A leucine-rich repeat receptor-like kinase gene is involved in the specification of outer cell layers in rice roots.

Root outer cell layers of Oryza sativa (rice), which comprise the epidermis, exodermis and sclerenchyma, play an important role in protecting the roots from various stresses in soil, but the molecular mechanisms for the specification of these cell layers are poorly understood. In this work, we report on defective in outer cell layer specification 1 (Docs1), which is involved in the specification of outer cell layers in rice roots. Docs1 was isolated by map-based cloning using a mutant (c68) defective in the outer cell layers of primary roots. It encodes a leucine-rich repeat receptor-like kinase (LRR RLK). Docs1 mRNA was expressed in all tissues including roots, leaf blades and sheaths, and flowers. Immunostaining with an anti-Docs1 antibody showed that Docs1 was localized at the epidermis and exodermis, depending on the root region. Furthermore, Docs1 showed polar localization at the distal side. Subcellular examination showed that Docs1 was localized to the plasma membrane. Comparison of genome-wide transcriptional profiles between the wild-type and the knock-out mutant roots using microarray analysis showed that 61 and 41 genes were up- and downregulated in the mutant, including genes encoding putative transcription factors and genes potentially involved in cell wall metabolism. These results suggest that Docs1 might directly or indirectly regulate multiple genes involved in the proper development of root outer cell layers in rice.

Molecular identification of a major quantitative trait locus, qLTG3-1, controlling low-temperature germinability in rice.

Tolerance to abiotic stress is an important agronomic trait in crops and is controlled by many genes, which are called quantitative trait loci (QTLs). Identification of these QTLs will contribute not only to the understanding of plant biology but also for plant breeding, to achieve stable crop production around the world. Previously, we mapped three QTLs controlling low-temperature tolerance at the germination stage (called low-temperature germinability). To understand the molecular basis of one of these QTLs, qLTG3-1 (quantitative trait locus for low-temperature germinability on chromosome 3), map-based cloning was performed, and this QTL was shown to be encoded by a protein of unknown function. The QTL qLTG3-1 is strongly expressed in the embryo during seed germination. Before and during seed germination, specific localization of beta-glucuronidase staining in the tissues covering the embryo, which involved the epiblast covering the coleoptile and the aleurone layer of the seed coat, was observed. Expression of qLTG3-1 was tightly associated with vacuolation of the tissues covering the embryo. This may cause tissue weakening, resulting in reduction of the mechanical resistance to the growth potential of the coleoptile. These phenomena are quite similar to the model system of seed germination presented by dicot plants, suggesting that this model may be conserved in both dicot and monocot plants.

Two adjacent nucleotide-binding site-leucine-rich repeat class genes are required to confer Pikm-specific rice blast resistance.

The rice blast resistance gene Pikm was cloned by a map-based cloning strategy. High-resolution genetic mapping and sequencing of the gene region in the Pikm-containing cultivar Tsuyuake narrowed down the candidate region to a 131-kb genomic interval. Sequence analysis predicted two adjacently arranged resistance-like genes, Pikm1-TS and Pikm2-TS, within this candidate region. These genes encoded proteins with a nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) and were considered the most probable candidates for Pikm. However, genetic complementation analysis of transgenic lines individually carrying these two genes negated the possibility that either Pikm1-TS or Pikm2-TS alone was Pikm. Instead, it was revealed that transgenic lines carrying both of these genes expressed blast resistance. The results of the complementation analysis and an evaluation of the resistance specificity of the transgenic lines to blast isolates demonstrated that Pikm-specific resistance is conferred by cooperation of Pikm1-TS and Pikm2-TS. Although these two genes are not homologous with each other, they both contain all the conserved motifs necessary for an NBS-LRR class gene to function independently as a resistance gene.

From laboratory to field: OsNRAMP5-knockdown rice is a promising candidate for Cd phytoremediation in paddy fields.

Previously, we reported that OsNRAMP5 functions as a manganese, iron, and cadmium (Cd) transporter. The shoot Cd content in OsNRAMP5 RNAi plants was higher than that in wild-type (WT) plants, whereas the total Cd content (roots plus shoots) was lower. For efficient Cd phytoremediation, we produced OsNRAMP5 RNAi plants using the natural high Cd-accumulating cultivar Anjana Dhan (A5i). Using a positron-emitting tracer imaging system, we assessed the time-course of Cd absorption and accumulation in A5i plants. Enhanced 107Cd translocation from the roots to the shoots was observed in A5i plants. To evaluate the phytoremediation capability of A5i plants, we performed a field experiment in a Cd-contaminated paddy field. The biomass of the A5i plants was unchanged by the suppression of OsNRAMP5 expression; the A5i plants accumulated twice as much Cd in their shoots as WT plants. Thus, A5i plants could be used for rapid Cd extraction and the efficient phytoremediation of Cd from paddy fields, leading to safer food production.

Control of root system architecture by DEEPER ROOTING 1 increases rice yield under drought conditions.

The genetic improvement of drought resistance is essential for stable and adequate crop production in drought-prone areas. Here we demonstrate that alteration of root system architecture improves drought avoidance through the cloning and characterization of DEEPER ROOTING 1 (DRO1), a rice quantitative trait locus controlling root growth angle. DRO1 is negatively regulated by auxin and is involved in cell elongation in the root tip that causes asymmetric root growth and downward bending of the root in response to gravity. Higher expression of DRO1 increases the root growth angle, whereby roots grow in a more downward direction. Introducing DRO1 into a shallow-rooting rice cultivar by backcrossing enabled the resulting line to avoid drought by increasing deep rooting, which maintained high yield performance under drought conditions relative to the recipient cultivar. Our experiments suggest that control of root system architecture will contribute to drought avoidance in crops.

Characterizing the role of rice NRAMP5 in Manganese, Iron and Cadmium Transport.

Metals like manganese (Mn) and iron (Fe) are essential for metabolism, while cadmium (Cd) is toxic for virtually all living organisms. Understanding the transport of these metals is important for breeding better crops. We have identified that OsNRAMP5 contributes to Mn, Fe and Cd transport in rice. OsNRAMP5 expression was restricted to roots epidermis, exodermis, and outer layers of the cortex as well as in tissues around the xylem. OsNRAMP5 localized to the plasma membrane, and complemented the growth of yeast strains defective in Mn, Fe, and Cd transport. OsNRAMP5 RNAi (OsNRAMP5i) plants accumulated less Mn in the roots, and less Mn and Fe in shoots, and xylem sap. The suppression of OsNRAMP5 promoted Cd translocation to shoots, highlighting the importance of this gene for Cd phytoremediation. These data reveal that OsNRAMP5 contributes to Mn, Cd, and Fe transport in rice and is important for plant growth and development.

Loss of function of a proline-containing protein confers durable disease resistance in rice.

Blast disease is a devastating fungal disease of rice, one of the world's staple foods. Race-specific resistance to blast disease has usually not been durable. Here, we report the cloning of a previously unknown type of gene that confers non-race-specific resistance and its successful use in breeding. Pi21 encodes a proline-rich protein that includes a putative heavy metal-binding domain and putative protein-protein interaction motifs. Wild-type Pi21 appears to slow the plant's defense responses, which may support optimization of defense mechanisms. Deletions in its proline-rich motif inhibit this slowing. Pi21 is separable from a closely linked gene conferring poor flavor. The resistant pi21 allele, which is found in some strains of japonica rice, could improve blast resistance of rice worldwide.

Molecular breeding of a novel herbicide-tolerant rice by gene targeting.

We have previously reported the production of a rice cell line tolerant to the acetolactate synthase (ALS)-inhibiting herbicide bispyribac (BS), and demonstrated that the BS-tolerant phenotype was due to a double mutation in the rice ALS gene. We further indicated that while changing either of the two amino acids (W548 L or S627I) individually resulted in a BS-tolerant phenotype, conversion of both amino acids simultaneously conferred increased tolerance to BS. As the BS-tolerant cell line had lost the ability to regenerate during two years of tissue culture selection, we attempted to introduce these two point mutations into the rice ALS gene via gene targeting (GT). Using our highly efficient Agrobacterium-mediated transformation system in rice, we were able to regenerate 66 independent GT rice plants from 1500 calli. Furthermore, two-thirds of these plants harbored the two point mutations exclusively, without any insertion of foreign DNA such as border sequences of T-DNA. The GT plants obtained in the present study are therefore equivalent to non-GM herbicide-tolerant rice plants generated by conventional breeding approaches that depend on spontaneous mutations. Surprisingly, GT rice homozygous for the modified ALS locus showed hyper-tolerance to BS when compared to BS-tolerant plants produced by a conventional transgenic system; ALS enzymatic activity in plants homozygous for the mutated ALS gene was inhibited only by extremely high concentrations of BS. These results indicate that our GT method has successfully created novel herbicide-tolerant rice plants that are superior to those produced by conventional mutation breeding protocols or transgenic technology.

Rice WRKY45 plays a crucial role in benzothiadiazole-inducible blast resistance.

Benzothiadiazole (BTH) is a so-called plant activator and protects plants from diseases by activating the salicylic acid (SA) signaling pathway. By microarray screening, we identified BTH- and SA-inducible WRKY transcription factor (TF) genes that were upregulated within 3 h after BTH treatment. Overexpression of one of them, WRKY45, in rice (Oryza sativa) markedly enhanced resistance to rice blast fungus. RNA interference-mediated knockdown of WRKY45 compromised BTH-inducible resistance to blast disease, indicating that it is essential for BTH-induced defense responses. In a transient expression system, WRKY45 activated reporter gene transcription through W-boxes. Epistasis analysis suggested that WRKY45 acts in the SA signaling pathway independently of NH1, a rice ortholog of Arabidopsis thaliana NPR1, which distinguishes WRKY45 from known Arabidopsis WRKY TFs. Two defense-related genes, encoding a glutathione S-transferase and a cytochrome P450, were found to be regulated downstream of WRKY45 but were not regulated by NH1, consistent with the apparent independence of the WRKY45- and NH1-dependent pathways. Defense gene expression in WRKY45-overexpressed rice plants varied with growth conditions, suggesting that some environmental factor(s) acts downstream of WRKY45 transcription. We propose a role for WRKY45 in BTH-induced and SA-mediated defense signaling in rice and its potential utility in improving disease resistance of rice, an importance food resource worldwide.

Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice.

Several approaches have recently been adopted to improve Agrobacterium-mediated transformation of rice, both to generate the large number of T-DNA insertion plants needed for functional analysis of the rice genome, and for production of rice with additional agronomical value. However, about 3 months of in vitro culture is still required for isolation of transgenic rice plants. Here, we report the competency of scutellum tissue from 1-day pre-cultured seeds for Agrobacterium-mediated transformation. Furthermore, early infection of rice seeds with Agrobacterium enhanced efficient selection of transformed calli. Using our system, we successfully regenerated transgenic rice plantlets within a month of the start of the aseptic culture of mature seeds. Our new system should reduce the somaclonal variation accompanying prolonged culture of rice cells in the dedifferentiated state and facilitate the molecular breeding of rice.