BcLTF1 is required for full virulence.

<p>(<b>A</b>) Overexpression of <i>bcltf1</i> affects the formation of infection structures. Onion epidermal strips were inoculated with conidial suspensions (on the left) or non-sporulating mycelia (on the right), and incubated for 24 h in DD. Conidia and hyphae on the surface were stained with lactophenol blue whereas invasively growing hyphae remain colorless. Scale bars, 50 µm. (<b>B</b>) Deletion of <i>bcltf1</i> impairs the capability to colonize primary leaves of <i>P. vulgaris</i>. Leaves of living plants were inoculated with conidial suspensions and incubated in LD. White scale bars, 1 cm; black scale bars, 2 mm. (<b>C</b>) Spreading lesions provoked by Δ<i>bcltf1</i> mutants are characterized by increased H₂O₂ accumulation. Primary leaves were inoculated with conidial suspensions, detached 1 to 4 dpi, and incubated for 2 h in DAB solution. Plant tissues were decolorized prior to microscopy. (<b>D</b>) Addition of ascorbic acid restores virulence of Δ<i>bcltf1</i> mutants. Conidia were suspended in GB5 (gray) or in GB5 supplemented with 5 g/l ascorbic acid (black). Mean values and standard deviations of lesion diameters were calculated from eight lesions per strain and condition at 3 dpi. Asterisks indicate significant differences compared to WT:B05.10 in each condition (p<0.001). Pictures of lesions derived from ascorbic acid-supplemented conidial suspensions were taken 3 dpi.</p

Differentially expressed genes may encode regulators of light-dependent differentiation.

<p>Relative expressions of the genes (i.e. log2-normalized intensities scaled by gene) in the four conditions (16 hybridizations) were clustered and depicted by color scale, where shades of green and red represent under- and overexpressed genes, respectively. Homologues in <i>N. crassa</i> were identified by a bidirectional best-hit (BDBH) approach. Light-induced genes in <i>B. cinerea</i> and <i>N. crassa</i> are marked with orange asterisks. For more details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004040#pgen.1004040.s013" target="_blank">Table S3</a>. (<b>A</b>) Expression of most photoreceptor-encoding genes is similarly induced by light in wild type and Δ<i>bcltf1</i> strains. All predicted photoreceptors were clustered. (<b>B</b>) Expression levels of 45 TF-encoding genes are altered in response to light or deletion of <i>bcltf1</i>. TFs that were differentially expressed in at least one condition were subjected to clustering.</p

BcLTF1 modulates the transcriptional responses to light.

<p>Wild type B05.10 and the Δ<i>bcltf1</i> mutant were grown on solid CM for 52 h in DD; then, cultures were exposed for 60 min to light (+LP) or kept for additional 60 min in darkness (D). Four biological replicates were performed. RNA samples were labeled and hybridized to microarrays with all predicted genes of <i>B. cinerea</i>. Statistical analysis revealed 2,074 differentially expressed genes. Relative expressions of the genes (i.e. log2-normalized intensities scaled by gene) in the four conditions (16 hybridizations) were clustered, and 11 of the 14 theoretically possible expression profiles were found (for more details, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004040#s4" target="_blank">Materials and Methods</a>). This figure represents the centroid views of the 11 expression profiles (x = hybridization and y = log2-normalized intensity scaled by gene). The percentages among the 2,074 genes are indicated for each profile. Genes whose expression is only affected by the deletion of <i>bcltf1</i> belong to profiles 1 (748 genes) and 2 (791 genes). Profiles 3 (94 genes) and 4 (5 genes) comprise genes whose expression is affected by light in a similar manner in both genomic backgrounds (WT, Δ<i>bcltf1</i>). Genes (436) belonging to the other profiles are affected by light treatment and the deletion of <i>bcltf1</i>.</p

Functional categories that are enriched due to light treatment or deletion of BcLTF1.

<p>Gene sets were as previously defined by Amselem et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004040#pgen.1004040-Amselem1" target="_blank">[2]</a> or defined in the present study. GSEA (<a href="http://www.broadinstitute.org/gsea/" target="_blank">http://www.broadinstitute.org/gsea/</a>) toolkit was used to determine whether gene sets show statistically significant, concordant differences due either to light treatment or BcLTF1 deletion. Normalized Enrichment Scores (NES) and p-values (in brackets) are indicated for cases where p-values <0.05.</p

<i>B. cinerea</i> strains used in this study.

<p><i>B. cinerea</i> strains used in this study.</p

BcLTF1-GFP fusion proteins are localized in the nuclei.

<p>(<b>A</b>) Subcellular localization of BcLTF1-GFP does not depend on illumination conditions. The fusion protein was expressed from the constitutive <i>A. nidulans oliC</i> promoter in the Δ<i>bcltf1</i> background. Conidia were incubated on microscope slides for 18 h in DD (dark), or 12 h in DD followed by 6 h in LL (light). Nuclei were visualized using the fluorescent dye Hoechst 33342. Scale bars, 5 µm. (<b>B</b>) BcLTF1-GFP localizes to the numerous nuclei in infectious hyphae. Onion epidermis strips were inoculated with conidial suspensions and incubated for 24 h in DD. Conidia and hyphae on the surface were stained with the fluorescent dye calcofluor white (CFW). Sites of penetration are indicated by black arrows. Scale bars, 20 µm.</p

Photoresponses in <i>B. cinerea</i> are modulated by the light-responsive transcription factor BcLTF1.

<p>Light determines the mode of asexual reproduction (conidia vs. sclerotia), is required for differentiation of the fruiting bodies (apothecia) and is further considered to set the circadian clock. The cellular redox status is affected by emerging singlet oxygen and other ROS; compensation is achieved by induction of antioxidant systems including enzymes and carotenoids. <i>Bcltf1</i> represents one of the 244 light-induced genes, and its absence affects several light-dependent features such as the formation of reproductive structures. The deletion of BcLTF1 leads to an unbalanced ROS status (net overproduction of H₂O₂), possibly in the mitochondria, as suggested by the upregulation of the alternative respiration pathway. The altered ROS status is considered to cause the inability of the deletion mutant to tolerate additional oxidative stress that arises during illumination (singlet oxygen), exposure to ROS (H₂O₂, menadione) and during host infection (oxidative burst).</p

BcLTF1 is needed to cope with oxidative stress and regulates the generation of ROS.

<p>(<b>A</b>) Radial growth rates of Δ<i>bcltf1</i> mutants are affected by supplements. Strains were incubated in LL (white), LD (gray), or DD (black) on solid CM (control), and CM with 0.7 M NaCl, 0.7 M sorbitol, 300 µM menadione, 7.5 mM H₂O₂, 800 µM DTT (dithiothreitol) or 28 mM AA (ascorbic acid). Mean values and standard deviations were calculated from five colonies per strain and condition. Asterisks indicate significant differences compared to WT:B05.10 in each condition (p<0.001). (<b>B</b>) Salt and oxidative stress prevent growth of Δ<i>bcltf1</i> mutants even in the absence of light. Pictures were taken after 2 d in DD. (<b>C</b>) Ascorbic acid and DTT restore light tolerance but not the proliferation of aerial hyphae. Pictures were taken after 2 d in LL. Hardly visible Δ<i>bcltf1</i> colonies are highlighted by dashed lines. (<b>D</b>) <i>Bcltf1</i> mutants are affected in generation of H₂O₂. Strains were incubated for 2 d in DD on CM or CM supplemented with 750 µM DTT. Then, colonies were flooded with DAB solution. DTT prevents oxidation of DAB in the range of wild-type colonies but not in the range of Δ<i>bcltf1</i> colonies (on the left). Identical quantities of fresh mycelia of B05.10, Δ<i>bcltf1</i> (A6, B1) and OE::<i>bcltf1</i> (T6, T7) were incubated in DAB solution (on the right). (<b>E</b>) The cytosolic glutathione redox potential is not significantly affected in the absence of <i>bcltf1</i>. Reactions of redox-sensitive reporter roGFP2 expressed in Δ<i>bcltf1</i> and wild type were compared. Reduction of roGFP2 is displayed after an oxidation event provoked by addition of 10 mM H₂O₂ after 2 min. Indicated values are the means of triplicates; standard deviations are shown. For details, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004040#s4" target="_blank">Materials and Methods</a>.</p

Mutations of <i>bcltf1</i> alter light-dependent growth and differentiation programs.

<p>(<b>A</b>) BcLTF1 is essential for allowing growth on minimal medium. Indicated strains were incubated for 2 d on solid CD. Scale bar, 1 cm. (<b>B</b>) Exposure to light negatively affects radial growth rates of Δ<i>bcltf1</i> mutants on synthetic complete medium. Strains were incubated on solid CM in LL (white bars), LD (gray bars), and DD (black bars). Colony diameters (growth in cm) were determined after 3 d of incubation. Mean values and standard deviations were calculated from five colonies per strain and light condition. Asterisks indicate significant differences compared to WT:B05.10 in each condition (p<0.001). (<b>C</b>) Both overexpression and deletion of BcLTF1 modulate the differentiation program in DD. Strains were incubated for 14 d on solid CM. Close-up views of the cultures incubated in DD are shown in the lower panel. Scale bar, 5 mm.</p

Impact of BcVEL1 on asexual and sexual reproduction and conidial germination.

<p>(A) Numbers of conidia (per Petri dish) produced by the strains in light-dark (LD) conditions and in continuous darkness (DD), respectively. Data represent mean values of the results from three Petri dishes per strain and condition. (B) Numbers of sclerotia (per Petri dish) produced by the strains in continuous darkness. Data represent mean values of the results from three Petri dishes per strain and condition. (C) Apothecia derived from crossing of sclerotia of strain SAS405 (<i>MAT1-2</i>) with microconidia of mutant B05.10:Δ<i>bcvel1</i> (<i>MAT1-1</i>). Picture was taken after 14 weeks of joining sclerotia and microconidia. (D) Germination on hydrophobic surfaces. Conidia were suspended in water and incubated for 24 h on polypropylene. The experiments were done in triplicates, each time hundred conidia were counted. Short germ tubes do not exceed the length of the conidia while long germ tubes do. Scale bar represents 20 µm.</p