Figure 1.
Phenotypic analysis of cluster 19A mutants.
A) Schematic representation of the cluster 19A region in the genome of U. maydis. Arrows indicate open reading frames (ORFs), labeled by the respective gene numbers. Related gene families are indicated in orange, yellow, blue, green and brown and the percentages of amino acid similarity are given below. Unique effector genes are shown in grey. Genes encoding proteins with a predicted secretion signal are depicted in orange, yellow, blue, green, brown or grey while open arrows indicate genes encoding proteins without a prediction for secretion. The left half of cluster 19A was designated as 19A-1 and the right half was designated 19A-2 as indicated. B) Biotrophic development of SG200 and SG200Δ19A. Maize seedlings were infected with SG200 or SG200Δ19A and development was monitored microscopically. At 1 dpi penetrating hyphae on the leaf surface were visualized by calcofluor staining. Proliferating biotrophic hyphae were observed after chlorazole-black E staining at 13 dpi. At 30 dpi mature teliospores were observed indicated by yellow arrows. Bar = 100 µm. C) Macroscopic symptoms of representative leaves infected with SG200, SG200Δ19A, SG200Δ19A-1 and SG200Δ19A-2 at 12 dpi. Note the absence of anthocyanin pigment accumulation in infections with SG200Δ19A and SG200Δ19A-1. D) Quantification of fungal biomass by qPCR. Genomic DNA was extracted from the leaves infected with SG200, SG200Δ19A, SG200Δ19A-1 and SG200Δ19A-2 at 2 and 6 dpi and used for qPCR. Relative fungal biomass was calculated by the comparison between U. maydis peptidylprolyl isomerase gene (ppi) and Z. mays glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH). Error bars indicate standard deviation. ** p<0.01. p-values were calculated by Student's t-test.
Figure 2.
Expression data compilation for cluster 19A genes and presence of orthologous genes in other smut fungi.
Expression data for cluster 19A genes were compiled from the following sources: a) Gene expressed in young seedlings [14], b) Gene expressed in tassel [14], c) Gene expressed in tumor tissue [12], d) Gene down-regulated in response to b inactivation during biotrophic development [20] and nd) no expression detected. Orthologs in S. reilianum and U. hordei are listed, and in these columns ND indicates that no orthologs were detected at a cut-off of e-value 10e−10. The adopted color scheme corresponds to the scheme used in Figures 1, 3 and 4. Please note that the order of genes was changed in several cases and does not follow the order in cluster 19A (*).
Figure 3.
Mapping of effector genes responsible for the virulence phenotype of cluster 19A mutant.
A) Schematic representation of analyzed sub-deletions. The region of 19A-1 was divided into 4 parts, 19A-1b, 19A-1a, 19A-1c and 19A-1d according to the distribution of gene families. In the second half of the cluster (19A-2) only the 19A-2e region was deleted. B) Quantitative evaluation of virulence of cluster 19A mutants. The scoring scheme follows the one described by Kaemper et al. [12] with severity of virulence corresponding to the color intensity. The numbers on top of respective bars indicate the total number of infected plants. Results from at least 3 independent experiments are combined. Dots above respective panels indicate that at least one of symptoms (ligula swelling, small tumors, normal tumors, heavy tumors, stunted or dead) was significantly changed in the mutant relative to SG200 by Student's t-test. C) Quantification of infected plants showing anthocyanin accumulation. In the same experiments as in B) the percentage of plants showing anthocyanin accumulation was scored.
Figure 4.
Virulence of tin gene deletion mutants and complemented strains.
A) Schematic representation of the deleted tin genes in cluster 19A. tin1-1 to tin1-5 correspond to the 5 genes represented by 19A-1b, the other tin genes lie in region 19A-1c (tin2), region 19A-1d (tin3), and region 19A-2e (tin4 and tin5). B) Quantitative evaluation of virulence of tin gene deletion mutants and complemented strains. Virulence of the displayed deletion mutants and respective complementation strains (indicated as Δ and C, respectively below each bar) was determined as described in Figure 3B. C) Quantification of infected plants showing anthocyanin accumulation after infection with tin2 mutant and control strains. Plants were infected with the indicated strains and the percentage of plants showing anthocyanin accumulation was scored 12 dpi.
Figure 5.
Plant responses to cluster 19A mutant strains.
A) Hierarchical clustering visualizing the relative expression of the 1816 maize genes transcriptionally regulated 4 days after infection by U. maydis strain SG200Δ19A. X-axis depicts clustering of the microarray samples, with three biological replicates per strain. Y-axis shows clustering of the regulated maize transcripts based on the similarity of their expression patterns. B) Venn diagram showing numbers of maize transcripts differentially expressed 4 days after infection with SG200Δ19A (red circle) as compared to the combined deregulated transcripts of all individual tin mutants (green circle). For the tin mutants, all genes regulated in at least one of the mutant infections have been combined. Red numbers indicate induced expression; numbers of down-regulated transcripts are given in blue. C) Venn diagram showing maize transcripts differentially expressed 4 days after infection by the U. maydis mutants SG200Δ19A-1b (Δ19A-1b), SG200Δtin3 (Δtin3), SG200Δtin4 (Δtin4), SG200Δtin5 (Δtin5), respectively.
Figure 6.
Graphic representation of Gene Ontology terms.
Hierarchical presentation of Gene Ontology [43] terms showing molecular functions significantly enriched amongst upregulated maize transcripts in tin mutant infected tissue. Numbers give p-values for enrichment of the respective GO. p1: p-value of enrichment in upregulated genes after infection by SG200Δtin3 (pink boxes); p2: p-value of enrichment in upregulated maize genes after infection by SG200Δtin4 (green box); p3: p-value of enrichment in upregulated maize genes after infection by SG200Δtin5 (blue boxes). Box in yellow indicates significant enrichment in samples p1 and p2; box in dark pink indicates significant enrichment in p1 and p3; boxes in turquoise indicate significant enrichment in p2 and p3 and darker color symbolizes more significant enrichment. Red arrows indicate hierarchical connections between (significantly enriched) Gene Ontology terms. Dotted arrows represent GO terms not enriched and therefore spared out from the figure.