Fig 1.
Mating type determination in Tetrahymena.
(A) Life cycle of Tetrahymena. Tetrahymena grows asexually by binary fission (left panel). A mature cell expresses only 1 mating type during vegetative growth. The micro- and macronucleus are indicated by the small and large circle, respectively. Two sexually matured cells of different mating type can form pairs and enter the conjugation process when starved (right panel). The mating partners exchange meiotic products of the parental micronuclei and form zygotic nuclei that further divide and differentiate into the new micro- and macronuclei. Parental macronuclei degrade at the end of this process. Paired cells separate and divide to generate 4 progeny cells. (B) The mat locus of the micronucleus (upper panel) and the macronucleus (bottom panel) of a mating type VI cell. The connecting lines indicate DNA rearrangements including the deletion of IESs and the removal of all other mating-type gene pairs, presumably by recombination between the homologous sequences present in the MTA/MTB C-terminal exons [10]. (C) Selfing occurs in a ΔTKU80 strain after starvation. Normal clonal cells do not form mating pairs (left panel), but mating pairs were observed within a clone of the ΔTKU80 strain after starvation (right panel). Scale bar = 10 μm. IES, internal eliminated sequence; Mac, macronucleus; Mic, micronucleus.
Table 1.
Selfer ratio at different fission number.
Table 2.
Selfers can produce nonselfers.
Fig 2.
Selfers contain multiple mating-type genes in the macronucleus.
(A) Analysis of the macronuclear MTA and MTB complete C-terminal exons. Left panels illustrate the macronuclear mat locus of 6 mating types and the primers used for detecting the MTA complete C-terminal exons (blue arrows) and the MTB complete C-terminal exons (green arrows). Black vertical lines within the gene indicate the IESs deletion junctions. DNA of 6 normal strains (strain BII, MT3, CU438, MT5, CU427, and CU428; they are of mating type II, III, IV, V, VI, and VII, respectively) and 7 ΔTKU80 selfers were examined by PCR. The normal strains contain 1 properly rearranged pair of the MTA and MTB complete C-terminal exons. However, multiple MTA and MTB complete C-terminal exons were detected in the ΔTKU80 selfers. Notably, the detected exon pairs were not of the same mating type in some selfers. (B) Examination of mat locus of MT IV and VI cells by Southern hybridization. Left panels illustrate the mat locus of MT IV and VI cells. IESs are indicated by gray rectangles. Probes used for detecting MT IV and VI are indicated by black bars. Xs indicate the Xba I sites. Cellular DNA from the ΔTKU80 selfer k4, 8 stabilized subclones, 8 selfer subclones, and control cells was analyzed. DNA fragments corresponding to both MT IV (pink arrow) and VI (blue arrow) were detected in selfer k4 and the 8 k4 selfer subclones, whereas only the fragment corresponding to MT IV was detected in the 8 stabilized MT IV subclones. The bracket indicates nonspecific bands, and open arrowheads indicate the expected micronuclear form DNA. (C) Unusual mating-type genes are detected in ΔTKU80 selfers. DNA of ΔTKU80 selfers and control cells was analyzed by PCR using 3 primer pairs (blue arrows) that cannot detect the properly rearranged macronuclear forms. One pair (a+b) can detect the micronuclear form (containing IESs indicated by gray rectangles, upper panel). The unusual products presumably derived from the mixed-type gene pairs of II+V (amplified by primer a and b), II+IV (amplified by primer a and c), and IV+V (amplified by primer d and b) were detected in the selfers but not in the control cells or the mixture of 2 mating types. The expected micronuclear form was detected when using primer pair a and b in normal cells (and was likely outcompeted by the much more abundant unusual product in selfers). These unusual products are likely generated from the macronuclear forms shown in the lower left panels and are verified by DNA sequencing. (D) Examination of the mat locus in MT II and II+V cells by Southern hybridization. Left panels illustrate the mat locus of the micronucleus and the normal (MT II type) and the unusual (II+V mixed-type) mat genes in the macronucleus. IESs in micronucleus are indicated by gray rectangles. The probe used is indicated by the black bar. Xs indicate the Xba I sites. Cellular DNA from the ΔTKU80 selfer E4, l3, b3, 7 stabilized MT II subclones of b3, 8 selfer subclones of b3, and control cells was analyzed. Unusual product corresponding to the mixed-type genes of II+V (black arrowhead) was detected in selfer E4, l3, and b3. The selfer b3 contained both fragments corresponding to MT II (yellow arrow) and II+V, which appeared to segregate in its subclones. All 8 b3 selfer subclones maintained the II+V type DNA, whereas the 7 stabilized MT II subclones contained only MT II type DNA and had lost the unusual product. (E) Examination of the mat locus in MT IV cells and related selfers by Southern hybridization. Left panels illustrate the mat locus in the micronucleus and the macronucleus in MT IV cells and selfers with the unusual II+IV type. IESs in micronucleus are indicated by gray rectangles. The probe is indicated by the black bar. Xs indicate Xba I sites. Cellular DNA from the ΔTKU80 selfer m3, u4, k4, and control cells was analyzed. The unusual product corresponding to mixed-type II+IV (black arrowhead) was detected in selfer m3 and u4. Selfer u4 contains both DNA fragments corresponding to MT IV (pink arrow) and II+IV. Raw images associated with this figure can be found in S1 Raw Images. IES, internal eliminated sequence; Mac, macronucleus; Mic, micronucleus; MT, mating type.
Fig 3.
Unusual mat gene structures are formed during conjugation.
(A) Detection of the unusual II+IV and IV+V mixed-type genes in the macronucleus during conjugation. Left panels illustrate the presumed unusual mat gene structures and the PCR primer locations. Cellular DNA of ΔTKU80 selfers, normal cells, conjugating ΔTKU80 normal cells at different time points (0, 8, 10, 12, 14, 16, 18, 20, 24, and 36 hours post-mixing) and after conjugation and feeding (3 and 6 hours) was examined by PCR. These unusual products were detected at late conjugation in both normal and mutant cells, suggesting that they could be unfinished mat rearrangement intermediates [10]. (B) Unusual mat gene products persist long after conjugation in mutants but not normal cells. Left panels illustrate the unusual mat gene structures and the locations of the PCR primers. DNA of ΔTKU80, normal cells, conjugating ΔTKU80 and normal cells at different hours during conjugation (16 and 24 hours post-mixing) and at different numbers of fission after conjugation (1F, 4F, 10F, 22F, 34F, 40F, 52F, 64F, 82F, and 100F) was examined by PCR. α-tub gene served as the template control. These likely unfinished intermediates were detected at late conjugation and were lost later during vegetative growth in normal cells. In ΔTKU80 cells, these unusual structures were maintained in vegetative growth. (C) Examination of unusual mat genes during and after conjugation by Southern hybridization. Left panels illustrate the mat locus in the micronucleus and the normal and unusual structures in the macronucleus. IESs in the micronucleus are indicated by gray rectangles and the probe by the black bar. Bs and Ps indicate the Bgl II and Pst I sites, respectively. Cellular DNA from normal cells, TKU80 germline KO strains, ΔTKU80 selfer E4, and normal and TKU80 mutant cells during and after conjugation was analyzed. In normal cells, these unusual structures were observed in late conjugation and disappeared at approximately 10 fissions after conjugation and feeding. However, they were abundantly present and did not disappear in progeny of ΔTKU80 cells. Raw images associated with this figure can be found in S1 Raw Images. Conj, conjugation; F, fissions; IES, internal eliminated sequence; Mac, macronucleus; Mic, micronucleus; MT, mating type.
Fig 4.
Selfers are generated by silencing TKU70-2 or TKU80 after conjugation.
Hairpin RNA to induce gene silencing was induced at approximately 1 fission time and terminated at approximately 22 fissions or continuously during vegetative growth. The ratios of selfing cells without silencing, temporary silencing (1–22 fissions), and continuous silencing are measured and shown in the boxplot. Selfing was examined after 69 vegetative fissions. Selfers increased significantly after silencing TKU70-2 or TKU80 (n = 5; p-values: *p < 0.05; **p < 0.01, Wilcoxon rank-sum test) but not TKU70-2 or the GFP control. The data underlying this figure can be found in S1 Data. Thick horizontal lines represent medians of each distribution, the open box shows the middle 2 quartiles, and the circles show outliers. GFP, green fluorescent protein.
Fig 5.
Phenotypic assortment was not significantly affected in other loci in the absence of TKU80.
Caryonides containing heterozygous Chx and Mpr were examined in serial vegetative fissions. The cumulative proportions of drug-sensitive clones in different fissions are illustrated in up (cycloheximide) and down (6-methylpurine) panels with the regression lines (gray dash) and regression equations. The difference of the slops was not significant between control and ΔTKU80 cells in Chx (p = 0.4797, t test) and Mpr (p = 0.2229, t test). n = 54 (control cells); n = 104 (ΔTKU80 cells). The data underlying this figure can be found in S1 Data. Chx, cycloheximide resistance; Mpr, 6-methylpurine resistance.
Fig 6.
Mating compatibility of the selfers.
Tester cells of 6 different MTs were labeled with NHS-Rhodamine. After starvation, selfers were mixed with tester cells at the ratio of 2:1. Selfing and/or nonselfing (pairing with tester) pairs were fixed and analyzed under a microscope at 4 hours post-mixing. (A) Selfer k4 (containing both MT IV and MT VI genes) formed mating pairs with all 6 tester cells. (B) Selfer E4 (containing mixed-type II+V genes) formed mating pairs with 5 tester cells but not with MT V cells. (C and D) Subclones of selfer b3 that containing only the mixed-type II+V genes showed the same mating compatibility as selfer E4. (D) Subclones of selfer b3 that contained both MT II and the mixed-type II+V genes could form mating pairs with all strains including MT V cells. Pair ratio is indicated in the parentheses. n = 100 pairs. The data underlying this figure can be found in S1 Data. MT, mating type.
Fig 7.
Predicted model of mating compatibility in T. thermophila.
The 2-component model of self/nonself-recognition for mating is illustrated. MTA of 1 mating partner interacts with different mating-type MTB of the other partner reciprocally (dash line). Cells of the same mating type cannot form mating pairs because of the lack of either interaction (solid X mark). Although there was only 1 interaction of MTA and MTB, loose mating pairs formed if cells possessing different MTB (hollow X mark); no mating pair formed if cells possessing the same MTB (solid X mark). When the selfer II+V (MTA2/MTB5) cells encounter normal MT II (MTA2/MTB2) cells, the active form of MTB5 (without MTA5 inhibition) forms stable interaction with MTA2 of MT II, which allows loose pairs to form. When the selfer II+V (MTA2/MTB5) encountering normal MT V (MTA5/MTB5) cells, no interaction occurs between the selfer MTB5 and the MT V MTA5. Therefore, the MTB5 of MT V is inhibited by MTA5, and thus, no pairing occurs. Wt represents normal mating-type strains. MT IV/VI represents multiple mat alleles of both MT IV and MT VI. MTA and MTB are represented by A and B, respectively. Gray stars mark the inactive MTBs. The matings involving MTA/MTB knockout strains (second row) are taken from a previous report [10]. MT, mating type.