Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice

Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot‐and‐mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound‐ and pathogen‐inducible mpi promoter. The mpi‐pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi‐pci rice, compared with larvae fed on wild‐type plants, was observed. Expression of the mpi‐pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi‐pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi‐pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi‐pci fusion gene for dual resistance against insects and pathogens in rice plants.     

Effects of diapause on post‐diapause reproductive investment in the moth Ostrinia scapulalis

Diapause is a strategy used by many insect species to survive adverse environmental conditions. However, diapause incurs costs that may have adverse effects on post‐diapause development and reproduction. We herein investigated the effects of diapause on the post‐diapause reproductive investment of males and females in a multivoltine moth, the adzuki bean borer, Ostrinia scapulalis (Walker) (Lepidoptera: Crambidae). We found that (1) post‐diapause males and females were smaller and had lower mating success than non‐diapause individuals, (2) post‐diapause females had lower fecundity and shorter longevity than non‐diapause females, (3) post‐diapause males transferred similar numbers of eupyrene and apyrene sperm as non‐diapause males, (4) the fecundity and longevity of non‐diapause females mated with post‐diapause males and those mated with non‐diapause males were not significantly different, and (5) no significant relationship was found between diapause duration (short and long) and post‐diapause reproductive investments in both males and females. These results suggest that post‐diapause males did not reduce reproductive investment in spite of the cost of diapause, and the significant decline in reproductive output in post‐diapause females was due to their reduced body weight and longevity, which appeared to be direct consequences of the cost of diapause.     

The role of capsid maturation on adenovirus priming for sequential uncoating

Adenovirus assembly concludes with proteolytic processing of several capsid and core proteins. Immature virions containing precursor proteins lack infectivity because they cannot properly uncoat, becoming trapped in early endosomes. Structural studies have shown that precursors increase the network of interactions maintaining virion integrity. Using different biophysical techniques to analyze capsid disruption in vitro, we show that immature virions are more stable than the mature ones under a variety of stress conditions and that maturation primes adenovirus for highly cooperative DNA release. Cryoelectron tomography reveals that under mildly acidic conditions mimicking the early endosome, mature virions release pentons and peripheral core contents. At higher stress levels, both mature and immature capsids crack open. The virus core is completely released from cracked capsids in mature virions, but it remains connected to shell fragments in the immature particle. The extra stability of immature adenovirus does not equate with greater rigidity, because in nanoindentation assays immature virions exhibit greater elasticity than the mature particles. Our results have implications for the role of proteolytic maturation in adenovirus assembly and uncoating. Precursor proteins favor assembly by establishing stable interactions with the appropriate curvature and preventing premature ejection of contents by tightly sealing the capsid vertices. Upon maturation, core organization is looser, particularly at the periphery, and interactions preserving capsid curvature are weakened. The capsid becomes brittle, and pentons are more easily released. Based on these results, we hypothesize that changes in core compaction during maturation may increase capsid internal pressure to trigger proper uncoating of adenovirus.     

Effects of gamma-irradiation on midgut proteolytic activity of the mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae)

The Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann), is a key pest of citrus in Spain because of significant yield losses and to quarantine restrictions. Biologically based control methods, such as the Sterile Insect Technique (SIT), which relies on the sterilization by irradiation of large numbers of insects, is gaining an increasing role in the control of medfly in Mediterranean areas. However, gamma-irradiation might damage the midgut epithelium cells, causing a lowering of nutritive assimilation that can negatively affect adult performance. Irradiation effects on digestive physiology are well established for a number of insect pests, but there is no information on medfly. Our aim was to determine the effects of gamma-irradiation on C. capitata digestive protease activity. Both larvae and adults were found to use a similar proteolytic system based on aspartyl-, trypsin-, chymotrypsin-, amino peptidase-, and carboxypeptidase A- and B-like activities. Pupae of the Vienna-7 (tsl) strain were irradiated at 70 or 140 Gy, two days before emergence, and the adults fed during 5 days on sugar-protein (4:1) diets. Protease activity was measured in midgut extracts and compared with males non-irradiated reared in the same conditions. The results showed that the irradiation doses tested had no effect on the digestive proteolytic activities of medfly adults. Moreover, the longevity of irradiated medflies at the highest dose (140 Gy) was similar to that of controls.     

美国不同棱型大麦种质资源品质分析

对新引进的300份美国大麦种质资源不同棱型间蛋白质、赖氨酸和淀粉含量的鉴定结果分析表明:①二棱大麦蛋白质总体方差与六棱大麦具有极显著的差异,其频率分布分别是以含量12.5%和17.0%为中心的双峰分布,这可能反映了二棱大麦利用上的新特点;淀粉含量二棱大麦显著高于六棱;六棱大麦当蛋白质作为固定变量时,赖氨酸与淀粉的相关性不显著.②提出了促进SPSS统计分析软件在农业科研上的开发应用研究的建议.     

Attenuation of dengue virus infection by adeno-associated virus-mediated siRNA delivery

BACKGROUND: The need for safe and effective treatment of dengue virus (DEN), a class A agent that causes dengue hemorrhagic fever/dengue shock syndrome, has been a critical global priority. An effective vaccine for DEN is not yet available. In this study the possibility of attenuating DEN infection using adeno-associated virus (AAV)-encoded short interfering RNAs (siRNA) was examined in Vero cells and human dendritic cells (DCs). METHODS: A cassette encoding siRNA targeted to a 3' untranslated sequence common to all DEN serotypes was designed and tested for its ability to attenuate DEN infection by use of AAV delivery. RESULTS: Vero cells or DCs infected with AAV-siRNA showed a significant, dose-dependent reduction in DEN infection. Treatment of DCs with AAV-siRNA also decreased the DEN-induced apoptosis of DCs and did not induce significant inflammation. CONCLUSION: These results demonstrate that AAV-mediated siRNA delivery is capable of reducing DEN infection in cells and may be useful in decreasing DEN replication in humans.     

Mesoscopic gel at low agarose concentration in water: a dynamic light scattering study.

Previous work in our laboratory has shown that at very low agarose concentration in water gelation still occurs within mutually disconnected, high concentration regions generated by spinodal demixing. The freely diffusing particles obtained in these conditions are studied in the present work by depolarized dynamic light scattering and probe diffusion experiments. These particles are found to behave as large (in fact, mesoscopic) polymer fibers entangled in a continuously rearranged mesh with scaling parameters typical of partially flexible, neutral chains. The present results allow specifying the notion of mesoscopic gelation. They also reveal that the same symmetry-breaking mechanism that allows macroscopic gelation at polymer concentrations well below the threshold for random cross-link percolation generates additional and unexpected phenomena.     

Effect of different levels of NADH availability on metabolite distribution in <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentation in minimal and complex media

A range of intracellular NADH availability was achieved by combining external and genetic strategies. The effect of these manipulations on the distribution of metabolites in Escherichia coli was assessed in minimal and complex medium under anoxic conditions. Our in vivo system to increase intracellular NADH availability expressed a heterologous NAD⁺-dependent formate dehydrogenase (FDH) from Candida boidinii in E. coli. The heterologous FDH pathway converted 1 mol formate into 1 mol NADH and carbon dioxide, in contrast to the native FDH where cofactor involvement was not present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 mol to 4 mol NADH/mol glucose consumed. In the current study, we found that yields of greater than 4 mol NADH were achieved when carbon sources more reduced than glucose were combined with our in vivo NADH regeneration system. This paper demonstrates experimentally that different levels of NADH availability can be achieved by combining the strategies of feeding the cells with carbon sources which have different oxidation states and regenerating NADH through the heterologous FDH pathway. The general trend of the data is substantially similar for minimal and complex media. The NADH availability obtained positively correlates with the proportion of reduced by-products in the final culture. The maximum theoretical yield for ethanol is obtained from glucose and sorbitol in strains overexpressing the heterologous FDH pathway.