Data_Sheet_1_Bioavailability of Nutritional Resources From Cells Killed by Oxidation Supports Expansion of Survivors in Ustilago maydis Populations.DOCX

<p>After heavy exposure of Ustilago maydis cells to clastogens, a great increase in viability was observed if the treated cells were kept under starvation conditions. This restitution of viability is based on cell multiplication at the expense of the intracellular compounds freed from the damaged cells. Analysis of the effect of the leaked material on the growth of undamaged cells revealed opposing biological activity, indicating that U. maydis must possess cellular mechanisms involved not only in reabsorption of the released compounds from external environment but also in contending with their treatment-induced toxicity. From a screen for mutants defective in the restitution of viability, we identified four genes (adr1, did4, kel1, and tbp1) that contribute to the process. The mutants in did4, kel1, and tbp1 exhibited sensitivity to different genotoxic agents implying that the gene products are in some overlapping fashion involved in the protection of genome integrity. The genetic determinants identified by our analysis have already been known to play roles in growth regulation, protein turnover, cytoskeleton structure, and transcription. We discuss ecological and evolutionary implications of these results.</p

The Telomerase Reverse Transcriptase Subunit from the Dimorphic Fungus <i>Ustilago maydis</i>

<div><p>In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus <i>Ustilago maydis</i>. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting <i>trt1</i> had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in <i>est2</i> budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of <i>trt1</i> crosses <i>in planta</i> suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.</p></div

Telomere repeat amplification protocol (TRAP) analysis in <i>U. maydis</i>.

<p>Telomerase activity in wild-type and mutant strains was determined. The absorbance data were used to construct a graphical representation of the telomerase activity for the sporidia of <i>U. maydis</i> strains (either wild-type or <i>trt</i>^-). Tumor cells derived from the 521×520 cross and a plant control were included to evaluate and detect telomerase activity. The medians of the telomerase-positive control cells (HEK293) and the 521 wild-type strain were significantly different from the median of the treated negative controls (P<0.05); however, no significant differences were detected between the negative controls and the <i>trt1</i>-disrupted mutants. The samples heated to 85°C are indicated with Δ, and the RNase-treated samples are designated as RNase. Telomerase activity was also determined in tumors and maize leaves.</p

Determination of telomerase activity in <i>U. maydis</i> strains.

<p>The average absorbance values yielded by the telomerase activity in cell extracts are numerically expressed and shown in the chart. The absorbance for each sample was calculated according to the manufacturer's instructions and is shown. Heat- (85°C) and RNase-treated samples were used as negative controls. Telomerase activity was measured under the same conditions in the positive controls and tested samples. Telomerase activity was measured only in the mutant trt1-1; telomerase-negative samples (either from mutants, tumors, or plants) were not treated with heat or RNase, and their activity was not determined (N.D.). All of the experiments were performed at least three times.</p><p>* Media of at least three repetitions.</p><p>Determination of telomerase activity in <i>U. maydis</i> strains.</p

Structure of the <i>U. maydis</i> um11198 locus.

<p>The illustrative representation of the locus encoding the putative telomerase reverse transcriptase subunit (Trt1) of <i>U. maydis</i> is shown. (A) The open reading frame is depicted as a box, and the TERT domains are colored. The thin black lines represent the non-coding sequences located up- and downstream of the gene. (B) The conserved GQ (blue), RBD (red), and TR (green) domains of Trt1 are indicated above each highlighted alignment. The conserved residues are colored as in A. The sequences are from the representative organisms <i>Homo sapiens</i> (accession NP_937983.2), <i>Arabidopsis thaliana</i> (accession AF172097_1), and <i>Saccharomyces cerevisiae</i> (accession AAB64520.1), where the motif E does not align with other TERTs (asterisk). The aligned sequences used to define the motifs include at least 12 species, but only the representative organisms are shown.</p

<i>U. maydis</i> strains used.

<p>^*Partially characterized.</p><p><i>U. maydis</i> strains used.</p

Analysis of the effects of <i>trt</i>+ restoration in pTrt1 <i>U. maydis</i> transformants.

<p>The telomere length distribution in the telomerase-deficient strain W204 was assessed by Southern blotting TRF after reintroduction (200 doubling periods) of <i>tert1</i> in the pTrt1 <i>U. maydis</i> transformants as described above. (A) The TRF hybridization pattern of parental 521 (lane 1), 520 (lane 2), <i>trt1</i>-disrupted mutants trt1-1 (lane 3) and trt1-2 (lane 4) strains, the progeny derivative W204 (lane 5), and five of its W204-derived clones (T1 to T5, lanes 6 to 10) were analyzed using telomere sequences (TTAGGG) that were ³²P- labeled at 17 kBq/ml as probes. (C) The filter was stripped and re-hybridized to TR-p + TR-d sequences ³²P- labeled as a probe. The strain names are shown above the autoradiography. A molecular weight marker is shown on the left.</p

Time course of maize infection with mutant and wild-type strains of <i>U. maydis</i>.

<p>The wild-type 520 and 521 strains and their compatible derivatives trt1-1 and trt1-53 were used to perform <i>in planta</i> mating to determine the effects of the mutant <i>trt1</i> gene on cell cycle completion. The infectious process is listed on the left along with the disease signs and symptoms and lesion characteristics. The names of the compatible strains are shown above each column pair, and the mating genotype is displayed below. Column pairs were used for each compatible mating to show the percentages of plants assayed on the day of sign or symptom appearance.</p>a<p>Wild-type X Wild-type.</p>b<p>Mutant X Wild-type.</p>c<p>Mutant X Mutant.</p><p>Time course of maize infection with mutant and wild-type strains of <i>U. maydis</i>.</p

Terminal restriction fragment (TRF) analysis of a <i>trt1</i>-disrupted <i>U. maydis</i> mutant.

<p>(A) Samples of total DNA from the <i>U. maydis</i> 521 strain (150 ng) and trt1-1 mutant clone (500 ng) were digested with <i>Pst</i>I and quantified. The wild-type 521 strain was used as a control. A 36-generation series of samples was acquired from the trt1-1 mutant culture at the following population doubling times: 76 (lane 1), 112 (lane 2), 148 (lane 3), 184 (lane 4), 220 (lane 5), 256 (lane 6), 292 (lane 7), and 328 (lane 8). The DNA samples were hybridized to a TTAGGG probe as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109981#s4" target="_blank">Materials and Methods</a>. (B) The nylon membrane was stripped and re-hybridized to UT4-a probe, an <i>Eco</i>RI/<i>Bam</i>HI fragment from the subtelomeric <i>UTASa</i> sequence, at high stringency conditions. The sizes (kb) are indicated on the left, and the arrows indicate the DNA bands showing changes in mobility or appearance.</p

Extrachromosomal <i>trt1</i> complementation and growth rate restoration.

<p>After restoring the replicative potential by <i>trt1</i> reintroduction, the growth rate was analyzed in the transformants with the same method used for the non-transformed controls. The strains analyzed are shown on the left, and the plasmids used to transform these strains are indicated at the top. In parentheses, minutes were changed to hours.</p><p>Extrachromosomal <i>trt1</i> complementation and growth rate restoration.</p