In vitro insect-feeding bioassay to determine the resistance of transgenic rice plants transformed with insect resistance genes against striped stem borer (Chilo suppressalis)

Summary To determine the degree of insect resistance in transgenic plants, different bioassays are used which typically use either whole plant or small pieces of leaves or stems of transgenic plants, following culture under greenhouse conditions. An in vitro insect-feeding bioassay is presented which permits the infestation of transgenic plantlets with newly hatched larvae from the striped stem borer. The bioassay consists of the germination of rice seeds in vitro using Murashige and Skoog medium in test tubes, and then infestation of each 3–4 cm long seedling with one neonate larva obtained from surfacesterilized eggs of Chilo suppressalis. The infested in vitro plantlets are kept in culture rooms at 25°C for several days and then the seedling damage and the growth of the larvae are analyzed. Senia (japonica variety) homozygous transgenic rice plants were used for these experiments. The plants were transformed with either the cry1B or the maize proteinase inhibitor (mpi) genes. Both genes confer resistance to Chilo suppressalis. With non-transformed plants the larvae grew and developed normally, feeding on the small rice plantlets. In contrast, with cry1B plants, the neonate larvae died during the first days of the infestation. These plantlets recovered completely and developed similarly to the non-infested control plants. With transgenic plants transformed with the mpi gene, the neonate larvae did not die but grew more slowly compared with the controls. Thus, this in vitro insect-feeding bioassay is a rapid and easy method to detect the resistance of cry and mpi transgenic plants to stem borers such as Chilo suppressalis.     

The conserved factor DE-ETIOLATED 1 cooperates with CUL4-DDB1DDB2 to maintain genome integrity upon UV stress

Plants and many other eukaryotes can make use of two major pathways to cope with mutagenic effects of light, photoreactivation and nucleotide excision repair (NER). While photoreactivation allows direct repair by photolyase enzymes using light energy, NER requires a stepwise mechanism with several protein complexes acting at the levels of lesion detection, DNA incision and resynthesis. Here we investigated the involvement in NER of DE-ETIOLATED 1 (DET1), an evolutionarily conserved factor that associates with components of the ubiquitylation machinery in plants and mammals and acts as a negative repressor of light-driven photomorphogenic development in Arabidopsis. Evidence is provided that plant DET1 acts with CULLIN4-based ubiquitin E3 ligase, and that appropriate dosage of DET1 protein is necessary for efficient removal of UV photoproducts through the NER pathway. Moreover, DET1 is required for CULLIN4-dependent targeted degradation of the UV-lesion recognition factor DDB2. Finally, DET1 protein is degraded concomitantly with DDB2 upon UV irradiation in a CUL4-dependent mechanism. Altogether, these data suggest that DET1 and DDB2 cooperate during the excision repair process.     

Fertilizing Methods and Nutrient Balance at the End of Traditional Organic Agriculture in the Mediterranean Bioregion: Catalonia (Spain) in the 1860s

By reconstructing the nutrient balance of a Catalan village circa 1861–65 we examine the sustainability of organic agricultural systems in the northwest Mediterranean bioregion prior to the green revolution and the question of whether the nutrients extracted from the soil were replenished. With a population density of 59 inhabitants per square km, similar to other northern European rural areas at that time, and a lower livestock density per cropland unit, this village experienced a manure shortage. The gap was filled by other labour-intensive ways of transferring nutrients from uncultivated areas into the cropland. Key elements in this agricultural system were vineyards because they have few nutrient requirements, and woodland and scrublands as sources of relevant amounts of nutrients collected in several ways.     

Use of RT-Defective HIV Virions: New Tool to Evaluate Specific Response in Chronic Asymptomatic HIV-Infected Individuals

Background

Generation of new reagents that can be used to screen or monitor HIV-1-specific responses constituted an interesting field in the development of HIV vaccines to improve their efficacy.

Methods

We have evaluated the specific T cell response against different types of NL4-3 virions (including NL4-3 aldrithiol-2 treated, NL4-3/ΔRT and R5 envelopes: NL4-3/ΔRT/ΔEnv[AC10] and NL4-3/ΔRT/ΔEnv[Bal]) and against pools of overlapping peptides (15 mer) encompassing the HIV-1 Gag and Nef regions. Cryopreserved PBMC from a subset of 69 chronic asymptomatic HIV positive individuals have been employed using different techniques including IFN-γ ELISPOT assay, surface activation markers and intracellular cytokine staining (ICS) by flow cytometry.

Results

The differential response obtained against NL4-3 aldrithiol-2 treated and NL4-3/ΔRT virions (25% vs 55%, respectively) allow us to divide the population in three groups: “full-responders” (positive response against both viral particles), “partial-responders” (positive response only against NL4-3/ΔRT virions) and “non-responders” (negative responses). There was no difference between X4 and R5 envelopes. The magnitude of the total responses was higher against NL4-3/ΔRT and was positively correlated with gender and inverse correlated with viral load. On the contrary CD4+ T cell count was not associated with this response. In any case responses to the viruses tended to be lower in magnitude than those detected by the overlapping peptides tested. Finally we have found an increased frequency of HLA-B27 allele (23% vs 9%) and a significant reduction in some activation markers (CD69 and CD38) on T cells surface in responders vs non-responders individuals.

Conclusions

In summary these virions could be considered as alternative and useful reagents for screening HIV-1-specific T cell responses in HIV exposed uninfected people, HIV infected patients and to assess immunogenicity of new prototypes both in vitro and in vaccine trials, by a feasible, simply, effective and low cost assay.     

Effects of Disinfection on Legionella spp., Eukarya, and Biofilms in a Hot Water System

Legionella species are frequently detected in hot water systems, attached to the surface as a biofilm. In this work, the dynamics of Legionella spp. and diverse bacteria and eukarya associated together in the biofilm, coming from a pilot scale 1 system simulating a real hot water system, were investigated throughout 6 months after two successive heat shock treatments followed by three successive chemical treatments. Community structure was assessed by a fingerprint technique, single-strand conformation polymorphism (SSCP). In addition, the diversity and dynamics of Legionella and eukarya were investigated by small-subunit (SSU) ribosomal cloning and sequencing. Our results showed that pathogenic Legionella species remained after the heat shock and chemical treatments (Legionella pneumophila and Legionella anisa, respectively). The biofilm was not removed, and the bacterial community structure was transitorily affected by the treatments. Moreover, several amoebae had been detected in the biofilm before treatments (Thecamoebae sp., Vannella sp., and Hartmanella vermiformis) and after the first heat shock treatment, but only H. vermiformis remained. However, another protozoan affiliated with Alveolata, which is known as a host cell for Legionella, dominated the eukaryal species after the second heat shock and chemical treatment tests. Therefore, effective Legionella disinfection may be dependent on the elimination of these important microbial components. We suggest that eradicating Legionella in hot water networks requires better study of bacterial and eukaryal species associated with Legionella in biofilms.