Partial peptides from rice defensin OsAFP1 exhibited antifungal activity against the rice blast pathogen Pyricularia oryzae.

Rice blast caused by Pyricularia oryzae is one of the most devastating diseases worldwide. This study aimed to investigate the antifungal activity of rice defensin OsAFP1 and its partial peptides against P. oryzae. The partial peptides near the N- and C-terminal regions of OsAFP1 exhibited approximately the same antifungal activity as the entire protein against P. oryzae. These partial peptides have the potential to be used as fungicides.

The superwoman1-cleistogamy2 mutant is a novel resource for gene containment in rice.

Outcrossing between cultivated plants and their related wild species may result in the loss of favourable agricultural traits in the progeny or escape of transgenes in the environment. Outcrossing can be physically prevented by using cleistogamous (i.e. closed-flower) plants. In rice, flower opening is dependent on the mechanical action of fleshy organs called lodicules, which are generally regarded as the grass petal equivalents. Lodicule identity and development are specified by the action of protein complexes involving the SPW1 and OsMADS2 transcription factors. In the superwoman1-cleistogamy1 (spw1-cls1) mutant, SPW1 is impaired for heterodimerization with OsMADS2 and consequently spw1-cls1 shows thin, ineffective lodicules. However, low temperatures help stabilise the mutated SPW1/OsMADS2 heterodimer and lodicule development is restored when spw1-cls1 is grown in a cold environment, resulting in the loss of the cleistogamous phenotype. To identify a novel, temperature-stable cleistogamous allele of SPW1, targeted and random mutations were introduced into the SPW1 sequence and their effects over SPW1/OsMADS2 dimer formation were assessed in yeast two-hybrid experiments. In parallel, a novel cleistogamous allele of SPW1 called spw1-cls2 was isolated from a forward genetic screen. In spw1-cls2, a mutation leading to a change of an amino acid involved in DNA binding by the transcription factor was identified. Fertility of spw1-cls2 is somewhat decreased under low temperatures but unlike for spw1-cls1, the cleistogamous phenotype is maintained, making the line a safer and valuable genetic resource for gene containment.

A decrease in phytic acid content substantially affects the distribution of mineral elements within rice seeds.

Phytic acid (myo-inositol hexakisphosphate; InsP6) is the storage compound of phosphorus and many mineral elements in seeds. To determine the role of InsP6 in the accumulation and distribution of mineral elements in seeds, we performed fine mappings of mineral elements through synchrotron-based X-ray microfluorescence analysis using developing seeds from two independent low phytic acid (lpa) mutants of rice (Oryza sativa L.). The reduced InsP6 in lpa seeds did not affect the translocation of mineral elements from vegetative organs into seeds, because the total amounts of phosphorus and the other mineral elements in lpa seeds were identical to those in the wild type (WT). However, the reduced InsP6 caused large changes in mineral localization within lpa seeds. Phosphorus and potassium in the aleurone layer of lpa greatly decreased and diffused into the endosperm. Zinc and copper, which were broadly distributed from the aleurone layer to the inner endosperm in the WT, were localized in the narrower space around the aleurone layer in lpa mutants. We also confirmed that similar distribution changes occurred in transgenic rice with the lpa phenotype. Using these results, we discussed the role of InsP6 in the dynamic accumulation and distribution patterns of mineral elements during seed development.

Transgenic strategies to confer resistance against viruses in rice plants.

Rice (Oryza sativa L.) is cultivated in more than 100 countries and supports nearly half of the world's population. Developing efficient methods to control rice viruses is thus an urgent necessity because viruses cause serious losses in rice yield. Most rice viruses are transmitted by insect vectors, notably planthoppers and leafhoppers. Viruliferous insect vectors can disperse their viruses over relatively long distances, and eradication of the viruses is very difficult once they become widespread. Exploitation of natural genetic sources of resistance is one of the most effective approaches to protect crops from virus infection; however, only a few naturally occurring rice genes confer resistance against rice viruses. Many investigators are using genetic engineering of rice plants as a potential strategy to control viral diseases. Using viral genes to confer pathogen-derived resistance against crops is a well-established procedure, and the expression of various viral gene products has proved to be effective in preventing or reducing infection by various plant viruses since the 1990s. RNA interference (RNAi), also known as RNA silencing, is one of the most efficient methods to confer resistance against plant viruses on their respective crops. In this article, we review the recent progress, mainly conducted by our research group, in transgenic strategies to confer resistance against tenuiviruses and reoviruses in rice plants. Our findings also illustrate that not all RNAi constructs against viral RNAs are equally effective in preventing virus infection and that it is important to identify the viral "Achilles' heel" gene to target for RNAi attack when engineering plants.

Agronomic traits and gene containment capability of cleistogamous rice lines with the superwoman1-cleistogamy mutation.

Pollen-mediated transgene flow is a major concern for the production of genetically modified (GM) rice. Cleistogamy is a useful tool for preventing this form of gene flow. We previously identified the cleistogamous rice mutant superwoman1-cleistogamy (spw1-cls) and determined its molecular genetic mechanism. In the present study, we cultivated spw1-cls over five years to examine effects of cleistogamy on agronomic traits. Simultaneously, we cultivated cleistogamous backcross lines created by continuous backcrossing with "Yumeaoba" (a japonica cultivar) as the recurrent parent and by application of a DNA marker. In these experimental cultivations, spw1-cls and its backcross lines showed almost equal or slightly lower, but acceptable, agronomic traits compared with each control line. We also conducted natural crossing tests in paddy fields to assess the gene containment capability of spw1-cls. In a series of field experiments, there was no natural crossing between spw1-cls (pollen donor) and pollen recipient lines, but the wild-type donor and recipient lines were crossed. Thus, the cleistogamy of the spw1-cls mutation is able to inhibit natural crossing effectively, without significant loss of commercial benefits, such as yield. We conclude that spw1-cls cleistogamy is a practical tool for gene containment in GM rice cultivation.

Phytochrome A requires jasmonate for photodestruction.

The plant photoreceptor phytochrome is organised in a small gene family with phytochrome A (phyA) being unique, because it is specifically degraded upon activation by light. This so called photodestruction is thought to be important for dynamic aspects of sensing such as measuring day length or shading by competitors. Signal-triggered proteolytic degradation has emerged as central element of signal crosstalk in plants during recent years, but many of the molecular players are still unknown. We therefore analyzed a jasmonate (JA)-deficient rice mutant, hebiba, that in several aspects resembles a mutant affected in photomorphogenesis. In this mutant, the photodestruction of phyA is delayed as shown by in vivo spectroscopy and Western blot analysis. Application of methyl-JA (MeJA) can rescue the delayed phyA photodestruction in the mutant in a time- and dose-dependent manner. Light regulation of phyA transcripts thought to be under control of stable phytochrome B (phyB) is still functional. The delayed photodestruction is accompanied by an elevated sensitivity of phytochrome-dependent growth responses to red and far-red light.

A large-scale collection of phenotypic data describing an insertional mutant population to facilitate functional analysis of rice genes.

In order to facilitate the functional analysis of rice genes, we produced about 50,000 insertion lines with the endogenous retrotransposon Tos17. Phenotypes of these lines in the M2 generation were observed in the field and characterized based on 53 phenotype descriptors. Nearly half of the lines showed more than one mutant phenotype. The most frequently observed phenotype was low fertility, followed by dwarfism. Phenotype data with photographs of each line are stored in the Tos17 mutant panel web-based database with a dataset of sequences flanking Tos17 insertion points in the rice genome (http://tos.nias.affrc.go.jp/). This combination of phenotypic and flanking sequence data will stimulate the functional analysis of rice genes.