Four LS regions that lack similarity to the genome of Vaa but are enriched for TEs are highlighted in red ovals and numbered as LS1, 2, 3, and 4.
Table 3.
Predicted proteins present in Vd, Vaa and F. oxysporum but absent in F. solani, F. graminearum, and F. verticilliodes.
Figure 5.
A maximum-likelihood tree including four glucosyltransferase proteins from the plant pathogens V. dahliae (VDAG_02071.1), V. albo-atrum (VDBG_03162.1), Fusarium oxysporum f. sp. lycopersici (FOXG_02706.2), and the insect pathogen Metarhizium anisopliae ARSEF 23 (Genbank accession EFY99848), and their relationship to bacterial homologs.
The tree was constructed employing a maximum likelihood-based package, PhyML and the branch lengths in substitutions per site were calculated using the WAG evolutionary model (methods).
Figure 6.
Targeted knock-out of the putative glucan glucosyltransferase in Verticillium dahliae, strain VdLs.17, results in reduced fungal virulence on Nicotiana benthamiana.
The picture shows non-inoculated N. benthamiana plants (mock) and N. benthamiana plants inoculated with the VdLs.17 wild-type strain or two independent glucan glucosyltransferase gene (VDAG_02071) knock out strains (ΔGT-A and ΔGT-B), at 18 days postinoculation (dpi). The assay was performed three times with similar results.