Characterization of vacuolar amino acid transporter from <i>Fusarium oxysporum</i> in <i>Saccharomyces cerevisiae</i>

<p><i>Fusarium oxysporum</i> causes wilt disease in many plant families, and many genes are involved in its development or growth in host plants. A recent study revealed that vacuolar amino acid transporters play an important role in spore formation in <i>Schizosaccharomyces pombe</i> and <i>Saccharomyces cerevisiae</i>. To investigate the role of vacuolar amino acid transporters of this phytopathogenic fungus, the <i>FOXG_11334</i> (<i>FoAVT3</i>) gene from <i>F. oxysporum</i> was isolated and its function was characterized. Transcription of <i>FoAVT3</i> was upregulated after rapamycin treatment. A green fluorescent protein fusion of FoAvt3p was localized to vacuolar membranes in both <i>S. cerevisiae</i> and <i>F. oxysporum</i>. Analysis of the amino acid content of the vacuolar fraction and amino acid transport activities using vacuolar membrane vesicles from <i>S. cerevisiae</i> cells heterologously expressing <i>FoAVT3</i> revealed that FoAvt3p functions as a vacuolar amino acid transporter, exporting neutral amino acids. We conclude that the <i>FoAVT3</i> gene encodes a vacuolar neutral amino acid transporter.</p> <p>Localization of vacuolar neutral amino acid transporter, GFP-FoAvt3p, in <i>Fusarium oxysporum</i>. FoAvt3p (green) functions as transporter of neutral amino acids from vacuole (blue) to cytosol.</p

Isolation of GOFAs by overexpression profiling.

A) “Overexpression profiling” for identifying GOFAs developed in this study. The detail is explained in the text. B) A proof of concept for overexpression profiling: identification of GOFA under 250 μM methotrexate. The bar plot and the numbers on the bars show occupancies of the DFR1 with reads per million reads (RPM). C) The time course of plasmid occupancy under heat stress. One of the four replicates (Pool_a-2) at 40°C in YPD for 80 generations (samples were analyzed every eight generations). Occupancies of each plasmid are shown with reads per million reads (RPM). The orange and red areas correspond to NCS2 and NCS6 reads, respectively. D-H) Fold changes of plasmid occupancies after the cultivation (upper) and Venn diagrams of hits in replicates (lower, FDR ≤ 0.05 and log2FC ≥ 5) under well-studied stresses; YPD at 30°C (D, control), 37°C (E) and 40°C (F) as the heat stresses, 1 M NaCl (G) as the salt stress, 2 mM H2O2 (H) as the oxidative stress. The log2FC is plotted along the y-axis as a function of the 5,751 overexpressed genes ordered by ORF names. Hits were shown as red-filled symbols. Hit genes are summarized in S2 Table.</p

Mitochondria appear to be a key target in the enhancement of salt tolerance by adding calcium.

A) Expression of ENA1 under salt stress is not enhanced by CaCl2 addition. The ENA1 promoter activity was detected by Western blotting of EGFP under the control of the ENA1 promoter under three conditions: YPD, 1 M NaCl (Na), and 1 M NaCl with 5 mM CaCl2/YPD (Na/Ca). The lower panel shows the EGFP level in Na/Ca relative to Na during the logarithmic growth phase. The lower panel shows the EGFP level in Na/Ca relative to Na during the logarithmic growth phase. The error bar indicates the SD of relative values (n = 3). The p-value was calculated using Welch’s t-test. B) A scheme of systematic analysis for relative fitness of gene knockouts. The detail is explained in the text. C) Comparing relative knockouts’ fitness (Z) between Na and Na/Ca. The blue cycles indicate knockouts with reduced fitness (FDR ≤ 0.05 and ΔZ ≤ 1, Welch’s t-test, and the Benjamini-Hochberg correction, n = 3). D) Enriched gene ontology (GO) terms in "cellular component" in the 296 knockouts with reduced fitness under Na/Ca (p ≤ 0.05, Holm-Bonferroni correction). The bar plot shows the number of genes with indicated GO terms. Other categories of enriched GO terms are shown in S5 Table. E) The distribution of fitness was corrected by YPD (ZNa−ZYPD). The solid and the dashed line indicate mitochondria (Mito) genes and the other genes, respectively. The orange area represents Group I Mito. genes (ZNa−ZYPD ≥ 1), and the purple area means Group II Mito. genes (ZNa−ZYPD ≤ –1). F) The distribution of relative knockouts’ fitness of Group I (orange), Group II (purple), and the others (grey, 4,052 genes) under each condition. The p-values are from Welch’s t-test by comparison with Other. G-H) Comparisons of relative knockouts’ fitness between Na and YPD (G) and Na and Na/Ca (H) are shown. The purple and orange cycles indicate the knockouts belonging to Group I and Group II, respectively. The vertical and horizontal dashed lines indicate Z = 0. I) Enriched gene ontology (GO) terms in "biological function" of the knockouts belonging to Group I (upper, orange) and Group II (bottom, purple) (p ≤ 0.05, Holm-Bonferroni correction). A complete set of enriched GO terms can be found in S6 Table. J) The Group I and Group II Mito. genes have separate functions in the mitochondrial respiratory chain. The complexes or proteins within Group I and Group II Mito. genes are colored orange and purple, respectively. K) Microscopic images of the cells with mitochondria and their reactive oxygen species (ROS) level under four conditions. Plus or minus of "Na" indicate YPD with or without 1 M NaCl, and plus or minus of "Ca" indicate with or without 5 mM CaCl2. The green color shows the mitochondria inner membrane observed with Tim50-GFP. The red color indicates the mitochondrial ROS level stained by MitoTracker Red CM-H2Xros.</p

Strain-dependent requirements of calcium and potassium for the salt stress reflect strain-dependent GOFAs.

A) Construction of the ENA1 co-overexpression (-coe) library by mating. The detail is explained in the text. B) Fold change of plasmid occupancy after the 16 generations in CEN.PK2 with ENA1-oe under 1 M NaCl (upper), and a Venn diagram of hits in replicates (lower, FDR ≤ 0.05 and log2FC ≥ 5). The log2FC is plotted along the y-axis as a function of the 5,751 overexpressed genes ordered by ORF names. These data are summarized in S4 Table. C) A comparison of the mean fold change of plasmid occupancy with and without ENA1-coe under 1 M NaCl. The colored circles indicate triple hit genes in three replicates: without ENA1-core (blue), with ENA1-core (red), and both (purple). The dashed lines represent the threshold of hits as log2FC ≥ 5. D) Growth rates of CEN.PK2-1C under 1 M NaCl with supplements. N.D means not detected. Error bars indicate SD. All 15 pairs differed significantly (Welch’s t-test and Benjamini-Hochberg correction, FDR ≤ 0.05, n = 3). The value of N.D is set to 0 for the statistical test. E-F) Fitness landscapes of BY4741 (E) and CEN.PK2-1C (F) under 1 M NaCl with various KCl and CaCl2 levels. The downward triangle points to 1 M NaCl/YPD, with increasing amounts of KCl or CaCl2, added along the x- or y-axes. The growth rates at each KCl and CaCl2 addition are represented as the z-axis and colored as a purple-to-orange heat map, corresponding to the relative growth rate. G-H) A diagram of the expected relationship between slopes on fitness landscapes and GOFAs in BY4741 (G) and CEN.PK2-1C (H). Arrows indicate the correspondence between Ca2+ or K+ requirement and each GOFA. I and J) Effects of CaCl2 addition on the growth rates of CEN.PK cells overexpressing ENA1 (ENA1-oe) and ECM27 (ECM27-oe). ENA1 and ECM27 were overexpressed using pTOW48036 and pRS423nz, respectively. The Vector/Vector cells without CaCl2 addition did not grow, but the growth rate was set to 0 for convenience in I and shown as N.D in J. Error bars indicate SD (n = 3). All 6 pairs with 0 mM CaCl2 and 5 pairs with 50 mM CaCl2 were significantly different (Welch’s t-test and Benjamini-Hochberg correction, FDR ≤ 0.05, n = 3). A pair with no significance is shown in the figure. The value of N.D is set to 0 for the statistical test.</p

Effects of <i>avt3</i>⁺ expression on the vacuolar amino acid composition of <i>S</i>. <i>cerevisiae</i>.

<p>The vacuolar pools of <i>S</i>. <i>cerevisiae</i> were prepared and analyzed using an amino acid analyzer. The results represent the mean ± SD based on at least three independent experiments: wild-type cells carrying an empty plasmid (<i>white bar</i>), <i>avt3</i>Δ<i>avt4</i>Δ cells carrying an empty plasmid (<i>black bar</i>), pGPD-GFP-<i>avt3</i>⁺ (<i>light gray bar</i>), and pGPD-GFP-<i>avt3</i>^<i>E469A</i> (<i>dark gray bar</i>).</p

Predicted topology model and intracellular localization of SpAvt3p.

<p>(A)<i>Top</i>, predicted topology model of SpAvt3p. <i>Bottom</i>, sequence alignments of SpAvt3p (Q10074.1) in TM6 (amino acids 451–477) and analogous regions in the homologs according to CLUSTALW: <i>Saccharomyces cerevisiae</i> Avt3p and Avt4p (P36062 and P50944, respectively), <i>Arabidopsis thaliana</i> At5G65990 (ABH04593), and human hPAT1and hPAT2 (AAI36439 and AAI01104, respectively). Identical and similar residues are denoted by <i>black boxes</i> and <i>gray boxes</i>, respectively. The conserved glutamate residue is indicated by an asterisk. (B) The <i>avt3</i>Δ mutant cells expressing GFP-SpAvt3p fusion protein were subjected to fluorescence microscopy. Vacuolar membranes were stained with FM4-64. BF, bright field; bar, 5 μm.</p

SpAvt3p-dependent extrusion of amino acids by vacuolar membrane vesicles.

<p>(A) Immunoblot analysis of GFP-SpAvt3p and GFP-SpAvt3p^E469A in the vacuolar membrane vesicles isolated from <i>S</i>. <i>cerevisiae avt1</i>Δ<i>avt3</i>Δ<i>avt4</i>Δ mutant cells. Vacuolar membrane vesicles were prepared and analyzed by immunoblotting using anti-GFP serum and anti-Pho8 antibody. Pho8p was detected as the loading control. (B) Alanine and tyrosine export by vacuolar membrane vesicles. [¹⁴C]-labeled amino acids were preloaded into the vacuolar membrane vesicles isolated from <i>avt1</i>Δ<i>avt3</i>Δ<i>avt4</i>Δ cells carrying an empty plasmid (<i>circles</i>), pGPD-GFP-<i>avt3</i>⁺ (<i>triangles</i>), or pGPD-GFP-<i>avt3</i>^<i>E469A</i> (<i>diamonds</i>). The export assay was performed in the presence (<i>black symbols</i>) or absence (<i>white symbols</i>) of 2 mM ATP. Preloaded vesicles were removed immediately before (0 min) or at 1, 2, 4, and 8 min after the addition of ATP, and collected on cellulose acetate membrane filters. The amount of preloaded [¹⁴C]-labeled amino acids at 0 min was taken as 100%. The relative amounts trapped on the filters are shown. The values represent the mean ± SD based on at least three independent experiments. (C) Effects of CCA on ATP-driven alanine and tyrosine export. The experiments were performed as described above. Vacuolar membrane vesicles were incubated with 1 μM CCA for 10 min before loading with [¹⁴C]-labeled amino acids. The amount of preloaded [¹⁴C]-labeled amino acids at 0 min was taken as 100%. The relative amounts trapped on the filters at 8 min after the addition of ATP are shown. The values represent the mean ± SD based on at least three independent experiments: <i>avt1</i>Δ<i>avt3</i>Δ<i>avt4</i>Δ cells carrying an empty plasmid without (<i>white bar</i>) or with (<i>black bar</i>) CCA, and pGPD-GFP-<i>avt3</i>⁺ without (<i>light gray bar</i>) or with CCA (<i>dark gray bar</i>).</p

Effects of <i>avt3</i>⁺ expression on the ATP-dependent uptake of basic amino acids by vacuolar membrane vesicles.

<p>Vacuolar membrane vesicles were isolated from the wild-type cells carrying an empty plasmid (<i>squares</i>), the <i>avt3</i>Δ<i>avt4</i>Δ cells carrying an empty plasmid (<i>circles</i>), pGPD-GFP-<i>avt3</i>⁺ (<i>triangles</i>), and pGPD-GFP-<i>avt3</i>^<i>E469A</i> (<i>diamonds</i>). The amino acid uptake assay was performed with (<i>black symbols</i>) or without (<i>white symbols</i>) 2 mM ATP. The values represent the mean ± SD based on at least three independent experiments.</p

Vacuolar morphology of <i>S</i>. <i>pombe</i> ARC039 (wild-type), <i>avt3</i>Δ, <i>avt3</i>Δ/pTN54, and <i>avt3</i>Δ/pTN54-<i>avt3</i>⁺.

<p>Cells were grown in MM medium without thiamine for 20 h and the vacuolar membranes were then stained with FM4-64. BF, bright field; bar, 5 μm.</p