Fig 1.
Hos2 is important for pathogenicity.
(A) Phylogenetic tree of class I and II HDACs in Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Candida albicans (Ca) and Ustilago maydis (Um). The scale bar represents an evolutionary distance of 0.2 amino acid substitutions per site. (B) Quantification of plant symptoms infected with the indicated strains 14 days post-infection (dpi). Mean values of at least three independent experiments are shown. The total number of infected plants is indicated above each column. Statistically significant differences of each mutant cross compared to the wild-type cross are indicated (**** and ** denote a p<0.0001 and p<0.01 respectively, Mann-Whitney test) (C) Representative images of plants with infected wild-type or Δhos2 mutant strains 14 dpi. Scale bar = 1 cm. (D) Spore production, visualised by light microscopy, in the indicated HDAC mutants 21 dpi. Scale bar = 20 μm.
Fig 2.
(A) Light microscopy images of the indicated HDAC Ustilago maydis mutants in a FB1 background. Cells were grown in rich YEPSL media until exponential phase. (B) Quantification of the number of buds per cell in the indicated strains. Total number of cells counted for each strain is indicated above each pair of columns. Mean values and standard deviations (SDs) from three independent experiments are shown. Asterisks indicate statistically significant differences compared to wild-type (Fisher’s exact test. **** denotes a p<0.0001). (C) Mating crosses between wild-type FB1 and FB2 or Δhos2 mutant strains grown on charcoal-containing PD (left, PD) or CM (right, CM) plates for 24 hours at 25°C. (D) Filamentation of the indicated solopathogenic strains grown on PD charcoal plates for 24 h at 25°C. (E) Quantification of symptoms in maize plants infected with the indicated strains at 14 dpi. Mean values of three independent experiments are shown. The total number of infected plants is indicated above each column. **** indicates statistically significant difference with p<0.0001 (Mann-Whitney test). (F) Representative images of plants infected with SG200 or SG200Δhos2 as indicated. Scale bar = 1 cm.
Fig 3.
b gene induction rescues Δhos2 filamentation defects.
(A) The filament forming capacity of AB31 and AB31Δhos2 strains. Expression of a compatible bE/bW heterodimer under the control of the crg1 promoter was induced by shifting from glucose to arabinose containing CM media. Filaments were observed 8 hours after induction. (B) Quantification of the filamentation phenotype of the indicated strains 8 hours after b gene induction. Mean values and SDs from three independent experiments are shown. The total number of filaments counted is indicated above each column. ns denotes not statistically significant difference (t-test, p>0.05) (C) bE1 expression levels, relative to the constitutively expressed ppi gene, in AB31 and AB31Δhos2 strains growing in CMD or CMA 8 hours post-induction. Mean values and SDs from three independent experiments, each containing three technical replicates, are shown. Relative values are shown normalised to one of the biological replicates of the sample with the lowest expression level (AB31/CMD in this case) that is assigned a value of 1. Statistically significant (*) and not significant (ns) differences are shown (Duncan’s new multiple range test, p<0.05) (D) Constitutive expression of b genes restores the filamentation defect of SG200Δhos2 mutants. The filamentation phenotypes of HA103 and HA103Δhos2 strains grown on PD charcoal plates for 24h. SG200 and SG200Δhos2 strains were used as controls. (E) bE1 expression levels in SG200 and SG200Δhos2 strains grown on CM charcoal plates for 24 hours. Mean bE1 expression values relative to ppi and SDs from three independent experiments, each containing three technical replicates, are shown. Values are normalised to one of the biological replicates of the sample with the lowest expression value (SG200Δhos2) that is assigned a value of 1. t-test retrieved a statistically significant difference in bE1 expression level between SG200 and SG200Δhos2, with p<0.0001 (denoted by ****).
Fig 4.
Hos2 is required for conjugation tube formation upon pheromone stimulation.
(A) Representative image of conjugation tube formation upon a2 pheromone stimulation in FB1 and FB1Δhos2 strains. Images were taken 5 hours post-pheromone addition. The pheromone solvent, DMSO, was used instead of pheromone as a negative control. (B) Quantification of the pheromone defective phenotype of Δhos2 cells 5 hours post-pheromone stimulation. Mean values and SDs from three independent experiments are shown. The total number of cells counted is indicated above each column. t-test retrieved a statistically significant difference in conjugation tube formation between the FB1 and FB1Δhos2, with p<0.0001 (showed as ****). (C) mfa1 expression levels relative to ppi. Three independent experiments, each with three technical replicates were performed. Mean values and SDs for these experiments are shown. Values are normalised to one of the biological replicates of the sample with the lowest expression level (SG200Δhos2) that is assigned a value of 1. An asterisk denotes a statistically significant difference with a p < 0.05 (t-test).
Fig 5.
Hos2 is not downstream of the pheromone responsive Fuz7 MAPK cascade.
(A) Schematic representation of the pheromone signalling pathway via the MAP kinase cascade. The events that following pheromone-receptor recognition are numbered. (B) Induced expression of the constitutively active Fuz7 MAPK kinase allele, fuz7DD, restores conjugation tube formation in Δhos2 mutants. fuz7DD induction was performed by shifting from glucose (non-inducing) to arabinose (inducing) containing CM media. Images shown were taken 5 hours after induction. (C) Quantification of fuz7DD-induced conjugation tube formation in the indicated strains 5 hours post-induction. Mean values and SDs from three independent experiments are shown. The total number of cells counted is indicated above each column. ns denotes not statistically significant differences (t-test, p>0.05) (D-G) Expression levels of the indicated genes, relative to ppi, in FB1Pcrg1fuz7DD and the corresponding hos2 mutant in glucose and arabinose containing CM media, 5 hours post fuz7DD induction. Mean values and SDs from three independent experiments, each with three technical replicates, are shown. Values are normalised to one biological replicate of the sample with the lowest expression value, that is assigned a value of 1. One asterisk denotes p<0.05 (Duncan’s new multiple range test). ns denotes not statistically significant differences.
Fig 6.
Hos2 controls virulence independently of Tup1.
(A) Filamentation phenotypes of the SG200 strain and its derivative single and double mutants for Δhos2 and Δtup1 on PD charcoal plates. (B) Quantification of the virulence phenotype of the indicated strains 14 dpi. Mean values for three independent experiments are shown. The total number of infected plants is indicated above each column. **** means a statistical significant difference with p<0.0001 (Mann-Whitney test). (C) Tumours of maize plants induced by infections with the indicated strains 14 dpi. Scale bar = 1 cm.
Fig 7.
Hos2 is required for cAMP-induced expression of mating-type genes.
(A-C) Expression level of the indicated genes relative to ppi. Strains were grown in PD broth for 8 hours with (+) or without (−) 6 mM cAMP. Mean values and SDs from three independent experiments, each containing three technical replicates, are shown. Values are normalised to one of the biological replicates of the sample with the lowest expression value that is assigned a value of 1. Statistically significant (*) and not significant (ns) differences are shown (Duncan’s new multiple range test, p<0.05).
Fig 8.
Hos2 directly binds to mating genes.
(A) ChIP analysis using an anti-HA antibody on chromatin extracts from either an untagged strain or a Hos2-HA3 strain, grown in PD. Inmunoprecipitated DNA was analysed by qPCR, amplifying open reading frames or specific regions within the ORFs (5’ or 3’) indicated below the graph. Values correspond to the amount of DNA recovered in the HA IP divided by the amount of DNA in the corresponding input extract. Mean values and SDs from four independent experiments, each with three technical replicates are shown. Statistically significant binding of Hos2-HA3 (red) to each locus by comparison to the untagged (blue) strain is indicated with * (Duncan’s new multiple range test, p<0.05). All pairwise comparisons involving Hos2-HA3 at bE1 locus were statistically significant (Duncan’s new multiple range test, p<0.05), except when compared to pra1 or 01779. The same was true for comparisons involving Hos2-HA3 at locus 01779. (B) ChIP analysis was performed and analysed as in (A), except that strains were grown in PD with or without the addition of 6 mM cAMP for 8 hours. For simplicity, values for the untagged strains are not shown, but were identical to those shown in A and did not vary upon cAMP addition. Statistically significant differences regarding the effect of cAMP addition in Hos2 binding to each locus are denoted with * (Duncan’s new multiple range test, p<0.05). Numbers in green indicate the fold expression increase in of the corresponding gene upon cAMP addition, as measured by RT-qPCR and shown in Fig 7 (note the Log10 in y axis of Fig 7).
Fig 9.
Hda1 and Hda2, are not redundant with Hos2 in the control of dimorphism and virulence.
(A) Quantification of infection symptoms caused by the indicated strains on maize plants 14 dpi. Mean values of three independent experiments are shown. Total number of infected plants are indicated above each column. Statistically significant differences are indicated with asterisks (Mann-Whitney test; the number of asterisks are used for the following p-values: * for p<0.05, ** for p<0.01, *** for p<0.001 and **** for p<0.0001. Asterisks placed above each column and bellow the total number of infected plants correspond to comparisons with the SG200 wild-type strain. (B) Representative image of tumours induced by wild-type and hos2 mutant strains 14 dpi. Scale bar = 1 cm. (C) Filamentation on PD charcoal plates of the indicated strains after 48 hours incubation at 25°C.
Fig 10.
Hos2 and Clr3 genetically interact in the control of virulence.
(A) Filamentation on PD charcoal plates of SG200 and its derivative mutants for hos2 and clr3 growing for 24 hours at 25°C. (B) Quantification of the virulence capacity of the indicated strains on maize plants 14 dpi. Mean values of three independent experiments are shown. Total number of infected plants are indicated above each column. All pairwise comparisons are statistically significant (**** denotes p<0.0001, Mann-Whitney test), except when comparing Δhos2 with Δclr3 single mutants. (C) Images of tumours induced by the indicated strains 14 dpi. (D) Expression of bE1 relative to ppi in the indicated strains. Mean values and SDs from three independent experiments, each containing three technical replicates, are shown. Values are normalised to the sample with the lowest expression value (SG200Δhos2Δclr3) that is assigned a value of 1. Data for wild-type and Δhos2 mutants are shown in Fig 3. (E-G) Expression level of the indicated genes relative to ppi. Mean values and SDs from three independent experiments, each containing three technical replicates, are shown. Values are normalised to one biological replicate of the sample with the lowest expression value (FB1Δhos2Δclr3 without cAMP) that is assigned a value of 1. Statistically significant (*) or not significant (ns) differences for pairwise comparisons involving clr3 and hos2-clr3 genetic interaction are indicated (Duncan’s multiple range test, p<0.05). Data and statistical analysis for wild-type and Δhos2 mutant are shown in Fig 7.