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

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

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 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

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

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

Signaling Governed by G Proteins and cAMP Is Crucial for Growth, Secondary Metabolism and Sexual Development in <em>Fusarium fujikuroi</em>

<div><p>The plant-pathogenic fungus <i>Fusarium fujikuroi</i> is a notorious rice pathogen causing hyper-elongation of infected plants due to the production of gibberellic acids (GAs). In addition to GAs, <i>F. fujikuroi</i> produces a wide range of other secondary metabolites, such as fusarins, fusaric acid or the red polyketides bikaverins and fusarubins. The recent availability of the fungal genome sequence for this species has revealed the potential of many more putative secondary metabolite gene clusters whose products remain to be identified. However, the complex regulation of secondary metabolism is far from being understood. Here we studied the impact of the heterotrimeric G protein and the cAMP-mediated signaling network, including the regulatory subunits of the cAMP-dependent protein kinase (PKA), to study their effect on colony morphology, sexual development and regulation of bikaverins, fusarubins and GAs. We demonstrated that fusarubin biosynthesis is negatively regulated by at least two Gα subunits, FfG1 and FfG3, which both function as stimulators of the adenylyl cyclase FfAC. Surprisingly, the primary downstream target of the adenylyl cyclase, the PKA, is not involved in the regulation of fusarubins, suggesting that additional, yet unidentified, cAMP-binding protein(s) exist. In contrast, bikaverin biosynthesis is significantly reduced in <i>ffg1</i> and <i>ffg3</i> deletion mutants and positively regulated by FfAC and FfPKA1, while GA biosynthesis depends on the active FfAC and FfPKA2 in an FfG1- and FfG3-independent manner. In addition, we provide evidence that G Protein-mediated/cAMP signaling is important for growth in <i>F. fujikuroi</i> because deletion of <i>ffg3</i>, <i>ffac</i> and <i>ffpka1</i> resulted in impaired growth on minimal and rich media. Finally, sexual crosses of <i>ffg1</i> mutants showed the importance of a functional FfG1 protein for development of perithecia in the mating strain that carries the MAT1-1 idiomorph.</p> </div

Gibberellic acid (GA) content of cAMP pathway mutants compared to WT.

<p>The WT and cAMP pathway mutants (Δ<i>ffg1,</i> Δ<i>ffg3,</i> Δ<i>ffac,</i> Δ<i>ffpka1,</i> Δ<i>ffpka2</i>) were grown for 7 days in synthetic ICI medium under GA biosynthesis-favoring conditions (6 mM glutamine). GA₃ and GA_4/7 were extracted from 20 ml liquid culture (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058185#s2" target="_blank">material and methods</a>). For comparison, GA content produced by the WT was set to 100%. Experiments were performed in triplicate.</p

Analysis of growth behavior of the cAMP pathway mutants on different media compared to the WT.

<p>A) WT and cAMP pathway mutants (Δ<i>ffg1,</i> Δ<i>ffg3,</i> Δ<i>ffac,</i> Δ<i>ffpka1,</i> Δ<i>ffpka2</i>) were grown on solidified ICI medium with 6 mM sodium nitrate as sole nitrogen source. B) WT and cAMP pathway mutants on solidified complete medium (CM) and Czapek dox (CD, minimal medium). After 7 days radial growth rates of the respective mutants was determined. Experiments were done in triplicate. C) Radial growth of the respective mutants grown on CM or CD medium was measured [mm]. Mean values and standard deviations are shown in the diagram.</p