Osamu Yatou

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.

Characterization of a rice (Oryza sativa L.) mutant deficient in the heme domain of nitrate reductase

SummaryBiochemical and genetical characterization of a rice nitrate reductase (NR)-deficient mutant, M819, which had been isolated as a chlorate-resistant mutant, was carried out. In M819, leaf NADH-NR activity was found to be about 10% of that of the wild-type cv ‘Norin 8’, while NADPH-NR activity was higher than that in the wild-type; FMNH2-NR and MV-NR activities were also 10% of those of the wild type; BPB-NR activity was higher than that of the wild type; and xanthine dehydrogenase activity was revealed to be present in both. These results suggest that the mutant line M819 lacks the functional heme domain of the NADH-NR polypeptide due to a point mutation or a small deletion within the coding region of the structural gene. Chlorate resistance in M819 was transmitted by a single recessive nuclear gene.

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.

Cleistogamy decreases the effect of high temperature stress at flowering in rice.

Abstract Rice sterility due to a high temperature at flowering is a serious agricultural problem that has been associated with global warming. The flowering stage in rice plants is most vulnerable to high temperature stress. Closed flowering rice plants may better withstand high temperature stress. The aim of this study was to determine the role of cleistogamy (closed flowering) in avoiding high temperature-induced sterility. Cleistogamy was induced by moderate heat treatment at 30°C during the panicle development stage. Both cleistogamous and chasmogamous (ordinary open flowering) rice plants, which possess the same genetic background, were subjected to 38°C or 36°C for 4 h just before flowering, and the percentage of fertility, number of pollen grains on a stigma, number of germinated pollen grains on a stigma, and temperatures inside and outside of the closed spikelets were examined. The cleistogamous rice plants showed a higher fertility percentage and a larger number of germinated pollen grains on a stigma than the chasmogamous rice plants. The temperature inside the closed flowering spikelets was 1.8°C lower than that outside the spikelets. The cleistogamous rice plants thus showed avoidance to high temperature stress at 38°C at flowering. On the basis of these results, we concluded that cleistogamy was advantageous to rice pollination and fertilization at high temperatures because of glume cooling.

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

Summary 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.

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.

In vitro but not in planta encapsidation of Rice gall dwarf virus core particles by the outer capsid P8 protein of Rice dwarf virus expressed in transgenic rice plants

Transencapsidation of the Rice gall dwarf virus (RGDV) inner core by the Rice dwarf virus (RDV) outer capsid P8 protein was examined in vitro and in planta. When RGDV core particles were incubated with an extract from RDV P8-transgenic rice leaf tissue, RDV P8 encapsidated the RGDV core particles to form double-shelled virus-like particles in vitro. In contrast, when RDV P8-transgenic rice plants were inoculated with RGDV, progeny RGDV particles contained RGDV P8 but RDV P8 was not detectable in the virions. No significant differences were found in acquisition by the vector insects and subsequent transmission rates between RGDV infecting nontransgenic rice plants and those infecting RDV P8-transgenic rice plants. These results indicate that mechanisms of and/or requirements for interactions between P8 and the inner core particles of phytoreoviruses differ between in vitro and in planta.