| 1. |
T Kazama, K Toriyama, A pentatricopeptide repeat-containing gene that promotes the processing of aberrant atp6 RNA of cytoplasmic male-sterile rice, FEBS LETTERS, 10.1016/S0014-5793(03)00480-0, 544, 1-3, 99-102, 2003.06, A fertility restorer gene (Rf-1) of [ms-bo] cytoplasmic male sterility (CMS) in rice has been reported to be responsible for the processing of RNA of aberrant atp6 of mitochondria. We have carried out map-based cloning of the Rf-1 gene and found that a 4.7-kb genomic fragment of a restorer line promoted the processing of aberrant atp6 RNA when introduced into a CMS line. The genomic fragment contained a single open reading frame encoding 18 repeats of the 35 amino acid pentatricopeptide repeat (PPR) motif. The cloned PPR gene is a possible candidate of Rf-1. A non-restoring genotype was identified to have deletions within the coding region. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.. |
| 2. |
Jong-In Park, Makoto Endo, Tomohiko Kazama, Hiroshi Saito, Hirokazu Hakozaki, Yoshinobu Takada, Makiko Kawagishi-Kobayashi, Masao Watanabe, Molecular characterization of two anther-specific genes encoding putative RNA-binding proteins, AtRBP45s, in Arabidopsis thaliana, GENES & GENETIC SYSTEMS, 10.1266/ggs.81.355, 81, 5, 355-359, 2006.10, RRM (RNA-recognition motif) domain is important for the post-transcriptional regulation of gene expression including RNA processing. In our previous study, we found one anther- and/or pollen-specific gene (LjRRM1, previously named as LjMfb-U93) in model legume, Lotus japonicus. Because of the richness of genomic information of another model plant, Arabidopsis thaliana, for functional analysis, we identified and characterized the orthologous genes in A. thaliana. By comparison of the partial nucleotide sequence of LjRRM1 to the public database, we identified three homologous genes (AtRBP45a, AtRBP45b, and AtRBP45c) in A. thaliana genome. Based on promoter analysis, both AtRBP45a and AtRBP45c were specifically expressed in immature anther tissues (tapetum cells) and mature pollen grains of transgenic plants. This expression pattern of AtRBP45a and AtRBP45c is quite similar to that of LjRRM1, indicating that AtRBP45a and AtRBP45c would be orthologous to LjRRM1. Because in another previous experiment, it was shown that proteins having RRM domains were related to pre-mRNA maturation, and as a conclusion, it is possible that LjRRM1, AtRBP45a, and AtRBP45c genes encoding RNA-binding proteins are functionally involved in the repression of translation in mature pollen grains in L. japonicus and A. thaliana.. |
| 3. |
Tokunori Hobo, Keita Suwabe, Koichiro Aya, Go Suzuki, Kentaro Yano, Takeshi Ishimizu, Masahiro Fujita, Shunsuke Kikuchi, Kazuki Hamada, Masumi Miyano, Tomoaki Fujioka, Fumi Kaneko, Tomohiko Kazama, Yoko Mizuta, Hirokazu Takahashi, Katsuhiro Shiono, Mikio Nakazono, Nobuhiro Tsutsumi, Yoshiaki Nagamura, Nori Kurata, Masao Watanabe, Makoto Matsuoka, Various spatiotemporal expression profiles of anther-expressed genes in rice., Plant & cell physiology, 10.1093/pcp/pcn128, 49, 10, 1417-28, 2008.10, The male gametophyte and tapetum play different roles during anther development although they are differentiated from the same cell lineage, the L2 layer. Until now, it has not been possible to delineate their transcriptomes due to technical difficulties in separating the two cell types. In the present study, we characterized the separated transcriptomes of the rice microspore/pollen and tapetum using laser microdissection (LM)-mediated microarray. Spatiotemporal expression patterns of 28,141 anther-expressed genes were classified into 20 clusters, which contained 3,468 (12.3%) anther-enriched genes. In some clusters, synchronous gene expression in the microspore and tapetum at the same developmental stage was observed as a novel characteristic of the anther transcriptome. Noteworthy expression patterns are discussed in connection with gene ontology (GO) categories and gene annotations, which are related to important biological events in anther development, such as pollen maturation, pollen germination, pollen tube elongation and pollen wall formation.. |
| 4. |
Tomohiko Kazama, Takahiro Nakamura, Masao Watanabe, Mamoru Sugita, Kinya Toriyama, Suppression mechanism of mitochondrial ORF79 accumulation by Rf1 protein in BT-type cytoplasmic male sterile rice, PLANT JOURNAL, 10.1111/j.1365-313X.2008.03529.x, 55, 4, 619-628, 2008.08, In BT-type cytoplasmic male sterile rice (Oryza sativa L.) with Chinsurah Boro II cytoplasm, cytoplasmic male sterility (CMS) is caused by an accumulation of the cytotoxic peptide ORF79. The ORF79 protein is expressed from a dicistronic gene atp6-orf79, which exists in addition to the normal atp6 gene in the BT-type mitochondrial genome. The CMS is restored by a PPR (pentatricopeptide-repeat) gene, Rf1, via RNA processing. However, it has not yet been elucidated how the accumulation of ORF79 is reduced by the action of the Rf1 protein. Here, we report that the level of processed orf79 transcripts in the restorer line was reduced to 50% of the unprocessed atp6-orf79 transcripts in the CMS line. Ninety percent of the processed orf79 transcripts, which remained after degradation, were not associated with the ribosome for translation. Our data suggests that the processing of atp6-orf79 transcripts diminishes the expression of orf79 by the translational reduction and degradation of the processed orf79 transcripts.. |
| 5. |
Keita Suwabe, Go Suzuki, Hirokazu Takahashi, Katsuhiro Shiono, Makoto Endo, Kentaro Yano, Masahiro Fujita, Hiromi Masuko, Hiroshi Saito, Tomoaki Fujioka, Fumi Kaneko, Tomohiko Kazama, Yoko Mizuta, Makiko Kawagishi-Kobayashi, Nobuhiro Tsutsumi, Nori Kurata, Mikio Nakazono, Masao Watanabe, Separated transcriptomes of male gametophyte and tapetum in rice: validity of a laser microdissection (LM) microarray., Plant & cell physiology, 10.1093/pcp/pcn124, 49, 10, 1407-16, 2008.10, In flowering plants, the male gametophyte, the pollen, develops in the anther. Complex patterns of gene expression in both the gametophytic and sporophytic tissues of the anther regulate this process. The gene expression profiles of the microspore/pollen and the sporophytic tapetum are of particular interest. In this study, a microarray technique combined with laser microdissection (44K LM-microarray) was developed and used to characterize separately the transcriptomes of the microspore/pollen and tapetum in rice. Expression profiles of 11 known tapetum specific-genes were consistent with previous reports. Based on their spatial and temporal expression patterns, 140 genes which had been previously defined as anther specific were further classified as male gametophyte specific (71 genes, 51%), tapetum-specific (seven genes, 5%) or expressed in both male gametophyte and tapetum (62 genes, 44%). These results indicate that the 44K LM-microarray is a reliable tool to analyze the gene expression profiles of two important cell types in the anther, the microspore/pollen and tapetum.. |
| 6. |
Toriyama K, Fujii S, Toda T, Itabashi E, Yamada M, Kazama T, Moleculae analysis of CW-type cytoplasmic male sterility and Rf17-mediated fertility restoration for hybeid rice breeding., SARAO Journal of Breeding and Genetics, Vol. 41, Special Supplement August 2009 ISSN 1029-7073 CD, 2009.08, Proceeding of The 14th Australian Plant Breeding Conference and 11th Society for the Advancement of Breeding Research in Asia and Oceania (SABRAO) Congress 2009. |
| 7. |
Etsuko Itabashi, Tomohiko Kazama, Kinya Toriyama, Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm, PLANT CELL REPORTS, 10.1007/s00299-008-0625-7, 28, 2, 233-239, 2009.02, Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of 'Lead Rice' and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6-orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of 'Chinsurah Boro II'. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6-orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.. |
| 8. |
Sota Fujii, Tomohiko Kazama, Mari Yamada, Kinya Toriyama, Discovery of global genomic re-organization based on comparison of two newly sequenced rice mitochondrial genomes with cytoplasmic male sterility-related genes, BMC GENOMICS, 10.1186/1471-2164-11-209, 11, 209-224, 2010.03, Background: Plant mitochondrial genomes are known for their complexity, and there is abundant evidence demonstrating that this organelle is important for plant sexual reproduction. Cytoplasmic male sterility (CMS) is a phenomenon caused by incompatibility between the nucleus and mitochondria that has been discovered in various plant species. As the exact sequence of steps leading to CMS has not yet been revealed, efforts should be made to elucidate the factors underlying the mechanism of this important trait for crop breeding.
Results: Two CMS mitochondrial genomes, LD-CMS, derived from Oryza sativa L. ssp. indica (434,735 bp), and CW-CMS, derived from Oryza rufipogon Griff. (559,045 bp), were newly sequenced in this study. Compared to the previously sequenced Nipponbare (Oryza sativa L. ssp. japonica) mitochondrial genome, the presence of 54 out of 56 protein-encoding genes (including pseudo-genes), 22 tRNA genes (including pseudo-tRNAs), and three rRNA genes was conserved. Two other genes were not present in the CW-CMS mitochondrial genome, and one of them was present as part of the newly identified chimeric ORF, CW-orf307. At least 12 genomic recombination events were predicted between the LD-CMS mitochondrial genome and Nipponbare, and 15 between the CW-CMS genome and Nipponbare, and novel genetic structures were formed by these genomic rearrangements in the two CMS lines. At least one of the genomic rearrangements was completely unique to each CMS line and not present in 69 rice cultivars or 9 accessions of O. rufipogon.
Conclusion: Our results demonstrate novel mitochondrial genomic rearrangements that are unique in CMS cytoplasm, and one of the genes that is unique in the CW mitochondrial genome, CW-orf307, appeared to be the candidate most likely responsible for the CW-CMS event. Genomic rearrangements were dynamic in the CMS lines in comparison with those of rice cultivars, suggesting that 'death' and possible 'birth' processes of the CMS genes occurred during the breeding history of rice.. |
| 9. |
Etsuko Itabashi, Natsuko Iwata, Sota Fujii, Tomohiko Kazama, Kinya Toriyama, The fertility restorer gene, Rf2, for Lead Rice-type cytoplasmic male sterility of rice encodes a mitochondrial glycine-rich protein, PLANT JOURNAL, 10.1111/j.1365-313X.2010.04427.x, 65, 3, 359-367, 2011.02, P>Cytoplasmic male sterility (CMS) is associated with a mitochondrial mutation that causes an inability to produce fertile pollen. The fertility of CMS plants is restored in the presence of a nuclear-encoded fertility restorer (Rf) gene. In Lead Rice-type CMS, discovered in the indica variety 'Lead Rice', fertility of the CMS plant is restored by the single nuclear-encoded gene Rf2 in a gametophytic manner. We performed map-based cloning of Rf2, and proved that it encodes a protein consisting of 152 amino acids with a glycine-rich domain. Expression of Rf2 mRNA was detected in developing and mature anthers. An RF2-GFP fusion was shown to be targeted to mitochondria. Replacement of isoleucine by threonine at amino acid 78 of the RF2 protein was considered to be the cause of functional loss in the rf2 allele. As Rf2 does not encode a pentatricopeptide repeat protein, unlike a majority of previously identified Rf genes, the data from this study provide new insights into the mechanism for restoring fertility in CMS.. |
| 10. |
Toda T, Fujii S, Noguchi K, Kazama T, Toriyama K, Rice MPR25 encodes a pentatricopeptide repeat protein and is essential for RNA editing of nad5 transcripts in mitochondria, Plant J., 10.1111/j.1365-313X.2012.05091.x., 2012.07. |
| 11. |
Kenji Osabe, Takahiro Kawanabe, Taku Sasaki, Ryo Ishikawa, Keiichi Okazaki, Elizabeth S. Dennis, Tomohiko Kazama, Ryo Fujimoto, Multiple Mechanisms and Challenges for the Application of Allopolyploidy in Plants, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 10.3390/ijms13078696, 13, 7, 8696-8721, 2012.07, An allopolyploid is an individual having two or more complete sets of chromosomes derived from different species. Generation of allopolyploids might be rare because of the need to overcome limitations such as co-existing populations of parental lines, overcoming hybrid incompatibility, gametic non-reduction, and the requirement for chromosome doubling. However, allopolyploids are widely observed among plant species, so allopolyploids have succeeded in overcoming these limitations and may have a selective advantage. As techniques for making allopolyploids are developed, we can compare transcription, genome organization, and epigenetic modifications between synthesized allopolyploids and their direct parental lines or between several generations of allopolyploids. It has been suggested that divergence of transcription caused either genetically or epigenetically, which can contribute to plant phenotype, is important for the adaptation of allopolyploids.. |
| 12. |
Keiko Kobayashi, Masuyo Kawabata, Keizo Hisano, Tomohiko Kazama, Ken Matsuoka, Mamoru Sugita, Takahiro Nakamura, Identification and characterization of the RNA binding surface of the pentatricopeptide repeat protein, NUCLEIC ACIDS RESEARCH, 10.1093/nar/gkr1084, 40, 6, 2712-2723, 2012.03, The expressions of chloroplast and mitochondria genes are tightly controlled by numerous nuclear-encoded proteins, mainly at the post-transcriptional level. Recent analyses have identified a large, plant-specific family of pentatricopeptide repeat (PPR) motif-containing proteins that are exclusively involved in RNA metabolism of organelle genes via sequence-specific RNA binding. A tandem array of PPR motifs within the protein is believed to facilitate the RNA interaction, although little is known of the mechanism. Here, we describe the RNA interacting framework of a PPR protein, Arabidopsis HCF152. First, we demonstrated that a Pfam model could be relevant to the PPR motif function. A series of proteins with two PPR motifs showed significant differences in their RNA binding affinities, indicating functional differences among PPR motifs. Mutagenesis and informatics analysis putatively identified five amino acids organizing its RNA binding surface [the 1st, 4th, 8th, 12th and 'ii'(-2nd) amino acids] and their complex connections. SELEX (Systematic evolution of ligands by exponential enrichment) and nucleobase preference assays determined the nucleobases with high affinity for HCF152 and suggested several characteristic amino acids that may be involved in determining specificity and/or affinity of the PPR/RNA interaction.. |
| 13. |
Masayuki Okazaki, Tomohiko Kazama, Hayato Murata, Keiji Motomura, Kinya Toriyama, Whole mitochondrial genome sequencing and transcriptional analysis to uncover an RT102-Type cytoplasmic male sterility-associated candidate gene derived from oryza rufipogon, Plant and Cell Physiology, 10.1093/pcp/pct102, 54, 9, 1560-1568, 2013.09, Cytoplasmic male sterility (CMS) is a maternally inherited trait in which plants fail to produce functional pollen and is associated with the expression of a novel open reading frame (orf) gene encoded by the mitochondrial genome. An RT102A CMS line and an RT102C fertility restorer line were obtained by successive backcrossing between Oryza rufipogon W1125 and O. sativa Taichung65. Using next-generation pyrosequen-cing, we determined whole-genome sequences of the mitochondria in RT102-CMS cytoplasm. To identify candidates for the CMS-associated gene in RT102 mitochondria, we screened the mitochondrial genome for the presence of specific orf genes that were chimeric or whose products carried predicted transmembrane domains. One of these orf genes, orf352, which showed different transcript sizes depending on whether the restorer of fertility (Rf) gene was present or not, was identified. The orf352 gene was co-transcribed with the ribosomal protein gene rpl5, and the 2.8 kb rpl5-orf352 transcripts were processed into 2.6 kb transcripts with a cleavage at the inside of the orf352 coding region in the presence of the Rf gene. The orf352 gene is an excellent candidate for the CMS-associated gene for RT102-CMS. © The Author 2013.. |
| 14. |
Keisuke Igarashi, Tomohiko Kazama, Keiji Motomura, Kinya Toriyama, Whole Genomic Sequencing of RT98 Mitochondria Derived from Oryza rufipogon and Northern Blot Analysis to Uncover a Cytoplasmic Male Sterility-Associated Gene, Plant and Cell Physiology, 10.1093/pcp/pcs177, 54, 2, 237-243, 2013.02, Cytoplasmic male sterility (CMS) is a maternally inherited trait resulting in the failure to produce functional pollen and is often observed when an alien cytoplasm is transferred into a cultivated species. An RT98A CMS line and an RT98C fertility restorer line were obtained by successive backcrossing between Oryza rufipogon W1109 and Oryza sativa cultivar Taichung 65. To uncover the CMS-associated mitochondrial genes, we determined the complete sequence of the RT98-CMS mitochondrial genome using next-generation pyrosequencing, and searched new open reading frames (orfs) absent in a reported mitochondrial genome of O. sativa Nipponbare. Then, six candidates were selected for the CMS-associated genes based on the criteria in which they were chimeric in structure or encoded a peptide with transmembrane domains. One of the candidates, orf113, showed different transcript sizes between RT98A and RT98C on Northern blot analysis. The orf113 gene was shown to be co-transcribed with atp4 and cox3 encoding ATP synthase F0 subunit 4 and Cyt c oxidase subunit 3, respectively, and their transcripts were distinctly processed in the presence of a fertility restorer gene. Our results indicate that orf113 is a CMS-associated gene of RT98-CMS. © 2012 The Author 2012. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved.. |
| 15. |
Tomohiko Kazama, Yusuke Yagi, Kinya Toriyama, Takahiro Nakamura, Heterogeneity of the 5 '-end in plant mRNA may be involved in mitochondrial translation, FRONTIERS IN PLANT SCIENCE, 10.3389/fpls.2013.00517, 4, 517, 2013.12. |
| 16. |
Shinya Fujii, Tomohiko Kazama, Yukihiro Ito, Soichi Kojima, Kinya Toriyama, A candidate factor that interacts with RF2, a restorer of fertility of Lead rice-type cytoplasmic male sterility in rice, RICE, 10.1186/s12284-014-0021-6, 7, 21, 2014.10, Background: The pollen function of cytoplasmic male sterile (CMS) plants is often recovered by the Restorer of fertility (Rf) gene encoded by the nuclear genome. An Rf gene of Lead rice type CMS, Rf2, encodes a small mitochondrial glycine-rich protein. RF2 is expected to function by interacting with other proteins, because RF2 has no motifs except for glycine-rich domain.
Findings: To elucidate the protein that interacts with RF2, we performed yeast two-hybrid screening. We identified four genes and named RF2-interacting candidate factors (RIF1 to RIF4). A study of subcellular localization demonstrated that only RIF2 was targeted to mitochondria. A pull-down assay using E. coli-produced recombinant GST-tagged RF2 and His-tagged RIF2 confirmed that RF2 interacted with RIF2. RIF2 encodes ubiquitin domain-containing protein.
Conclusions: These results suggest that RIF2 is a candidate factor of a fertility restoration complex of RF2.. |
| 17. |
Aaron Yap, Peter Kindgren, Catherine Colas des Francs-Small, Tomohiko Kazama, Sandra K. Tanz, Kinya Toriyama, Ian Small, AEF1/MPR25 is implicated in RNA editing of plastid atpF and mitochondrial nad5, and also promotes atpF splicing in Arabidopsis and rice, PLANT JOURNAL, 10.1111/tpj.12756, 81, 5, 661-669, 2015.03, RNA editing is an essential mechanism that modifies target cytidines to uridine in both mitochondrial and plastid mRNA. Target sites are recognized by pentatricopeptide repeat (PPR) proteins. Using bioinformatics predictions based on the code describing sequence recognition by PPR proteins, we have identified an Arabidopsis editing factor required for editing of atpF in plastids. A loss-of-function mutation in ATPF EDITING FACTOR1 (AEF1, AT3G22150) results in severe variegation, presumably due to decreased plastid ATP synthase levels. Loss of editing at the atpF site is coupled with a large decrease in splicing of the atpF transcript, even though the editing site is within an exon and 53 nucleotides distant from the splice site. The rice orthologue of AEF1, MPR25, has been reported to be required for editing of a site in mitochondrial nad5 transcripts, and we confirm that editing of the same site is affected in the Arabidopsis aef1 mutant. We also show that splicing of chloroplast atpF transcripts is affected in the rice mpr25 mutant. AEF1 is thus highly unusual for an RNA editing specificity factor in that it has functions in both organelles.
Significance Statement The RNA editing factor AEF1/MPR25 is a sequence-specific RNA binding protein with an unusual multi-functional role in editing and splicing of the chloroplast atpF transcript. It is unique (so far) amongst editing specificity factors in also acting in mitochondria, where it recognizes a similar sequence to induce editing of the nad5 transcript.. |
| 18. |
Shiro Mitsuya, Norifumi Murakami, Tadashi Sato, Tomohiko Kazama, Kinya Toriyama, Nicola Stephanie Skoulding, Mana Kano-Nakata, Akira Yamauchi, Evaluation of rice grain yield and yield components of Nona Bokra chromosome segment substitution lines with the genetic background of Koshihikari, in a saline paddy field., AoB PLANTS, 10.1093/aobpla/plz040, 11, 5, plz040, 2019.10, The ability to tolerate salt differs with the growth stages of rice and thus the yield components that are determined during various growth stages, are differentially affected by salt stress. In this study, we utilized chromosome segment substitution lines (CSSLs) from Nona Bokra, a salt-tolerant indica landrace, with the genetic background of Koshihikari, a salt-susceptible japonica variety. These were screened to find superior CSSLs under long-term saline conditions that showed higher grain yield and yield components in comparison to Koshihikari. One-month-old seedlings were transplanted into a paddy field without salinity. These were allowed to establish for 1 month further, then the field was flooded, with saline water maintained at 7.41 dS m-1 salinity until harvest. The experiments were performed twice, once in 2015 and a targeted study in 2016. Salt tolerance of growth and reproductive stage parameters was evaluated as the Salt Effect Index (SEI) which was computed as the difference in each parameter within each line between control and saline conditions. All CSSLs and Koshihikari showed a decrease in grain yield and yield components except panicle number under salinity. SL538 showed a higher SEI for grain yield compared with Koshihikari under salinity throughout the two experiments. This was attributed to the retained grain filling and harvest index, yet the mechanism was not due to maintaining Na+, Cl- and K+ homeostasis. Few other CSSLs showed greater SEI for grain weight under salinity compared with Koshihikari, which might be related to low concentration of Na+ in leaves and panicles. These data indicate that substitution of different Nona Bokra chromosome segments independently contributed to the maintenance of grain filling and grain weight of Koshihikari under saline conditions.. |
| 19. |
Toriyama K, Kazama T, Sato T, Fukuta Y, Oka M, Development of cytoplasmic male sterile lines and restorer lines of various elite Indica Group rice cultivars using CW-CMS/Rf17 system., Rice (New York, N.Y.), 10.1186/s12284-019-0332-8, 12, 1, 73, 2019.09. |
| 20. |
Chihiro Suketomo, Tomohiko Kazama, Kinya Toriyama, Fertility restoration of Chinese wild rice-type cytoplasmic male sterility by CRISPR/Cas9-mediated genome editing of nuclear-encoded RETROGRADE-REGULATED MALE STERILITY, PLANT BIOTECHNOLOGY, 10.5511/plantbiotechnology.20.0326b, 37, 3, 285-292, 2020.09, Cytoplasmic male sterility (CMS) is a trait that produces nonfunctional pollen caused by the interaction between mitochondrial and nuclear genes. In Chinese-wild (CW) type CMS, CWA, in rice (Oryza sativa L.), its mitochondria enhance the expression of the nuclear gene RETROGRADE-REGULATED MALE STERILITY (RMS), which causes pollen abortion. Fertility is recovered when its expression decreases in a restorer line, CWR. The expression of RMS is controlled by the single nucleotide polymorphism (SNP) located in the promoter region 2,286 bp upstream of the start codon of RMS. However, another gene, PPR2, which encodes pentatricopeptide repeat-domain containing protein, is predicted in the reverse strand of this region and a premature stop codon is created in CWR by the SNP. To prove RMS is directly involved in restoring fertility of CW-CMS, we introduced mutations into RMS and PPR2 using CRISPR/Cas9. Fertility was recovered in the genome-edited CMS plants with reduced expression of RMS and unaltered expression of PPR2, when the mutation was introduced in the promoter regions of RMS within or outside the coding sequence (CDS) of PPR2. Fertility restoration was not obtained when the mutation was introduced within the CDS of RMS. Our results demonstrated that PPR2 is not responsible for fertility restoration, and fertility was recovered by reduced expression of RMS, providing us with a new artificial fertility restorer line for agronomical use.. |
| 21. |
Shiho Omukai, Shin-ich Arimura, Kinya Toriyama, Tomohiko Kazama, Disruption of mitochondrial open reading frame 352 partially restores pollen development in cytoplasmic male sterile rice, PLANT PHYSIOLOGY, 10.1093/plphys/kiab236, 187, 1, 236-246, 2021.09, Plant mitochondrial genomes sometimes carry cytoplasmic male sterility (CMS)-associated genes. These genes have been harnessed in various crops to produce high-yielding F-1 hybrid seeds. The gene open reading frame 352 (orf352) was reported to be an RT102-type CMS gene in rice (Oryza sativa), although the mechanism underlying its role in CMS is unknown. Here, we employed mitochondrion-targeted transcription activator-like effector nucleases (mitoTALENs) to knockout orf352 from the mitochondrial genome in the CMS rice RT102A. We isolated 18 independent transformation events in RT102A that resulted in genome editing of orf352, including its complete removal from the mitochondrial genome in several plants. Sequence analysis around the mitoTALEN target sites revealed their induced double-strand breaks were repaired via homologous recombination. Near the 5'-target site, repair involved sequences identical to orf284, while repair of the 3'-target site yielded various new sequences that generated chimeric genes consisting of orf352 fragments. Plants with a chimeric mitochondrial gene encoding amino acids 179-352 of ORF352 exhibited the same shrunken pollen grain phenotype as RT102A, whereas plants either lacking orf352 or harboring a chimeric gene encoding amino acids 211-352 of ORF352 exhibited partial rescue of pollen viability and germination, although these plants failed to set seed. These results demonstrated that disruption of orf352 partially restored pollen development, indicating that amino acids 179-210 from ORF352 may contribute to pollen abortion.. |
| 22. |
Ayumu Takatsuka, Tomohiko Kazama, Kinya Toriyama, Cytoplasmic Male Sterility-Associated Mitochondrial Gene orf312 Derived from Rice (Oryza sativa L.) Cultivar Tadukan, RICE, 10.1186/s12284-021-00488-7, 14, 1, 2021.12, Background Cytoplasmic male sterility (CMS) is a trait associated with non-functional pollen or anthers, caused by the interaction between mitochondrial and nuclear genes. Findings A Tadukan-type CMS line (TAA) and a restorer line (TAR) were obtained by successive backcrossing between the Oryza sativa cultivars Tadukan (a cytoplasmic donor) and Taichung 65 (a recurrent pollen parent). Using Illumina HiSeq, we determined whole-genome sequences of the mitochondria of TAA and screened the mitochondrial genome for the presence of open reading frame (orf) genes specific to this genome. One of these orf genes, orf312, showed differential expression patterns in TAA and TAR anthers at the meiotic and mature stages, with transcript amounts in TAR being less than those in TAA. The orf312 gene is similar to the previously described orf288, a part of which is among the components comprising WA352, a chimeric CMS-associated gene of wild-abortive-type CMS. Conclusions The orf312 gene is a promising candidate for CMS-associated gene in TAA.. |
| 23. |
Ayumu Takatsuka, Tomohiko Kazama, Shin-ichi Arimura, Kinya Toriyama, TALEN-mediated depletion of the mitochondrial gene orf312 proves that it is a Tadukan-type cytoplasmic male sterility-causative gene in rice, PLANT JOURNAL, 10.1111/tpj.15715, 110, 4, 994-1004, 2022.05, Cytoplasmic male sterility (CMS) is a trait that causes pollen or anther dysfunctions, resulting in the lack of seed setting. CMS is considered to be caused by the expression of a unique mitochondrial open reading frame referred to as CMS-associated gene. orf312 has been reported as a CMS-associated gene of Tadukan-type CMS (TAA) in rice (Oryza sativa L.), which exhibits impaired anther dehiscence; however, evidence thereof has not yet been reported. Here, we took a loss-of-function approach, using a mitochondria-targeted transcription activator-like effector nuclease (mitoTALEN) designed to knock out orf312 in TAA, to prove that orf312 indeed is a CMS-causative gene. Out of 28 transgenic TAA plants harboring the mitoTALEN expression vector, deletion of orf312 was detected in 24 plants by PCR, Southern blot, and sequencing analyses. The 24 plants were grouped into three groups based on the deleted regions. All orf312-depleted TAA plants exhibited recovery of anther dehiscence and seed setting. The depletion of orf312 and fertility restoration was maintained in the next generation, even in mitoTALEN expression cassette null segregants. In contrast, orf312-retaining plants were sterile. These results provide robust evidence that orf312 is a Tadukan-type CMS-causative gene.. |
| 24. |
Toriyama, K., Kazama, T., Sato, T., Fukuta, Y. & Oka, M., Development of cytoplasmic male sterile lines and restorer lines of various elite Indica Group rice cultivars using CW-CMS/Rf17 system, Rice, 12, 1, 73, 2019.12. |
| 25. |
Mitsuya, S., Murakami, N., Sato, T., Kazama, T., Toriyama, K., Skoulding, N. S., Kano-Nakata, M., Yamauchi, A. & Ismail, A. M., Evaluation of rice grain yield and yield components of Nona Bokra chromosome segment substitution lines with the genetic background of Koshihikari, in a saline paddy field, AoB PLANTS, 11, 5, 2019.10. |
| 26. |
Takatsuka, A., Kazama, T., Arimura, S. I. & Toriyama, K., TALEN-mediated depletion of the mitochondrial gene orf312 proves that it is a Tadukan-type cytoplasmic male sterility-causative gene in rice, PLANT JOURNAL, 10.1111/tpj.15715, 110, 4, 994-1004, 2022.05. |
| 27. |
Omukai, S., Arimura, S. I., Toriyama, K. & Kazama, T.,, Disruption of mitochondrial open reading frame 352 partially restores pollen development in cytoplasmic male sterile rice, PLANT PHYSIOLOGY, 10.1093/plphys/kiab236, 187, 1, 236-246, 2021.09. |
| 28. |
Ayumu Takatsuka, Tomohiko Kazama, Kinya Toriyama, Cytoplasmic Male Sterility-Associated Mitochondrial Gene orf312 Derived from Rice (Oryza sativa L.) Cultivar Tadukan, Rice, doi.org/10.1186/s12284-021-00488-7, 2021.05. |
| 29. |
Suketomo C., Kazama T., Toriyama K., Fertility restoation of Chinese wild rice-type cytoplasmic male sterility by CRISPR/Cas9-mediated genome editing of nuclear-encoded RETROGRADE-REGULATED MALE STERILITY., Plant Biotechnology, vol. 37, 285-292, 2020.09. |
| 30. |
Morita Ryouhei, Ichida Hiroyuki, Ishii Kotaro, Hayashi Yoriko, Abe Hiroshi, Shirakawa Yuki, Ichinose Katsunori, Tsuneizumi Kazuhide, Kazama Tomohiko, Toriyama Kinya, Sato Tadashi, Abe Tomoko, LONG GRAIN 1: a novel gene that regulates grain length in rice, MOLECULAR BREEDING, 10.1007/s11032-019-1032-1, 39, 9, Article number: 135, 2019.09. |
| 31. |
Kazama T, Okuno M, Watari Y, Yanase S, Koizuka C, Tsuruta Y, Sugaya H, Toyoda A, Itoh T, Tsutsumi N, Toriyama K, Koizuka N, Arimura SI, Curing cytoplasmic male sterility via TALEN-mediated mitochondrial genome editing., Nature plants, 10.1038/s41477-019-0459-z, 5, 7, 722-730, 2019.07. |
| 32. |
Tetsuya Murakami, Tomohiko Kazama, Kinya Toriyama, Genetic analysis of male sterility obtained from a rice cultivar Lebed backcrossed with Taichung 65, Rice, 10.1186/s12284-018-0222-5, 11, 1, 2018.12, Background: Male sterility is a useful agronomic trait for breeding of self-pollinating crops and is often observed in the progenies of hybrids of distantly related species, for example, Oryza sativa L. subsp. indica and O. sativa L. subsp. japonica. To explore new male sterile lines in rice, we performed successive backcrosses using a japonica cultivar Taichung 65 (T65) as a recurrent pollen parent and various indica cultivars as the initial female parents. Findings: We observed male sterile plants in the backcross progeny from an indica cultivar, Lebed. Both fertile and sterile plants were present in the BC4F1 generation. The sterile plants segregated for fertile and sterile plants when backcrossed with T65 in BC5F1, BC6F1 and BC7F1 with a ratio of 1:1. Conversely, all the backcross progenies from the fertile BC4F1 were consistently fertile. Anthers of the male sterile line were stunted and did not shed pollen
cross-sectional observations revealed defects in sporophytic cells. The male sterility appears to be caused by heterozygous alleles derived from T65 and Lebed. A male sterility gene was mapped between two INDEL markers on the long arm of chromosome 10, which corresponded to a 407 kb region in the Nipponbare genome. Conclusions: Since the heterozygous Lebed allele acts as dominant sporophytic pollen killer, it would be useful for recurrent selection breeding of japonica rice.. |
| 33. |
Keisuke Igarashi, Tomohiko Kazama, Kinya Toriyama, A Gene Encoding Pentatricopeptide Repeat Protein Partially Restores Fertility in RT98-Type Cytoplasmic Male-Sterile Rice, PLANT AND CELL PHYSIOLOGY, 10.1093/pcp/pcw135, 57, 10, 2187-2193, 2016.10, Cytoplasmic male sterility (CMS) lines in rice, which have the cytoplasm of a wild species and the nuclear genome of cultivated rice, are of value for the study of genetic interactions between the mitochondrial and nuclear genomes. The RT98-type CMS line RT98A and the fertility restorer line RT98C carry the cytoplasm of the wild species Oryza rufipogon and the nuclear genome of the Taichung 65 cultivar (Oryza sativa L.). Based on a classical crossing experiment, fertility is reported to be restored gametophytically by the presence of a tentative single gene, designated Rf98, which is derived from the cytoplasm donor. Fine mapping of Rf98 revealed that at least two genes, which are closely positioned, are required for complete fertility restoration in RT98A. Here, we identified seven pentatricopeptide repeat (PPR) genes that are located within a 170 kb region as candidates for Rf98. Complementation tests revealed that the introduction of one of these PPR genes, PPR762, resulted in the partial recovery of fertility with a seed setting rate up to 9.3%. We conclude that PPR762 is an essential fertility restorer gene for RT98-type CMS. The low rate of seed setting suggested that some other genes near the Rf98 locus are also necessary for the full recovery of seed setting.. |
| 34. |
Toriyama* K, Kazama T, Development of cytoplasmic male sterile IR24 and IR64 using CW-CMS/Rf17 system, Rice, 10.1186/s12284-016-0097-2, 9, 22, 1-4, 2016.05, IF=3.739. |
| 35. |
Tomohiko Kazama, Etsuko Itabashi, Shinya Fujii, Takahiro Nakamura, Kinya Toriyama, Mitochondrial ORF79 levels determine pollen abortion in cytoplasmic male sterile rice, PLANT JOURNAL, 10.1111/tpj.13135, 85, 6, 707-716, 2016.03, Cytoplasmic male sterility (CMS) is an important agricultural trait characterized by lack of functional pollen, and caused by ectopic and defective mitochondrial gene expression. The pollen function in CMS plants is restored by the presence of nuclear-encoded restorer of fertility (Rf) genes. Previously, we cloned Rf2, which restores the fertility of Lead Rice (LD)-type CMS rice. However, neither the function of Rf2 nor the identity of the mitochondrial gene causing CMS has been determined in LD-CMS rice. Here, we show that the mitochondrial gene orf79 acts as a CMS-associated gene in LD-CMS rice, similar to its role in BT-CMS rice originating from Chinsurah Boro II, and Rf2 weakly restores fertility in BT-CMS rice. We also show that RF2 promotes degradation of atp6-orf79 RNA in a different manner from that of RF1, which is the Rf gene product in BT-CMS rice. The amount of ORF79 protein in LD-CMS rice was one-twentieth of the amount in BT-CMS rice. The difference in ORF79 protein levels probably accounts for the mild and severe pollen defects in LD-CMS and BT-CMS rice, respectively. In the presence of Rf2, accumulation of ORF79 was reduced to almost zero and 25% in LD-CMS and BT-CMS rice, respectively, which probably accounts for the complete and weak fertility restoration abilities of Rf2 in LD-CMS and BT-CMS rice, respectively. These observations indicate that the amount of ORF79 influences the pollen fertility in two strains of rice in which CMS is induced by orf79.
Significance Statement In plants, incompatibility between nuclear and mitochondrial genotypes causes cytoplasmic male sterility (CMS). Here, we show that RF2, a nuclear-encoded restorer of fertility gene, degrades transcripts of a CMS causative gene, orf79, and that differences in the amount of ORF79 influence pollen fertility in different strains of orf79-induced CMS rice.. |
| 36. |
Tomohiko Kazama, Kinya Toriyama, Whole Mitochondrial Genome Sequencing and Re-Examination of a Cytoplasmic Male Sterility-Associated Gene in Boro-Taichung-Type Cytoplasmic Male Sterile Rice, PLOS ONE, 10.1371/journal.pone.0159379, 11, 7, Article number e0159379, 2016.07, Nuclear genome substitutions between subspecies can lead to cytoplasmic male sterility (CMS) through incompatibility between nuclear and mitochondrial genomes. Boro-Taichung (BT)-type CMS rice was obtained by substituting the nuclear genome of Oryza sativa subsp. indica cultivar Chinsurah Boro II with that of Oryza sativa subsp. japonica cultivar Taichung 65. In BT-type CMS rice, the mitochondrial gene orf79 is associated with male sterility. A complete sequence of the Boro-type mitochondrial genome responsible for BT-type CMS has not been determined to date. Here, we used pyrosequencing to construct the Boro-type mitochondrial genome. The contiguous sequences were assembled into five circular DNA molecules, four of which could be connected into a single circle. The two resulting subgenomic circles were unable to form a reliable master circle, as recombination between them was scarcely detected. We also found an unequal abundance of DNA molecules for the two loci of atp6. These results indicate the presence of multi-partite DNA molecules in the Borotype mitochondrial genome. Expression patterns were investigated for Boro-type mitochondria- specific orfs, which were not found in the mitochondria from the standard japonica cultivar Nipponbare. Restorer of fertility 1 (RF1)-dependent RNA processing has been observed in orf79-containing RNA but was not detected in other Boro-type mitochondria-specific orfs, supporting the conclusion that orf79 is a unique CMS-associated gene in Boro-type mitochondria.. |
