Hos2 directly binds to mating genes.

<p>(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, <i>p</i><0.05). All pairwise comparisons involving Hos2-HA3 at <i>bE1</i> locus were statistically significant (Duncan’s new multiple range test, <i>p</i><0.05), except when compared to <i>pra1</i> or <i>01779</i>. The same was true for comparisons involving Hos2-HA3 at locus <i>01779</i>. (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, <i>p</i><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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005134#ppat.1005134.g007" target="_blank">Fig 7</a> (note the Log₁₀ in <i>y</i> axis of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005134#ppat.1005134.g007" target="_blank">Fig 7</a>).</p

Hos2 is important for pathogenicity.

<p>(A) Phylogenetic tree of class I and II HDACs in <i>Saccharomyces cerevisiae</i> (Sc), <i>Schizosaccharomyces pombe</i> (Sp), <i>Candida albicans</i> (Ca) and <i>Ustilago maydis</i> (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 <i>p</i><0.0001 and <i>p</i><0.01 respectively, Mann-Whitney test) (C) Representative images of plants with infected wild-type or Δ<i>hos2</i> 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.</p

<i>b</i> gene induction rescues Δ<i>hos2</i> filamentation defects.

<p>(A) The filament forming capacity of AB31 and AB31Δ<i>hos2</i> strains. Expression of a compatible bE/bW heterodimer under the control of the <i>crg1</i> 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 <i>b</i> gene induction. Mean values and SDs from three independent experiments are shown. The total number of filaments counted is indicated above each column. <i>ns</i> denotes not statistically significant difference (t-test, <i>p</i>>0.05) (C) <i>bE1</i> expression levels, relative to the constitutively expressed <i>ppi</i> gene, in AB31 and AB31Δ<i>hos2</i> 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 (<i>ns</i>) differences are shown (Duncan’s new multiple range test, <i>p</i><0.05) (D) Constitutive expression of <i>b</i> genes restores the filamentation defect of SG200Δ<i>hos2</i> mutants. The filamentation phenotypes of HA103 and HA103Δ<i>hos2</i> strains grown on PD charcoal plates for 24h. SG200 and SG200Δ<i>hos2</i> strains were used as controls. (E) <i>bE1</i> expression levels in SG200 and SG200Δ<i>hos2</i> strains grown on CM charcoal plates for 24 hours. Mean <i>bE1</i> expression values relative to <i>ppi</i> 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 (SG200Δ<i>hos2</i>) that is assigned a value of 1. <i>t</i>-test retrieved a statistically significant difference in <i>bE1</i> expression level between SG200 and SG200Δ<i>hos2</i>, with <i>p</i><0.0001 (denoted by ****).</p

Hos2 is required for cAMP-induced expression of mating-type genes.

<p>(A-C) Expression level of the indicated genes relative to <i>ppi</i>. 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 (<i>ns</i>) differences are shown (Duncan’s new multiple range test, <i>p</i><0.05).</p

Hos2 is required for mating.

<p>(A) Light microscopy images of the indicated HDAC <i>Ustilago maydis</i> 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 <i>p</i><0.0001). (C) Mating crosses between wild-type FB1 and FB2 or Δ<i>hos2</i> 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 <i>p</i><0.0001 (Mann-Whitney test). (F) Representative images of plants infected with SG200 or SG200Δ<i>hos2</i> as indicated. Scale bar = 1 cm.</p

Hos2 is required for conjugation tube formation upon pheromone stimulation.

<p>(A) Representative image of conjugation tube formation upon a2 pheromone stimulation in FB1 and FB1Δ<i>hos2</i> 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 Δ<i>hos2</i> 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 FB1Δ<i>hos2</i>, with <i>p</i><0.0001 (showed as ****). (C) <i>mfa1</i> expression levels relative to <i>ppi</i>. 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 (SG200Δ<i>hos2</i>) that is assigned a value of 1. An asterisk denotes a statistically significant difference with a <i>p</i> < 0.05 (t-test).</p

Hos2 is not downstream of the pheromone responsive Fuz7 MAPK cascade.

<p>(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, <i>fuz7DD</i>, restores conjugation tube formation in Δ<i>hos2</i> mutants. <i>fuz7DD</i> 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 <i>fuz7DD</i>-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. <i>ns</i> denotes not statistically significant differences (t-test, <i>p</i>>0.05) (D-G) Expression levels of the indicated genes, relative to <i>ppi</i>, in FB1<i>Pcrg1fuz7DD</i> and the corresponding <i>hos2</i> mutant in glucose and arabinose containing CM media, 5 hours post <i>fuz7DD</i> 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 <i>p</i><0.05 (Duncan’s new multiple range test). <i>ns</i> denotes not statistically significant differences.</p

Hda1 and Hda2, are not redundant with Hos2 in the control of dimorphism and virulence.

<p>(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 <i>p</i>-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 <i>hos2</i> 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.</p

Expression of <i>fuz7DD</i> in the Δpmt4 strain partially restores the tumor induction in maize.

<p><b>A.</b> Disease progression in plant infections with the strains indicated. No infection symptoms were detected on plants inoculated with the SG200Δpmt4 strain. The SG200fuz7DDΔpmt4 induces anthocyanin production and small tumors formation. Bar represents 2 cm. <b>B.</b> Plants were infected with the indicated strains and symptoms were scored 12 days post-infection. <i>N</i> indicates the total number of plants evaluated in each case.</p

Model of the Pmt4 roles developed during <i>U. maydis</i> pathogenic development.

<p>Pmt4 has a major function in appressorium biology: Pmt4 O-mannosylates Msb2, a membrane-sensor that regulates appressorium development upstream of the MAP kinase cascade. The deletion of <i>pmt4</i> severely affects appressorium formation, probably due to a deficient glycosylation of Msb2. During appressorium-mediated penetration Pmt4 also probably acts above the MAP kinase cascade. Furthermore, Pmt4 plays a role <i>in planta</i> by O-mannosylation of proteins required for tumor formation.</p