The immune enhancer, thymoquinone, and the hope of utilizing the immune system of Aedes caspius against disease agents

Insects have developed an efficient defence system against  microorganisms, which involves both humoral and cellular mechanisms. Recent studies on insect defence system are aimed at utilizing it inthe battle against mosquito-borne diseases. However, mounting immune responses of insects has proved to impose fitness costs. The current study was conducted to test the costs of enhancing the immune responses of Aedes caspius by oral administration of thymoquinone, the active ingredient of the black seed oil, Nigella sativa L. (Ranunculaceae). The obtained results showed an effective humoral activity against Micrococcus luteus (NCTC 2665, Sigma-Aldrich, UK) and Bacillus cereus when mosquitoes were injected with lipopolysaccharide (LPS), Bacillus thuringiensis or B. cereus. However, this activity showed no effect against B. thuringiensis. Furthermore, oral administration of thymoquinone enhanced the humoral activity against B. cereus but not against B. thuringiensis. On the other hand, thymoquinone-enhanced immunity imposed reproductive costs in terms of higher percentages of follicular apoptosis and resorption. This observation has been confirmed by the semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis, which showed a relatively higher expression of selected caspases genes, namely: CASP 18, CASP 19 and CASP 20, but not CASP 21, transcripts in immune enhanced mosquitoes compared to non-enhanced ones. Based on these results, this study suggests that enhancing the humoral activity by thymoquinone proved to be costly in terms of triggering follicular apoptosis and resorption. Thus, as part of the scenario ofimmunity-reproduction conflict, it was concluded that the impact of immune stimulation and/or enhancement on the vector reproduction constitutes a limiting factor to the utilization of thymoquinone in the immuno-control strategy against mosquito-borne diseases

Regulation of the Fruit-Specific PEP Carboxylase SlPPC2 Promoter at Early Stages of Tomato Fruit Development

The SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. We recently showed that a 1966 bp DNA fragment located upstream of the ATG codon of the SlPPC2 gene (GenBank AJ313434) confers appropriate fruit-specificity in transgenic tomato. In this study, we further investigated the regulation of the SlPPC2 promoter gene by analysing the SlPPC2 cis-regulating region fused to either the firefly luciferase (LUC) or the β-glucuronidase (GUS) reporter gene, using stable genetic transformation and biolistic transient expression assays in the fruit. Biolistic analyses of 5′ SlPPC2 promoter deletions fused to LUC in fruits at the 8th day after anthesis revealed that positive regulatory regions are mostly located in the distal region of the promoter. In addition, a 5′ UTR leader intron present in the 1966 bp fragment contributes to the proper temporal regulation of LUC activity during fruit development. Interestingly, the SlPPC2 promoter responds to hormones (ethylene) and metabolites (sugars) regulating fruit growth and metabolism. When tested by transient expression assays, the chimeric promoter:LUC fusion constructs allowed gene expression in both fruit and leaf, suggesting that integration into the chromatin is required for fruit-specificity. These results clearly demonstrate that SlPPC2 gene is under tight transcriptional regulation in the developing fruit and that its promoter can be employed to drive transgene expression specifically during the cell expansion stage of tomato fruit. Taken together, the SlPPC2 promoter offers great potential as a candidate for driving transgene expression specifically in developing tomato fruit from various tomato cultivars

S-nitrosylation of NADPH oxidase regulates cell death in plant immunity

Changes in redox status are a conspicuous feature of immune responses in a variety of eukaryotes, but the associated signalling mechanisms are not well understood. In plants, attempted microbial infection triggers the rapid synthesis of nitric oxide and a parallel accumulation of reactive oxygen intermediates, the latter generated by NADPH oxidases related to those responsible for the pathogen-activated respiratory burst in phagocytes. Both nitric oxide and reactive oxygen intermediates have been implicated in controlling the hypersensitive response, a programmed execution of plant cells at sites of attempted infection. However, the molecular mechanisms that underpin their function and coordinate their synthesis are unknown. Here we show genetic evidence that increases in cysteine thiols modified using nitric oxide, termed S-nitrosothiols, facilitate the hypersensitive response in the absence of the cell death agonist salicylic acid and the synthesis of reactive oxygen intermediates. Surprisingly, when concentrations of S-nitrosothiols were high, nitric oxide function also governed a negative feedback loop limiting the hypersensitive response, mediated by S-nitrosylation of the NADPH oxidase, AtRBOHD, at Cys 890, abolishing its ability to synthesize reactive oxygen intermediates. Accordingly, mutation of Cys 890 compromised S-nitrosothiol- mediated control of AtRBOHD activity, perturbing the magnitude of cell death development. This cysteine is evolutionarily conserved and specifically S-nitrosylated in both human and fly NADPH oxidase, suggesting that this mechanism may govern immune responses in both plants and animals