Trichoderma reesei Rad51 tolerates mismatches in hybrid meiosis with diverse genome sequences

Abstract

Most eukaryotes possess two RecA-like recombinases (ubiquitous Rad51 and meiosis-specific Dmc1) to promote interhomolog recombination during meiosis. However, some eukaryotes have lost Dmc1. Given that mammalian and yeast Saccharomyces cerevisiae (Sc) Dmc1 have been shown to stabilize recombination intermediates containing mismatches better than Rad51, we used the Pezizomycotina filamentous fungus Trichoderma reesei to address if and how Rad51-only eukaryotes conduct interhomolog recombination in zygotes with high sequence heterogeneity. We applied multidisciplinary approaches (next- and third-generation sequencing technology, genetics, cytology, bioinformatics, biochemistry, and single-molecule biophysics) to show that T. reesei Rad51 (TrRad51) is indispensable for interhomolog recombination during meiosis and, like ScDmc1, TrRad51 possesses better mismatch tolerance than ScRad51 during homologous recombination. Our results also indicate that the ancestral TrRad51 evolved to acquire ScDmc1-like properties by creating multiple structural variations, including via amino acid residues in the L1 and L2 DNA-binding loops.

Keywords: Dmc1; Rad51; Trichoderma; homologous recombination; meiosis.

Fig. 1.

T. reesei QM6a/CBS999.97(MAT1-1) hybrid meiosis generates interhomolog recombination products. (A) NGS-based mapping of meiotic recombination products. The first trace represents a graph of GC content (window size 5,000 bp) for the telomere-to-telomere sequence of the first QM6a chromosome. Nucleotide sequences of QM6a (in blue), CBS999.97(MAT1-1) (in red) and the four representative F1 progeny are shown. Due to the short lengths of NGS reads, it is difficult to accurately assemble the nucleotide sequences of chromosome regions (in white) hosting repetitive and/or high AT-biased sequences. (B) TGS-based mapping of meiotic recombination products using the newly developed software program TSETA (31). The first two horizontal rows of sequence data represent the full-length sequences of the third chromosomes (ChIII) of QM6a (in cyan) and CBS999.97(MAT1-1) (in magenta). The next four horizontal rows of sequence data represent full-length ChIII of the four representative F1 progeny, respectively. Nucleotide sequences identical to those of parental QM6a and CBS999.97(MAT1-1) are also indicated in cyan and magenta, respectively. COs are located where 2:2 markers undergo a reciprocal genotype change. The strain-specific or gapped (deletion) regions are colored white. The positions of COs, 0:4, 1:3, 3:1, and 4:0 SNP or InDel markers, as well as RIP mutations, IMs, and IDs (i.e., 1n:3, 2n:2, 3n:1, or 4n:0 segregation markers) are indicated by vertical lines (31).

Fig. 2.

Comparative analysis of all COs and NCOs in the three QM6a/CBS999.97(MAT1-1) hybrid asci. The NGS results of 23 different SK1/S288c tetrads published previously (11) were reanalyzed here. (A) Cumulative distribution of the GC lengths associated with CO and/or NCO during hybrid meiosis of QM6a/CBS999.97(MAT1-1) and SK1/S288c. TSETA detected 1,715 putative NCO products (with one or more 0:4, 1:3, 3:1, or 4:0 markers) in the first asci (Dataset S2). However, the majority of these are “false-positive” NCOs due to either RIP or InDels associated with poly-A/T or poly-G/C low-complexity regions. Accordingly, only NCOs with ≥2 APDs (i.e., the sum of SNPs + InDels) and ≥1 SNP are analyzed here (SI Appendix, Table S14). (B) Cumulative distribution of APD density in the CO-associated GC tracts is shown. Two-sample Kolmogorov–Smirnov tests (two-sided) confirmed that both cumulative distributions are significantly different (***P < 0.001).

Fig. 3.

TrRad51 is essential for DSB repair during vegetative growth and meiosis. (A) Generation of T. reesei rad51Δ mutants. The cassettes for removal of rad51. The protein-encoding regions and the hygromycin selectable marker (hph) are indicated. Shaded boxes represent the upstream and downstream sequences of the protein-encoding genes. The restriction enzyme sites are indicated by italics. The three DNA probes for Southern hybridization are indicated by black boxes. For Southern hybridization, genomic DNA was isolated, digested by the indicated restriction enzyme(s), and then visualized by Southern blotting. (B) Western blot analysis. Total cell lysate of indicated T. reesei haploid strains was prepared by using the trichloroacetic acid precipitation protocol we developed previously (39). Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Anti-TrRad51 monoclonal antibodies were used to visualize TrRad51 proteins by Western blotting. (C) MMS resistance. Spot assay showing fivefold serial dilutions of indicated conidia (asexual spores) grown on MEA plates with no or 0.015% (wt/vol) MMS. (D) TrRad51 is dispensable for the formation of stromata. Photographs of representative developing rad51Δ homozygous stromata at indicated day (D) after induction of sexual development. (E) Visualization of asci from WT and rad51Δ homozygous zygotes. Rosettes of asci were dissected from developing fruiting bodies (n > 10), stained with 4′,6-diamidino-2-phenylindole (DAPI), and then visualized by fluorescence microscopy. Representative differential interference contrast (DIC) and DAPI fluorescent images are shown. (F) Percentage of rad51Δ asci with one, two, three, or four DAPI fluorescent spots.

Fig. 4.

TrRad51 can process strand exchange with mismatched substrate. Fluorescence-based strand-exchange assay reveals a better mismatch tolerance of TrRad51 for mismatched substrate in strand exchange compared to ScRad51. (A) Schematic of the fluorescence-based strand-exchange assay. Successful strand-exchange events separate two fluorophores, resulting in increased Cy3 signal (see SI Appendix, Materials and Methods). (B) Schematics of homologous and mismatched substrates. (C) ScRad51 (I)- or ScDmc1 (II)-mediated strand exchange with homologous (blue) or mismatched (red) substrates was monitored by Cy3 emission signal at the indicated reaction time. Experimental repeats are presented as dotted curves and statistical analysis of all repeats is reported in E. (D) TrRad51-mediated strand exchange with all substrates was monitored by Cy3 emission signal at the indicated reaction time. (E) The mismatch tolerances of all recombinases with mismatched/homologous substrates were determined at 120 min. **P < 0.01; ****P < 0.0001. Data shown are average values ± SEM from three independent experiments. Statistics was performed by one-way ANOVA with Tukey’s post hoc test.

Fig. 5.

TrRad51 stabilizes mismatched triplex-state DNA. Single-molecule triplex-state stability experiments reveal similar stabilities of TrRad51 on sequences with full homology and sequences containing mismatched DNA. (A) Schematic of single-molecule triplex-state stability experiment. (B–D) Kinetics of triplex-state dissociation of 15-nt full homologous (in orange), 15-nt containing one mismatched sequence (in blue), and 12-nt full homology DNA substrates (in green) measured for ScDmc1 (B), ScRad51 (C), and TrRad51 (D), respectively. The proportions of triplex state DNA are plotted in natural log-scale, and the slope of the plot represents the dissociation rate. Error bars represent the SEM of three replicates. The dissociation rates are listed in SI Appendix, Table S17.

Fig. 6.

Chimeric ScRad51 with TrRad51 L2-specific amino acids can partially rescue the low spore viability phenotype of S288c/SK1 dmc1Δ hed1Δ hybrid diploid cells. (A) Comparison of Rad51 L1 and L2 amino acid sequences from “dual-RecA” and “Rad51-only” sexual eukaryotes. Species names in red indicate “Rad51-only” Pezizomycotina filamentous fungi; species names in blue indicate “Rad51-only” animals. L1/L2 amino acids that are ScRad51-specific and TrRad51-specific are highlighted in blue and red, respectively. Yeast and human Dmc1 were added to the alignment to aid comparison. Amino acids that are completely or partially conserved in these recombinase proteins are shaded in black or gray, respectively. (B) Schematic representation of S. cerevisiae mutant Rad51 proteins (ScRad51) carrying amino acid substitutions derived from T. reesei L1 (TrL1) and/or L2 (TrL2). Amino acids marked in red and blue represent residues with or without substitutions, respectively (SI Appendix, Table S18). (C) MMS resistance. Spot assay showing fivefold serial dilutions of indicated strains grown on YPD plates with 0, 0.01%, or 0.02% MMS. (D) Spore viability. Spore viability of indicated S288c/SK1 hybrid diploid cells was analyzed after 3 d on sporulation media at 30 °C. To score spore viability, only tetrads (but not dyads or triads) were dissected on YPD. Asterisks indicate spore viability of the rad51 mutants that is significantly different from that of WT under the same genetic background with P values calculated using Z-tests of two proportions (*P < .05 and ***P < .001). n.d. (not determined).