Table_2_Functional analysis of the bZIP-type transcription factors AtfA and AtfB in Aspergillus nidulans.DOC

Transcription factors (TFs) with the basic leucin zipper domain are key elements of the stress response pathways in filamentous fungi. In this study, we functionally characterized the two bZIP type TFs AtfA and AtfB by deletion (Δ) and overexpression (OE) of their encoding genes in all combination: ΔatfA, ΔatfB, ΔatfAΔatfB, ΔatfAatfBOE, ΔatfBatfAOE, atfAOE, atfBOE and atfAOEatfBOE in Aspergillus nidulans. Based on our previous studies, ΔatfA increased the sensitivity of the fungus to oxidative stress mediated by menadione sodium bisulfite (MSB) and tert-butylhydroperoxide (tBOOH), while ΔatfB was not sensitive to any oxidative stress generating agents, namely MSB, tBOOH and diamide at all. Contrarily, the ΔatfB mutant was sensitive to NaCl, but tolerant to sorbitol. Overexpression of atfB was able to compensate the MSB sensitivity of the ΔatfA mutant. Heavy metal stress elicited by CdCl2 reduced diameter of the atfBOE and atfAOEatfBOE mutant colonies to about 50% of control colony, while the cell wall stress generating agent CongoRed increased the tolerance of the ΔatfA mutant. When we tested the heat stress sensitivity of the asexual spores (conidiospores) of the mutants, we found that conidiospores of ΔatfAatfBOE and ΔatfBatfAOE showed nearly 100% tolerance to heat stress. Asexual development was negatively affected by ΔatfA, while atfAOE and atfAOE coupled with ΔatfB increased the number of conidiospores of the fungus approximately 150% compared to the control. Overexpression of atfB led to a 25% reduction in the number of conidiospores, but increased levels of abaA mRNA and size of conidiospores. Sexual fruiting body (cleistothecium) formation was diminished in the ΔatfA and the ΔatfAΔatfB mutants, while relatively elevated in the ΔatfB and the ΔatfBatfAOE mutants. Production of the mycotoxin sterigmatocystin (ST) was decreased to undetectable levels in the ΔatfA mutant, yet ST production was restored in the ΔatfAΔatfB mutant, suggesting that ΔatfB can suppress ST production defect caused by ΔatfA. Levels of ST were also significantly decreased in the ΔatfAatfBOE, ΔatfBatfAOE and atfAOEatfBOE mutants.</p

Table_1_Functional analysis of the bZIP-type transcription factors AtfA and AtfB in Aspergillus nidulans.DOC

Transcription factors (TFs) with the basic leucin zipper domain are key elements of the stress response pathways in filamentous fungi. In this study, we functionally characterized the two bZIP type TFs AtfA and AtfB by deletion (Δ) and overexpression (OE) of their encoding genes in all combination: ΔatfA, ΔatfB, ΔatfAΔatfB, ΔatfAatfBOE, ΔatfBatfAOE, atfAOE, atfBOE and atfAOEatfBOE in Aspergillus nidulans. Based on our previous studies, ΔatfA increased the sensitivity of the fungus to oxidative stress mediated by menadione sodium bisulfite (MSB) and tert-butylhydroperoxide (tBOOH), while ΔatfB was not sensitive to any oxidative stress generating agents, namely MSB, tBOOH and diamide at all. Contrarily, the ΔatfB mutant was sensitive to NaCl, but tolerant to sorbitol. Overexpression of atfB was able to compensate the MSB sensitivity of the ΔatfA mutant. Heavy metal stress elicited by CdCl2 reduced diameter of the atfBOE and atfAOEatfBOE mutant colonies to about 50% of control colony, while the cell wall stress generating agent CongoRed increased the tolerance of the ΔatfA mutant. When we tested the heat stress sensitivity of the asexual spores (conidiospores) of the mutants, we found that conidiospores of ΔatfAatfBOE and ΔatfBatfAOE showed nearly 100% tolerance to heat stress. Asexual development was negatively affected by ΔatfA, while atfAOE and atfAOE coupled with ΔatfB increased the number of conidiospores of the fungus approximately 150% compared to the control. Overexpression of atfB led to a 25% reduction in the number of conidiospores, but increased levels of abaA mRNA and size of conidiospores. Sexual fruiting body (cleistothecium) formation was diminished in the ΔatfA and the ΔatfAΔatfB mutants, while relatively elevated in the ΔatfB and the ΔatfBatfAOE mutants. Production of the mycotoxin sterigmatocystin (ST) was decreased to undetectable levels in the ΔatfA mutant, yet ST production was restored in the ΔatfAΔatfB mutant, suggesting that ΔatfB can suppress ST production defect caused by ΔatfA. Levels of ST were also significantly decreased in the ΔatfAatfBOE, ΔatfBatfAOE and atfAOEatfBOE mutants.</p

Image_1_Functional analysis of the bZIP-type transcription factors AtfA and AtfB in Aspergillus nidulans.JPEG

Transcription factors (TFs) with the basic leucin zipper domain are key elements of the stress response pathways in filamentous fungi. In this study, we functionally characterized the two bZIP type TFs AtfA and AtfB by deletion (Δ) and overexpression (OE) of their encoding genes in all combination: ΔatfA, ΔatfB, ΔatfAΔatfB, ΔatfAatfBOE, ΔatfBatfAOE, atfAOE, atfBOE and atfAOEatfBOE in Aspergillus nidulans. Based on our previous studies, ΔatfA increased the sensitivity of the fungus to oxidative stress mediated by menadione sodium bisulfite (MSB) and tert-butylhydroperoxide (tBOOH), while ΔatfB was not sensitive to any oxidative stress generating agents, namely MSB, tBOOH and diamide at all. Contrarily, the ΔatfB mutant was sensitive to NaCl, but tolerant to sorbitol. Overexpression of atfB was able to compensate the MSB sensitivity of the ΔatfA mutant. Heavy metal stress elicited by CdCl2 reduced diameter of the atfBOE and atfAOEatfBOE mutant colonies to about 50% of control colony, while the cell wall stress generating agent CongoRed increased the tolerance of the ΔatfA mutant. When we tested the heat stress sensitivity of the asexual spores (conidiospores) of the mutants, we found that conidiospores of ΔatfAatfBOE and ΔatfBatfAOE showed nearly 100% tolerance to heat stress. Asexual development was negatively affected by ΔatfA, while atfAOE and atfAOE coupled with ΔatfB increased the number of conidiospores of the fungus approximately 150% compared to the control. Overexpression of atfB led to a 25% reduction in the number of conidiospores, but increased levels of abaA mRNA and size of conidiospores. Sexual fruiting body (cleistothecium) formation was diminished in the ΔatfA and the ΔatfAΔatfB mutants, while relatively elevated in the ΔatfB and the ΔatfBatfAOE mutants. Production of the mycotoxin sterigmatocystin (ST) was decreased to undetectable levels in the ΔatfA mutant, yet ST production was restored in the ΔatfAΔatfB mutant, suggesting that ΔatfB can suppress ST production defect caused by ΔatfA. Levels of ST were also significantly decreased in the ΔatfAatfBOE, ΔatfBatfAOE and atfAOEatfBOE mutants.</p

Comparison of NH-water exchange rates measured in CLEANEX NMR experiments.

<p>The NH-water exchange rates (s^-1) are shown for PAF (green) and PAF^D19S (blue) at 298 K and pH = 6.0. Note that the very slow exchange rates from residues located in β-strands are not available by this technique, while measurement of fast exchange is inaccurate as shown by the error bars. Some Asn side-chain carboxamides are separately shown (to the right from the dashed line from residue 55: N18, N33, N40, N49, N50).</p

Viability of <i>N</i>. <i>crassa</i> conidia exposed to 32 μM PAF and PAF^D19S.

<p>(<b><i>A</i></b>) Colony forming units were determined by counting the colonies emerging on agar plates after conidia had been treated for 1, 4 and 24 h, respectively, plated in appropriate dilutions and incubated for 24 h (untreated controls) or 48 h. Values are given in %-survival (untreated controls were set to be 100%) and represent the mean of three experiments ± standard errors. <b><i>(B</i></b><i>)</i> Untreated conidia and conidia exposed to ethanol served as negative and positive staining controls, respectively. Conidia exposed to BODIPY-PAF (<b><i>C</i></b>) and BODIPY-PAF^D19S (<b><i>D</i></b>) for 1, 4 and 24 h were co-stained with PI. Scale bar = 30 μm.</p

The structural backbone and surface charge of PAF and PAF^D19S.

<p>(<b><i>A</i></b>) Amino acid sequence of mature PAF and PAF^D19S showing the β-strands (red arrows) and the site of amino acid exchange. (<b><i>B</i></b>) Backbone of the structural ensemble of PAF (left) and PAF^D19S (right). Arrows indicate the β-strands that are connected by loops, the Asp/Ser19 exchange is highlighted with red "sticks", respectively. (<b><i>C</i></b>) Surface representation of PAF (left) and PAF^D19S (right) coloured according to electrostatic potential calculated in vacuum (blue: electropositive; red: electronegative). The position of amino acid exchange is indicated by an arrow.</p

The localization of BODIPY-labelled PAF and PAF^D19S in 6 h-old <i>N</i>. <i>crassa</i> germlings.

<p>After the treatment for 1 h with 32 μM BODIPY-PAF the conidial cell wall, vacuoles and the cytoplasm were stained. PAF treated hyphae showed large vacuoles (asterisks, left panels). Only weak signals at the outer layers and in the cells could be observed with BODIPY-PAF^D19S. Scale bar = 20 μm.</p

The effect of ion supplementation of the culture medium on the growth-inhibitory activity of PAF and PAF^D19S in <i>N</i>. <i>crassa</i>.

<p>The effect of ion supplementation of the culture medium on the growth-inhibitory activity of PAF and PAF^D19S in <i>N</i>. <i>crassa</i>.</p

The effect of the addition of ions to the growth medium on binding and uptake of BODIPY-labelled PAF and PAF^D19S.

<p>Conidia of <i>N</i>. <i>crassa</i> were germinated in the presence of 4 μM BODIPY-PAF or BODIPY-PAF^D19S for 6 h in liquid medium supplemented with 1 mM NaCl, MgCl₂ and CaCl₂, respectively. In the control the 0.2 x Vogel’s growth medium was without ion supplementation. Scale bar = 30 μm.</p

The effect of PAF and PAF^D19S on the [Ca²⁺]_c resting level of 6 h-old <i>N</i>. <i>crassa-AEQ</i> germlings.

<p>Proteins were added (arrow at time point 0) at a final concentration of 3.2 and 32 μM to the growth medium (<b><i>A</i></b>, <b><i>C</i></b>) and in medium pre-treated with 5 mM BAPTA (<b><i>B</i></b>, <b><i>D</i></b>). Top panels for PAF and bottom panels for PAF^D19S. Measurements were taken every 40 sec over a period of 60 min. Samples treated with medium without the antifungal protein served as controls. Values represent the mean of three samples ± standard errors.</p