Table 1.
Wolbachia putative Type IV effector proteins screened in this study.
Fig 1.
wBm genes inhibit yeast growth upon expression.
(A) BY4742 yeast strains genetically modified with GEV for β-estradiol-dependent induction of GAL promoters (Materials and Methods) and harboring the GAL-inducible control plasmid pYES2/NT A, or pYES2/NT A containing the specified wBm open reading frame were grown to saturation in CSM-uracil medium containing 2% glucose, and each culture was diluted to OD600 = 1.0 in sterile 0.9% NaCl. 10-fold serial dilutions were spotted onto CSM-uracil containing 2% glucose with and without 1 μM β-estradiol. Plates were incubated for 48 h at 30°C. (B) Strains and dilutions from (A) were spotted to CSM-uracil media containing 2% glucose supplemented with 7.5 mM ZnCl2, 5 mM caffeine, and with or without 1 μM β-estradiol. Plates were incubated for 72 h at 30°C. Images are representative of three independent experiments.
Fig 2.
Normal vacuole dynamics are altered upon wBm gene expression.
BY4742 yeast strains modified with GEV for β-estradiol-dependent induction of GAL promoters (Materials and Methods), and harboring GAL-inducible pYES2/NT A control plasmid or pYES2/NT A containing an individual wBm open reading frame were grown to saturation in CSM-uracil medium, subcultured to CSM-uracil and grown for 6h at 30°C with or without the addition of 1 μM β-estradiol. Cells were stained for 20 minutes with 10 μM FM4-64 at 30°C followed by a 1.5 h chase in CSM-uracil at 30°C. Vacuoles were visualized via microscopy, and cells displaying abnormal vacuole morphologies were counted. Percentage and standard deviation of cells displaying the corresponding abnormal vacuolar morphology is noted and determined from three independent experiments; n ≥ 100 cells per experiment. Bar = 3 μ.
Fig 3.
wBm0152 expression inhibits endosomal traffic to the yeast vacuole.
BY4742 yeast strains modified with GEV for β-estradiol-dependent induction of GAL promoters (Materials and Methods), and harboring (A) GFP-CPS plasmid or (B) Sna3-GFP plasmid in addition to pYES2/NT A control plasmid or pYES2/NT A wBm0152 were grown to saturation in CSM-lysine-uracil medium. Cells were subcultured to CSM-lysine-uracil with and without the addition of 1 μM β-estradiol and grown for 6h at 30°C. Cells were stained with 10 μM FM4-64 dye for 20 minutes at 30°C and chased 1.5h in CSM-lysine-uracil medium at 30°C. Cells were visualized, and representative images are shown. Bar = 3 μ. Inset in (A) magnifies the cell denoted by the arrow to better visualize the GFP-CPS trafficking defect in cells expressing wBm0152; images are representative of two independent experiments.
Fig 4.
wBm0076 is a WAS(p)-family protein.
Domain structure of wBm0076 orthologs, including: human N-WASp (hu N-WASp; BAA20128.1), Brugia malayi WSP-1 (Bma WSP-1; CRZ22528.1), S. cerevisiae Las17p (Sce Las17p, NP_014824.1), wBm0076 (WP_011256278.1), wMel WD_0811 (WD_0811; AAS14498), Rickettsia conorii RickA (Rc RickA; WP_041471735.1), and Listeria monocygenes ActA (Lm ActA; ABC40914.2). Domains including poly-proline motifs (PP), the WH2/verprolin-like (V), central α-helical (C), and acidic regions (A) are depicted. (B) Sequence alignment of the conserved VCA domains of the proteins in (A). Completely conserved residues are highlighted with a black box and residues conserved in more than half of the sequences are denoted with an asterisk. Only the first WH2 domains of hu N-WASP and Bma WSP-1 are aligned for simplicity.
Fig 5.
wBm0076 expression alters yeast actin dynamics in vivo.
S288C yeast strains modified with GEV for β-estradiol-dependent induction of GAL promoters (Materials and Methods), expressing (A) Abp1-RFP and harboring either the pYES2/NT A control plasmid or pYES2/NT A wBm0076 were grown to saturation in CSM-uracil medium, subcultured to CSM-uracil, with or without 1 μM β-estradiol, for 6h at 30°C. Cells were harvested at indicated time points and visualized. Bar = 5 μ. (B-D) The average ± standard deviation Abp1-containing punctae are plotted per cell (B), cell diameter (C), and estimated cell volume / punctae (D, assume cell is a sphere during volume calculation) were measured from cells grown in (A), and ≥100 cells each per three independent experiments were measured; ns = P > 0.05 (not significant); (*) = P ≤ 0.05; (**) = P ≤ 0.01; (****) = P ≤ 0.0001. (E) As in (A), except yeast cells harbored Abp140-3xGFP instead of Abp1-RFP.
Fig 6.
Deletion of ABP1 reduces the toxicity of wBm0076 expression.
(A) BY4742 yeast strains deleted for the indicated gene and harboring either pYES2/NT A or pYES2/NT A wBm0076 (0076) were grown overnight in CSM medium lacking uracil. Cultures were diluted to an OD600 = 1.0 in sterile 0.9% NaCl, then spotted in 10-fold dilutions on plates containing 1% raffinose and either 2% glucose or 2% galactose to induce wBm0076 expression. Plates were incubated for 72 h at 30°C and imaged. (B) BY4742 yeast strains modified with GEV for β-estradiol-dependent induction of GAL promoters and harboring either pYES2/NT A (pYES), pYES2/NT A wBm0076 (0076), or pYES2/NT A wBm0076-mRuby2 (0076-mRuby) were grown spotted as in (A), except that plates either contained or lacked 1 μM β-estradiol to induce wBm0076 expression. Plates were incubated for 48 h and imaged. (C) Yeast strains harboring Abp140-3xGFP and pYES2/NT A wBm0076-mRuby2 were grown overnight in CSM medium lacking uracil and subcultured to fresh CSM-Ura containing 1 μM β-estradiol. After 5 h outgrowth at 30°C, cells were harvested and visualized. Images are representative of three independent experiments; bar = 5μ.
Table 2.
Summary of aberrant yeast phenotypes observed in this study.