The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens

Abstract

The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G2 of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency.

Fig 1. Growth responses of P. patens to treatment with bleomycin.

(A) Chronic exposure. Tissue explants were inoculated on BCDAT medium supplemented with bleomycin at 0, 0.32, 1.6, 8, 40, 200 and 1000 ng.ml^-1 bleomycin as indicated, and incubated for 10 days under standard growth conditions. (B) Chronic exposure. Colony areas (mean ± SD) for plants exposed to bleomycin. Colony areas in the plates illustrated in Fig 1a were determined by image analysis of digital photographs of the individual plates, and are presented as the mean colony areas normalized to the area of the culture dish for each treatment. (C) Acute treatment. Tissue was incubated for 24h in BCDAT liquid medium supplemented with bleomycin at the concentrations indicated. Explants were then inoculated on drug-free medium and incubated under standard growth conditions for 23d and photographed at 9, 16 and 23d for image analysis. Mean colony areas (± SD, n = 12) were normalized to the area of the culture dish.

Fig 2. Transcriptional responses of PpRad51 genes to bleomycin treatment.

Tissue was incubated for 24 hours in BCDAT liquid medium supplemented with bleomycin at concentrations of 1.6, 8, 40 and 200ng.ml^-1 and 1 and 5 μg.ml^-1 respectively, as indicated. RNA was extracted for determination of transcript levels by real-time qPCR. The fold changes in transcript abundance relative to control (drug-free) treatments are shown (means ± SEM). Black bars: Rad51-1 mRNA; Grey bars: Rad51-2 mRNA.

Fig 3. Time-course of the transcriptional response of selected DNA repair genes.

Tissue was incubated in BCDAT medium supplemented with 200ng.ml^-1 for 1, 4, 8, 16 and 24 hours prior to harvest. RNA was extracted for determination of transcript levels by real-time qPCR. The fold changes in transcript abundance relative to control (drug-free) treatments are shown (means ± SEM). (A) Rad51-1 mRNA (black bars); Rad51-2 mRNA (grey bars). (B) Ku70 (black bars); Ku80 (grey bars). (C) PARP-1 (Pp3c22_13240V3.1) (black bars); PARP-2 (Pp3c8_17220V3.1) (grey bars); (D) SRS2-like helicase (Pp3c1_29170V3.1) (black bars); Alc1-like helicase (Pp3c10_6710V3.1) (grey bars).

Fig 4. Bleomycin sensitivity of P. patens mutants.

Tissue explants of wild-type and mutant protonemal tissue were inoculated on BCDAT agar medium, supplemented with a 5-fold dilution series of bleomycin (1.6ng.ml^-1 – 200ng.ml^-1). Plants were grown for a period of approximately 3 weeks, and plant growth was determined by image analysis of digital photographs taken at intervals to generate the Growth Index. WT and mutant plants (8–12 explants) were inoculated on the same plates for direct comparison. Because individual experiments took place at intervals over a period of 2 years, direct comparisons cannot be made between each pair of panels, as different batches of bleomycin with varying potency were used during this time. Mutants analysed (from top) were Ppsrs2-KO; Pprtel1-KO (note the 5-fold difference in the amplitudes of the y-axes); Ppteb-KO and Pctip-KO. Representative images of plant growth are shown in S9 Fig.