Figure 1.
The “mirror” capability of ballistospore-forming yeast species.
(A) The plate on the left was inoculated with Sporobolomyces sp. wild type strain IAM 13481, and inverted onto an uninoculated plate on the right. (B) Diagram of the steps in the formation of a ballistospore from a yeast cell, based on [2], [10]. (C) The 18 mirror mutants isolated in this study growing on the top plate (left) and their replication to a bottom plate (right). The wild type strain is in the top left hand corner of the bottom plate.
Table 1.
Strains used in this study.
Figure 2.
Loss of PHS1 impairs the ability to produce ballistospores.
(A) The structure of the PHS1 gene, and the T-DNA insertion position within it in strain GI209. Exons are in black and introns in white, with the 5′ and 3′ untranslated regions in darker grey. The two primers, ALID1288 and ALI1289, were used to amplify the wild type copy for gene complementation. (B) DNA sequence alignments of the wild type or T-DNA insertional mutant strain. The 15-bp black region in the wild type is replaced with the T-DNA molecule, whose left and right border sequences are in bold italics. (C) The loss of PHS1 can be complemented by reintroducing a wild type copy of the PHS1 gene. Yeast nitrogen base (YNB) without uracil is the selection medium for transformation. Ballistospores were fired from the top plate onto the bottom plate, and colonies allowed to grow. (D) An inhibitor of VLCFA synthesis, cafenstrole, reduces ballistospore production. Wild type Sporobolomyces were 10-fold serially diluted, and grown on YPD supplemented with cafenstrole or the solvent DMSO for two days (top plate) inverted over the bottom plates. The bottom plates were cultured two additional days.
Figure 3.
Sporobolomyces PHS1 complements loss-of-function of the S. cerevisiae phs1 homolog.
Five strains (numbered 1–5) were cultured on different media (A) and the presence or absence of the native PHS1 or the Sporobolomyces PHS1 gene in S. cerevisiae tested by PCR (B). (A) Ten fold serial dilutions of yeast strains. YPD allows equal growth of the strains. G418 selects for strains carrying a phs1::KanMX allele. The YNB – uracil selects for strains carrying a plasmid with the URA3 selectable marker. 5-FOA is used to counter-select or “cure” plasmids containing the URA3 marker from a ura3 mutant strain background. Strain 1 is a diploid with both copies of PHS1. Strain 2 is a heterozygote strain with one wild type and one mutated copy of the PHS1 gene. Strain 3 is strain 2 expressing the Sporobolomyces PHS1 gene from a 2 µ plasmid. Strain 4 is a haploid progeny from meiosis of strain 3. Strain 5 is a control haploid strain with the 2 µ plasmid. (B) The top panel shows PCR of the wild type allele of S. cerevisiae PHS1 (near the 0.85 kb ladder marker) or the replacement allele phs1::KanMX (near to 1.6 kb ladder marker). The bottom panel is amplification of the Sporobolomyces PHS1 gene from the same strains.
Figure 4.
The phs1 mutation in Sporobolomyces causes reduction in 3-hydroxyacyl-CoA dehydratase activity.
(A) Sporobolomyces (Sp. sp.) wild type (IAM 13481), mutant phs1 (GI209), ura5 auxotroph (AIS2), uracil prototroph phs1+ URA5 (AIS13), and complementing strain phs1 + PHS1 (AIS15) cells were grown in YPD medium at 25°C. As controls, S. cerevisiae (Sa. ce.) wild type (R1158) and pTetO7-PHS1 (TH-3237) were grown in YPD medium containing 10 µg/ml doxycyclin for 6.5 h at 30°C. Total membranes (4 µg) prepared from the strains were incubated for 15 min at 37°C [14C]3-hydroxypalmitoyl-CoA (3.6 µM; 10 nCi/µl). After termination of the reactions, lipids were saponified, acidified, extracted, and separated by normal phase TLC. (B) The radioactivities associated with the reaction product 2,3-trans hexadecenoic acid were quantified using a bioimaging analyzer BAS-2500. Values represent the mean ± S.D. from three independent experiments. Statistically significant differences are indicated (**p<0.01; t-test).
Figure 5.
The phs1 mutation causes reduction in elongation of C16:0-CoA.
(A) Sporobolomyces (Sp. sp.) ura5 auxotroph (AIS2), uracil prototroph phs1+ URA5 (AIS13) and complementing strain phs1 + PHS1 (AIS15) cells were grown in YPD medium at 25°C. S. cerevisiae (Sa. ce.) wild type (R1158) and pTetO7-PHS1 (TH-3237) strains were grown in YPD medium containing 10 µg/ml doxycyclin for 6.5 h at 30°C. Total membranes (20 µg) prepared from the strains were incubated with C16:0-CoA (20 µM) and 100 µM (0.075 µCi) [14C]malonyl-CoA in the presence of 1 mM NADPH for 1 h at 37°C. (A) After termination of the reactions, lipids were saponified, acidified, extracted, and separated by normal-phase TLC, followed by detection by autoradiography. (B) After termination of the reactions, lipids were saponified, acidified, and extracted. The resulting fatty acids were then converted to fatty acid methyl ester, extracted, separated by reverse-phase TLC, and detected by autoradiography. [3H]Palmitic acid (C16 FA) and [14C]3-hydroxypalmitic acid (C16 3-OH FA) were used as standards.
Figure 6.
The phs1 mutation affects sphingolipid synthesis.
Sporobolomyces (Sp. sp.) wild type (IAM 13481), mutant phs1 (GI209), ura5 auxotroph (AIS2), uracil prototroph phs1+ URA5 (AIS13), and complementing strain phs1 + PHS1 (AIS15) cells and S. cerevisiae (Sa. ce.) wild type (R1158) were grown in YPD medium medium (A and B) or in SC medium lacking inositol (C) at 25°C. Cells were labeled with [3H]palmitic acid (A), [3H]dihydrosphingosine (B), or [3H]inositol (C) for 2 h. Lipids were extracted, treated with nothing or alkaline solution, and separated by normal phase TLC, followed by detection by autoradiography. Abbreviations are CER, ceramide; DHS, dihydrosphingosine; FA, fatty acid; PE, phosphatidylethanolamine; PHS, phytosphingosine; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; IPC, inositolphosphorylceramide; MIPC, mannosylinositol phosphorylceramide; LCBP, long chain base phospholipid; M(IP)2C, mannosyldiinositol phosphorylceramide. The asterisks indicate unidentified lipids.
Figure 7.
Stress phenotypes of the phs1 mutant of Sporobolomyces suggest that defects in other cellular processes can impair the formation of spores.
10-fold serial dilutions of overnight cultures of Sporobolomyces wild type (IAM 13481), mutant phs1 (GI209), complementing strain phs1 + PHS1 (AIS15) and uracil prototroph phs1+ URA5 (AIS13) were plated onto YPD media without or with stress agents, and cultured 3–4 days. Stress agents were caspofungin (50 µg/ml), congo red (0.4%), sodium dodecyl sulfate (SDS, 0.015%) and sodium chloride (NaCl, 1 M).