Characterization of two carbonyl reductases from Ogataea polymorpha NBRC 0799.

The enzyme responsible for the enantioselective production of (S)-1,1,1-trifluoro-2-propanol ((S)-TFP) from 1,1,1-trifluoroacetone (TFA) has been identified in Ogataea polymorpha NBRC 0799. We purified two carbonyl reductases, OpCRD-A and OpCRD-B from this strain, and revealed their characteristics. Both enzymes were specific to NADH, but the following characteristics were different: The molecular mass of subunit OpCRD-A was 40 kDa and that of OpCRD-B was 43 kDa. Amino acid sequences of both enzymes were only 21% identical. OpCRD-B contained 4 mol of zinc per mole of enzyme, but OpCRD-A did not. The optimal pH, temperature, pH stability, thermostability, and inhibitor specificity were also remarkably different. With regard to substrate specificity, both enzymes exhibited high reductase activity toward a wide variety of ketones, aldehydes and fluoroketones, and dehydrogenase activity toward 2-propanol and 2-butanol. The reductase activity was much higher than the dehydrogenase activity at acidic pH. OpCRD-A enantioselectively produced (S)-TFP from TFA, but OpCRD-B preferentially produced (R)-TFP. Thus, we concluded that OpCRD-A plays the main role in the production of (S)-TFP by a reaction of O. polymorpha NBRC 0799 cells and that OpCRD-A has great potential for efficient production of (S)-TFP, as it is an S-specific enzyme and does not catalyze the dehydrogenation of (S)-TFP.

Fluorescent reporter analysis revealed the timing and localization of AVR-Pia expression, an avirulence effector of Magnaporthe oryzae.

In order to facilitate infection, the rice blast pathogen Magnaporthe oryzae secretes an abundance of proteins, including avirulence effectors, to diminish its host's defences. Avirulence effectors are recognized by host resistance proteins and trigger the host's hypersensitive response, which is a rapid and effective form of innate plant immunity. An understanding of the underlying molecular mechanisms of such interactions is crucial for the development of strategies to control disease. However, the expression and secretion of certain effector proteins, such as AVR-Pia, have yet to be reported. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that AVR-Pia was only expressed during infection. Fluorescently labelled AVR-Pia indicated that AVR-Pia expression was induced during appressorial differentiation in the cells of both rice and onion, as well as in a penetration-deficient (Δpls1) mutant capable of developing melanized appressoria, but unable to penetrate host cells, suggesting that AVR-Pia expression is independent of fungal penetration. Using live-cell imaging, we also documented the co-localization of green fluorescent protein (GFP)-labelled AVR-Pia and monomeric red fluorescent protein (mRFP)-labelled PWL2, which indicates that AVR-Pia accumulates in biotrophic interfacial complexes before being delivered to the plant cytosol. Together, these results suggest that AVR-Pia is a cytoplasmic effector that is expressed at the onset of appressorial differentiation and is translocated to the biotrophic interfacial complex, and then into the host's cytoplasm.

Mechanism by Which Magnesium Oxide Suppresses Tablet Hardness Reduction during Storage.

This study investigated how the inclusion of magnesium oxide (MgO) maintained tablet hardness during storage in an unpackaged state. Tablets were prepared with a range of MgO levels and stored at 40°C with 75% relative humidity for up to 14 d. The hardness of tablets prepared without MgO decreased over time. The amount of added MgO was positively associated with tablet hardness and mass from an early stage during storage. Investigation of the water sorption properties of the tablet components showed that carmellose water sorption correlated positively with the relative humidity, while MgO absorbed and retained moisture, even when the relative humidity was reduced. In tablets prepared using only MgO, a petal- or plate-like material was observed during storage. Fourier transform infrared spectrophotometry showed that this material was hydromagnesite, produced when MgO reacts with water and CO2. The estimated level of hydromagnesite at each time-point showed a significant negative correlation with tablet porosity. These results suggested that MgO suppressed storage-associated softening by absorbing moisture from the environment. The conversion of MgO to hydromagnesite results in solid bridge formation between the powder particles comprising the tablets, suppressing the storage-related increase in volume and increasing tablet hardness.

Heterologous production, purification, and immunodetection of Magnaporthe oryzae avirulence protein AVR-Pia.

The avirulence gene AVR-Pia of Magnaporthe oryzae, which induces a hypersensitive reaction in rice cultivars containing the resistance gene Pia, was expressed in Escherichia coli. AVR-Pia protein was collected as inclusion bodies, denatured, and refolded. Finally, recombinant AVR-Pia (rAVR-Pia) protein was purified by column chromatography. Infiltration of rAVR-Pia triggered cell browning in the leaves of rice cultivar Aichiasahi (Pia), with accumulation of H2O2 and induction of PR1a expression in rice. On the other hand, these reactions were not observed in Shin-2 (pia) leaves after the same treatment. This observation indicated that rAVR-Pia had the function of an avirulence protein. rAVR-Pia was used for immunization of a rabbit, and anti-AVR-Pia antiserum was prepared. The specificity of this antibody was appraised by detecting native AVR-Pia in the inoculated leaf sheath extract using Western blotting in combination with immunoprecipitation. Native AVR-Pia was successfully detected, and its molecular weight was estimated to be 7.4 kDa, indicating signal peptide cleavage. Additionally, secreted native AVR-Pia was quantified as 3.7 ng/g rice sheath.

Homologous recombination causes the spontaneous deletion of AVR-Pia in Magnaporthe oryzae.

AVR-Pia, an avirulence gene in the genome of the rice blast fungus Magnaporthe oryzae, triggers a hypersensitive reaction in rice cultivars harbouring the resistance gene Pia. The copy number of AVR-Pia was revealed to vary from one to three among M. oryzae isolates avirulent to Pia rice, and three copies of the gene were located on a single chromosome in strain Ina168, from which the gene was originally cloned. The spontaneous avr-Pia mutant originated from Ina168, named Ina168m95-1, which lacks the AVR-Pia gene, and was therefore used to elucidate the molecular mechanism of the deletion of all three copies of AVR-Pia. Screening and analysis of cosmid clones indicated that two copies of the DNA-type transposon Occan (Occan(9E12) and Occan(3A3) ) were located on the same chromosome, and three copies of AVR-Pia were located in between the two Occan elements. Ina168m95-1 contains a conserved Occan element, named Occan(m95-1) , between sequences homologous to the 5'-flanking region of Occan(3A3) and the 3'-flanking region of Occan(9E12) . In addition, sequence polymorphisms indicated a homologous recombination between Occan(3A3) and Occan(9E12) , which resulted in Occan(m95-1) . Based on these observations, we propose the hypothesis that homologous recombination in the two Occan elements leads to the deletion of AVR-Pia in Ina168m95-1.

Molecular cloning and characterization of the AVR-Pia locus from a Japanese field isolate of Magnaporthe oryzae.

In order to clone and analyse the avirulence gene AVR-Pia from Japanese field isolates of Magnaporthe oryzae, a mutant of the M. oryzae strain Ina168 was isolated. This mutant, which was named Ina168m95-1, gained virulence towards the rice cultivar Aichi-asahi, which contains the resistance gene Pia. A DNA fragment (named PM01) that was deleted in the mutant and that co-segregated with avirulence towards Aichi-asahi was isolated. Three cosmid clones that included the regions that flanked PM01 were isolated from a genomic DNA library. One of these clones (46F3) complemented the mutant phenotype, which indicated clearly that this clone contained the avirulence gene AVR-Pia. Clone 46F3 contained insertions of transposable elements. The 46F3 insert was divided into fragments I-VI, and these were cloned individually into a hygromycin-resistant vector for the transformation of the mutant Ina168m95-1. An inoculation assay of the transformants revealed that fragment V (3.5 kb) contained AVR-Pia. By deletion analysis of fragment V, AVR-Pia was localized to an 1199-bp DNA fragment, which included a 255-bp open reading frame with weak homology to a bacterial cytochrome-c-like protein. Restriction fragment length polymorphism analysis of this region revealed that this DNA sequence co-segregated with the AVR-Pia locus in a genetic map that was constructed using Chinese isolates.