Calcium remediated the growth defect in the <i>sltA</i> deletion mutant.

<p>Photographs of point-inoculated colonies of <i>A. nidulans</i> ∆<i>sltA, veA</i>⁺ (RSS1.6P) and its control strain (FGSC4). The strains were grown on GMM, GMM supplemented with 200 mM KCl, or GMM supplemented with 10 mM CaCl₂. The plates were incubated for 5 days at 37°C.</p

Expression of <i>brlA</i> is affected by SltA.

<p>A) <i>brlA</i> expression analysis by qRT-PCR. The values were normalized to the wild-type (grown on GMM -24 h) levels considered as 100. Strains were initially grown in liquid medium in a shaker incubator for 24 hours at 250 rpm. Then equal amounts of mycelia were homogeneously spread onto solid medium GMM, or GMM, supplemented with 200 mM KCl or with 10 mM CaCl₂ (time point - 0 h) and further incubated at 37°C. Total RNA was extracted using Trizol® from mycelial samples collected at 24 and 48 hours after the shift. B) Quantification of conidia produced by ∆<i>sltA, veA</i>⁺ (RSS1.6P) and its wild-type control (FGSC4) under the same experimental conditions. An 8-mm-diameter core was extracted from the cultures at 24 and 48 hours after the shift and homogenized in water. Spores were counted with a hemocytometer. Values are means of three replicates. The error bar indicates standard error. Asterisk indicates not detected.</p

SltA affects <i>aflR</i> and <i>stcU</i> expression.

<p>A) Graph representing the relative expression levels of <i>aflR</i> analyzed by qRT-PCR. Strains were grown in liquid GMM, GMM supplemented with 200 mM KCl or 10 mM CaCl₂ in a shaker incubator for 24 and 48 hours respectively at 250 rpm at 37°C. Total RNA was extracted using Trizol®, from mycelial samples collected at 24 and 48 hours from the liquid cultures. The values were normalized to the wild-type (grown on GMM -24h) levels considered as 100. B) Expression analysis of <i>steA</i> by qRT-PCR. The values were normalized to the wild type (grown on GMM -24 h) levels considered as 100. C) TLC analysis of ST produced in wild-type (FGSC4) and ∆<i>sltA, veA</i>⁺ (RSS1.6P) cultures under the same experimental conditions. D) & E) Densitometry displaying the relative intensity of ST bands. Values are the means of two replicates. The error bar represents standard error.</p

SltA is required for normal Hülle cell production and regulates <i>nsdD</i> and <i>steA</i> expression.

<p>A) Quantification of Hülle cells produced by wild-type (FGSC4) and ∆<i>sltA, veA</i>⁺ strains (RSS1.6P). The strains were first grown in liquid shaken cultures for 24 hours at 250 rpm. Then equal amounts of mycelia were homogeneously spread onto solid GMM, GMM supplemented with 200 mM KCl, or GMM supplemented with 10 mM CaCl₂ (time point - 0 h) and further incubated at 37°C. B) and C) Expression levels of <i>nsdD</i> and <i>steA</i>, respectively, analyzed by qRT-PCR. Total RNA was extracted using Trizol®, from mycelial samples collected at 24 and 48 hours after the shift. The values were normalized to the wild-type (grown on GMM -24 h) levels considered as 100. The error bar represents standard error. Values are the means of three replicates.</p

Effect of <i>sltA</i> deletion on <i>A. nidulans</i> cleistothecial production.

<p>Micrographs of fruiting bodies (cleistothecia) in ∆<i>sltA, veA</i>⁺ (RSS1.6P) and wild type (FGSC4). Strains were top-inoculated with 5 x10⁶ conidia per plate on GMM, GMM supplemented with KCl (200 mM or 400 mM), or GMM supplemented with 10 mM CaCl₂, and incubated at 37°C for 10 days. Plates were wrapped to promote sexual development. Cultures were sprayed with 70% ethanol to facilitate the visualization of cleistothecia. Arrows indicate cleistothecia in the ∆<i>sltA, veA</i>⁺ culture. Magnification: x50.</p

SltA is necessary for normal growth and conidiation.

<p>A) Wild-type (FGSC4) and ∆<i>sltA</i>, <i>veA</i>^<i>+</i> (RSS1.6P) point-inoculated cultures containing GMM, or GMM supplemented with 200 mM or 400 mM KCl were incubated at 37°C for 5 days. B) & C) Measurement of the radial colony growth. D) & E) Quantification of conidial production from top-agar inoculated cultures (5 x 10⁶ spores/plate). Values are means of three replicates. The error bar indicates standard error.</p

Expression profile of <i>sltA</i> during vegetative growth, asexual and sexual-developmental phases in a wild-type strain.

<p>A wild-type <i>velvet</i> strain (<i>veA</i>⁺, strain WIM126) was cultured in supplemented liquid GMM for vegetative mycelial growth. Samples were collected at the times indicated on top of the figure. For induction of asexual or sexual development, vegetative mycelia were collected by filtration after 24 hours of culture in liquid GMM and placed on top of solid GMM. Samples were collected at the time points indicated on top of the figure. Extracted total RNAs were probed with a radioactively labeled <i>sltA</i> coding region. The 26S ribosomal RNA is shown as loading control.</p

SltA is necessary for normal ST biosynthesis particularly in the presence of potassium while calcium sustains toxin production in the absence of SltA.

<p>A) Wild type (FGSC4) and ∆<i>sltA, veA</i>⁺ (RSS1.6P) strains were top-agar inoculated with 5 x 10⁶ conidia per plate on GMM, GMM supplemented with KCl (200 and 400 mM) or CaCl₂ (10 mM), followed by incubation at 37°C for 5 days. ST toxin was extracted and analyzed by thin layer chromatography (TLC) as described in the experimental procedure section. B) Densitometry displaying the intensity of the ST bands. The densitometry was carried out using the Scion Image 4.03 software. Values are the means of two replicates. The error bar represents standard error.</p