Wheat Ym2 originated from Aegilops sharonensis and confers resistance to soil-borne Wheat yellow mosaic virus infection to the roots.

Wheat yellow mosaic virus (WYMV) is a pathogen transmitted into its host's roots by the soil-borne vector Polymyxa graminis. Ym1 and Ym2 genes protect the host from the significant yield losses caused by the virus, but the mechanistic basis of these resistance genes remains poorly understood. Here, it has been shown that Ym1 and Ym2 act within the root either by hindering the initial movement of WYMV from the vector into the root and/or by suppressing viral multiplication. A mechanical inoculation experiment on the leaf revealed that the presence of Ym1 reduced viral infection incidence, rather than viral titer, while that of Ym2 was ineffective in the leaf. To understand the basis of the root specificity of the Ym2 product, the gene was isolated from bread wheat using a positional cloning approach. The candidate gene encodes a CC-NBS-LRR protein and it correlated allelic variation with respect to its sequence with the host's disease response. Ym2 (B37500) and its paralog (B35800) are found in the near-relatives, respectively, Aegilops sharonensis and Aegilops speltoides (a close relative of the donor of bread wheat's B genome), while both sequences, in a concatenated state, are present in several accessions of the latter species. Structural diversity in Ym2 has been generated via translocation and recombination between the two genes and enhanced by the formation of a chimeric gene resulting from an intralocus recombination event. The analysis has revealed how the Ym2 region has evolved during the polyploidization events leading to the creation of cultivated wheat.

Organ-enriched gene expression during floral morphogenesis in wild barley.

Floral morphology varies considerably between dicots and monocots. The ABCDE model explaining how floral organ development is controlled was formulated using core eudicots and applied to grass crops. Barley (Hordeum. vulgare) has unique floral morphogenesis. Wild barley (H. vulgare ssp. spontaneum), which is the immediate ancestor of cultivated barley (H. vulgare ssp. vulgare), contains a rich reservoir of genetic diversity. However, the wild barley genes involved in floral organ development are still relatively uncharacterized. In this study, we generated an organ-specific transcriptome atlas for wild barley floral organs. Genome-wide transcription profiles indicated that 22 838 protein-coding genes were expressed in at least one organ. These genes were grouped into seven clusters according to the similarities in their expression patterns. Moreover, 5619 genes exhibited organ-enriched expression, 677 of which were members of 47 transcription factor families. Gene ontology analyses suggested that the functions of the genes with organ-enriched expression influence the biological processes in floral organs. The co-expression regulatory network showed that the expression of 690 genes targeted by MADS-box proteins was highly positively correlated with the expression of ABCDE model genes during floral morphogenesis. Furthermore, the expression of 138 genes was specific to the wild barley OUH602 genome and not the Morex genome; most of these genes were highly expressed in the glume, awn, lemma, and palea. This study revealed the global gene expression patterns underlying floral morphogenesis in wild barley. On the basis of the study findings, a molecular mechanism controlling floral morphology in barley was proposed.

Identification of the causal mutation in early heading mutant of bread wheat (Triticum aestivum L.) using MutMap approach.

In bread wheat (Triticum aestivum L.), fine-tuning the heading time is essential to maximize grain yield. Photoperiod-1 (Ppd-1) and VERNALIZATION 1 (Vrn-1) are major genes affecting photoperiod sensitivity and vernalization requirements, respectively. These genes have predominantly governed heading timing. However, Ppd-1 and Vrn-1 significantly impact heading dates, necessitating another gene that can slightly modify heading dates for fine-tuning. In this study, we developed an early heading mutant from the ethyl methanesulfonate-mutagenized population of the Japanese winter wheat cultivar "Kitahonami." MutMap analysis identified a nonsense mutation in the clock component gene Wheat PHYTOCLOCK 1/LUX ARRHYTHMO (WPCL-D1) as the probable SNP responsible for the early heading mutant on chromosome 3D. Segregation analysis using F2 and F3 populations confirmed that plants carrying the wpcl-D1 allele headed significantly earlier than those with the functional WPCL-D1. The early heading mutant exhibited increased expression levels of Ppd-1 and circadian clock genes, such as WPCL1 and LATE ELONGATED HYPOCOTYL (LHY). Notably, the transcript accumulation levels of Ppd-A1 and Ppd-D1 were influenced by the copy number of the functional WPCL1 gene. These results suggest that a loss-of-function mutation in WPCL-D1 is the causal mutation for the early heading phenotype. Adjusting the functional copy number of WPCL1 will be beneficial in fine-tuning of heading dates. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01478-5.

Series of Resistance Genes in Barley (Hordeum vulgare) that Control Barley Yellow Mosaic Virus Multiplication and the Root-to-Leaf Systemic Movement.

The infection of young winter barley (Hordeum vulgare L.) root system in winter by barley yellow mosaic virus (BaYMV) can lead to high yield losses. Resistance breeding is critical for managing this virus, but there are only a few reports on resistance genes that describe how the genes control BaYMV propagation and the systemic movement from the roots to the leaves. Here we report a real-time quantitative PCR analysis of the virus in barley roots and leaves carrying BaYMV resistance genes (rym1 to rym15 and an unknown gene) to elucidate the molecular mechanisms underlying the barley response to BaYMV. The resistance mechanism directly targets the virus. Moreover, the resistance genes/cultivars were classified into the following three groups according to their BaYMV titer: (i) immune (BaYMV was undetectable in the roots or leaves), (ii) partially immune (BaYMV was detected in the roots but not in the leaves), and (iii) susceptible (BaYMV was detected in the roots and leaves). Our results clarified the functions of the resistance genes in barley roots and leaves following a BaYMV infection. We anticipate our analysis to be a starting point for more understanding of the correspondence between resistance genes of Triticeae and the soil-borne viruses.

Mutations within the miR172 target site of wheat AP2 homoeologs regulate lodicule size and rachis internode length.

Closed fertilization in flowers, or cleistogamy, reduces the risk of fungal infection in Triticeae crops. In barley (Hordeum vulgare), cleistogamy is determined by a single recessive gene, cly1, which results from a single nucleotide polymorphism within the microRNA172 target site of the Apetala2 (AP2) transcription factor gene. The recessive cly1 allele negatively regulates the development of lodicules, keeping florets closed at anthesis. However, cleistogamy is not evident in hexaploid wheat (Triticum aestivum) cultivars. This study aimed at identifying mutations in wheat AP2 orthologs by ethyl methane sulfonate-induced mutagenesis and high-resolution melt analysis. Although flowers of AP2 mutants induced in the A and D genomes opened at anthesis, their lodicule size was significantly smaller, especially in the direction of depth, than that of wild-type plants. One of the mutants that carried a nucleotide replacement in AP2 from the D genome produced a compact spike caused by a substantial decrease in rachis internode length, analogous to the barley dense spike. Cleistogamous hexaploid wheat might be generated by combining effective mutant alleles of AP2-homoeologous genes.

Genome sequencing-based coverage analyses facilitate high-resolution detection of deletions linked to phenotypes of gamma-irradiated wheat mutants.

BACKGROUND: Gamma-irradiated mutants of Triticum aestivum L., hexaploid wheat, provide novel and agriculturally important traits and are used as breeding materials. However, the identification of causative genomic regions of mutant phenotypes is challenging because of the large and complicated genome of hexaploid wheat. Recently, the combined use of high-quality reference genome sequences of common wheat and cost-effective resequencing technologies has made it possible to evaluate genome-wide polymorphisms, even in complex genomes. RESULTS: To investigate whether the genome sequencing approach can effectively detect structural variations, such as deletions, frequently caused by gamma irradiation, we selected a grain-hardness mutant from the gamma-irradiated population of Japanese elite wheat cultivar "Kitahonami." The Hardness (Ha) locus, including the puroindoline protein-encoding genes Pina-D1 and Pinb-D1 on the short arm of chromosome 5D, primarily regulates the grain hardness variation in common wheat. We performed short-read genome sequencing of wild-type and grain-hardness mutant plants, and subsequently aligned their short reads to the reference genome of the wheat cultivar "Chinese Spring." Genome-wide comparisons of depth-of-coverage between wild-type and mutant strains detected ~ 130 Mbp deletion on the short arm of chromosome 5D in the mutant genome. Molecular markers for this deletion were applied to the progeny populations generated by a cross between the wild-type and the mutant. A large deletion in the region including the Ha locus was associated with the mutant phenotype, indicating that the genome sequencing is a powerful and efficient approach for detecting a deletion marker of a gamma-irradiated mutant phenotype. In addition, we investigated a pre-harvest sprouting tolerance mutant and identified a 67.8 Mbp deletion on chromosome 3B where Viviparous-B1 and GRAS family transcription factors are located. Co-dominant markers designed to detect the deletion-polymorphism confirmed the association with low germination rate, leading to pre-harvest sprouting tolerance. CONCLUSIONS: Short read-based genome sequencing of gamma-irradiated mutants facilitates the identification of large deletions linked to mutant phenotypes when combined with segregation analyses in progeny populations. This method allows effective application of mutants with agriculturally important traits in breeding using marker-assisted selection.

Two dominant genes in barley (Hordeum vulgare L.) complementarily encode perfect resistance to Japanese soil-borne wheat mosaic virus.

Japanese soil-borne wheat mosaic virus (Furovirus) is a damaging pathogen of wheat and barley. This virus can survive in the soil for several decades, so the deployment of resistant cultivars represents the only practical control measure. Here, a genetic analysis has identified two regions of the barley genome-one on chromosome 2H and the other on chromosome 3H-as harboring gene(s) encoding resistance to this virus. The joint presence of both loci, termed Jmv1 and Jmv2, made the plants essentially immune, with resistance being dominant over susceptibility at each locus. Phylogenetic analysis showed that the virus is not closely related to the type Furovirus species Soil-borne wheat mosaic virus. There was a difference between the RNA1- and RNA2-based phylogenies of the virus species in Furovirus implying the independent segregation of the virus subgenomes.

Genome-Wide Analysis of Snf2 Gene Family Reveals Potential Role in Regulation of Spike Development in Barley.

Sucrose nonfermenting 2 (Snf2) family proteins, as the catalytic core of ATP-dependent chromatin remodeling complexes, play important roles in nuclear processes as diverse as DNA replication, transcriptional regulation, and DNA repair and recombination. The Snf2 gene family has been characterized in several plant species; some of its members regulate flower development in Arabidopsis. However, little is known about the members of the family in barley (Hordeum vulgare). Here, 38 Snf2 genes unevenly distributed among seven chromosomes were identified from the barley (cv. Morex) genome. Phylogenetic analysis categorized them into 18 subfamilies. They contained combinations of 21 domains and consisted of 3 to 34 exons. Evolution analysis revealed that segmental duplication contributed predominantly to the expansion of the family in barley, and the duplicated gene pairs have undergone purifying selection. About eight hundred Snf2 family genes were identified from 20 barley accessions, ranging from 38 to 41 genes in each. Most of these genes were subjected to purification selection during barley domestication. Most were expressed abundantly during spike development. This study provides a comprehensive characterization of barley Snf2 family members, which should help to improve our understanding of their potential regulatory roles in barley spike development.

Genome-wide analysis of rice cis-natural antisense transcription under cadmium exposure using strand-specific RNA-Seq.

BACKGROUND: The elucidation of novel transcripts and their expression in response to various stress conditions is necessary to understand the transcriptional network of plants as an adaptation to biotic and abiotic stresses. We performed strand-specific RNA-Seq (ssRNA-Seq) on rice exposed to cadmium (Cd) for 24 h and investigated the expression of cis-natural antisense transcripts (cis-NATs), a class of endogenous coding or non-protein-coding RNAs with sequence complementarity to the opposite strands of RAP transcripts. RESULTS: Many RAP transcripts possessed cis-NATs and these cis-NATs were responsive to some extent. Cis-NATs were upregulated from 26, 266 and 409 RAP gene loci, while 2054, 2501 and 2825 RAP transcripts were upregulated from 38,123 RAP loci under high Cd exposure in roots at 1, 12 and 24 h, respectively. In addition, most of the upregulated cis-NATs showed little upregulation under ABA or cold treatment. A number of cis-NATs were upregulated from less than 35 RAP gene loci in different tissue and time-point combinations under low Cd exposure, suggesting that cis-NATs respond to environmental stress. Furthermore, 409 RAP transcripts with upregulated cis-NATs were classified into three groups based on the expression of the RAP transcripts from the opposite DNA strand, including 138 upregulated, 128 invariable, and 143 downregulated transcripts, although the responses of cis-NATs and RAP transcripts were not always correlated. CONCLUSIONS: We have shown that the cis-NATs identified by ssRNA-Seq analysis are novel genes and that some of them are stress-specific and show different responses depending on the degree of stress and tissue. These results improve our understanding of the complete molecular mechanism of plant adaptation to Cd exposure.

TENOR: Database for Comprehensive mRNA-Seq Experiments in Rice.

Here we present TENOR (Transcriptome ENcyclopedia Of Rice, http://tenor.dna.affrc.go.jp), a database that encompasses large-scale mRNA sequencing (mRNA-Seq) data obtained from rice under a wide variety of conditions. Since the elucidation of the ability of plants to adapt to various growing conditions is a key issue in plant sciences, it is of great interest to understand the regulatory networks of genes responsible for environmental changes. We used mRNA-Seq and performed a time-course transcriptome analysis of rice, Oryza sativa L. (cv. Nipponbare), under 10 abiotic stress conditions (high salinity; high and low phosphate; high, low and extremely low cadmium; drought; osmotic; cold; and flood) and two plant hormone treatment conditions (ABA and jasmonic acid). A large number of genes that were responsive to abiotic stresses and plant hormones were detected by differential expression analysis. Furthermore, several responsive genes were found to encode transcription factors that could control the transcriptional network of stress responses, but the timing of the induction of these genes was not uniform across conditions. A significant number of cis-regulatory elements were enriched in the promoter regions of the responsive genes and were shared among conditions. These data suggest that some key components of gene regulation networks are shared between different stress signaling pathways. All the resources (novel genes identified from mRNA-Seq data, expression profiles, co-expressed genes and cis-regulatory elements) can be searched for and are available in TENOR.

Variation of DNA methylation patterns associated with gene expression in rice (Oryza sativa) exposed to cadmium.

We report genome-wide single-base resolution maps of methylated cytosines and transcriptome change in Cd-exposed rice. Widespread differences were identified in CG and non-CG methylation marks between Cd-exposed and Cd-free rice genomes. There are 2320 non-redundant differentially methylated regions detected in the genome. RNA sequencing revealed 2092 DNA methylation-modified genes differentially expressed under Cd exposure. More genes were found hypermethylated than those hypomethylated in CG, CHH and CHG (where H is A, C or T) contexts in upstream, gene body and downstream regions. Many of the genes were involved in stress response, metal transport and transcription factors. Most of the DNA methylation-modified genes were transcriptionally altered under Cd stress. A subset of loss of function mutants defective in DNA methylation and histone modification activities was used to identify transcript abundance of selected genes. Compared with wide type, mutation of MET1 and DRM2 resulted in general lower transcript levels of the genes under Cd stress. Transcripts of OsIRO2, OsPR1b and Os09g02214 in drm2 were significantly reduced. A commonly used DNA methylation inhibitor 5-azacytidine was employed to investigate whether DNA demethylation affected physiological consequences. 5-azacytidine provision decreased general DNA methylation levels of selected genes, but promoted growth of rice seedlings and Cd accumulation in rice plant.

A BAC physical map of aus rice cultivar 'Kasalath', and the map-based genomic sequence of 'Kasalath' chromosome 1.

Comparative analysis using available genomic resources within closely related species is an effective way to investigate genomic sequence and structural diversity. Rice (Oryza sativa L.) has undergone significant physiological and morphological changes during its domestication and local adaptation. We present a complete bacterial artificial chromosome (BAC) physical map for the aus rice cultivar 'Kasalath', which covers 90% of the sequence of temperate japonica rice cultivar 'Nipponbare'. Examination of physical distances between computational and experimental measurements of 'Kasalath' BAC insert size revealed the presence of more than 500 genomic regions that appear to have significant chromosome structural changes between the two cultivars. In particular, a genomic region on the long arm of 'Kasalath' chromosome 11 carrying a disease-resistance gene cluster was greatly expanded relative to the 'Nipponbare' genome. We also decoded 41.37 Mb of high-quality genomic sequence from 'Kasalath' chromosome 1. Extensive comparisons of chromosome 1 between 'Kasalath' and 'Nipponbare' led to the discovery of 317,843 single-nucleotide polymorphisms (SNPs) and 66,331 insertion/deletion (indel) sites. Nearly two-thirds of the expressed genes on rice chromosome 1 carried natural variations involving SNPs and/or indels that resulted in substitutions, insertions or deletions of amino acids in one cultivar relative to the other. We also observed gain and loss of genes caused by large indels. This study provides an important framework and an invaluable dataset for further understanding of the molecular mechanisms underlying the evolution and functions of the rice genome.

RNA sequencing-mediated transcriptome analysis of rice plants in endoplasmic reticulum stress conditions.

BACKGROUND: The endoplasmic reticulum (ER) stress response is widely known to function in eukaryotes to maintain the homeostasis of the ER when unfolded or misfolded proteins are overloaded in the ER. To understand the molecular mechanisms of the ER stress response in rice (Oryza sativa L.), we previously analyzed the expression profile of stably transformed rice in which an ER stress sensor/transducer OsIRE1 was knocked-down, using the combination of preliminary microarray and quantitative RT-PCR. In this study, to obtain more detailed expression profiles of genes involved in the initial stages of the ER stress response in rice, we performed RNA sequencing of wild-type and transgenic rice plants produced by homologous recombination in which endogenous genomic OsIRE1 was replaced by missense alleles defective in ribonuclease activity. RESULTS: At least 38,076 transcripts were investigated by RNA sequencing, 380 of which responded to ER stress at a statistically significant level (195 were upregulated and 185 were downregulated). Furthermore, we successfully identified 17 genes from the set of 380 ER stress-responsive genes that were not included in the probe set of the currently available microarray chip in rice. Notably, three of these 17 genes were non-annotated genes, even in the latest version of the Rice Annotation Project Data Base (RAP-DB, version IRGSP-1.0). CONCLUSIONS: Therefore, RNA sequencing-mediated expression profiling provided valuable information about the ER stress response in rice plants and led to the discovery of new genes related to ER stress.

Genome-wide expression analysis of reactive oxygen species gene network in Mizuna plants grown in long-term spaceflight.

BACKGROUND: Spaceflight environment have been shown to generate reactive oxygen species (ROS) and induce oxidative stress in plants, but little is known about the gene expression of the ROS gene network in plants grown in long-term spaceflight. The molecular response and adaptation to the spaceflight environment of Mizuna plants harvested after 27 days of cultivation onboard the International Space Station (ISS) were measured using genome-wide mRNA expression analysis (mRNA-Seq). RESULTS: Total reads of transcripts from the Mizuna grown in the ISS as well as on the ground by mRNA-Seq showed 8,258 and 14,170 transcripts up-regulated and down-regulated, respectively, in the space-grown Mizuna when compared with those from the ground-grown Mizuna. A total of 20 in 32 ROS oxidative marker genes were up-regulated, including high expression of four hallmarks, and preferentially expressed genes associated with ROS-scavenging including thioredoxin, glutaredoxin, and alternative oxidase genes. In the transcription factors of the ROS gene network, MEKK1-MKK4-MPK3, OXI1-MKK4-MPK3, and OXI1-MPK3 of MAP cascades, induction of WRKY22 by MEKK1-MKK4-MPK3 cascade, induction of WRKY25 and repression of Zat7 by Zat12 were suggested. RbohD and RbohF genes were up-regulated preferentially in NADPH oxidase genes, which produce ROS. CONCLUSIONS: This large-scale transcriptome analysis revealed that the spaceflight environment induced oxidative stress and the ROS gene network activation in the space-grown Mizuna. Among transcripts altered in expression by space conditions, some were common genes response to abiotic and biotic stress. Furthermore, certain genes were exclusively up-regulated in Mizuna grown on the ISS. Surprisingly, Mizuna grew in space normally, as well as on the ground, demonstrating that plants can acclimate to long-term exposure in the spaceflight environment by reprogramming the expression of the ROS gene network.

Identification of Barley yellow mosaic virus Isolates Breaking rym3 Resistance in Japan.

In early spring 2018, significant mosaic disease symptoms were observed for the first time on barley leaves (Hordeum vulgare L., cv. New Sachiho Golden) in Takanezawa, Tochigi Prefecture, Japan. This cultivar carries the resistance gene rym3 (rym; resistance to yellow mosaic). Through RNA-seq analysis, Barley yellow mosaic virus (BaYMV-Takanezawa) was identified in the roots of all five plants (T01-T05) in the field. Phylogenetic analysis of RNA1, encompassing known BaYMV pathotypes I through V, revealed that it shares the same origin as isolate pathotype IV (BaYMV-Ohtawara pathotype). However, RNA2 analysis of isolates revealed the simultaneous presence of two distinct BaYMV isolates, BaYMV-Takanezawa-T01 (DRR552862, closely related to pathotype IV) and BaYMV-Takanezawa-T02 (DRR552863, closely related to pathotype III). The amino acid sequences of the BaYMV-Takanezawa isolates displayed variations, particularly in the VPg and N-terminal region of CP, containing mutations not found in other domains of the virus genome. Changes in the CI (RNA1 amino acid residue 459) and CP (RNA1 amino acid residue 2138) proteins correlated with pathogenicity. These findings underscore the importance of monitoring and understanding the genetic diversity of BaYMV for effective disease management strategies in crop breeding.

Characterisation of the wheat (Triticum aestivum L.) transcriptome by de novo assembly for the discovery of phosphate starvation-responsive genes: gene expression in Pi-stressed wheat.

BACKGROUND: Phosphorus (P) is an essential macronutrient for plant growth and development. To modulate their P homeostasis, plants must balance P uptake, mobilisation, and partitioning to various organs. Despite the worldwide importance of wheat as a cultivated food crop, molecular mechanisms associated with phosphate (Pi) starvation in wheat remain unclear. To elucidate these mechanisms, we used RNA-Seq methods to generate transcriptome profiles of the wheat variety 'Chinese Spring' responding to 10 days of Pi starvation. RESULTS: We carried out de novo assembly on 73.8 million high-quality reads generated from RNA-Seq libraries. We then constructed a transcript dataset containing 29,617 non-redundant wheat transcripts, comprising 15,047 contigs and 14,570 non-redundant full-length cDNAs from the TriFLDB database. When compared with barley full-length cDNAs, 10,656 of the 15,047 contigs were unalignable, suggesting that many might be distinct from barley transcripts. The average expression level of the contigs was lower than that of the known cDNAs, implying that these contigs included transcripts that were rarely represented in the full-length cDNA library. Within the non-redundant transcript set, we identified 892-2,833 responsive transcripts in roots and shoots, corresponding on average to 23.4% of the contigs not covered by cDNAs in TriFLDB under Pi starvation. The relative expression level of the wheat IPS1 (Induced by Phosphate Starvation 1) homologue, TaIPS1, was 341-fold higher in roots and 13-fold higher in shoots; this finding was further confirmed by qRT-PCR analysis. A comparative analysis of the wheat- and rice-responsive transcripts for orthologous genes under Pi-starvation revealed commonly upregulated transcripts, most of which appeared to be involved in a general response to Pi starvation, namely, an IPS1-mediated signalling cascade and its downstream functions such as Pi remobilisation, Pi uptake, and changes in Pi metabolism. CONCLUSIONS: Our transcriptome profiles demonstrated the impact of Pi starvation on global gene expression in wheat. This study revealed that enhancement of the Pi-mediated signalling cascade using IPS1 is a potent adaptation mechanism to Pi starvation that is conserved in both wheat and rice and validated the effectiveness of using short-read next-generation sequencing data for wheat transcriptome analysis in the absence of reference genome information.

Diversity in the complexity of phosphate starvation transcriptomes among rice cultivars based on RNA-Seq profiles.

Rice has developed several morphological and physiological strategies to adapt to phosphate starvation in the soil. In order to elucidate the molecular basis of response to phosphate starvation, we performed mRNA sequencing of 4 rice cultivars with variation in growth response to Pi starvation as indicated by the shoot/root dry weight ratio. Approximately 254 million sequence reads were mapped onto the IRGSP-1.0 reference rice genome sequence and an average of about 5,000 transcripts from each cultivar were found to be responsive under phosphate starvation. Comparative analysis of the RNA-Seq profiles of the 4 cultivars revealed similarities as well as distinct differences in expression of these responsive transcripts. We elucidated a set of core responsive transcripts including annotated and unannotated transcripts commonly expressed in the 4 cultivars but with different levels of expression. De novo assembly of unmapped reads to the Nipponbare genome generated a set of sequence contigs representing potential new transcripts that may be involved in tolerance to phosphate starvation. This study can be used for identification of genes and gene networks associated with environmental stress and the development of novel strategies for improving tolerance to phosphate starvation in rice and other cereal crops.

Genetic resistance in barley against Japanese soil-borne wheat mosaic virus functions in the roots.

Infection by the Japanese soil-borne wheat mosaic virus (JSBWMV) can lead to substantial losses in the grain yield of barley and wheat crops. While genetically based resistance to this virus has been documented, its mechanistic basis remains obscure. In this study, the deployment of a quantitative PCR assay showed that the resistance acts directly against the virus rather than by inhibiting the colonization of the roots by the virus' fungal vector Polymyxa graminis. In the susceptible barley cultivar (cv.) Tochinoibuki, the JSBWMV titre was maintained at a high level in the roots during the period December-April, and the virus was translocated from the root to the leaf from January onwards. In contrast, in the roots of both cv. Sukai Golden and cv. Haruna Nijo, the titre was retained at a low level, and translocation of the virus to the shoot was strongly suppressed throughout the host's entire life cycle. The roots of wild barley (Hordeum vulgare ssp. spontaneum) accession H602 responded in the early stages of infection similarly to those of the resistant cultivated forms, but the host was unable to suppress the translocation of the virus to the shoot from March onwards. The virus titre in the root was presumed to have been restricted by the action of the gene product of Jmv1 (on chromosome 2H), while the stochastic nature of the infection was suppressed by the action of that of Jmv2 (on chromosome 3H), a gene harbored by cv. Sukai Golden but not by either cv. Haruna Nijo or accession H602.

Expression of ER quality control-related genes in response to changes in BiP1 levels in developing rice endosperm.

Binding protein (BiP) is the key chaperone involved in folding of secretory proteins such as seed storage proteins in the ER lumen. To obtain functional information about BiP1, a gene that is predominantly expressed during rice seed maturation, we generated several transgenic rice plants in which various levels of BiP1 protein accumulated in an endosperm-specific manner. Severe suppression (BiP1 KD) or significant over-expression (BiP1 OEmax) of BiP1 not only altered seed phenotype and the intracellular structure of endosperm cells, but also reduced seed storage protein content, starch accumulation and grain weight. Microarray and RT-PCR analyses indicated that expression of many chaperone and co-chaperone genes was induced in transgenic plants, with more prominent expression in the BiP1 KD line than in the BiP1 OEmax line. Transcriptional induction of most chaperones was observed in calli treated with dithiothreitol or tunicamycin, treatments that trigger ER stress, indicating that induction of the chaperone genes in transgenic rice was caused by an ER stress response. In transient assays using rice protoplasts, the ortholog (Os06g0622700) of the AtbZIP60 transcription factor was shown to be involved in activation of some chaperone genes. Slight increases in the BiP1 level compared with wild-type, accompanied by increased levels of calnexin and protein disulfide isomerase-like proteins, resulted in significant enhancement of seed storage protein content, without any change in intracellular structure or seed phenotype. Judicious modification of BiP1 levels in transgenic rice can provide suitable conditions for the production of secretory proteins by alleviating ER stress.

Massive parallel sequencing of mRNA in identification of unannotated salinity stress-inducible transcripts in rice (Oryza sativa L.).

BACKGROUND: Microarray technology is limited to monitoring the expression of previously annotated genes that have corresponding probes on the array. Computationally annotated genes have not fully been validated, because ESTs and full-length cDNAs cannot cover entire transcribed regions. Here, mRNA-Seq (an Illumina cDNA sequencing application) was used to monitor whole mRNAs of salinity stress-treated rice tissues. RESULTS: Thirty-six-base-pair reads from whole mRNAs were mapped to the rice genomic sequence: 72.0% to 75.2% were mapped uniquely to the genome, and 5.0% to 5.7% bridged exons. From the piling up of short reads mapped on the genome, a series of programs (Bowtie, TopHat, and Cufflinks) comprehensively predicted 51,301 (shoot) and 54,491 (root) transcripts, including 2,795 (shoot) and 3,082 (root) currently unannotated in the Rice Annotation Project database. Of these unannotated transcripts, 995 (shoot) and 1,052 (root) had ORFs similar to those encoding the amino acid sequences of functional proteins in a BLASTX search against UniProt and RefSeq databases. Among the unannotated genes, 213 (shoot) and 436 (root) were differentially expressed in response to salinity stress. Sequence-based and array-based measurements of the expression ratios of previously annotated genes were highly correlated. CONCLUSION: Unannotated transcripts were identified on the basis of the piling up of mapped reads derived from mRNAs in rice. Some of these unannotated transcripts encoding putative functional proteins were expressed differentially in response to salinity stress.