Systematic approaches to using the FOX hunting system to identify useful rice genes

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23 000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis . This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.     

The FOX hunting system: an alternative gain-of-function gene hunting technique

We have developed a novel gain-of-function system that we have named the FOX hunting system (Full-length cDNA Over-eXpressing gene hunting system). We used normalized full-length cDNA and introduced each cDNA into Arabidopsis by in planta transformation. About 10 000 independent full-length Arabidopsis cDNAs were expressed independently under the CaMV 35S promoter in Arabidopsis. Each transgenic Arabidopsis contained on average 2.6 cDNA clones and was monitored under various categories such as morphological changes, fertility and leaf color. We found 1487 possible morphological mutants from 15 547 transformants. When 115 pale green T(1) mutants were analyzed, 59 lines represented the mutant phenotypes in more than 50% of the T(2) progeny. Characterization of two leaf color mutants revealed the significance of this approach. We also document mutants from several categories and their corresponding full-length cDNAs.     

Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice

Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.     

A genome-wide gain-of-function analysis of rice genes using the FOX-hunting system

The latest report has estimated the number of rice genes to be approximately 32,000. To elucidate the functions of a large population of rice genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the Full-length cDNA Over-eXpresser (FOX) gene-hunting system. This system is very useful for analyzing various gain-of-function phenotypes from large populations of transgenic plants overexpressing cDNAs of interest and others with unknown or important functions. We collected the plasmid DNAs of 13,980 independent full-length cDNA (FL-cDNA) clones to produce a FOX library by placing individual cDNAs under the control of the maize Ubiquitin-1 promoter. The FOX library was transformed into rice by Agrobacterium-mediated high-speed transformation. So far, we have generated approximately 12,000 FOX-rice lines. Genomic PCR analysis indicated that the average number of FL-cDNAs introduced into individual lines was 1.04. Sequencing analysis of the PCR fragments carrying FL-cDNAs from 8615 FOX-rice lines identified FL-cDNAs in 8225 lines, and a database search classified the cDNAs into 5462 independent ones. Approximately 16.6% of FOX-rice lines examined showed altered growth or morphological characteristics. Three super-dwarf mutants overexpressed a novel gibberellin 2-oxidase gene,confirming the importance of this system. We also show here the other morphological alterations caused by individual FL-cDNA expression. These dominant phenotypes should be valuable indicators for gene discovery and functional analysis.     

Overexpression of TIFY genes promotes plant growth in rice through jasmonate signaling

Because environmental stress can reduce crop growth and yield, the identification of genes that enhance agronomic traits is increasingly important. Previous screening of full-length cDNA overexpressing (FOX) rice lines revealed that OsTIFY11b, one of 20 TIFY proteins in rice, affects plant size, grain weight, and grain size. Therefore, we analyzed the effect of OsTIFY11b and nine other TIFY genes on the growth and yield of corresponding TIFY-FOX lines. Regardless of temperature, grain weight and culm length were enhanced in lines overexpressing TIFY11 subfamily genes, except OsTIFY11e. The TIFY-FOX plants exhibited increased floret number and reduced days to flowering, as well as reduced spikelet fertility, and OsTIFY10b, in particular, enhanced grain yield by minimizing decreases in fertility. We suggest that the enhanced growth of TIFY-transgenic rice is related to regulation of the jasmonate signaling pathway, as in Arabidopsis. Moreover, we discuss the potential application of TIFY overexpression for improving crop yield.     

FOX-superroots of Lotus corniculatus, overexpressing Arabidopsis full-length cDNA, show stable variations in morphological traits

Using the full-length cDNA overexpressor (FOX) gene-hunting system, we have generated 130 Arabidopsis FOX-superroot lines in bird's-foot trefoil (Lotus corniculatus) for the systematic functional analysis of genes expressed in roots and for the selection of induced mutants with interesting root growth characteristics. We used the Arabidopsis-FOX Agrobacterium library (constructed by ligating pBIG2113SF) for the Agrobacterium-mediated transformation of superroots (SR) and the subsequent selection of gain-of-function mutants with ectopically expressed Arabidopsis genes. The original superroot culture of L. corniculatus is a unique host system displaying fast root growth in vitro, allowing continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions. Several of the Arabidopsis FOX-superroot lines show interesting deviations from normal growth and morphology of roots from SR-plants, such as differences in pigmentation, growth rate, length or diameter. Some of these mutations are of potential agricultural interest. Genomic PCR analysis revealed that 100 (76.9%) out of the 130 transgenic lines showed the amplification of single fragments. Sequence analysis of the PCR fragments from these 100 lines identified full-length cDNA in 74 of them. Forty-three out of 74 full-length cDNA carried known genes. The Arabidopsis FOX-superroot lines of L. corniculatus, produced in this study, expand the FOX hunting system and provide a new tool for the genetic analysis and control of root growth in a leguminous forage plant.     

Evidence for a disease-resistance pathway in rice similar to the NPR1-mediated signaling pathway in Arabidopsis

The Arabidopsis NPR1/NIM1 gene is a key regulator of systemic acquired resistance (SAR). Over-expression of NPR1 leads to enhanced resistance in Arabidopsis. To investigate the role of NPR1 in monocots, we over-expressed the Arabidopsis NPR1 in rice and challenged the transgenic plants with Xanthomonas oryzae pv. oryzae (Xoo), the rice bacterial blight pathogen. The transgenic plants displayed enhanced resistance to Xoo. RNA blot hybridization indicates that enhanced resistance requires expression of NPR1 mRNA above a threshold level in rice. To identify components mediating the resistance controlled by NPR1, we used NPR1 as bait in a yeast two-hybrid screen. We isolated four cDNA clones encoding rice NPR1 interactors (named rTGA2.1, rTGA2.2, rTGA2.3 and rLG2) belonging to the bZIP family. rTGA2.1, rTGA2.2 and rTGA2.3 share 75, 76 and 78% identity with Arabidopsis TGA2, respectively. In contrast, rLG2 shares highest identity (81%) to the maize liguleless (LG2) gene product, which is involved in establishing the leaf blade-sheath boundary. The interaction of NPR1 with the rice bZIP proteins in yeast was impaired by the npr1-1 and npr1-2 mutations, but not by the nim1-4 mutation. The NPR1-rTGA2.1 interaction was confirmed by an in vitro pull-down experiment. In gel mobility shift assays, rTGA2.1 binds to the rice RCH10 promoter and to a cis-element required sequence-specifically for salicylic acid responsiveness. This is the first demonstration that the Arabidopsis NPR1 gene can enhance disease resistance in a monocot plant. These results also suggest that monocot and dicot plants share a conserved signal transduction pathway controlling NPR1-mediated resistance.     

Engineering OsBAK1 gene as a molecular tool to improve rice architecture for high yield

Generating a new variety of plant with erect-leaf is a critical strategy to improve rice grain yield, as plants with this trait can be dense-planted. The erect-leaf is a significant morphological trait partially regulated by Brassinosteroids (BRs) in rice plants. So far, only a few genes can be used for molecular breeding in rice. Here, we identified OsBAK1 as a potential gene to alter rice architecture. Based on rice genome sequences, four closely related homologs of Arabidopsis BAK1 ( AtBAK1 ) gene were amplified. Phylogenetic analysis and suppression of a weak Arabidopsis mutant bri1-5 indicated that OsBAK1 (Os08g0174700) is the closest relative of AtBAK1. Genetic, physiological, and biochemical analyses all suggest that the function of OsBAK1 is conserved with AtBAK1 . Overexpression of a truncated intracellular domain of OsBAK1 , but not the extracellular domain of OsBAK1 , resulted in a dwarfed phenotype, similar to the rice BR-insensitive mutant plants. The expression of OsBAK1 changed important agricultural traits of rice such as plant height, leaf erectness, grain morphologic features, and disease resistance responses. Our results suggested that a new rice variety with erect-leaf and normal reproduction can be generated simply by suppressing the expression level of OsBAK1 . Therefore, OsBAK1 is a potential molecular breeding tool for improving rice grain yield by modifying rice architecture.     

Genomic basis for cell-wall diversity in plants. A comparative approach to gene families in rice and Arabidopsis

Monocotyledons and dicotyledons are distinct, not only in their body plans and developmental patterns, but also in the structural features of their cell walls. The recent completion of the rice (Oryza sativa) genomic sequence and publication of the sequence data, together with the completed database of the Arabidopsis thaliana genome, provide the first opportunity to compare the full complement of cell-wall-related genes from the two distinct classes of flowering plants. We made this comparison by exploiting the fact that Arabidopsis and rice have type I and type II walls, respectively, and therefore represent the two extremes in terms of the structural features of plant cell walls. In this review article, we classify all cell-wall-related genes into 32 gene families, and generate their phylogenetic trees. Using these data, we can phylogenetically compare individual genes of particular interest between Arabidopsis and rice. This comparative genome approach shows that the differences in wall architecture in the two plant groups actually mirror the diversity of the individual gene families involved in the cell-wall dynamics of the respective plant species. This study also identifies putative rice orthologs of genes with well-defined functions in Arabidopsis and other plant species.     

Rice-<Emphasis Type="Italic">Arabidopsis</Emphasis> FOX line screening with FT-NIR-based fingerprinting for GC-TOF/MS-based metabolite profiling

The full-length cDNA over-expressing (FOX) gene hunting system is useful for genome-wide gain-of-function analysis. The screening of FOX lines requires a high-throughput metabolomic method that can detect a wide range of metabolites. Fourier transform-near-infrared (FT-NIR) spectroscopy in combination with the chemometric approach has been used to analyze metabolite fingerprints. Since FT-NIR spectroscopy can be used to analyze a solid sample without destructive extraction, this technique enables untargeted analysis and high-throughput screening focusing on the alteration of metabolite composition. We performed non-destructive FT-NIR-based fingerprinting to screen seed samples of 3000 rice-Arabidopsis FOX lines; the samples were obtained from transgenic Arabidopsis thaliana lines that overexpressed rice full-length cDNA. Subsequently, the candidate lines exhibiting alteration in their metabolite fingerprints were analyzed by gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) in order to assess their metabolite profiles. Finally, multivariate regression using orthogonal projections to latent structures (O2PLS) was used to elucidate the predictive metabolites obtained in FT-NIR analysis by integration of the datasets obtained from FT-NIR and GC-TOF/MS analyses. FT-NIR-based fingerprinting is a technically efficient method in that it facilitates non-destructive analysis in a high-throughput manner. Furthermore, with the integrated analysis used here, we were able to discover unique metabotypes in rice-Arabidopsis FOX lines; thus, this approach is beneficial for investigating the function of rice genes related to metabolism.     

Functional characterisation of OsCPK21, a calcium-dependent protein kinase that confers salt tolerance in rice

Calcium acts as a messenger in various signal transduction pathways in plants. Calcium-dependent protein kinases (CDPKs) play important roles in regulating downstream components in calcium signaling pathways. In rice, the CDPKs constitute a large multigene family consisting of 29 genes, but the biological functions and functional divergence or redundancy of most of these genes remain unclear. Using a mini-scale full-length cDNA overexpressor (FOX) gene hunting system, we generated 250 independent transgenic rice plants overexpressing individual rice CDPKs (CDPK FOX-rice lines). These CDPK FOX-rice lines were screened for salt stress tolerance. The survival rate of the OsCPK21-FOX plants was higher than that of wild-type (WT) plants grown under high salinity conditions. The inhibition of seedling growth by abscisic acid (ABA) treatment was greater in the OsCPK21-FOX plants than in WT plants. Several ABA- and high salinity-inducible genes were more highly expressed in the OsCPK21-FOX plants than in WT plants. These results suggest that OsCPK21 is involved in the positive regulation of the signaling pathways that are involved in the response to ABA and salt stress.     

Screening of abiotic stress‐responsive cotton genes using a cotton full‐length cDNA overexpressing Arabidopsis library

Cotton (Gossypium hirsutum L.) is a major crop and the main source of natural fiber worldwide. Because various abiotic and biotic stresses strongly influence cotton fiber yield and quality, improved stress resistance of this crop plant is urgently needed. In this study, we used Gateway technology to construct a normalized full‐length cDNA overexpressing (FOX) library from upland cotton cultivar ZM12 under various stress conditions. The library was transformed into Arabidopsis to produce a cotton‐FOX‐Arabidopsis library. Screening of this library yielded 6,830 transgenic Arabidopsis lines, of which 757 were selected for sequencing to ultimately obtain 659 cotton ESTs. GO and KEGG analyses mapped most of the cotton ESTs to plant biological process, cellular component, and molecular function categories. Next, 156 potential stress‐responsive cotton genes were identified from the cotton‐FOX‐Arabidopsis library under drought, salt, ABA, and other stress conditions. Four stress‐related genes identified from the library, designated as GhCAS, GhAPX, GhSDH, and GhPOD, were cloned from cotton complementary DNA, and their expression patterns under stress were analyzed. Phenotypic experiments indicated that overexpression of these cotton genes in Arabidopsis affected the response to abiotic stress. The method developed in this study lays a foundation for high‐throughput cloning and rapid identification of cotton functional genes.     

Construction of a full-length cDNA library from young spikelets of hexaploid wheat and its characterization by large-scale sequencing of expressed sequence tags

The polyploid nature of wheat is a key characteristic of the plant. Full-length complementary DNAs (cDNAs) provide essential information that can be used to annotate the genes and provide a functional analysis of these genes and their products. We constructed a full-length cDNA library derived from young spikelets of common wheat, and obtained 24056 expressed sequence tags (ESTs) from both ends of the cDNA clones. These ESTs were grouped into 3605 contigs using the phrap method, representing expressed loci from each of the three genomes. Using BLAST, 3605 contigs were grouped into 1902 gene clusters, showing that loci of the three genomes are not always expressed. A homology search of these gene clusters against a wheat EST database (15964 gene clusters) and a rice full-length cDNA database (21447 gene clusters) revealed that a quarter of the wheat full-length cDNAs were novel. A protein database of Arabidopsis was used to examine the functional classification of these gene clusters. The GC-content in the 5 -UTR region of wheat cDNAs was compared to that of rice. Forty-three genes (3.5% of wheat cDNAs homologous to those of rice) possessed distinct GC-content in the 5 -UTR region, suggesting different breeding behaviors of wheat and rice.     

Maize orthologs of rice GS5 and their transregulator are associated with kernel development

Genome information from model species such as rice can assist in the cloning of genes in a complex genome,such as maize.Here,we identified a maize ortholog of rice GS5 that contributes to kernel development in maize.The genomewide association analysis of the expression levels of ZmGS5,and 15 of its 26 paralogs,identified a trans-regulator on chromosome 7,which was a BAKi-like gene.This gene that we named as ZmBAK1-7 could regulate the expression of ZmGS5 and three of the paralogs.Candidate-gene association analyses revealed that these five genes were associated with maize kernel development-related traits.Linkage analyses also detected that ZmGSs and ZmBAK1-7 co-localized with mapped QTLs.A transgenic analysis of ZmGS5 in Arabidopsis thaliana L.showed a significant increase in seed weight and cell number,suggesting that ZmGS5 may have a conserved function among different plant species that affects seed development.     

转拟南芥NPR1基因桂99 T3代植株的抗病性及农艺性状表现

以转拟南芥AtNPR1基因的恢复系品种桂99T3代纯合株系为材料,考查其农艺性状及其抗病性,并比较转基因植株与桂99侵染水稻白叶枯病菌后的农艺性状。结果表明:转基因植株表现出对水稻白叶枯病的抗性显著增强77%以上;穗长、剑叶长、有效穗数、一次枝梗数、每穗实粒数、单株产量和谷粒宽等农艺性状与未转基因桂99无显著差别。在受到水稻白叶枯病菌侵染后,转基因植株的一次枝梗数、每穗粒数、每穗实粒数和单株产量等方面均比对照桂99高出13%～78%。说明AtNPR1基因增强了水稻的抗病能力,从而降低了病害引起的产量损失。转基因植株的恢复力不受影响,稻米品质比桂99更加优良。本工作为转基因水稻抗病育种的研究奠定了基础。     

SRWD: a novel WD40 protein subfamily regulated by salt stress in rice (OryzasativaL.)

By analysis with microarray data, we found that a gene encoding a novel protein containing five WD40 repeats, was regulated by salt stress in rice and named as SRWD1 (Salt responsive WD40 protein 1). By database searching, additional four SRWD1-like genes (SRWD2-SRWD5) were found in rice genome, and these five SRWD genes formed a novel WD40 subfamily. Phylogenetic analysis showed that plant SRWD proteins divided into four groups. The significant functional divergences during SRWD evolution were found. The tissue-specific and salt responsive expression profiling for SRWD genes was investigated based on microarray data. It was found that all five SRWD genes in rice were regulated by salt stress. Further, we found that SRWD1 was regulated with different patterns by salt stress in two rice cultivars responding differently to salt stress. Our study correlates WD40 proteins with salt stress in plants and provides fundamental information for the further investigation of plant SRWD proteins.