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Rice (Oryza sativa) plants carrying the Pi-i resistance gene to blast fungus Magnaporthe oryzae restrict invaded fungus in infected tissue via hypersensitive reaction or response (HR), which is accompanied by rapid ethylene production and formation of small HR lesions. Ethylene biosynthesis has been implicated to be important for blast resistance; however, the individual roles of ethylene and cyanide, which are produced from the precursor 1-aminocyclopropane-1-carboxylic acid, remain unevaluated. In this study, we found that Pi-i-mediated resistance was compromised in transgenic rice lines, in which ethylene biosynthetic enzyme genes were silenced and then ethylene production was inhibited. The compromised resistance in transgenic lines was recovered by exogenously applying cyanide but not ethephon, an ethylene-releasing chemical in plant tissue. In a susceptible rice cultivar, treatment with cyanide or 1-aminocyclopropane-1-carboxylic acid induced the resistance to blast fungus in a dose-dependent manner, while ethephon did not have the effect. Cyanide inhibited the growth of blast fungus in vitro and in planta, and application of flavonoids, secondary metabolites that exist ubiquitously in the plant kingdom, enhanced the cyanide-induced inhibition of fungal growth. These results suggested that cyanide, whose production is triggered by HR in infected tissue, contributes to the resistance in rice plants via restriction of fungal growth.

The role of ethylene (ET) in resistance to infection with blast fungus (Magnaporthe grisea) in rice (Oryza sativa) is poorly understood. To study it, we quantified ET levels after inoculation, using young rice plants at the four-leaf stage of rice cv Nipponbare (wild type) and its isogenic plant (IL7), which contains the Pi-i resistance gene to blast fungus race 003. Small necrotic lesions by hypersensitive reaction (HR) were formed at 42 to 72 h postinoculation (hpi) in resistant IL7 leaves, and whitish expanding lesions at 96 hpi in susceptible wild-type leaves. Notable was the enhanced ET emission at 48 hpi accompanied by increased 1-aminocyclopropane-1-carboxylic acid (ACC) levels and highly elevated ACC oxidase (ACO) activity in IL7 leaves, whereas only an enhanced ACC increase at 96 hpi in wild-type leaves. Among six ACC synthase (ACS) and seven ACO genes found in the rice genome, OsACS2 was transiently expressed at 48 hpi in IL7 and at 96 hpi in wild type, and OsACO7 was expressed at 48 hpi in IL7. Treatment with an inhibitor for ACS, aminooxyacetic acid, suppressed enhanced ET emission at 48 hpi in IL7, resulting in expanding lesions instead of HR lesions. Exogenously supplied ACC compromised the aminooxyacetic acid-induced breakdown of resistance in IL7, and treatment with 1-methylcyclopropene and silver thiosulfate, inhibitors of ET action, did not suppress resistance. These findings suggest the importance of ET biosynthesis and, consequently, the coproduct, cyanide, for HR-accompanied resistance to blast fungus in young rice plants and the contribution of induced OsACS2 and OsACO7 gene expression to it.

The E2F family plays a pivotal role in cell cycle control and is conserved among plants and animals, but not in fungi. This provides for the possibility that the E2F family was integrated during the development of higher organisms, but little is known about this. We examined the effect of E2F ectopically expressed in transgenic tobacco (Nicotiana tabacum) plants on growth and development using E2Fa (AtE2F3) and DPa from Arabidopsis. E2Fa-DPa double transgenic lines exhibited altered phenotypes with curled leaves, round shaped petals, and shortened pistils. In mature but not immature leaves of the double transgenic lines, there were enlarged nuclei with increasing ploidy levels accompanied by the ectopic expression of S phase- but not M phase-specific genes. This indicates that a high expression of E2F promotes endoreduplication by accelerating S phase entry in terminally differentiated cells with limited mitotic activity. Furthermore, mature leaves of the transgenic plants contained increased numbers of small cells, especially on the palisade (adaxial) side of the outer region toward the edge, and the leaf strips exhibited hormone-independent callus formation when cultured in vitro. These observations suggest that an enhanced E2F activity modulates cell cycle in a cell type-specific manner and affects plant morphology depending on a balance between activities for committing to S phase and M phase, which likely differ between organs or tissues.

E2F transcription factors are required for the progression and arrest of the cell cycle in animals. Like animals, plants have evolved to conserve the E2F family. The Arabidopsis genome encodes E2F and DP proteins that share a high similarity with the animal E2F and DP families. Here, we show that Arabidopsis E2F and DP proteins are not predominantly localized to the nucleus in analyses with green fluorescent protein, and that the complete nuclear localization of some members is driven by the co-expression of their specific partner proteins. Both AtE2F1 and AtE2F3 were translocated to the nucleus and transactivate an E2F reporter gene when co-expressed with DPa but not DPb. In contrast, AtE2F2 was inactive for both nuclear translocation and transactivation even when Dpa or DPb was co-expressed. Because the DNA binding activities of the three E2Fs are equally stimulated by the interaction with DPa or DPb in vitro, the observed transactivation of AtE2F1 and AtE2F3 is DPa specific and nuclear import dependent. A green fluorescent protein fusion with an AtE2F3 mutant, in which a conserved nuclear export signal-like sequence in the dimerization domain was deleted, was localized to the nucleus. Thus, the concomitant nuclear translocation seems to be conferred by the DPa interaction to release an activity that inhibits an intrinsic nuclear import activity of AtE2Fs. Furthermore, the nuclear translocation of AtE2F3 stimulated by DPa was abolished by the deletion of the N-terminal region of AtE2F3, which is conserved among all the E2F proteins identified in plants to date. Replacement of the N-terminal region of AtE2F3 with a canonical nuclear localization signal only partially mimicked the effect of the DPa co-expression, demonstrating the function of plant E2F distinct from that observed for animal E2Fs. These observations suggest that the function of plant E2F and DP proteins is primarily controlled by their nuclear localization mediated by the interaction with specific partner proteins.