Fig 1.
Synteny map around the ter1 locus in Ustilaginales species.
Representation of the genes that encode proteins that are located in a widely preserved region around the intergenic area where the ter1 gene is located (indicated by a blue rectangle). The region shows high conservation, where the same two ORFs flank ter1 in every examined organism (except of the 3’ ORF in P. flocculosa).
Fig 2.
Preserved elements identified within the ter1 locus.
(A) Representation of the chromosomal context of ter1. The image shows the region corresponding to both isoforms of the UMAG_03168 locus, the deleted region and the location of preserved motifs that include essential elements for TER activity obtained from multiple sequence alignment. (B) Multiple alignments of preserved motifs within orthologous intergenic regions of ter1. The intergenic regions contain characteristic TER sequences. The sequence of the putative template domain is conserved in all examined organisms. At the 5’ end of the region, three preserved elements were identified (CE1–3). Toward the 3’ end of the region, the consensus sequence AAU5-6CY was identified, which resembles the Sm-binding site of TLC1 in S. cerevisiae and thus could correspond to the end of the mature ter1 transcript.
Fig 3.
RNA-seq data analysis of the ter1 locus.
(A) Genomic context and conserved elements (upper panel) and density of RNA-seq reads mapped to the ter1 locus (lower panel). (B) Transcripts of the ter1 locus originating upstream of UMAG_03168 (represented by white rectangles) reconstructed in this work. (C) Analysis of the relative expression of the isoforms of ter1. Quantification of normalized counts was carried out with the EdgeR method for each isoform of ter1 and of isoforms of housekeeping genes.
Table 1.
Differential expression analysis of ter1 isoforms.
Fig 4.
Confirmation of transcription from the ter1 locus occurs.
(A) Upper panel. Schematic representation of ter1 locus and alignment sites of primers used for RT-PCR assays located upstream of the putative Sm site (red asterisk) and polyadenylation signals (green asterisks), reverse primers used for cDNA synthesis are represented by blue arrows. (B–D). Combination of primer pairs used and expected amplicon sizes for each combination. (B’–D’) Agarose gel electrophoresis of PCR products obtained from cDNA synthesis with the reverse primers indicated at the top of each lane, genomic DNA was used as a positive control. (B and B’). Confirmation of transcription of the ter1 locus containing at least one fragment from the 3’ end of UMAG_03168 and of the existence of the ter1-i4 isoform, evidenced by the presence of a 637 bp fragment unable to amplify in the positive control, top panel, and bottom panel, respectively (C, C’ and D, D’). The long ter1-i1 isoform allowed the amplification of the 418 bp fragment with the ter-i1A-5a and ter-i4D-3b primer pair and revealed at least two isoforms processed by alternative splicing. (E) A 405 bp amplified fragment of α-Tubulin transcript was used as a gene expression control.
Fig 5.
Phenotypic alterations caused by the interruption of ter1 in U. maydis.
(A) TRF analysis of the WT 518 strain and ter1 mutants. The loss of TER triggers a progressive shortening of TRF and eventually leads to the emergence of surviving cells showing the amplification of a 350 bp fragment. (B) Serial 10-fold dilutions of cultures were spotted on YEPS or MC plates and incubated for 2 days at 28°C; ter1 mutants showed dramatic decreases in colony formation and colony size, consistent with the loss of telomerase activity described in other organisms. (C) Cell morphology of strain WT 518 and mutants ter1-02 and ter1-24. The interruption of ter1 causes alterations in cell morphology, and the mutants have irregularly edged cells and an increase in the presence of elongated cells with alterations in the budding angle, suggesting defects in cell cycle regulation. (D and E) Growth kinetics of the WT strain and ter1 mutants. Strains were grown in YEPS broth (28°C) and sampled every two hours. Samples were used for measurements of OD600 or were diluted and plated. Colonies were counted after 24 h of incubation at 28°C and plotted. The results of triplicate assays are shown.
Fig 6.
Electrophoretic karyotype and percentage of cells exhibiting nuclear aberrations and alterations in the budding angle.
(A) Electrophoretic karyotype of WT 521 strain and four telomerase negative mutants trt1-1 and trt1-2 are TERT-disrupted mutants (trt1 Δ) and ter1-02 and ter1-24 are TER-disrupted mutants (ter1Δ); trt1-1 and ter1-02 strains (lanes 2nd and 4th respectively) show increases in chromosome size, possibly due to UTASa amplification, both strains also exhibit fuzzy bands along the entire lane possibly associated to DNA DSBs, which coincided with the formation of nuclear aberrations; in contrast, the trt1-2 and ter1-24 strains had a similar karyotype to that of the WT strain. (B) trt1-disrupted mutants also show slight changes in the budding angle, which in the ter1-disrupted strains is noticeable, mainly as polar growth, perhaps as consequence of alterations in cell cycle regulation. (C) Interruption of ter1 triggers an increase in the formation of nuclear aberrations and chromosome bridges, suggesting DNA damage response system; in contrast, trt1 mutants exhibit an only slight increase in the formation of these structures.
Fig 7.
Development of infection in maize plants.
(A) Quantification of symptoms in maize plants infected with the indicated crosses at 21 dpi. The mean values of three independent experiments are shown. (B) Details of the leaves and the stem at 21 dpi of the plants inoculated with the wild crosses and heterozygous crosses ter1+/ter1–. (C and D) Stem detail and anthocyanin formation at 21 dpi of plants inoculated with the wild crosses and heterozygous ter1+/ter1– crosses, respectively. (E) tumor developed by wild crosses at 21 dpi.