Difference between revisions of "Os09g0104200"
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| − | + | OsRAD51D plays a critical role in reproductive growth in rice. <ref name="ref1" /> | |
==Annotated Information== | ==Annotated Information== | ||
===Function=== | ===Function=== | ||
| + | [[File: Shijc-Os09g0104200-Fig1.png|right|thumb|200px|'' Phenotypic characterization of vegetative and reproductive organs of osrad51d mutant and Ubi:RNAi-OsRAD51D transgenic rice plants. (from reference <ref name="ref1" />).'']] | ||
| + | Osrad51d knock-out and RNAi-mediated knock-down transgenic (Ubi:RNAi-OsRAD51D) plants revealed that suppression of OsRAD51D exerted negligible effects on the vegetative growth of rice plants. In contrast, loss of OsRAD51D caused defects in reproductive growth. Homozygous mutant flowers displayed impaired development of pollen, lemma and palea, stamen, and carpel, which resulted in sterile flowers. The abnormalities of heterozygous mutant and Ubi:RNAi-OsRAD51D plants were intermediate between wild type and homozygous mutant plants. | ||
| + | [[File: Shijc-Os09g0104200-Fig2.png|right|thumb|200px|'' Cytogenetic analysis of root tips, PMCs, and pollen cells from wild type (WT) andosrad51d mutant rice plants. (from reference <ref name="ref1" />).'']] | ||
| + | Furthermore, previous studies in Arabidopsis indicate that AtRAD51B, AtRAD51D, and AtXRCC2 are not essential for meiosis <ref name="ref2" /><ref name="ref3" /><ref name="ref4" />. Triple, as well as single/double, mutations of AtRAD51B, AtRAD51D, and AtXRCC2 result in normal growth and fertility <ref name="ref5" />. These results are in sharp contrast with our results in that loss of OsRAD51D function caused severe errors in floral development and infertility. Cytogenetic analysis showed that osrad51d PMCs were unable to form normal homologous chromosome pairings and most mutant chromosomes were fragmented and scattered throughout the cells during meiosis I. Due to these meiotic anomalies, homozygous osrad51d pollen cells contained numerous micro-nuclei and formed atypical tetrads, resulting in malfunctioning pollen. Taken together, our results are consistent with the view that, unlike Arabidopsis AtRAD51D, OsRAD51D is critically involved in meiosis and is an essential factor for reproductive growth in rice plants. Nevertheless, it is still possible that absence of OsRAD51D could be affecting cellular stability of other OsRAD51 paralogs and thus producing an indirect effect on meiosis and floral development. A previous study of RAD51 in moss Physcomitrella patens suggested that there are fundamental differences in the use of the HR pathway in different plant species, and that an essential role of RAD51 in viability does not correlate with organism taxonomic complexity<ref name="ref6" />. | ||
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| + | ===Mutation=== | ||
| + | [[File: Shijc-Os09g0104200-Fig3.png|right|thumb|200px|'' Molecular characterization of T-DNA osrad51d knock-out mutant and Ubi:RNAi-OsRAD51D knock-down transgenic rice plants. (from reference <ref name="ref1" />).'']] | ||
| + | A single loss-of-function rice mutant line that contained a T-DNA insertion in the OsRAD51D gene was obtained (Yi and An, 2013; http://signal.salk.edu/cgi-bin/RiceGE). This mutant was named osrad51d. The T-DNA insertion was mapped to the third exon in OsRAD51D located on chromosome 9 (line PFG_4A-00300.R). A homozygous line for the T-DNA insertion was identified by genomic PCR with FW2/RV3 and RB/RV3 primer sets. Disruption of OsRAD51D by T-DNA insertion was further verified by RT-PCR. The results showed that the homozygous osrad51d mutant leaves contained undetectable amounts of all three OsRAD51D isoform transcripts under the experimental conditions, whereas the heterozygous osrad51d line possessed reduced levels (approximately 50%) of the transcripts. Genomic Southern blot analysis indicated that the osrad51d mutant progeny contained a single T-DNA integration into the OsRAD51D gene. Because only one allele of the osrad51d loss-of-function mutant plant was obtained, RNAi-mediated knock-down transgenic rice lines (Ubi:RNAi-OsRAD51D) were generated. RT-PCR revealed that the mRNA levels of OsRAD51D.1/OsRAD51D.3 and OsRAD51D.2 significantly decreased in the independent T2 Ubi:RNAi-OsRAD51D lines #1 and #2. Genomic Southern blotting demonstrated that these two RNAi-transgenic plants were independent lines . | ||
===Expression=== | ===Expression=== | ||
| − | + | [[File: Shijc-Os09g0104200-Fig4.png|right|thumb|200px|'' Identification and expression of OsRAD51D in rice. (from reference <ref name="ref1" />).'']] | |
| − | + | OsRAD51D.1/OsRAD51D.3 and OsRAD51D.2 isoforms were expressed in all tissues examined in rice plants. A FW1/RV1 primer set detected both OsRAD51D.1 and OsRAD51D.3 mRNAs due to their similar structure. Yeast two-hybrid assay showed that human RAD51B and RAD51D interacted in the presence of RAD51C. In vivo immuno-precipitation results indicated that human AD51 paralogs form the RAD51B-RAD51C-RAD51D-XRCC2 complex. Arabidopsis AtRAD51B and AtRAD51C also interact in yeast cells <ref name="ref7" />. Yeast two-hybrid studies showed that rice OsRAD51D.1 interacted with OsRAD51B and OsRAD51C, whereas OsRAD51D.2 and OsRAD51D.3 were unable to associate with other OsRAD51 paralogs. In vitro pull-down assays also indicated that bacterially expressed OsRAD51D.1, but not OsRAD51D.2 and OsRAD51D.3, was associated with OsRAD51B and OsRAD51C. These results raise the possibility that the OsRAD51D.1 isoform is the major OsRAD51D protein in rice plants. | |
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| − | + | ===Splicing Variants=== | |
| + | There are five RAD51 paralogs, OsRAD51B, OsRAD51C, OsRAD51D, OsXRCC2, and OsXRCC3, in rice. The rice OsRAD51D gene is 5804 bp in length and composed of nine exons and eight introns. Three splicing variants of OsRAD51D were predicted and termed OsRAD51D.1 (GenBank accession no. KJ472480), OsRAD51D.2 (GenBank accession no. KJ472481), and OsRAD51D.3 (GenBank accession no. KJ472482). The coding regions of OsRAD51D.1, OsRAD51D.2, and OsRAD51D.3 are 849 bp encoding 283 amino acids (30.3 kDa), 552 bp encoding 184 amino acids (19.8 kDa), and 768 bp encoding 256 amino acids (27.4 kDa), respectively All three deduced OsRAD51D proteins contained Walker A and Walker B motifs and a RecA domain in their central regions. Walker A and Walker B motifs function as nucleotide binding domains <ref name="ref8" />, whereas the RecA motif is an ATP hydrolysis domain <ref name="ref9" />. Both Walker A and Walker B motifs are highly conserved in plant (rice, wheat, maize, and Arabidopsis) and human RAD51D homologs. The RecA domain in OsRAD51D is 45–76% and 29% identical to those in plant and human RAD51D proteins, respectively. | ||
==Labs working on this gene== | ==Labs working on this gene== | ||
| − | + | 1. Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea | |
==References== | ==References== | ||
| − | + | <references> | |
| + | <ref name="ref1"> Byun, M. Y. and Kim, W. T. (2014), Suppression of OsRAD51D results in defects in reproductive development in rice (Oryza sativa L.). The Plant Journal, 79: 256–269. doi: 10.1111/tpj.12558 </ref> | ||
| + | <ref name="ref2">Bleuyard, J.Y., Gallego, M.E., Savigny, F. and White, C.I. (2005) Differing requirements for the Arabidopsis Rad51 paralogs in meiosis and DNA repair. Plant J. 41, 533–545. </ref> | ||
| + | <ref name="ref3">Durrant, W.E., Wang, S. and Dong, X. (2007) Arabidopsis SNI1 and RAD51D regulate both gene transcription and DNA recombination during the defense response. Proc. Natl Acad. Sci. USA, 104, 4223–4227. </ref> | ||
| + | <ref name="ref4">Da Ines, O., Degroote, F., Amiard, S., Goubely, C., Gallego, M.E. and White, C.I. (2013) Effects of XRCC2 and RAD51B mutations on somatic and meiotic recombination in Arabidopsis thaliana. Plant J. 74, 959–970. </ref> | ||
| + | <ref name="ref5">Wang, Y., Xiao, R., Wang, H., Cheng, Z., Li, W., Zhu, G., Wang, Y. and Ma, H. (2013) The Arabidopsis RAD51 paralogs RAD51B, RAD51D and XRCC2 play partially redundant roles in somatic DNA repair and gene regulation. New Phytol. 201, 292–304. </ref> | ||
| + | <ref name="ref6">Markmann-Mulisch, U., Wendeler, E., Zobell, O., Schween, G., Steinbiss, H.H. and Reissa, B. (2007) Differential requirements for RAD51 in Physcomitrella patens and Arabidopsis thaliana development and DNA damage repair. Plant Cell, 19, 3080–3089. </ref> | ||
| + | <ref name="ref7">Osakabe, K., Abe, K., Yamanouchi, H. et al. (2005) Arabidopsis Rad51B is important for double-strand DNA breaks repair in somatic cells. Plant Mol. Biol. 57, 819–833. </ref> | ||
| + | <ref name="ref8">Hanson, P.I. and Whiteheart, S.W. (2005) AAA+ proteins: have engine, will work. Nat. Rev. Mol. Cell Biol. 6, 519–529.</ref> | ||
| + | <ref name="ref9">Cox, M.M. (2007) Motoring along with the bacterial RecA protein. Nat. Rev. Mol. Cell Biol. 8, 127–138. </ref> | ||
| + | </references> | ||
==Structured Information== | ==Structured Information== | ||
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[[Category:Genes]] | [[Category:Genes]] | ||
[[Category:Japonica mRNA]] | [[Category:Japonica mRNA]] | ||
Latest revision as of 09:37, 12 June 2015
OsRAD51D plays a critical role in reproductive growth in rice. [1]
Contents
Annotated Information
Function
Osrad51d knock-out and RNAi-mediated knock-down transgenic (Ubi:RNAi-OsRAD51D) plants revealed that suppression of OsRAD51D exerted negligible effects on the vegetative growth of rice plants. In contrast, loss of OsRAD51D caused defects in reproductive growth. Homozygous mutant flowers displayed impaired development of pollen, lemma and palea, stamen, and carpel, which resulted in sterile flowers. The abnormalities of heterozygous mutant and Ubi:RNAi-OsRAD51D plants were intermediate between wild type and homozygous mutant plants.
Furthermore, previous studies in Arabidopsis indicate that AtRAD51B, AtRAD51D, and AtXRCC2 are not essential for meiosis [2][3][4]. Triple, as well as single/double, mutations of AtRAD51B, AtRAD51D, and AtXRCC2 result in normal growth and fertility [5]. These results are in sharp contrast with our results in that loss of OsRAD51D function caused severe errors in floral development and infertility. Cytogenetic analysis showed that osrad51d PMCs were unable to form normal homologous chromosome pairings and most mutant chromosomes were fragmented and scattered throughout the cells during meiosis I. Due to these meiotic anomalies, homozygous osrad51d pollen cells contained numerous micro-nuclei and formed atypical tetrads, resulting in malfunctioning pollen. Taken together, our results are consistent with the view that, unlike Arabidopsis AtRAD51D, OsRAD51D is critically involved in meiosis and is an essential factor for reproductive growth in rice plants. Nevertheless, it is still possible that absence of OsRAD51D could be affecting cellular stability of other OsRAD51 paralogs and thus producing an indirect effect on meiosis and floral development. A previous study of RAD51 in moss Physcomitrella patens suggested that there are fundamental differences in the use of the HR pathway in different plant species, and that an essential role of RAD51 in viability does not correlate with organism taxonomic complexity[6].
Mutation
A single loss-of-function rice mutant line that contained a T-DNA insertion in the OsRAD51D gene was obtained (Yi and An, 2013; http://signal.salk.edu/cgi-bin/RiceGE). This mutant was named osrad51d. The T-DNA insertion was mapped to the third exon in OsRAD51D located on chromosome 9 (line PFG_4A-00300.R). A homozygous line for the T-DNA insertion was identified by genomic PCR with FW2/RV3 and RB/RV3 primer sets. Disruption of OsRAD51D by T-DNA insertion was further verified by RT-PCR. The results showed that the homozygous osrad51d mutant leaves contained undetectable amounts of all three OsRAD51D isoform transcripts under the experimental conditions, whereas the heterozygous osrad51d line possessed reduced levels (approximately 50%) of the transcripts. Genomic Southern blot analysis indicated that the osrad51d mutant progeny contained a single T-DNA integration into the OsRAD51D gene. Because only one allele of the osrad51d loss-of-function mutant plant was obtained, RNAi-mediated knock-down transgenic rice lines (Ubi:RNAi-OsRAD51D) were generated. RT-PCR revealed that the mRNA levels of OsRAD51D.1/OsRAD51D.3 and OsRAD51D.2 significantly decreased in the independent T2 Ubi:RNAi-OsRAD51D lines #1 and #2. Genomic Southern blotting demonstrated that these two RNAi-transgenic plants were independent lines .
Expression
OsRAD51D.1/OsRAD51D.3 and OsRAD51D.2 isoforms were expressed in all tissues examined in rice plants. A FW1/RV1 primer set detected both OsRAD51D.1 and OsRAD51D.3 mRNAs due to their similar structure. Yeast two-hybrid assay showed that human RAD51B and RAD51D interacted in the presence of RAD51C. In vivo immuno-precipitation results indicated that human AD51 paralogs form the RAD51B-RAD51C-RAD51D-XRCC2 complex. Arabidopsis AtRAD51B and AtRAD51C also interact in yeast cells [7]. Yeast two-hybrid studies showed that rice OsRAD51D.1 interacted with OsRAD51B and OsRAD51C, whereas OsRAD51D.2 and OsRAD51D.3 were unable to associate with other OsRAD51 paralogs. In vitro pull-down assays also indicated that bacterially expressed OsRAD51D.1, but not OsRAD51D.2 and OsRAD51D.3, was associated with OsRAD51B and OsRAD51C. These results raise the possibility that the OsRAD51D.1 isoform is the major OsRAD51D protein in rice plants.
Splicing Variants
There are five RAD51 paralogs, OsRAD51B, OsRAD51C, OsRAD51D, OsXRCC2, and OsXRCC3, in rice. The rice OsRAD51D gene is 5804 bp in length and composed of nine exons and eight introns. Three splicing variants of OsRAD51D were predicted and termed OsRAD51D.1 (GenBank accession no. KJ472480), OsRAD51D.2 (GenBank accession no. KJ472481), and OsRAD51D.3 (GenBank accession no. KJ472482). The coding regions of OsRAD51D.1, OsRAD51D.2, and OsRAD51D.3 are 849 bp encoding 283 amino acids (30.3 kDa), 552 bp encoding 184 amino acids (19.8 kDa), and 768 bp encoding 256 amino acids (27.4 kDa), respectively All three deduced OsRAD51D proteins contained Walker A and Walker B motifs and a RecA domain in their central regions. Walker A and Walker B motifs function as nucleotide binding domains [8], whereas the RecA motif is an ATP hydrolysis domain [9]. Both Walker A and Walker B motifs are highly conserved in plant (rice, wheat, maize, and Arabidopsis) and human RAD51D homologs. The RecA domain in OsRAD51D is 45–76% and 29% identical to those in plant and human RAD51D proteins, respectively.
Labs working on this gene
1. Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea
References
- ↑ 1.0 1.1 1.2 1.3 1.4 Byun, M. Y. and Kim, W. T. (2014), Suppression of OsRAD51D results in defects in reproductive development in rice (Oryza sativa L.). The Plant Journal, 79: 256–269. doi: 10.1111/tpj.12558
- ↑ Bleuyard, J.Y., Gallego, M.E., Savigny, F. and White, C.I. (2005) Differing requirements for the Arabidopsis Rad51 paralogs in meiosis and DNA repair. Plant J. 41, 533–545.
- ↑ Durrant, W.E., Wang, S. and Dong, X. (2007) Arabidopsis SNI1 and RAD51D regulate both gene transcription and DNA recombination during the defense response. Proc. Natl Acad. Sci. USA, 104, 4223–4227.
- ↑ Da Ines, O., Degroote, F., Amiard, S., Goubely, C., Gallego, M.E. and White, C.I. (2013) Effects of XRCC2 and RAD51B mutations on somatic and meiotic recombination in Arabidopsis thaliana. Plant J. 74, 959–970.
- ↑ Wang, Y., Xiao, R., Wang, H., Cheng, Z., Li, W., Zhu, G., Wang, Y. and Ma, H. (2013) The Arabidopsis RAD51 paralogs RAD51B, RAD51D and XRCC2 play partially redundant roles in somatic DNA repair and gene regulation. New Phytol. 201, 292–304.
- ↑ Markmann-Mulisch, U., Wendeler, E., Zobell, O., Schween, G., Steinbiss, H.H. and Reissa, B. (2007) Differential requirements for RAD51 in Physcomitrella patens and Arabidopsis thaliana development and DNA damage repair. Plant Cell, 19, 3080–3089.
- ↑ Osakabe, K., Abe, K., Yamanouchi, H. et al. (2005) Arabidopsis Rad51B is important for double-strand DNA breaks repair in somatic cells. Plant Mol. Biol. 57, 819–833.
- ↑ Hanson, P.I. and Whiteheart, S.W. (2005) AAA+ proteins: have engine, will work. Nat. Rev. Mol. Cell Biol. 6, 519–529.
- ↑ Cox, M.M. (2007) Motoring along with the bacterial RecA protein. Nat. Rev. Mol. Cell Biol. 8, 127–138.
