A candidate factor that interacts with RF2, a restorer of fertility of Lead rice-type cytoplasmic male sterility in rice
© Fujii et al.; licensee Springer. 2014
Received: 29 May 2014
Accepted: 19 August 2014
Published: 7 October 2014
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.
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.
These results suggest that RIF2 is a candidate factor of a fertility restoration complex of RF2.
Cytoplasmic male sterility (CMS), which is caused by an aberrant mitochondrial gene, is unable to produce functional pollen. Pollen function is often recovered by a Restorer of fertility (Rf) gene encoded by the nuclear genome. Many Rf genes are reported to encode pentatricopeptide repeat (PPR) protein, which is involved in the processing of mitochondrial RNA (Fujii and Toriyama  for a review). Different from PPR-type Rf genes, Rf2, which is a fertility restorer gene of Lead Rice (LD) type CMS, encodes a mitochondria-targeted 152-amino acid protein containing glycine-rich domain (Itabashi et al. ). Rf2 is, therefore, considered to restore fertility by a novel mechanism. RF2 is expected to function by interacting with other proteins, because mature RF2 is a small protein that putatively consists of 80 amino acids after removing a predicted mitochondrial targeting signal sequence; it has no motifs except for a 29-amino acid long glycine-rich domain (Itabashi et al. ).
Genes identified by Y2H screening
Number of clones
RAP-DB Locus ID
cDNA accession No.
Conserved hypothetical protein
Ubiquitin domain containing protein
Similar to Sinapyl alcohol dehydrogenase
Conserved hypothetical protein
A fertility restoration factor is expected to function in mitochondria. We therefore investigated the subcellular localization of RIF1, RIF2, RIF3 and RIF4. Coding sequences of RIF proteins were PCR-amplified using primers listed in Additional file 1: Table S1 and using full-length cDNAs obtained from the Rice Genome Resource Center (National Institute of Agrobiological Sciences, Tsukuba, Japan). They were cloned into pENTR D-TOPO vector (Invitrogen, Tokyo, Japan), and then inserted between CaMV 35S promoter and GFP gene in pGWB5 vector (Nakagawa et al. ).
Another possibility for RIF2 function in regard to fertility restoration is that RIF2 plays a role in an RNA processing complex. In this case the complex would contain a factor that directly recognizes and processes a target RNA, since neither RF2 nor RIF2 harbors a motif associated with RNA processing. Formation of an RNA processing complex was reported in Hong-Lian CMS. In this CMS, GRP162, which contains a glycine-rich motif and an RNA recognition motif, has been reported to interact with a PPR protein RF5 and unknown components to form a fertility restoration-complex that mediates the processing of CMS-associated atp6-orf79 RNA (Hu et al. ). RF2 of LD-CMS showed limited amino acid sequence identity (28%) to GRP162 but lacks an RNA recognition motif. The RIF2, which encodes ubiquitin-domain containing protein, might be one of the components of a fertility restoration-complex in the LD-CMS/Rf2 system. Further study is now in progress to discover other components, which might include RNA recognition/binding proteins in the RF2 fertility restoration-complex that mediate processing of a CMS-associated mitochondrial RNA.
KT and TK conceived and designed the experiments. SF performed the experiments and drafted the manuscript. KT and YI revised the manuscript. YI and SK supervised Y2H experiments. All authors read and approved the final manuscript.
Cytoplasmic male sterility
Restorer of fertility
Yeast two hybrid
This work was supported by MEXT/JSPS KAKENHI Grant Numbers 2338002, 24117502 and 26292002, and by Science and technology research promotion program for agriculture, forestry, fisheries and food industry (No. 26010A).
- Arimura S, Tsutsumi N: A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division. Proc Natl Acad Sci USA 2002, 99: 5727–5731. doi:10.1073/pnas.082663299 doi:10.1073/pnas.082663299 10.1073/pnas.082663299PubMed CentralView ArticlePubMedGoogle Scholar
- Chen L, Shiotani K, Togashi T, Miki D, Aoyama M, Wong HL, Kawasaki T, Shimamoto K: Analysis of the Rac/Rop small GTPase family in rice: expression, subcellular localization and role in disease resistance. Plant Cell Physiol 2010, 51: 585–595. doi:10.1093/pcp/pcq024 doi:10.1093/pcp/pcq024 10.1093/pcp/pcq024View ArticlePubMedGoogle Scholar
- Fujii S, Toriyama K: Genome barriers between nuclei and mitochondria exemplified by cytoplasmic male sterility. Plant Cell Physiol 2008, 49: 1484–1494. doi:10.1093/pcp/pcn102 doi:10.1093/pcp/pcn102 10.1093/pcp/pcn102PubMed CentralView ArticlePubMedGoogle Scholar
- Fujii S, Yamada M, Toriyama K: Cytoplasmic male sterility-related protein kinase, OsNek3, is regulated downstream of mitochondrial protein phosphatase 2C, DCW11. Plant Cell Physiol 2009, 50: 828–837. doi:10.1093/pcp/pcp026 doi:10.1093/pcp/pcp026 10.1093/pcp/pcp026View ArticlePubMedGoogle Scholar
- Hu J, Wang K, Huang W, Liu G, Wang J, Huang Q, Ji Y, Qin X, Wan L, Zhu R, Li S, Yang D, Zhu Y: The rice pentatricopeptide repeat protein RF5 restores fertility in Hong-Lian cytoplasmic male-sterile lines via a complex with the glycine-rich protein GRP162. Plant Cell 2012, 24: 109–122. doi:10.1105/tpc.111.093211 doi:10.1105/tpc.111.093211 10.1105/tpc.111.093211PubMed CentralView ArticlePubMedGoogle Scholar
- Itabashi E, Iwata N, Fujii S, Kazama T, Toriyama K: The fertility restorer gene, Rf2, for Lead Rice-type cytoplasmic male sterility of rice encodes a mitochondrial glycine-rich protein. Plant J 2011, 65: 359–367. doi:10.1111/j.1365–313X.2010.04427.x doi:10.1111/j.1365-313X.2010.04427.x 10.1111/j.1365-313X.2010.04427.xView ArticlePubMedGoogle Scholar
- Ko HS, Uehara T, Tsuruma K, Nomura Y: Ubiquilin interacts with ubiquitylated proteins and prooteasome through its ubiquitin-associated and ubiquitin-like domains. FEBS Let 2004, 566: 110–114. doi:10.1016/j.febslet.2004.04.031 doi:10.1016/j.febslet.2004.04.031 10.1016/j.febslet.2004.04.031View ArticleGoogle Scholar
- Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, Toyooka K, Matsuoka K, Jinbo T, Kimura T: Development of series of Gateway Binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 2007, 104: 34–41. doi:10.1263/jbb.104.34 doi:10.1263/jbb.104.34 10.1263/jbb.104.34View ArticlePubMedGoogle Scholar
- Shanbhag R, Shi G, Rujiviphat J, McQuibban GA: The emerging role of proteolysis in mitochondrial quality control and the etiology of Parkinson's disease. Parkinson's Dis 2012, 2012: 382175. doi:10. 1155/2012/382175 doi:10. 1155/2012/382175Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.