The mitochondrial alternative oxidase Aox1 is needed to cope with respiratory stress but dispensable for pathogenic development in <i>Ustilago maydis</i>

<div><p>The mitochondrial alternative oxidase is an important enzyme that allows respiratory activity and the functioning of the Krebs cycle upon disturbance of the respiration chain. It works as a security valve in transferring excessive electrons to oxygen, thereby preventing potential damage by the generation of harmful radicals. A clear biological function, besides the stress response, has so far convincingly only been shown for plants that use the alternative oxidase to generate heat to distribute volatiles. In fungi it was described that the alternative oxidase is needed for pathogenicity. Here, we investigate expression and function of the alternative oxidase at different stages of the life cycle of the corn pathogen <i>Ustilago maydis</i> (Aox1). Interestingly, expression of Aox1 is specifically induced during the stationary phase suggesting a role at high cell density when nutrients become limiting. Studying deletion strains as well as overexpressing strains revealed that Aox1 is dispensable for normal growth, for cell morphology, for response to temperature stress as well as for filamentous growth and plant pathogenicity. However, during conditions eliciting respiratory stress yeast-like growth as well as hyphal growth is strongly affected. We conclude that Aox1 is dispensable for the normal biology of the fungus but specifically needed to cope with respiratory stress.</p></div

Expression of Aox1-Gfp.

<p><b>A)</b> Expression of Aox1-Gfp dependent on OD and growth phase. Aox1-Gfp is expressed in the stationary phase. Aox1-Gfp was detected by anti-Gfp. Tub1 serves as loading control. <b>B)</b> Expression of Aox1-Gfp is not dependent on acidification of the media. Western Blot, growth curve, and pH values of the media (unbuffered = CM; buffered = CM + 100 mM MOPS) depending on time. <b>C)</b> Sporidia of FB2, FB2Potef:aox1-Gfp, and FB2Potef:5'UTR-aox1-Gfp showing that aox1-Gfp under the control of the otef-promoter is expressed irrespective of the growth phase.</p

Validation of the function and localization of Aox1-Gfp and other mutants.

<p><b>A)</b> Schematic depiction of the genomic loci of the investigated strains. For deletion the ORF of <i>aox1</i> was replaced by a hygromycin resistance cassette. The strains with the genotype <i>aox1-Gfp</i> express a C-terminal Gfp fusion of <i>aox1</i> under control of the native <i>aox1</i> promoter. Ectopic expression of aox1-gfp was achieved by introducing a C-terminal fusion with Gfp in the defined <i>cbx</i> locus. These constructs were expressed by the constitutive active P_otef promoter. Where indicated, constructs included the 65 bp native 5' UTR of <i>aox1</i>. <b>B)</b> Representative respiration profiles of FB2 (<i>wt</i>) and <i>aox1Δ</i> sporidia in the stationary phase upon addition of respiratory inhibitors. Potassium cyanide (KCN), inhibits complex IV of the respiratory chain. Residual respiration is due to the activity of alternative oxidase (Aox1) and can be inhibited by addition of n-octylgallate (nOG). Note that FB2aox1Δ does not show any respiration after addition of CN. Furthermore, oxidase capacity of <i>aox1-Gfp</i> in FB2aox1-Gfp seems to be attenuated, due to lower oxygen consumption (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173389#pone.0173389.s003" target="_blank">S2 Fig</a>). <b>C)</b> Localization of Aox1-Gfp into the mitochondria of stationary phase sporidia of <i>U</i>. <i>maydis</i>. Sporidial cells were stained with Mitotracker Red and Aox1-Gfp. Fluorescence signals were analyzed by fluorescence microscopy. <b>D)</b> Aox1-Gfp signal can only be seen in the stationary phase of growth. Mixed culture of sporidial cells in exponential phase (OD600 ~0.5) labeled with biotin/avidin-TRITC, and a stationary phase culture of <i>U</i>. <i>maydis</i>.</p

Effect of the presence of Aox1 and antimycin A on the filamentous growth of <i>U</i>. <i>maydis</i>.

<p><b>A)</b> Filamentous growth of <i>U</i>. <i>may</i>dis AB33 wt and mutants in the presence or absence of antimycin A. <b>B)</b> Measurement of the length of the hyphae formed by <i>U</i>. <i>maydis</i> AB33 and derivatives in the presence or absence of antimycin A.</p

Effect of Aox1 on the sporidia growth of <i>U</i>. <i>maydis</i> and sensitivity to temperature.

<p><b>A)</b> Budding of yeast cells. Sporidia of FB2 (wt), FB2aox1Δ, FB2aox1-Gfp, and FB2Potef:aox1-Gfp. <b>B)</b> Time course of cell growth in rich medium as measured by the absorbance at 600 nm. <b>C)</b> Effect of temperature on the growth of sporidia. Growth plates with 1:5 dilutions (starting with OD = 0.5).</p

Aox1 is expressed in the stationary phase.

<p><b>A)</b> Representative oxygen consumption measurements of FB2 sporidia in exponential and stationary phases. Respiration resistant to potassium cyanide (KCN) in <i>U</i>. <i>maydis</i> was due to the alternative oxidase (Aox1), which was inhibited by the addition of 6.0 μM of n-octylgallate (nOG). <b>B)</b> Percent of Aox1 capacity of cells cultured in several carbon and nitrogen sources and harvested at the exponential and stationary phases. The respiratory activity was determined from the slope obtained before and after the addition of cyanide, as shown in Fig 1A. <b>C)</b> Western blot of the Aox1 in mitochondria of cells cultured in different carbon and nitrogen sources. The Coomassie stain shows similar protein load for the different lanes.</p

Effect of Aox1 on the growth of <i>U</i>. <i>maydis</i> and infectivity.

<p><b>A)</b> Filamentous growth of <i>U</i>. <i>maydis</i> AB33 derivatives 8 h.p,i., size bar, 10 μm. DIC and Gfp fluorescence images are depicted for each strain. Note that the Gfp-labelled images in AB33 and AB33aox1Δ depict unspecific fluorescence, which is detectable with low intensity in these control strains. <b>B)</b> Percentage of hyphae (8 h.p.i.): unipolarity, bipolarity, and septum formation was quantified (error bars, s.e.m.; n = 3 independent experiments; >100 hyphae were counted per experiment; note that septum formation is given relative to the values of unipolar or bipolar hyphae set to 100%). <b>C)</b> Results of plant infection experiments with wt strains (FB1 x FB2) and the <i>aox1Δ</i> strains (FB1aox1Δ x FB2aox1Δ). The percentage of plants with typical disease symptoms is given (two experiments, at least 50 plants infected with each strain).</p

Effect of the presence of Aox1 on the sensitivity of sporidia to antimycin A.

<p><b>A)</b> Sensitivity of <i>U</i>. <i>maydis</i> WT (FB2) and mutants (FB2aox1Δ and FB2aox1-Gfp) to antimycin A. 10 μl of antimycin A (10 mg/ml) was placed in the center of the plate with <i>U</i>. <i>maydis</i> cells, and the radius of inhibition of the growth for FB2, FB2aox1Δ and FB2aox1-Gfp was measured. <b>B)</b> Colony forming units obtained in presence or absence of antimycin A by cells harvested at the exponential and stationary phases. <b>C)</b> Induction of Aox1-Gfp expression by antimycin A (2 μM final concentration). The fluorescent signal was measured by flow cytometry. <b>D)</b> Sensitivity of <i>U</i>. <i>maydis</i> WT and mutants to dessication.</p