Additional File 2:

Table S5. Significantly enriched DAVID GO annotation clusters for three protein groups with different dN/dS estimates. (XLSX 17 kb

Additional File 1:

Figure S1, Supplementary Methods, Tables S1-S4. Additional methods and results of additional analyses as specified in the main text. (PDF 429 kb

The Ca²⁺/Calcineurin-Dependent Signaling Pathway in the Gray Mold <em>Botrytis cinerea</em>: The Role of Calcipressin in Modulating Calcineurin Activity

<div><p>In the gray mold fungus <em>Botrytis cinerea</em> the Gα subunit Bcg1 of a heterotrimeric G protein is an upstream activator of the Ca²⁺/calmodulin-dependent phosphatase calcineurin. In this study we focused on the functional characterization of the catalytic subunit of calcineurin (BcCnA) and its putative regulator calcipressin (BcRcn1). We deleted the genes encoding both proteins to examine their role concerning growth, differentiation and virulence. The Δ<em>bccnA</em> mutant shows a severe growth defect, does not produce conidia and is avirulent, while the loss of BcRcn1 caused retardation of hyphal growth and delayed infection of host plants, but had no impact on conidiation and sclerotia formation. Expression of several calcineurin-dependent genes and <em>bccnA</em> itself is positively affected by BcRcn1. Complementation of the Δ<em>bcrcn1</em> mutant with a GFP-BcRcn1 fusion construct revealed that BcRcn1 is localized in the cytoplasm and accumulates around the nuclei. Furthermore, we showed that BcCnA physically interacts with BcRcn1 and the regulatory subunit of calcineurin, BcCnB. We investigated the impact of several protein domains characteristic for modulation and activation of BcCnA via BcRcn1, such as the phosphorylation sites and the calcineurin-docking site, by physical interaction studies between BcCnA and wild-type and mutated copies of BcRcn1. Based on the observed phenotypes we conclude that BcRcn1 acts as a positive modulator of BcCnA and the Ca²⁺/calcineurin-mediated signal transduction in <em>B. cinerea</em>, and that both proteins regulate fungal development and virulence.</p> </div

Phenotypical characterization of <i>bccnA</i>^ΔAID and <i>bcrcn1</i> mutants.

<p>Analyzed strains: <i>B. cinerea</i> wild-type B05.10 (WT), <i>bccnA</i>^ΔAID mutant (expressing truncated version of <i>bccnA</i>), Δ<i>bcrcn1</i> mutant, Δ<i>bcrcn1</i> complementation strain (<i>bcrcn1</i>com). A: Growth on complete medium (CM) after 2 weeks in light/dark (12 h/12 h) regime for conidiogenesis and darkness (sclerotia formation). B: Pathogenicity assay: living bean leaves were inoculated with spore suspensions (2*10⁵ spores/ml). Pictures were taken 2–10 days post infection (dpi).</p

Characterization of calcipressin and calcineurin A interaction in <i>B. cinerea</i>. A:

<p>Alignments of the predicted CnA-docking motif and the putative Gsk3-binding domain in the BcRcn1 background (BcRcn1 protein: 255 aa). Mammalian and yeast as well as the synthetic peptide sequences derive from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041761#pone.0041761-Roy1" target="_blank">[45]</a>. Black letters/stars: highly conserved residues; dark grayish letters: amino acids with strongly similar properties; light gray letters: weakly similar properties. B: Phenotype of mutated strains: growth analyses with wild-type B05.10 (WT), Δ<i>bcrcn1</i>, Δ<i>bcrcn1</i> + <i>bcrcn1</i>mut^APPPA, Δ<i>bcrcn1</i> + <i>bcrcn1</i>mut^PVIVIT after 1 week on described media. Inoculation was made with agar plugs of equal size (5 mm). C: Yeast strain NMY51 was transformed with the bait vector containing the <i>bccnA</i> gene and different prey vectors: + control: pAI-Alg5, - control: pPR3-N, BcRcn1/BcCnB/BcRcn1mut^APPPA/BcRcn1mut^PVIVIT: pPR3-N_BcRcn1/_BcCnB/_BcRcn1mut^APPPA/_BcRcn1mut^PVIVIT. Strains were incubated in different concentrations (non-diluted, 1∶10, 1∶100, 1∶1000-diluted) on SD-L-W (selection for vectors) and on SD-L-W-H-adenin + X-Gal-plates (interaction of tested proteins). Percentage of growth was calculated by counting of colonies in comparison to number of colonies on SD-L-W. The test was performed three times.</p

Expression studies in the wild-type and the Δ<i>bcrcn1</i> mutant strain.

<p>The wild-type B05.10 and the Δ<i>bcrcn1</i> mutant were grown for 72 h in liquid culture (SISLER medium) and then moved to fresh cultures without (−) or with (+) 20 µg/ml of the calcineurin-inhibitor cyclosporine A (CsA). The northern blot was hybridized to radioactively labeled probes of several BcCnA/BcCrz1-dependent genes (CND-genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041761#pone.0041761-Viaud1" target="_blank">[30]</a>, CND5 =  <i>bcbot1</i> as a botrydial biosynthesis gene, <i>bcboa4</i> as a botcinic acid biosynthesis gene), <i>bccnA</i> and <i>bcrcn1</i>. Loading controls: <i>bcactA</i> and rRNA.</p

Phenotype of Δ<i>bccnA</i> mutants.

<p><b>A:</b> Colony growth on CM medium after 4 weeks post inoculation (LD: 12 h rhythm light/dark, D: constant darkness), respectively. Two different primary Δ<i>bccnA</i> mutants T1 and T2 were tested in comparison to the <i>B. cinerea</i> wild-type B05.10. <b>B:</b> Growth of Δ<i>bccnA</i> mutant after 6 weeks on CM medium. Arrows indicate sector formation and different morphologies within one colony (scale bar 2 mm). <b>C–D:</b> Hyphal morphology and nuclei staining of <i>B. cinerea</i> strains with reduced calcineurin activities. Wild-type conidia (WT) were incubated for 48 h in liquid CM medium with and without 10 µg/ml of the calcineurin inhibitor cyclosporine A (CsA). Δ<i>bccnA</i> mycelia were incubated for 3 to 4 weeks in liquid CM medium. Cell walls and septa were stained with the fluorescent dye Calcofluor White. Scale bar: 20 µm (C). Nuclei were visualized using the fluorescent dye Hoechst 33342 (for details see Materials and Methods). Scale bar: 50 µm (D). Wild-type conidia (WT) were incubated for 48 h in liquid CM medium with and without 10 µg/ml of the calcineurin inhibitor cyclosporine A (CsA). Δ<i>bccnA</i> mycelia were incubated for 4 weeks in liquid CM medium. E: Pathogenicity assay: living bean leaves were inoculated with agar plugs with non-sporulating mycelia of the wild-type (WT) and the Δ<i>bccnA</i> mutant. Images were taken 3 and 6 days post infection (dpi).</p

Localization of GFP-BcRcn1 in <i>B. cinerea</i>.

<p>The strain Δ<i>bcrcn1</i> was transformed with a <i>gfp</i>-<i>bcrcn1</i> fusion construct under the control of the constitutive <i>oliC</i>-promoter. Fluorescent protein was demonstrated to be homogenously distributed at a basal level in the cytoplasm but accumulated around or in the nuclei. Nuclei were visualized by using the fluorescent dye Hoechst 33342 (for details see Materials and Methods). Scale bar: 20 µm.</p

Phenotypical comparison of calcium signaling mutants.

<p>Pictures were taken after incubation of growing strains for 2 weeks on the indicated media. Glucose, sorbitol and NaCl were added with a molarity of 1 M to the basic CM medium, MgCl₂ with a concentration of 67 mM.</p

Germination capabilities of Δ<i>bcbem1</i> conidia are impaired.

<p>Germination rates (<b>A</b>) and the germ tube morphology (<b>B</b>) are altered on hydrophobic surfaces. Conidial suspensions of strains (1×10⁵/ml in H₂O) were incubated on polypropylene foil in a humid chamber for 24 h. Total numbers of germinated conidia and conidia forming elongated germ tubes were counted. Experiment was done in triplicates; mean values and standard deviations were calculated from 300 conidia per strain and condition. Scale bars  = 10 µm. (<b>C</b>) Nutrient-induced germination rates of Δ<i>bcbem1</i> conidia are significantly decreased. Conidia (5×10⁵/ml) were incubated for 24 h in liquid GB5 supplemented with the indicated carbon sources (10.5 M) on glass surfaces. Experiment was done in triplicates; mean values and standard deviations were calculated from 300 conidia per strain and condition. (<b>D</b>) Germination kinetics and germ tube morphology of Δ<i>bcbem1</i> conidia are different from wild-type conidia. Time course of nutrient-induced germination (in GB5 +2% glucose) is shown. Scale bars  = 10 µm.</p