Suppression of OsRAD51D results in defects in reproductive development in rice (Oryza sativa L.)

Abstract

SUMMARYThe cellular roles of RAD51 paralogs in somatic and reproductive growth have been extensively described in a wide range of animal systems and, to a lesser extent, in Arabidopsis, a dicot model plant. Here, the Os-RAD51D gene was identified and characterized in rice (Oryza sativa L.), a monocot model crop. In the rice genome, three alternative OsRAD51D mRNA splicing variants, OsRAD51D.1, OsRAD51D.2, and OsRAD51D.3, were predicted. Yeast two-hybrid studies, however, showed that only OsRAD51D.1 interacted with Os-RAD51B and OsRAD51C paralogs, suggesting that OsRAD51D.1 is a functional OsRAD51D protein in rice. Loss-of-function osrad51d mutant rice plants displayed normal vegetative growth. However, the mutant plants were defective in reproductive growth, resulting in sterile flowers. Homozygous osrad51d mutant flowers exhibited impaired development of lemma and palea and contained unusual numbers of stamens and stigmas. During early meiosis, osrad51d pollen mother cells (PMCs) failed to form normal homologous chromosome pairings. In subsequent meiotic progression, mutant PMCs represented fragmented chromosomes. The osrad51d pollen cells contained numerous abnormal micro-nuclei that resulted in malfunctioning pollen. The abnormalities of heterozygous mutant and T2 Ubi:RNAi-OsRAD51D RNAi-knock-down transgenic plants were intermediate between those of wild type and homozygous mutant plants. The osrad51d and Ubi:RNAi-OsRAD51D plants contained longer telomeres compared with wild type plants, indicating that OsRAD51D is a negative factor for telomere lengthening. Overall, these results suggest that OsRAD51D plays a critical role in reproductive growth in rice. This essential function of OsRAD51D is distinct from Arabidopsis, in which AtRAD51D is not an essential factor for meiosis or reproductive development.

OsDMC1 Is Not Required for Homologous Pairing in Rice Meiosis

“…Of these, mutations in OsRAD51C, OsRAD51D, or OsXRCC3 disturb homologous pairing, causing the production of large chromosome fragments at metaphase I. However, the roles of OsRAD51B and OsXRCC2 in rice during meiosis have not been demonstrated (Byun and Kim, 2014;Tang et al, 2014;Zhang et al, 2015). In Arabidopsis, mutations in AtRAD51C or AtXRCC3 lead to the presence of entangled chromosomes interconnected by chromatin bridges, while AtRAD51B, AtRAD51D, and AtXRCC2 have not been proven to participate in meiosis.…”

RAD54 is essential for RAD51-mediated repair of meiotic DSB in Arabidopsis

“…These three genes are highly expressed in meiotic tissues in animals [28–30] and plants [31–33]. In humans, mutation in XRCC2 has been linked to meiotic arrest, azoospermia and infertility [34] and absence of RAD51B or RAD51D lead to meiotic defects in the moss Physcomitrella patens and rice, respectively [35–37]. The Arabidopsis xrcc2 mutant and, to a lesser extent rad51b , have been associated with increased meiotic recombination rates, but all three mutants are fully fertile and present no detectable meiotic defects [24, 38–40].…”

Homologous U-box E3 Ubiquitin Ligases OsPUB2 and OsPUB3 Are Involved in the Positive Regulation of Low Temperature Stress Response in Rice (Oryza sativa L.)

“…Transformed callus was selected on hygromycin B (40 mg L -1 ) and carbenicillin (250 mg L -1 ) containing medium and then transferred to the regeneration medium (2 mg L -1 kinetin, 4% MS medium containing B5 vitamins, 1 mg L -1 NAA, 1.2% phytoagar, 2% sorbitol, and 5% sucrose, pH 5.8). All processes during rice transformation were followed as described previously (Byun and Kim, 2014). Transgenic T0 plants were transplanted to soil and independent T4 overexpressing (lines #1 and #2) and T3 RNAi knock-down (lines #1 and #2) transgenic rice plants were used for phenotypic analysis.…”

“…Total genomic DNA was extracted from developing leaves of wild-type and transgenic rice plants by using the CTAB (2% CTAB, 2% PVP-40, 1.4 M NaCl, 100 mM Tris-HCl, pH 8.0, and 20 mM EDTA) method as described by Byun and Kim (2014). Total genomic DNA (10 μg) was digested with Bam HI or Eco RI restriction enzyme (Thermo Scientific, USA) and separated by electrophoresis on 0.7% agarose gel.…”

“…Total genomic DNA (10 μg) was digested with Bam HI or Eco RI restriction enzyme (Thermo Scientific, USA) and separated by electrophoresis on 0.7% agarose gel. Separated DNA on the gel was transferred to Hybond-N nylon membrane, and the blot was hybridized with 32P-labeled hygromycin B phosphotransferase ( Hph ) probes under high stringency conditions (65°C) as described by Byun and Kim (2014). The autoradiography signals were visualized using the Bio-Imaging Analyzer (BAS2500; Fuji Film, Japan).…”