Differential requirements for RAD51 in Physcomitrella patens and Arabidopsis thaliana development and DNA damage repair

Abstract

RAD51, the eukaryotic homolog of the bacterial RecA recombinase, plays a central role in homologous recombination (HR) in yeast and animals. Loss of RAD51 function causes lethality in vertebrates but not in other animals or in the flowering plant Arabidopsis thaliana, suggesting that RAD51 is vital for highly developed organisms but not for others. Here, we found that loss of RAD51 function in the moss Physcomitrella patens, a plant of less complexity, caused a significant vegetative phenotype, indicating an important function for RAD51 in this organism. Moreover, loss of RAD51 caused marked hypersensitivity to the double-strand break-inducing agent bleomycin in P. patens but not in Arabidopsis. Therefore, HR is used for somatic DNA damage repair in P. patens but not in Arabidopsis. These data imply fundamental differences in the use of recombination pathways between plants. Moreover, these data demonstrate that the importance of RAD51 for viability is independent of taxonomic position or complexity of an organism. The involvement of HR in DNA damage repair in the slowly evolving species P. patens but not in fast-evolving Arabidopsis suggests that the choice of the recombination pathway is related to the speed of evolution in plants.

Figure 1.

Schematic Representation of P. patens RAD51 Genes and the Gene Targeting Strategy. Schematic representations of Pp RAD51A (A) and Pp RAD51B (B). The gene targeting vectors used to inactivate the genes are shown above (not to scale). Restriction sites used for cloning or DNA gel blot analysis are shown. The probes used for DNA gel blotting and fragment sizes of authentic and replaced genes are shown below the sketch. Black arrows indicate the RAD51 target gene coding regions. Regions of homology (given in base pairs) between target loci and replacement vectors are shown as small boxes. The probes used for DNA gel blotting are shown as black bars. Open arrows denote genes and promoters. PAC-GFP, fusion protein of chloroplast-targeted pale cress and GFP; 35S, cauliflower mosaic virus 35S promoter; nptII, kanamycin resistance gene; sul R, sulfonamide resistance gene.

Figure 2.

DNA Gel Blot Analysis of P. patens RAD51 Transformants. (A) Genomic DNA of transformants obtained by transformation of the wild type with pKOrad51A digested with BamHI, separated by agarose gel electrophoresis, blotted, and the blot hybridized with the probe shown in Figure 1A. Pp RAD51A is located on a 3.16-kb BamHI fragment that is split in fragments of 1.16 and 2.2 kb after gene replacement. Lane 1, transformant 2/1-4; lane 2, transformant 2/1-5; lane 3, transformant 2/1-6; lane 4, transformant 2/1-7, lane 5; the wild type; lane 6, transformant 2/1-9 (Pp rad51A). (B) Genomic DNA of transformants obtained with pKOrad51B in the wild type (transformant names starting with 1) and the Pp rad51A knockout line (transformant names starting with 7) was digested with HindIII, separated by agarose gel electrophoresis, blotted, and the blot hybridized with two different probes. Probe 1 covers the entire gene but mainly detects a 1.43-kb fragment of Pp rad51A in the wild type that shifts to 5.11 bp after replacement. Probe 2 corresponds to the region of Pp RAD51B that is deleted from the genome after gene replacement. Both probes cross-hybridize with Pp RAD51A and detect a 2.31-kb fragment in the wild type that is lost in Pp rad51A knockout lines. Lane 1, transformant 7/2 (Pp rad51AB); lane 2, 1/3 (Pp rad51B); lane 3, 1/7; lane 4, 1/5; lane 5, 1/1 (Pp rad51B-2); lane 6, 1/2; lane 7, 7/1; lane 8, 1/4; lane 9, 7/4; lane 10, 1/6; lane 11, 1/8; lane 12, Pp rad51A; lane 13, the wild type. Closed arrows indicate bands obtained by cross-hybridization with vector sequences. The open arrow indicates a vector cross-hybridizing band, except for lanes 3 and 6 in which the signal is specific for Pp RAD51B sequences.

Figure 3.

P. patens rad51 Double Knockout Mutants Are Affected in Growth. The average increase in size of young Pp rad51A, Pp rad51B, Pp rad51AB, and Pp rad51B-2 P. patens colonies is shown relative to the wild type.

Figure 4.

Vegetative Developmental Phenotypes of P. patens rad51 Mutants. (A) Morphology of wild-type, Pp rad51A, Pp rad51B, and Pp rad51AB P. patens colonies. The picture was taken after 2 weeks of growth. (B) Close-up view of colonies shown in (A) to show the morphology of protonema filaments.

Figure 5.

Phenotypes of P. patens rad51 Mutants at Later Developmental Stages. (A) Wild-type, Pp rad51A, Pp rad51B, and Pp rad51AB gametophores at the stage of spore capsule maturation. (B) Close-up view of the gametophores in (A), showing normal spore capsule maturation in Pp rad51A and Pp rad51B and a delay in development in Pp rad51AB. The inset in the Pp rad51AB panel shows a gametophore at a later stage that carries a normally matured spore capsule. (C) Spores obtained from mature wild-type, Pp rad51A, Pp rad51B, and Pp rad51AB spore capsules. (D) Germination efficiencies of spores obtained from Pp rad51A, Pp rad51B, and Pp rad51AB spore capsules.

Figure 6.

Resistance of P. patens rad51 Mutants to DNA Damage. (A) Resistance to bleomycin. Survival curves of P. patens wild-type, Pp rad51A, Pp rad51B, Pp rad51AB, and Pp rad51B-2 protonema fragments obtained after treatment with 0, 0.3, 1, 3, 10, and 30 units/liter of bleomycin. (B) Resistance to UV light. Survival curves obtained with 5-d-old protonema colonies of P. patens wild type, Pp rad51A, Pp rad51B, Pp rad51AB, and Pp rad51AB-2 after treatment with 0, 10, 50, 100, 175, and 250 mJ/cm² UV.

Figure 7.

Resistance of the Arabidopsis rad51-1 Mutant to DNA Damage. (A) Resistance to bleomycin. At rad51-1 and wild-type seedlings were germinated in the presence of 0, 0.3, 0.75, 1.5, 3.75, and 7.5 units/liter bleomycin, survival was scored, homozygous At rad51-1 individuals were detected by genotyping, and the data normalized and plotted. (B) Resistance to mitomycin C. At rad51-1 and wild-type seedlings were germinated in the presence of 0, 10, 40, 80, and 160 μM mitomycin C, survival was scored, homozygous At rad51-1 individuals were detected by genotyping, and the data normalized and plotted.