Figure 1.
Venn diagram illustrating overlaps between numbers of genes that are differentially regulated in response to plant surface-cues.
A. Overlaps of up-regulated (red) and down-regulated (blue) genes during differentiation from budding cells to filaments (HS vs GC), budding cells to appressoria (HS+FA vs GC) and filaments to appressoria (HS+FA vs HS). B. Overlaps of up-regulated genes (red) during differentiation from budding cells to filaments and appressoria (HS vs GC and HS+FA vs GC, respectively) and down-regulated genes (blue) in AM1Δsho1Δmsb2 (DSM) compared to AM1 (DSM vs AM1), both incubated on HS+FA. HS: Hydrophobic surface, FA: Fatty acid, GC: Glass control.
Figure 2.
Genes encoding secreted proteins are transcriptionally induced by hydrophobicity and hydroxy fatty acids.
A. Expression of genes encoding putative secreted proteins is significantly enriched during appressorium formation. For each gene set the number of genes encoding proteins predicted to be secreted is compared to the expected number of genes predicted from the whole genome sequence. *, ** and *** denote p-values (hypergeometric distribution) of p<0.01, p<0.001 and p<0.0001, respectively. B. The heat-map depicts the expression of genes encoding putative secreted proteins that were found to be up-regulated in at least one of the following comparisons: HS vs GC, HS+FA vs HS and HS+FA vs GC. Expression is visualized for the AM1 strain (GC, HS, HS+FA) and the AM11Δsho1Δmsb2 (DSM) strain (HS, HS+FA). The order of genes was defined by hierarchical clustering [42] resulting in the classification of three major groups: Group A: HS-induced and predominantly FA-repressed. Group B: HS and FA-induced. Group C: HS and/or FA-induced, sho1/msb2 dependent. All genes are listed in Table S3. HS: Hydrophobic surface, FA: Fatty acid, GC: Glass control, DSM: AM1Δsho1Δmsb2.
Figure 3.
Arabinofuranosidases function in plant penetration and are needed for full virulence.
A. Disease symptoms caused by SG200, afg single mutants and afg triple mutants as well as derivates of the latter strain, complemented with either afg1, afg2 or afg3. The indicated strains were injected into maize seedlings and symptoms were scored 12 days after infection according to severity; the color code for each category is given below. Three independent experiments were carried out and the average values are expressed as a percentage of the total number of infected plants (n), which is given above each column. *Significant difference for each category and pair given above (p<0.05, student's t) ns: not significant. Tumor formation is significantly reduced in SG200Δ3afg and this defect can be partially complemented by introducing either afg1, afg2 or afg3. B. Filament formation. SG200 and SG200Δ3afg were spotted on PD charcoal plates and incubated for 24 h at 28°C. The white fuzzy colonies reflect the formation of b-dependent filaments. C. Appressorium formation. AM1 and AM1Δ3afg were sprayed on ParafilmM with 100 µM HDA and incubated for 18 h at 28°C. Hyphae were stained with calcofluor and the average percentage of cells that expressed the AM1 marker was determined relative to the cells that had formed filaments. In three independent experiments more than 400 cells per strain were analyzed and error bars indicate standard error. D. Penetration efficiency. The indicated strains were injected into maize seedlings and 20 h after inoculation the number of appressoria that have penetrated the epidermis relative to the total number of appressoria (n) was determined. Three independent experiments were conducted and the error bars denote standard error. *Significant difference (p<0.05, student's t).
Figure 4.
Sho1 and Msb2 regulate the expression of the appressorium-specific transcription factors biz1 and hdp2.
A. Schematic overview of the transcriptional b-network (left) and its regulation during appressorium formation (right). All components of the b-cascade are significantly up-regulated in response to plant surface cues (HS+FA vs GC). Only appressorium-specific biz1 and hdp2 genes are down-regulated in AM1Δsho1Δmsb2 mutants compared to the AM1 control strain (DSM vs AM1). *Significant difference (see Materials and Methods for details) HS: Hydrophobic surface, FA: Fatty acid, GC: Glass control B. Symptoms caused by AN1, AN1Δhdp2 and the complemented strain AN1Δhdp2/hdp2. Strains were injected into maize seedlings and symptoms were scored 12 days after infection according to severity; the color code for each category is given below. Three independent experiments were carried out and the average values are expressed as a percentage of the total number of infected plants (n), which is given above each column. C. Filament formation. The indicated strains were spotted on PD charcoal plates and incubated for 24 h at 28°C. The white fuzzy colonies reflect the formation of b-dependent filaments. D. Appressorium formation. AN1 and the indicated derivatives were sprayed on ParafilmM with 100 µM HDA and incubated for 18 h at 28°C. Hyphae were stained with calcofluor and GFP fluorescence was monitored. Scale bar represents 10 µm. E. Quantification of appressoria from the indicated strains using the same conditions as in D. Average percentage of cells that expressed the appressoria-specific GFP-reporter was determined relative to the cells that had formed filaments. In three independent experiments more than 400 cells were analyzed and error bars indicate standard error.