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To understand the transcriptional regulatory mechanism of host genes during the activation of defense responses in rice, we isolated WRKY transcription factors whose expressions were altered upon attack of the fungal pathogen Magnaporthe grisea, the causal agent of the devastating rice blast disease. A systematic expression analysis of OsWRKYs (Oryza sativa L. WRKYs) revealed that among 45 tested genes the expression of 15 genes was increased remarkably in an incompatible interaction between rice and M. grisea. Twelve of the M. grisea-inducible OsWRKY genes were also differentially regulated in rice plants infected with the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). In experiments with defense signaling molecules, the expression of two genes, OsWRKY45 and OsWRKY62, was increased in salicylic acid (SA)-treated leaves and the expression of three genes, OsWRKY10, OsWRKY82, and OsWRKY85 was increased by jasmonic acid (JA) treatment. OsWRKY30 and OsWRKY83 responded to both SA- and JA treatments. The expression profiles suggest that a large number of WRKY DNA-binding proteins are involved in the transcriptional activation of defense-related genes in response to rice pathogens.

The Arabidopsis (Arabidopsis thaliana) hexokinase 1 (AtHXK1) is recognized as an important glucose (Glc) sensor. However, the function of hexokinases as Glc sensors has not been clearly demonstrated in other plant species, including rice (Oryza sativa).To investigate the functions of rice hexokinase isoforms, we characterized OsHXK5 and OsHXK6, which are evolutionarily related to AtHXK1. Transient expression analyses using GFP fusion constructs revealed that OsHXK5 and OsHXK6 are associated with mitochondria. Interestingly, the OsHXK5DmTP-GFP and OsHXK6DmTP-GFP fusion proteins, which lack N-terminal mitochondrial targeting peptides, were present mainly in the nucleus with a small amount of the proteins seen in the cytosol. In addition, the OsHXK5NLS-GFP and OsHXK6NLS-GFP fusion proteins harboring nuclear localization signals were targeted predominantly in the nucleus, suggesting that these OsHXKs retain a dual-targeting ability to mitochondria and nuclei. In transient expression assays using promoter::luciferase fusion constructs, these two OsHXKs and their catalytically inactive alleles dramatically enhanced the Glc-dependent repression of the maize (Zea mays) Rubisco small subunit (RbcS) and rice a-amylase genes in mesophyll protoplasts of maize and rice. Notably, the expression of OsHXK5, OsHXK6, or their mutant alleles complemented the Arabidopsis glucose insensitive2-1 mutant, thereby resulting in wild-type characteristics in seedling development, Glc-dependent gene expression, and plant growth. Furthermore, transgenic rice plants overexpressing OsHXK5 or OsHXK6 exhibited hypersensitive plant growth retardation and enhanced repression of the photosynthetic gene RbcS in response to Glc treatment. These results provide evidence that rice OsHXK5 and OsHXK6 can function as Glc sensors.In higher plants, sugars are known to function as signaling molecules in addition to being a fundamental source of fuel for carbon and energy metabolism. Indeed, sugars have been shown to regulate physiological processes during the entire plant life cycle, from germination to flowering and senescence, and to function during defense responses to biotic and abiotic

Hexokinase (HXK) is a dual-function enzyme that both phosphorylates hexose to form hexose 6-phosphate and plays an important role in sugar sensing and signaling. To investigate the roles of hexokinases in rice growth and development, we analyzed rice sequence databases and isolated ten rice hexokinase cDNAs, OsHXK1 (Oryza sativa Hexokinase 1) through OsHXK10. With the exception of the single-exon gene OsHXK1, the OsHXKs all have a highly conserved genomic structure consisting of nine exons and eight introns. Gene expression profiling revealed that OsHXK2 through OsHXK9 are expressed ubiquitously in various organs, whereas OsHXK10 expression is pollen-specific. Sugars induced the expression of three OsHXKs, OsHXK2, OsHXK5, and OsHXK6, in excised leaves, while suppressing OsHXK7 expression in excised leaves and immature seeds. The hexokinase activity of the OsHXKs was confirmed by functional complementation of the hexokinase-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). OsHXK4 was able to complement this mutant only after the chloroplast-transit peptide was removed. The subcellular localization of OsHXK4 and OsHXK7, observed with green fluorescent protein (GFP) fusion constructs, indicated that OsHXK4 is a plastid-stroma-targeted hexokinase while OsHXK7 localizes to the cytosol.

To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM) analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls, and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence, the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls. This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition, DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC), the gelatinization temperatures of endosperm starch were found to be 1-5 degrees C lower than those of the control. We propose a distinct role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice.

Recent evidence suggests that tau aggregates are not only neurotoxic, but also propagate in neurons acting as a seed for native tau aggregation. Prion-like tau transmission is now considered as an important pathogenic mechanism driving the progression of tau pathology in the brain. However, prion-like tau species have not been clearly characterized. To identify infectious tau conformers, here we prepared diverse tau aggregates and evaluated the effect on inducing intracellular tau-aggregation. Among tested, tau dimer containing P301L-mutation is identified as the most infectious form to induce tau pathology. Biochemical analysis reveals that P301L-tau dimer is covalently cross-linked with a disulfide bond. The relatively small and covalently cross-linked tau dimer induced tau pathology efficiently in primary neurons and also in tau-transgenic mice. So far, the importance of tau disulfide cross-linking has been overlooked in the study of tau pathology. Here our results suggested that tau disulfide cross-linking might play critical role in tau propagation by producing structurally stable and small tau conformers.

SUMMARY Alzheimer’s disease (AD) is closely associated with synaptic dysfunction and thus, current treatments often aim to stimulate neurotransmission to improve cognitive impairment. While the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for synaptic transmission, the correlation between SNAREs and AD neuropathology is unknown. Here we report that intracellular amyloid-β (Aβ) oligomers directly inhibit SNARE-mediated exocytosis by impairing SNARE complex formation. We observe abnormal reduction of SNARE complex levels in the brains of APP/PS1 transgenic (TG) mice compared to age-matched wild types. We demonstrate that Aβ oligomers block SNARE complex assembly through the direct interaction with a target membrane (t-)SNARE syntaxin 1a in vitro. Furthermore, the results of the in vitro single-vesicle content-mixing assay reveal that Aβ oligomers inhibit SNARE-mediated fusion pores. Thus, our study identifies a potential molecular mechanism by which intracellular Aβ oligomers hamper SNARE-mediated exocytosis, likely leading to AD-associated synaptic dysfunctions.

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