The Telomerase Reverse Transcriptase Subunit from the Dimorphic Fungus Ustilago maydis

In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 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 trt1 crosses in planta 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

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