Antimicrobial activity of UV-induced phenylamides from rice leaves.

Rice produces a wide array of phytoalexins in response to pathogen attacks and UV-irradiation. Except for the flavonoid sakuranetin, most phytoalexins identified in rice are diterpenoid compounds. Analysis of phenolic-enriched fractions from UV-treated rice leaves showed that several phenolic compounds in addition to sakuranetin accumulated remarkably in rice leaves. We isolated two compounds from UV-treated rice leaves using silica gel column chromatography and preparative HPLC. The isolated phenolic compounds were identified as phenylamide compounds: N-trans-cinnamoyltryptamine and N-p-coumaroylserotonin. Expression analysis of biosynthetic genes demonstrated that genes for arylamine biosynthesis were upregulated by UV irradiation. This result suggested that phenylamide biosynthetic pathways are activated in rice leaves by UV treatment. To unravel the role of UV-induced phenylamides as phytoalexins, we examined their antimicrobial activity against rice fungal and bacterial pathogens. N-trans-Cinnamoyltryptamine inhibited the growth of rice brown spot fungus (Bipolaris oryzae). In addition to the known antifungal activity to the blast fungus, sakuranetin had antimicrobial activity toward B. oryzae and Rhizoctonia solani (rice sheath blight fungus). UV-induced phenylamides and sakuranetin also had antimicrobial activity against rice bacterial pathogens for grain rot (Burkholderia glumae), blight (Xanthomonas oryzae pv. oryzae) and leaf streak (X. oryzae pv. oryzicola) diseases. These findings suggested that the UV-induced phenylamides in rice are phytoalexins against a diverse array of pathogens.

Epitope mapping of monoclonal antibodies for the Deinococcus radiodurans bacteriophytochome.

Bacteriophytochromes (BphP) are phytochrome-like light sensing proteins in bacteria, which use biliverdin as a chromophore. In order to study the biochemical properties of the DrBphP protein, five (2B8, 2C11, 3B2, 3D2, and 3H7) anti-DrBphP monoclonal antibodies were produced through the immunization of mice with purified full-length DrBphP and DrBphN (1-321 amino acid) proteins, and epitope mapping was then carried out. Among the five antibodies, 2B8 and 2C11 preferentially recognized the N-terminal region of BphP whereas 3B2, 3D2, and 3H7 showed preference for the C-terminal region. We performed further epitope mapping using recombinant truncated BphP proteins to narrow down their target sequences. The results demonstrated that each of the five monoclonal antibodies recognized different regions on the DrBphP protein. Additionally, epitopes of 2B8 and 3H7 antibodies were discovered to be shorter than 10 amino acids (2B8: RDPLPFFPP, 3H7: PGEIEEA). These two antibodies with such specific recognition epitopes could be especially valuable for developing new peptide tags for protein detection and purification.

Transcriptomic analysis of UV-treated rice leaves reveals UV-induced phytoalexin biosynthetic pathways and their regulatory networks in rice.

Rice produces diterpenoid and flavonoid phytoalexins for defense against pathogen attack. The production of phytoalexins in rice is also induced by UV-irradiation. To understand the metabolic networks involved in UV-induced phytoalexin biosynthesis and their regulation, phytochemical and transcriptomic analyses of UV-treated rice leaves were performed. In response to UV treatment, the accumulation of flavonoids was observed in rice leaves, which may serve as antioxidants against UV-induced oxidative stress. The phytochemical analysis confirmed sakuranetin accumulation and also demonstrated the induction of phenylamide synthesis in rice leaves by UV-irradiation. Transcriptomic analysis established that aromatic amino acid biosynthetic genes were immediately up-regulated after UV treatment. The genes involved in the phenylpropanoid pathway and flavonoid biosynthesis were also up-regulated. These findings suggest that the aromatic amino acid and flavonoid biosynthetic pathways are coordinately activated for the production of flavonoids and phenolic phytoalexins such as sakuranetin and phenylamides. An in silico analysis of UV-induced O-methyltransferase and acyltransferase genes suggested that these genes may be implicated in sakuranetin and phenylamide synthesis, respectively. The transcriptomic analysis also showed up-regulation of both methylerythritol phosphate pathway and the diterpenoid phytoalexin biosynthetic genes in response to UV treatment. A functional gene network analysis of phytoalexin biosynthetic and UV-induced genes for signaling components and transcription factors using RiceNet suggested that regulatory networks comprising signal perceiving receptor kinases, G-proteins, signal transducing mitogen-activated protein kinases and calcium signaling components, and transcription factors control flavonoid and phytoalexin biosynthesis in rice leaves under UV-C stress conditions.

Manipulation of triose phosphate/phosphate translocator and cytosolic fructose-1,6-bisphosphatase, the key components in photosynthetic sucrose synthesis, enhances the source capacity of transgenic Arabidopsis plants.

Photoassimilated carbons are converted to sucrose in green plant leaves and distributed to non-phototropic tissues to provide carbon and energy. In photosynthetic sucrose biosynthesis, the chloroplast envelope triose phosphate/phosphate translocator (TPT) and cytosolic fructose-1,6-bisphosphatase (cFBPase) are key components in photosynthetic sucrose biosynthesis. The simultaneous overexpression of TPT and cFBPase was utilized to increase the source capacity of Arabidopsis. The TPT and cFBPase overexpression lines exhibited enhanced growth with larger rosette sizes and increased fresh weights compared with wild-type (WT) plants. The simultaneous overexpression of TPT and cFBPase resulted in enhanced photosynthetic CO(2) assimilation rates in moderate and elevated light conditions. During the phototropic period, the soluble sugar (sucrose, glucose, and fructose) levels in the leaves of these transgenic lines were also higher than those of the WT plants. These results suggest that the simultaneous overexpression of TPT and cFBPase enhances source capacity and consequently leads to growth enhancement in transgenic plants.

Impaired function of the tonoplast-localized sucrose transporter in rice, OsSUT2, limits the transport of vacuolar reserve sucrose and affects plant growth.

Physiological functions of sucrose (Suc) transporters (SUTs) localized to the tonoplast in higher plants are poorly understood. We here report the isolation and characterization of a mutation in the rice (Oryza sativa) OsSUT2 gene. Expression of OsSUT2-green fluorescent protein in rice revealed that OsSUT2 localizes to the tonoplast. Analysis of the OsSUT2 promoter::ß-glucuronidase transgenic rice indicated that this gene is highly expressed in leaf mesophyll cells, emerging lateral roots, pedicels of fertilized spikelets, and cross cell layers of seed coats. Results of Suc transport assays in yeast were consistent with a H(+)-Suc symport mechanism, suggesting that OsSUT2 functions in Suc uptake from the vacuole. The ossut2 mutant exhibited a growth retardation phenotype with a significant reduction in tiller number, plant height, 1,000-grain weight, and root dry weight compared with the controls, the wild type, and complemented transgenic lines. Analysis of primary carbon metabolites revealed that ossut2 accumulated more Suc, glucose, and fructose in the leaves than the controls. Further sugar export analysis of detached leaves indicated that ossut2 had a significantly decreased sugar export ability compared with the controls. These results suggest that OsSUT2 is involved in Suc transport across the tonoplast from the vacuole lumen to the cytosol in rice, playing an essential role in sugar export from the source leaves to sink organs.

Identification and characterization of the duplicate rice sucrose synthase genes OsSUS5 and OsSUS7 which are associated with the plasma membrane.

Systematic searches using the complete genome sequence of rice (Oryza sativa) identified OsSUS7, a new member of the rice sucrose synthase (OsSUS) gene family, which shows only nine single nucleotide substitutions in the OsSUS5 coding sequence. Comparative genomic analysis revealed that the synteny between OsSUS5 and OsSUS7 is conserved, and that significant numbers of transposable elements are scattered at both loci. In particular, a 17.6-kb genomic region containing transposable elements was identified in the 5' upstream sequence of the OsSUS7 gene. GFP fusion experiments indicated that OsSUS5 and OsSUS7 are largely associated with the plasma membrane and partly with the cytosol in maize mesophyll protoplasts. RT-PCR analysis and transient expression assays revealed that OsSUS5 and OsSUS7 exhibit similar expression patterns in rice tissues, with the highest expression evident in roots. These results suggest that two redundant genes, OsSUS5 and OsSUS7, evolved via duplication of a chromosome region and through the transposition of transposable elements.

An ankyrin repeat protein is involved in anthocyanin biosynthesis in Arabidopsis.

The ankyrin domain is one of the most common protein motifs in eukaryotic proteins. Repeated ankyrin domains are ubiquitous and their mediation of protein-protein interactions is involved in a number of physiological and developmental responses such as the cell cycle, signal transduction and cell differentiation. A novel putative phytochrome-interacting ankyrin repeat protein 2 (PIA2) containing three repeated ankyrin domains was identified in Arabidopsis. An in vitro pull-down and phosphorylation assay revealed that PIA2 is phosphorylated and interacts directly with oat phytochrome A. The N-terminal domain of PIA2 was specifically phosphorylated, whereas interactions between the domains of PIA2 and phytochrome A had no Pr/Pfr preference. PIA2 was ubiquitously expressed in most tissues and was localized in both the nucleus and the cytoplasm independent of treatment with light of specific wavelengths. Anthocyanin accumulation in seedlings grown under far-red light, a typical phenotype of wild-type plants, was reduced in a loss-of-function mutant of PIA2 (pia2), whereas anthocyanin accumulation was increased in an overexpressing plant (PIA2-OX). The gene expression of UDP-flavonoid-3'-glucosyl-transferase (UF3GT), a major enzyme in the anthocyanin biosynthesis processes, was decreased in pia2 knockout plants suggesting that decreased anthocyanin was because of the decreased expression of UF3GT. Our results suggest that PIA2 plays a role in the anthocyanin biosynthesis during seedling development as a novel phytochrome-interacting protein.

Proteomic identification of rhythmic proteins in rice seedlings.

Many aspects of plant metabolism that are involved in plant growth and development are influenced by light-regulated diurnal rhythms as well as endogenous clock-regulated circadian rhythms. To identify the rhythmic proteins in rice, periodically grown (12h light/12h dark cycle) seedlings were harvested for three days at six-hour intervals. Continuous dark-adapted plants were also harvested for two days. Among approximately 3000 reproducible protein spots on each gel, proteomic analysis ascertained 354 spots (~12%) as light-regulated rhythmic proteins, in which 53 spots showed prolonged rhythm under continuous dark conditions. Of these 354 ascertained rhythmic protein spots, 74 diurnal spots and 10 prolonged rhythmic spots under continuous dark were identified by MALDI-TOF MS analysis. The rhythmic proteins were functionally classified into photosynthesis, central metabolism, protein synthesis, nitrogen metabolism, stress resistance, signal transduction and unknown. Comparative analysis of our proteomic data with the public microarray database (the Plant DIURNAL Project) and RT-PCR analysis of rhythmic proteins showed differences in rhythmic expression phases between mRNA and protein, suggesting that the clock-regulated proteins in rice are modulated by not only transcriptional but also post-transcriptional, translational, and/or post-translational processes.

Role of the plastidic glucose translocator in the export of starch degradation products from the chloroplasts in Arabidopsis thaliana.

In higher plants, the plastidic glucose translocator (pGlcT) is assumed to play a role in the export of starch degradation products, but this has not yet been studied in detail. To elucidate the role of pGlcT in the leaves of Arabidopsis thaliana, we generated single and double mutants lacking three plastidic sugar transporters, pGlcT, the triose-phosphate/phosphate translocator (TPT), and the maltose transporter (MEX1), and analyzed their growth phenotypes, photosynthetic properties and metabolite contents. In contrast to the pglct-1 and pglct-2 single mutants lacking a visible growth phenotype, the double mutants pglct-1/mex1 and tpt-2/mex1 displayed markedly inhibited plant growth. Notably, pglct-1/mex1 exhibited more severe growth retardation than that seen for the other mutants. In parallel, the most severe reductions in sucrose content and starch turnover were observed in the pglct-1/mex1 mutant. The concurrent loss of pGlcT and MEX1 also resulted in severely reduced photosynthetic activities and extreme chloroplast abnormalities. These findings suggest that pGlcT, together with MEX1, contributes significantly to the export of starch degradation products from chloroplasts in A. thaliana leaves, and that this starch-mediated pathway for photoassimilate export via pGlcT and MEX1 is essential for the growth and development of A. thaliana.

A small GTPase activator protein interacts with cytoplasmic phytochromes in regulating root development.

Phytochromes enable plants to sense light information and regulate developmental responses. Phytochromes interact with partner proteins to transmit light signals to downstream components for plant development. PIRF1 (phytochrome-interacting ROP guanine-nucleotide exchange factor (RopGEF 1)) functions as a light-signaling switch regulating root development through the activation of ROPs (Rho-like GTPase of plant) in the cytoplasm. In vitro pulldown and yeast two-hybrid assays confirmed the interaction between PIRF1 and phytochromes. PIRF1 interacted with the N-terminal domain of phytochromes through its conserved PRONE (plant-specific ROP nucleotide exchanger) region. PIRF1 also interacted with ROPs and activated them in a phytochrome-dependent manner. The Pr form of phytochrome A enhanced the RopGEF activity of PIRF1, whereas the Pfr form inhibited it. A bimolecular fluorescence complementation analysis demonstrated that PIRF1 was localized in the cytoplasm and bound to the phytochromes in darkness but not in light. PIRF1 loss of function mutants (pirf1) of Arabidopsis thaliana showed a longer root phenotype in the dark. In addition, both PIRF1 overexpression mutants (PIRF1-OX) and phytochrome-null mutants (phyA-211 and phyB-9) showed retarded root elongation and irregular root hair formation, suggesting that PIRF1 is a negative regulator of phytochrome-mediated primary root development. We propose that phytochrome and ROP signaling are interconnected through PIRF1 in regulating the root growth and development in Arabidopsis.

Improvement of plant protein solubilization and 2-DE gel resolution through optimization of the concentration of Tris in the solubilization buffer.

It is important to solubilize acetone-precipitated proteins before isoelectric focusing (IEF) to achieve high resolution 2-DE gels. To resolve the maximum possible number of plant protein spots, we developed an improved solubilization buffer for plant proteins. We demonstrated that the resolution of 2-DE gels increased dramatically as the concentration of Tris-base increased, with maximum solubilization obtained at 200 mM Tris-base (Ly200T). The Ly200T buffer was more effective than the commonly used solubilization buffer containing 40 mM Tris at solubilizing acetone-precipitated plant proteins. Use of the Ly200T buffer to solubilize proteins resulted in an increase in intensity of approximately 30% of plant protein spots in the larger-than-40 kDa region of the gel. The Ly200T buffer also improved the resolution of abundant and basic proteins. Thus, the Ly200T buffer can be used to achieve greater resolution of protein spots in plant proteomics research.

Starch biosynthesis in cereal endosperm.

Stored starch generally consists of two d-glucose homopolymers, the linear polymer amylose and a highly branched glucan amylopectin that connects linear chains. Amylopectin structurally contributes to the crystalline organization of the starch granule in cereals. In the endosperm, amylopectin biosynthesis requires the proper execution of a coordinated series of enzymatic reactions involving ADP glucose pyrophosphorylase (AGPase), soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE), whereas amylose is synthesized by AGPase and granule-bound starch synthase (GBSS). It is highly possible that plastidial starch phosphorylase (Pho1) plays an important role in the formation of primers for starch biosynthesis in the endosperm. Recent advances in our understanding of the functions of individual enzyme isoforms have provided new insights into how linear polymer chains and branch linkages are synthesized in cereals. In particular, genetic analyses of a suite of mutants have formed the basis of a new model outlining the role of various enzyme isoforms in cereal starch production. In our current review, we summarize the recent research findings related to starch biosynthesis in cereal endosperm, with a particular focus on rice.

Expression analysis and functional characterization of the monosaccharide transporters, OsTMTs, involving vacuolar sugar transport in rice (Oryza sativa).

In Arabidopsis, the compartmentation of sugars into vacuoles is known to be facilitated by sugar transporters. However, vacuolar sugar transporters have not been studied in detail in other plant species. To characterize the rice (Oryza sativa) tonoplast monosaccharide transporters, OsTMT1 and OsTMT2, we analysed their subcellular localization using green fluorescent protein (GFP) and expression patterns using reverse-transcription polymerase chain reaction (RT-PCR), performed histochemical beta-glucuronidase (GUS) assay and in situ hybridization analysis, and assessed sugar transport ability using isolated vacuoles. Expression of OsTMT-GFP fusion protein in rice and Arabidopsis revealed that the OsTMTs localize at the tonoplast. Analyses of OsTMT promoter-GUS transgenic rice indicated that OsTMT1 and OsTMT2 are highly expressed in bundle sheath cells, and in vascular parenchyma and companion cells in leaves, respectively. Both genes were found to be preferentially expressed in the vascular tissues of roots, the palea/lemma of spikelets, and in the main vascular tissues and nucellar projections on the dorsal side of the seed coats. Glucose uptake studies using vacuoles isolated from transgenic mutant Arabidopsis (tmt1-2-3) expressing OsTMT1 demonstrated that OsTMTs are capable of transporting glucose into vacuoles. Based on expression analysis and functional characterization, our present findings suggest that the OsTMTs play a role in vacuolar glucose storage in rice.

Evidence for a role of hexokinases as conserved glucose sensors in both monocot and dicot plant species.

The role of the hexokinases (HXKs) as glucose (Glc) sensors has been mainly demonstrated for Arabidopsis (Arabidopsis thaliana) HXK1 (AtHXK1) but has yet to be shown in other plant species. In our recent publication, we reported that two rice (Oryza sativa) HXKs, OsHXK5 and OsHXK6, also function as Glc sensors. These two enzymes harbor both mitochondrial targeting peptides (mTPs) and nuclear localization signals (NLSs), and we confirmed their dual-targeting ability to nuclei and mitochondria using GFP fusion experiments. Consistently, it has been previously known that AtHXK1 is predominantly associated with mitochondria but is also present in nuclei in vivo at appreciable levels. Notably, the expression of OsHXK5, OsHXK6, or their catalytically inactive mutant alleles complemented the Arabidopsis glucose insensitive2 (gin2) mutant. In addition, transgenic rice plants overexpressing OsHXK5 or OsHXK6 exhibited hypersensitive plant growth retardation and enhanced repression of the Rubisco small subunit (RbcS) gene in response to glucose treatment. Our results thus provided evidence that OsHXK5 and OsHXK6 can function as glucose sensors in rice. Hence, the available current data suggest that the role of the HXKs as Glc sensors may be conserved in both monocot and dicot plant species, and that the nuclear localization of AtHXK1, OsHXK5 and OsHXK6 may be critical for Glc sensing and signaling.

Altered expression of pyrophosphate: fructose-6-phosphate 1-phosphotransferase affects the growth of transgenic Arabidopsis plants.

Pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) catalyzes the reversible interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate, a key step in the regulation of the metabolic flux toward glycolysis or gluconeogenesis. To examine the role of PFP in plant growth, we have generated transgenic Arabidopsis plants that either overexpress or repress Arabidopsis PFP sub-unit genes. The overexpressing lines displayed increased PFP activity and slightly faster growth relative to wild type plants, although their photosynthetic activities and the levels of metabolites appeared not to have significantly changed. In contrast, the RNAi lines showed significantly retarded growth in parallel with the reduced PFP activity. Analysis of photosynthetic activity revealed that the growth retardation phenotype of the RNAi lines was accompanied by the reduced rates of CO(2) assimilation. Microarray analysis of our transgenic plants further revealed that the altered expression of AtPFPbeta affects the expression of several genes involved in diverse physiological processes. Our current data thus suggest that PFP is important in carbohydrate metabolism and other cellular processes.

Rice Pi5-mediated resistance to Magnaporthe oryzae requires the presence of two coiled-coil-nucleotide-binding-leucine-rich repeat genes.

Rice blast, caused by the fungus Magnaporthe oryzae, is one of the most devastating diseases of rice. To understand the molecular basis of Pi5-mediated resistance to M. oryzae, we cloned the resistance (R) gene at this locus using a map-based cloning strategy. Genetic and phenotypic analyses of 2014 F2 progeny from a mapping population derived from a cross between IR50, a susceptible rice cultivar, and the RIL260 line carrying Pi5 enabled us to narrow down the Pi5 locus to a 130-kb interval. Sequence analysis of this genomic region identified two candidate genes, Pi5-1 and Pi5-2, which encode proteins carrying three motifs characteristic of R genes: an N-terminal coiled-coil (CC) motif, a nucleotide-binding (NB) domain, and a leucine-rich repeat (LRR) motif. In genetic transformation experiments of a susceptible rice cultivar, neither the Pi5-1 nor the Pi5-2 gene was found to confer resistance to M. oryzae. In contrast, transgenic rice plants expressing both of these genes, generated by crossing transgenic lines carrying each gene individually, conferred Pi5-mediated resistance to M. oryzae. Gene expression analysis revealed that Pi5-1 transcripts accumulate after pathogen challenge, whereas the Pi5-2 gene is constitutively expressed. These results indicate that the presence of these two genes is required for rice Pi5-mediated resistance to M. oryzae.

Role of the rice hexokinases OsHXK5 and OsHXK6 as glucose sensors.

The Arabidopsis (Arabidopsis thaliana) hexokinase 1 (AtHXK1) is recognized as an important glucose (Glc) sensor. However, the function of hexokinases as Glc sensors has not been clearly demonstrated in other plant species, including rice (Oryza sativa). To investigate the functions of rice hexokinase isoforms, we characterized OsHXK5 and OsHXK6, which are evolutionarily related to AtHXK1. Transient expression analyses using GFP fusion constructs revealed that OsHXK5 and OsHXK6 are associated with mitochondria. Interestingly, the OsHXK5DeltamTP-GFP and OsHXK6DeltamTP-GFP fusion proteins, which lack N-terminal mitochondrial targeting peptides, were present mainly in the nucleus with a small amount of the proteins seen in the cytosol. In addition, the OsHXK5NLS-GFP and OsHXK6NLS-GFP fusion proteins harboring nuclear localization signals were targeted predominantly in the nucleus, suggesting that these OsHXKs retain a dual-targeting ability to mitochondria and nuclei. In transient expression assays using promoterluciferase fusion constructs, these two OsHXKs and their catalytically inactive alleles dramatically enhanced the Glc-dependent repression of the maize (Zea mays) Rubisco small subunit (RbcS) and rice alpha-amylase genes in mesophyll protoplasts of maize and rice. Notably, the expression of OsHXK5, OsHXK6, or their mutant alleles complemented the Arabidopsis glucose insensitive2-1 mutant, thereby resulting in wild-type characteristics in seedling development, Glc-dependent gene expression, and plant growth. Furthermore, transgenic rice plants overexpressing OsHXK5 or OsHXK6 exhibited hypersensitive plant growth retardation and enhanced repression of the photosynthetic gene RbcS in response to Glc treatment. These results provide evidence that rice OsHXK5 and OsHXK6 can function as Glc sensors.

Loss of cytosolic fructose-1,6-bisphosphatase limits photosynthetic sucrose synthesis and causes severe growth retardations in rice (Oryza sativa).

During photosynthesis, triose-phosphates (trioseP) exported from the chloroplast to the cytosol are converted to sucrose via cytosolic fructose-1,6-bisphosphatase (cFBPase). Expression analysis in rice suggests that OscFBP1 plays a major role in the cytosolic conversion of trioseP to sucrose in leaves during the day. The isolated OscFBP1 mutants exhibited markedly decreased photosynthetic rates and severe growth retardation with reduced chlorophyll content, which results in plant death. Analysis of primary carbon metabolites revealed both significantly reduced levels of sucrose, glucose, fructose and starch in leaves of these mutants, and a high accumulation of sucrose to starch in leaves of rice plants. In the oscfbp1 mutants, products of glycolysis and the TCA cycle were significantly increased. A partitioning experiment of (14)C-labelled photoassimilates revealed altered carbon distributions including a slight increase in the insoluble fraction representing transitory starch, a significant decrease in the neutral fraction corresponding to soluble sugars and a high accumulation of phosphorylated intermediates and carboxylic acid fractions in the oscfbp1 mutants. These results indicate that the impaired synthesis of sucrose in rice cannot be sufficiently compensated for by the transitory starch-mediated pathways that have been found to facilitate plant growth in the equivalent Arabidopsis mutants.

Molecular tagging of the Bph1 locus for resistance to brown planthopper (Nilaparvata lugens Stål) through representational difference analysis.

During brown planthopper (BPH) feeding on rice plants, we employed a modified representational difference analysis (RDA) method to detect rare transcripts among those differentially expressed in SNBC61, a BPH resistant near-isogenic line (NIL) carrying the Bph1 resistance gene. This identified 3 RDA clones: OsBphi237, OsBphi252 and OsBphi262. DNA gel-blot analysis revealed that the loci of the RDA clones in SNBC61 corresponded to the alleles of the BPH resistant donor Samgangbyeo. Expression analysis indicated that the RDA genes were up-regulated in SNBC61 during BPH feeding. Interestingly, analysis of 64 SNBC NILs, derived from backcrosses of Samgangbyeo with a BPH susceptible Nagdongbyeo, using a cleaved amplified polymorphic sequence (CAPS) marker indicated that OsBphi252, which encodes a putative lipoxygenase (LOX), co-segregates with BPH resistance. Our results suggest that OsBphi252 is tightly linked to Bph1, and may be useful in marker-assisted selection (MAS) for resistance to BPH.