Shunichi Kosugi

PCF1 and PCF2 Specifically Bind to cis Elements in the Rice Proliferating Cell Nuclear Antigen Gene

We have previously defined the promoter elements, sites IIa and IIb, in the rice proliferating cell nuclear antigen (PCNA) gene that are essential for meristematic tissue-specific expression. In this study, we isolated and characterized cDNAs encoding proteins that specifically bind to sites IIa and IIb. The two DNA binding proteins, designated PCF1 and PCF2, share > 70% homology in common conserved sequences at the N-terminal regions. The conserved regions are responsible for DNA binding and homodimer and heterodimer formation, and they contain a putative basic helix-loop-helix (bHLH) motif. The structure and DNA binding specificity of the bHLH motif are distinguishable from those of other known bHLH proteins that bind to the E-box. The motif is > 70% homologous to several expressed sequence tags from Arabidopsis and rice, suggesting that PCF1 and PCF2 are members of a novel family of proteins that are conserved in monocotyledons and dicotyledons. A supershift assay using an anti-PCF2 antibody showed the involvement of PCF2 in site IIa (site IIb) binding activities in rice nuclear extracts, particularly in meristematic tissues. PCF1 and PCF2 were also more likely to form heterodimers than homodimers. Our results suggest that PCF1 and PCF2 are involved in meristematic tissue-specific expression of the rice PCNA gene through binding to sites IIa and IIb and formation of homodimers or heterodimers.

Design of Peptide Inhibitors for the Importin α/β Nuclear Import Pathway by Activity-Based Profiling

Despite the current availability of selective inhibitors for the classical nuclear export pathway, no inhibitor for the classical nuclear import pathway has been developed. Here we describe the development of specific inhibitors for the importin alpha/beta pathway using a novel method of peptide inhibitor design. An activity-based profile was created via systematic mutational analysis of a peptide template of a nuclear localization signal. An additivity-based design using the activity-based profile generated two peptides with affinities for importin alpha that were approximately 5 million times higher than that of the starting template sequence. The high affinity of these peptides resulted in specific inhibition of the importin alpha/beta pathway. These peptide inhibitors provide a useful tool for studying nuclear import events. Moreover, our inhibitor design method should enable the development of potent inhibitors from a peptide seed.

Dissection of the Octoploid Strawberry Genome by Deep Sequencing of the Genomes of Fragaria Species

Cultivated strawberry (Fragaria x ananassa) is octoploid and shows allogamous behaviour. The present study aims at dissecting this octoploid genome through comparison with its wild relatives, F. iinumae, F. nipponica, F. nubicola, and F. orientalis by de novo whole-genome sequencing on an Illumina and Roche 454 platforms. The total length of the assembled Illumina genome sequences obtained was 698 Mb for F. x ananassa, and ∼200 Mb each for the four wild species. Subsequently, a virtual reference genome termed FANhybrid_r1.2 was constructed by integrating the sequences of the four homoeologous subgenomes of F. x ananassa, from which heterozygous regions in the Roche 454 and Illumina genome sequences were eliminated. The total length of FANhybrid_r1.2 thus created was 173.2 Mb with the N50 length of 5137 bp. The Illumina-assembled genome sequences of F. x ananassa and the four wild species were then mapped onto the reference genome, along with the previously published F. vesca genome sequence to establish the subgenomic structure of F. x ananassa. The strategy adopted in this study has turned out to be successful in dissecting the genome of octoploid F. x ananassa and appears promising when applied to the analysis of other polyploid plant species.

E2Ls, E2F-like Repressors of Arabidopsis That Bind to E2F Sites in a Monomeric Form

E2F transcription factors are major regulators of cell proliferation, and each factor contributes differently to cell cycle control. Arabidopsis contains six E2F homologs, of which three are proteins that exhibit an overall similarity to animal E2Fs and interact with DPa and DPb to stimulate DNA binding. Here we describe the other three E2F-like proteins fromArabidopsis, E2L1–3, which have two copies of a domain with a limited similarity only to the DNA binding domain of E2F. Unlike known E2Fs, the three E2L proteins failed to interact with DPa and DPb and could efficiently bind E2F sites in a monomeric form through the dual-type domain. Transfection assays revealed that E2Ls repress the transcription of reporter genes under the control of E2F-regulated promoters, indicating that E2Ls function to antagonize transactivation mediated by E2F·DP. When fused to green fluorescence protein, E2L1 and E2L3 were predominantly localized to the nucleus whereas E2L2 was detected in both the nucleus and cytoplasm. Because the transcripts of E2Ls were abundant in meristematic rather than fully differentiated tissues, E2Ls may balance the activities of E2F·DP and play a role in restraining cell proliferation.

Constitutive E2F Expression in Tobacco Plants Exhibits Altered Cell Cycle Control and Morphological Change in a Cell Type-Specific Manner

The E2F family plays a pivotal role in cell cycle control and is conserved among plants and animals, but not in fungi. This provides for the possibility that the E2F family was integrated during the development of higher organisms, but little is known about this. We examined the effect of E2F ectopically expressed in transgenic tobacco (Nicotiana tabacum) plants on growth and development using E2Fa (AtE2F3) and DPa from Arabidopsis. E2Fa-DPa double transgenic lines exhibited altered phenotypes with curled leaves, round shaped petals, and shortened pistils. In mature but not immature leaves of the double transgenic lines, there were enlarged nuclei with increasing ploidy levels accompanied by the ectopic expression of S phase- but not M phase-specific genes. This indicates that a high expression of E2F promotes endoreduplication by accelerating S phase entry in terminally differentiated cells with limited mitotic activity. Furthermore, mature leaves of the transgenic plants contained increased numbers of small cells, especially on the palisade (adaxial) side of the outer region toward the edge, and the leaf strips exhibited hormone-independent callus formation when cultured in vitro. These observations suggest that an enhanced E2F activity modulates cell cycle in a cell type-specific manner and affects plant morphology depending on a balance between activities for committing to S phase and M phase, which likely differ between organs or tissues.

Nuclear Export Signal Consensus Sequences Defined Using a Localization‐Based Yeast Selection System

Proteins bearing nuclear export signals (NESs) are translocated to the cytoplasm from the nucleus mainly through the CRM1‐dependent pathway. However, the NES consensus sequence remains poorly defined, and there are currently no high‐throughput methods for identifying NESs. In this study, we report the development of an efficient yeast selection system for detecting nuclear export activity as well as several reliable NES consensus sequences identified using this method. Our selection system is based on the nuclear export‐dependent rescue of Tys1p, an essential cytoplasmic protein that has been artificially localized to the nucleus in a haploid Δtys1 knockout strain. A screen of a random peptide library revealed 101 distinct CRM1‐dependent NESs, which were classified into six patterns according to the conserved hydrophobic spacing. By combining mutational analyses, we have defined new NES consensus sequences with more specific and redundant residues than the traditional consensus sequence, which are consistent with most experimentally confirmed NESs. These NES consensus sequences should help identify functional NESs, and our selection system can be used to identify other targeting signals or proteins imported to specific subcellular compartments.

A genetic mechanism for female-limited Batesian mimicry in Papilio butterfly

In Batesian mimicry, animals avoid predation by resembling distasteful models. In the swallowtail butterfly Papilio polytes, only mimetic-form females resemble the unpalatable butterfly Pachliopta aristolochiae. A recent report showed that a single gene, doublesex (dsx), controls this mimicry; however, the detailed molecular mechanisms remain unclear. Here we determined two whole-genome sequences of P. polytes and a related species, Papilio xuthus, identifying a single ∼130-kb autosomal inversion, including dsx, between mimetic (H-type) and non-mimetic (h-type) chromosomes in P. polytes. This inversion is associated with the mimicry-related locus H, as identified by linkage mapping. Knockdown experiments demonstrated that female-specific dsx isoforms expressed from the inverted H allele (dsx(H)) induce mimetic coloration patterns and simultaneously repress non-mimetic patterns. In contrast, dsx(h) does not alter mimetic patterns. We propose that dsx(H) switches the coloration of predetermined wing patterns and that female-limited polymorphism is tightly maintained by chromosomal inversion.

Interaction of the Arabidopsis E2F and DP Proteins Confers Their Concomitant Nuclear Translocation and Transactivation

E2F transcription factors are required for the progression and arrest of the cell cycle in animals. Like animals, plants have evolved to conserve the E2F family. The Arabidopsis genome encodes E2F and DP proteins that share a high similarity with the animal E2F and DP families. Here, we show that Arabidopsis E2F and DP proteins are not predominantly localized to the nucleus in analyses with green fluorescent protein, and that the complete nuclear localization of some members is driven by the co-expression of their specific partner proteins. Both AtE2F1 and AtE2F3 were translocated to the nucleus and transactivate an E2F reporter gene when co-expressed with DPa but not DPb. In contrast, AtE2F2 was inactive for both nuclear translocation and transactivation even when Dpa or DPb was co-expressed. Because the DNA binding activities of the three E2Fs are equally stimulated by the interaction with DPa or DPb in vitro, the observed transactivation of AtE2F1 and AtE2F3 is DPa specific and nuclear import dependent. A green fluorescent protein fusion with an AtE2F3 mutant, in which a conserved nuclear export signal-like sequence in the dimerization domain was deleted, was localized to the nucleus. Thus, the concomitant nuclear translocation seems to be conferred by the DPa interaction to release an activity that inhibits an intrinsic nuclear import activity of AtE2Fs. Furthermore, the nuclear translocation of AtE2F3 stimulated by DPa was abolished by the deletion of the N-terminal region of AtE2F3, which is conserved among all the E2F proteins identified in plants to date. Replacement of the N-terminal region of AtE2F3 with a canonical nuclear localization signal only partially mimicked the effect of the DPa co-expression, demonstrating the function of plant E2F distinct from that observed for animal E2Fs. These observations suggest that the function of plant E2F and DP proteins is primarily controlled by their nuclear localization mediated by the interaction with specific partner proteins.

MutMap accelerates breeding of a salt-tolerant rice cultivar.

445 two chromosome substitution lines (CSSLs), SL502 and SL503, harboring chromosomal segments of Nona Bokra in the genetic background of Koshihikari, a cultivar closely related to Hitomebore10,11. We compared the growth of hst1, SL502 and SL503 at 0.75% NaCl (Supplementary Fig. 2). The 18 and 43% improved growth of hst1 as compared to SL503 and SL502, respectively, showed that this was a good starting point for breeding a salt-tolerant rice cultivar. For rapid identification of the mutation conferring salinity tolerance in hst1, we used MutMap, a method based on wholegenome resequencing of bulked DNA of F2 segregants1. The hst1 line was crossed to WT Hitomebore to generate F1 progeny, and F2 progeny were derived from self-pollination of the F1 progeny. Two-week-old F2 seedlings were treated with water containing 0.75% NaCl. The progeny segregated in a 133:54 ratio for salinity-susceptible and salinitytolerant phenotypes, respectively, conforming to a 3:1 segregation ratio (chi-squared test: χ2 = 2.2 × 10-16, nonsignificant) and thereby indicating that the salinity tolerance of hst1 is conferred by a single recessive mutation. We combined DNA from 20 F2 progeny that had the salinity-tolerance phenotype and applied whole-genome resequencing using an Illumina GAIIx DNA sequencer. We obtained a total of 7.34 Gbp of short (75-bp) reads (Supplementary Table 1) that were aligned to the Hitomebore reference sequence (DDBJ Sequence Read Archive DRA000927), resulting in the identification of 1,005 SNP positions. For each SNP position, the value of SNP-index (the ratio of short reads harboring SNPs different from the reference1) was obtained and a graph relating SNP positions and SNP-index was generated for all 12 rice chromosomes (Fig. 2a, Supplementary Fig. 3). The causative SNP should be shared by all the mutant F2 plants and therefore have a SNP-index = 1, whereas SNPs unrelated to the mutant phenotype should segregate in a 1:1 ratio among the F2 progeny, resulting in a SNP-index of ~0.5. MutMap applied to hst1 To the Editor: Following the 2011 earthquake and tsunami that affected Japan, >20,000 ha of rice paddy field was inundated with seawater, resulting in salt contamination of the land. As local rice landraces are not tolerant of high salt concentrations, we set out to develop a salttolerant rice cultivar. We screened 6,000 ethyl methanesulfonate (EMS) mutant lines of a local elite cultivar, ‘Hitomebore’, and identified a salt-tolerant mutant that we name hitomebore salt tolerant 1 (hst1). In this Correspondence, we report how we used our MutMap method1 to rapidly identify a loss-of-function mutation responsible for the salt tolerance of hst1 rice. The salttolerant hst1 mutant was used to breed a salt-tolerant variety named ‘Kaijin’, which differs from Hitomebore by only 201 singlenucleotide polymorphisms (SNPs). Field trials showed that it has the same growth and yield performance as the parental line under normal growth conditions. Notably, production of this salt-tolerant mutant line ready for delivery to farmers took only two years using our approach. Although soluble salts, such as nitrates and potassium salts, are common components of soil and essential plant nutrients, their accumulation above specific threshold concentrations can substantially affect plant growth. There is considerable variation among plants with respect to their tolerance of salinity, and rice is considered the most sensitive of all the cereals2. Yields of paddy rice start to decline at salinity levels >3 dS m–1 (measured by the electrical conductivity of the extract, ECe), beyond which a 12% reduction in yield is expected for every 1 dS m–1 increase in ECe. Soil salinity affects >6% of world’s total land area, causing yield losses as a result of both osmotic and ionic stresses to crop plants2. Soil salinization due to the flooding of agricultural lands by seawater has become an additional concern since the 2004 Indian Ocean tsunami4. In 2011, Japan was hit by the Great Tohoku Earthquake, which triggered a devastating tsunami, altogether claiming the lives of more than 15,000 people. The tsunami extended more than 5 km inland on the Sendai Plain of Miyagi Prefecture, one of the main rice-production regions in Japan5. An environmental impact assessment study conducted in the same area over a period of 2–7 months after the tsunami revealed wide spatial variation in the salinity level of ponded water, with ECe ranging from 0.31 to 68.2 mS cm–1 (ref. 6). Although salt concentration gradually decreased, it was too high for rice production to resume in October, 2011. To restore rice production in tsunamiaffected areas of the Tohoku region of Japan, we set out to develop and deliver a salt-tolerant rice cultivar from a line suited to local agronomic conditions. First, we carried out a genetic screen for salt tolerance using seeds pooled from approximately 6,000 independent EMS-mutagenized lines of Hitomebore7, (Supplementary Fig. 1). We identified a mutant that survived with 1.5% NaCl supplied to the soil with irrigation water for 7 days, which we designated hitomebore salt tolerant 1 (hst1). Seeds from a self-pollinated hst1 plant were used to further test the performance of hst1 at different NaCl concentrations (Fig. 1a,b). The hst1 mutant grew better than wild-type (WT) Hitomebore plants at both 0.375% and 0.75% NaCl concentrations, as measured after 14 days of treatment. The 0.375% NaCl treatment caused reductions of 38.4% and 2.9% in the fresh weight of WT and hst1 plants, respectively. At 0.75% NaCl, WT plants dried out, with a 61.5% reduction in fresh weight, whereas hst1 plants remained green with only a 13.2% reduction in fresh weight compared with hst1 plants that received fresh water (Fig. 1b). Previously, the rice SHOOT K+ CONCENTRATION 1 (SKC1) gene, encoding a Na+ transporter, was identified as the main quantitative trait locus (QTL) conferring salt tolerance in the indica cultivar Nona Bokra8,9. This QTL has been used to develop MutMap accelerates breeding of a salt-tolerant rice cultivar CORRESPONDENCE

Computational analysis suggests that alternative first exons are involved in tissue-specific transcription in rice (Oryza sativa)

MOTIVATION Transcription start site selection and alternative splicing greatly contribute to diversifying gene expression. Recent studies have revealed the existence of alternative first exons, but most have involved mammalian genes, and as yet the regulation of usage of alternative first exons has not been clarified, especially in plants. RESULTS We systematically identified putative alternative first exon transcripts in rice, verified the candidates using RT-PCR, and searched for the promoter elements that might regulate the alternative first exons. As a result, we detected a number of unreported alternative first exons, some of which are regulated in a tissue-specific manner. SUPPLEMENTARY INFORMATION http://www.bioinfo.sfc.keio.ac.jp/research/intron.