Volume 16, Issue 3, Pages 479-485 (November 2004) The INO80 Protein Controls Homologous Recombination in Arabidopsis thaliana  Olivier Fritsch, Giovanna Benvenuto, Chris Bowler, Jean Molinier, Barbara Hohn  Molecular Cell  Volume 16, Issue 3, Pages 479-485 (November 2004) DOI: 10.1016/j.molcel.2004.09.034

Figure 1 The atino80-1 Mutant Is Defective in Homologous Recombination (A) The luciferase construct integrated in the genome of reporter line 50B consists of two inactive fragments (LU, UC) of a luciferase reporter gene sharing an identical stretch of 1147 bp (U). HR between the inverted repeats restores a functional luciferase gene (LUC+). (B) Luciferase sectors (yellow) visualizing recombination events on 50B plants. Scale bar = 1 cm. (C) Somatic HR sectors on 14-day-old in vitro-grown atino80-1 and 50B plants. Top, light image; bottom, luciferase-imaging picture. Scale bar = 2 cm. (D) The INO80 transcript level correlates positively with HR frequency. wt, wild-type out-segregants; +/−, heterozygous; −/−, homozygous for the ino80 mutation. Recombination spots were counted on 20-day-old plants, and HR frequency was normalized against mean value of that in wild-type plants. Error bars = SEM. (E) INO80 transcript level. Northern blot hybridization with RNA isolated from the corresponding plants shown in (D). Ribosomal RNA revealed by ethidium bromide staining of the gel before hybridization was used as a loading control. (F) Gene organization and position of the T-DNA insertions in atino80-1, atino80-2, and atino80-3. Black boxes, exons; INO-IR, sequence used for the RNAi constructs. Molecular Cell 2004 16, 479-485DOI: (10.1016/j.molcel.2004.09.034)

Figure 2 AtINO80 Is Responsible for the Mutant HR Phenotype (A) Construct used to downregulate AtINO80 expression level. MASp, mannopine synthase promoter; Fad2i, FAD2 intron; INO-IR, see Figure 1F. (B) Reduction of INO80 transcript level in pooled INO-IR plants as seen by Northern blot probed with DIG-labeled INO80 cDNA. wt, wild-type RNA; vector and INO-IR, RNA from pooled plants transgenic for the empty vector and the INO-IR construct, respectively, in the 50B background. Loading control as for Figure 1E. (C) The HR phenotype is reproducible and is not locus-dependent. Plants similar to those in (B) were tested for HR. In addition, the GUS HR reporter line 1445 was used in a similar experimental setup. After counting recombination spots, HR frequency in INO-IR plants was normalized to the mean value of that in 50B or 1445 vector control plants, respectively. Error bars = SEM. (D) atino80-1 has a mild branching phenotype. Development of atino80-1 homozygous plants compared with that of wild-type out-segregants (INO80+/+). Scale bar = 5 cm. (E) Quantification of the increased branching of ino80-1 plants. The number of lateral shoots emerging from the rosette (left) and the total length of these lateral shoots (right) was meseared for ino80-1 (n = 20) and wild-type out-segregants (wt, n = 19) plants. Values are the mean value ± SEM per plant for one representative experiment out of three. Molecular Cell 2004 16, 479-485DOI: (10.1016/j.molcel.2004.09.034)

Figure 3 AtINO80 Is the INO80 Arabidopsis Ortholog (A) Phylogenic tree of the INO80 SWI/SNF subfamily of proteins obtained by aligning the ATPase domains of proteins representative of the SWI/SNF subfamilies together with INO80 subfamily members. See Supplemental Experimental Procedures for tree building methods and accession numbers. At, Arabidopsis thaliana; Dm, Drosophila melanogaster; Hs, Homo sapiens; Os, Oriza sativa; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe. (B) Structure of AtINO80 protein. The ATPase motifs are represented in two blocks. QTELY-FD and VYR-RA, the two INO80-specific conserved regions; NLS, nuclear localization signal; vertical white lines, intron positions. (C) INO80 binds to mononucleosomes in vitro. SDS-PAGE analysis of binding of in vitro-translated 35S-labeled INO80 protein to resin-immobilized chicken erythrocyte (Chicken Nu) or plant (Plant Nu) mononucleosomes. IN, INO80 input. Molecular Cell 2004 16, 479-485DOI: (10.1016/j.molcel.2004.09.034)

Figure 4 In atIno80-1, 0.5% of the Transcriptome Is Misregulated, Whereas Genotoxic Stress Sensitivity and T-DNA Integration Are Not Affected (A) Treatment with the DSB-causing agent bleomycin. Seven-day-old in vitro-grown seedlings were submitted to various doses of bleomycin in liquid culture in 96-well plates. After 7 days, the fresh weight was measured for samples of 12 plants each. Values are the mean ± SD for one experiment out of three repeats. (B) Exposure to the genotoxic agent MMS. Plants were grown as above and exposed to MMS for 7 days in liquid culture in 24-well plates. Weight was measured as above; the graph represents one out of three repeats ± SD. (C) Root transformation assay. The percentage of root segments carrying tumors was counted 3 weeks after cocultivation with a tumor inducing Agrobacterium strain. Values are the mean ± SEM of one out of three repeats. The rat5 control is a mutant deficient in T-DNA integration. WS, Wassilewskija ecotype plants as control for the rat5 mutant (Mysore et al., 2000). (D) Full-genome microarray transcriptome analysis of 2-week-old ino80 mutant plants from the three alleles (in duplicate) compared with wild-type plants (four replicates). The repartition of genes with increased (white) or decreased (black) expression levels in functional categories is represented. Molecular Cell 2004 16, 479-485DOI: (10.1016/j.molcel.2004.09.034)