Dynamin-related protein 2 interacts with the membrane-associated methyltransferase domain of plantago asiatica mosaic virus replicase and promotes viral replication.

Positive-strand RNA viruses replicate their RNA in the viral replication complex, a spherical structure formed by remodeling of host intracellular membranes. This process also requires the interaction between viral membrane-associated replication proteins and host factors. We previously identified the membrane-associated determinant of the replicase of plantago asiatica mosaic virus (PlAMV), a positive-strand RNA virus of the genus Potexvirus, in its methyltransferase (MET) domain, and suggested that its interaction with host factors is required to establish viral replication. Here we identified Nicotiana benthamiana dynamin-related protein 2 (NbDRP2) as an interactor of the MET domain of the PlAMV replicase by co-immunoprecipitation (Co-IP) and mass spectrometry analysis. NbDRP2 is closely related to the DRP2 subfamily proteins in Arabidopsis thaliana, AtDRP2A and AtDRP2B. Confocal microscopy observation and Co-IP confirmed the interaction between the MET domain and NbDRP2. Also, the expression of NbDRP2 was induced by PlAMV infection. PlAMV accumulation was reduced when the expression of NbDRP2 gene was suppressed by virus-induced gene silencing. In addition, PlAMV accumulation was reduced in protoplasts treated with dynamin inhibitor. These results indicate a proviral role of the interaction of NbDRP2 with the MET domain in PlAMV replication.

Cooperative roles of introns 1 and 2 of tobacco resistance gene N in enhanced N transcript expression and antiviral defense responses.

The tobacco virus resistance gene N contains four introns. Transient expression of transcripts from an N transgene containing these introns and driven by the native promoter in the presence of the elicitor of tobacco mosaic virus resulted in its increased expression. The requirement of the native promoter, the elicitor, or the individual introns for enhanced expression of N has not been fully studied. Here, we determined that 35S promoter-driven N transcript expression could be enhanced in the presence of the four introns regardless of the co-expression of the virus elicitor in tobacco. Function analyses using a series of N transgenes with different combination of introns revealed that the presence of intron 1 more so than intron 2 allowed higher accumulation of premature and mature N transcripts; however, both introns were important for not only enhanced gene expression but also for induction of cell death in tobacco and induced local resistance to spread of virus in Nicotiana benthamiana. Our findings indicate that introns 1 and 2 cooperatively contribute to N expression and virus resistance.

Multiple effects of swelling by sodium bicarbonate after delignification on enzymatic saccharification of rice straw.

The multiple effects of pretreatments by chemical delignification using acidified sodium chlorite (ASC) and swelling using sodium bicarbonate (SB) for enzymatic saccharification of rice straw in bioethanol production have been investigated in this study. The treatment with the combination of ASC three times (3× ASC) first and SB later resulted in the significant reduction in Klason lignin content up to 90% (wt./wt.). By the saccharification of the pretreated rice straw with cellulase enzymes, it was confirmed that SB treatment was an important step in the pretreatment process not only to disintegrate the cellulose structure but also to facilitate the amorphization of the crystalline cellulose as well as the extended removal of integrated lignin. Furthermore, FTIR analyses revealed that the crystal type of cellulose appeared to be changed from type I to type II by SB treatment, thereby increasing the cellulose surface area and making it more accessible to the cellulase enzyme. Conversion rate to sugar was remarkably increased when 3× ASC + SB treatments were applied to untreated rice straw, even though the saccharification of the treated rice straw was performed at a low enzyme loading (1/100, wt.-enzymes/wt.-substrate). Conclusively, rice straw could be saccharified at high yield in short time at low cellulase loading, enables the enzymatic saccharification to be more feasible for practical bioethanol production using rice straw as a substrate.

Enhancement of xylose uptake in 2-deoxyglucose tolerant mutant of Saccharomyces cerevisiae.

Chemical mutation of Saccharomyces cerevisiae using ethyl methane sulfonate was performed to enhance its ability of xylose uptake for ethanol production from lignocellulose under microaerobic condition. Among the appeared mutants, the mutant no. 2 (M2) strain screened using inhibitory effects of 2-deoxyglucose (DOG) showed more than 4-fold high ability in xylose uptake compared with the wild type strain, under the presence of glucose. The catabolite repression by glucose was sufficiently reduced in M2 strain due to its tolerance to the high concentration of DOG (0.5%, wt./vol.). Metabolomic analyses of various sugars in the cell revealed that some of xylose was reduced to xylitol in M2 cell, providing the concentration gradient of xylose and more uptake of xylose. Xylulose-5-phosphate was significantly detected in the crude cell extract from M2 strain, indicating higher metabolic activity in pentose phosphate pathway. This was also confirmed by in vitro analyses of key enzymes involved in glucose and xylose metabolism, such as hexokinase, glucose-6-phosphate dehydrogenase and xylose reductase. Glucose uptake was moderately suppressed in the presence of trehalose-6-phosphate inhibiting the activation of hexokinase, resulting in more uptake of xylose through hexose transport system. To our knowledge, this study is the first report verifying that the mutation technique successfully enhances the xylose uptake by S. cerevisiae, particularly under the presence of glucose.

Enzymatic digestion of corncobs pretreated with low strength of sulfuric acid for bioethanol production.

In this study, the effect and the optimum pretreatment condition of corncobs using low strength of H2SO4 were investigated, in which H2SO4 was used to improve the enzymatic digestibility of corncobs for saccharification without degradation of sugars released. The optimum pretreatment condition was found to be the addition of 0.5% (vol./vol.) H2SO4 and autoclaving at 122°C for 20 min. Under this condition, the structural integrity of corncob was altered to make cellulose microfibrils more accessible for cellulase enzymes, and the enzymatic digestion of corncobs could be significantly enhanced. A high yield of sugar, 80% (wt./wt.), could be obtained at a low enzyme dosage of 0.024 g enzymes/g cobs, when pretreated. As a result, the ethanol production was obviously improved by the pretreatment, i.e., the ethanol yield of 77% (wt./wt.) was obtained within 36 h in the SSF fermentation using Saccharomyces cerevisiae NBRC2114.