Overexpression of wheat Na+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants

Transgenic Arabidopsis plants overexpressing the wheat vacuolarNa⁺/H⁺ antiporter TNHX1 and H⁺-PPase TVP1 are much more resistantto high concentrations of NaCl and to water deprivation thanthe wild-type strains. These transgenic plants grow well inthe presence of 200 mM NaCl and also under a water-deprivationregime, while wild-type plants exhibit chlorosis and growthinhibition. Leaf area decreased much more in wild-type thanin transgenic plants subjected to salt or drought stress. Theleaf water potential was less negative for wild-type than fortransgenic plants. This could be due to an enhanced osmoticadjustment in the transgenic plants. Moreover, these transgenicplants accumulate more Na⁺ and K⁺ in their leaf tissue thanthe wild-type plants. The toxic effect of Na⁺ accumulation inthe cytosol is reduced by its sequestration into the vacuole.The rate of water loss under drought or salt stress was higherin wild-type than transgenic plants. Increased vacuolar soluteaccumulation and water retention could confer the phenotypeof salt and drought tolerance of the transgenic plants. Overexpressionof the isolated genes from wheat in Arabidopsis thaliana plantsis worthwhile to elucidate the contribution of these proteinsto the tolerance mechanism to salt and drought. Adopting a similarstrategy could be one way of developing transgenic staple cropswith improved tolerance to these important abiotic stresses. Key words: H⁺-pyrophosphatase, Na⁺/H⁺ antiporter, salt and drought tolerance, sodium sequestration, transgenic Arabidopsis plants     

Comparative analysis of expressed genes from cacao meristems infected by Moniliophthora perniciosa

BACKGROUND AND AIMS: Witches' broom disease is caused by the hemibiotrophic basidiomycete Moniliophthora perniciosa, and is one of the most important diseases of cacao in the western hemisphere. Because very little is known about the global process of such disease development, expressed sequence tags (ESTs) were used to identify genes expressed during the Theobroma cacao-Moniliophthora perniciosa interaction. METHODS: Two cDNA libraries corresponding to the resistant (RT) and susceptible (SP) cacao-M. perniciosa interactions were constructed from total RNA, using the DB SMART Creator cDNA library kit (Clontech). Clones were randomly selected, sequenced from the 5' end and analysed using bioinformatics tools including in silico analysis of the differential gene expression. KEY RESULTS: A total of 6884 ESTs were generated from the RT and SP cDNA libraries. These ESTs were composed of 2585 singlets and 341 contigs for a total of 2926 non-redundant sequences. The redundancy of the libraries was low and their specificity high when compared with the few other cacao libraries already published. Sequence analysis allowed the assignment of a putative functional category for 54 % of sequences, whereas approx. 22 % of sequences corresponded to unknown function and approx. 24 % of sequences did not show any significant similarity with other proteins present in the database. Despite the similar overall distribution of the sequences in functional categories between the two libraries, qualitative differences were observed. Genes involved during the defence response to pathogen infection or in programmed cell death were identified, such as pathogenesis related-proteins, trypsin inhibitor or oxalate oxidase, and some of them showed an in silico differential expression between the resistant and the susceptible interactions. CONCLUSIONS: As far as is known this is the first EST resource from the cacao-M. perniciosa interaction and it is believed that it will provide a significant contribution to the understanding of the molecular mechanisms of the resistance and susceptibility of cacao to M. perniciosa, to develop strategies to control witches' broom, and as a source of polymorphism for molecular marker development and marker-assisted selection.     

Cell-permeable peptides induce dose- and length-dependent cytotoxic effects

We have explored the threshold of tolerance of three unrelated cell types to treatments with potential cytoprotective peptides bound to Tat_48-57 and Antp_43-58 cell-permeable peptide carriers. Both Tat_48-57 and Antp_43-58 are well known for their good efficacy at crossing membranes of different cell types, their overall low toxicity, and their absence of leakage once internalised. Here, we show that concentrations of up to 100 μM of Tat_48-57 were essentially harmless in all cells tested, whereas Antp_43-58 was significantly more toxic. Moreover, all peptides bound to Tat_48-57 and Antp_43-58 triggered significant and length-dependent cytotoxicity when used at concentrations above 10 μM in all but one cell types (208F rat fibroblasts), irrespective of the sequence of the cargo. Absence of cytotoxicity in 208F fibroblasts correlated with poor intracellular peptide uptake, as monitored by confocal laser scanning fluorescence microscopy. Our data further suggest that the onset of cytotoxicity correlates with the activation of two intracellular stress signalling pathways, namely those involving JNK, and to a lesser extent p38 mitogen-activated protein kinases. These responses are of particular concern for cells that are especially sensitive to the activation of stress kinases. Collectively, these results indicate that in order to avoid unwanted and unspecific cytotoxicity, effector molecules bound to Tat_48-57 should be designed with the shortest possible sequence and the highest possible affinity for their binding partners or targets, so that concentrations below 10 μM can be successfully applied to cells without harm. Considering that cytotoxicity associated to Tat_48-57- and Antp_43-58 bound peptide conjugates was not restricted to a particular type of cells, our data provide a general framework for the design of cell-penetrating peptides that may apply to broader uses of intracellular peptide and drug delivery.     

The pygmy hog is a unique genus: 19th century taxonomists got it right first time round

The pygmy hog, Sus salvanius, the smallest and rarest extant suid was first described as the only member of the genus Porcula. It is currently regarded as member of the genus Sus and a sister taxon of the domestic pig/Eurasian wild boar (Sus scrofa). Phylogenetic analyses of 2316 bp from three mtDNA loci (control-region, cytochrome b, 16S) by Bayesian inference and statistical testing of alternative phylogenetic hypotheses all support the original classification of the pygmy hog as a unique genus. Thus, we propose that the species name Porcula salvania should be resurrected. The reclassification will heighten awareness of the need for the future protection and survival of this unique species.     

FliK regulates flagellar hook length as an internal ruler

The mechanism of length control of the flagellar hook is under debate between two theories. One claims that the FliK directly measures the hook length as a molecular ruler, while the other claims that the cytoplasmic substructure measures the amount of hook subunits to determine the hook length. Both agree that the FliK C-terminal domain catalyses the substrate-specificity switch to terminate hook elongation. In this study, we systematically created fliK mutants with deletions and insertions at various sites within the FliK N-terminal domain and analysed their effects on the final hook length. Insertions of peptide fragments from the Yersinia YscP into FliK gave rise to hooks with defined lengths, which was proportional to the molecular size of the FliK-YscP chimeras. Among fliK deletion mutants, only those with small truncations in three specific sites of FliK produced hooks of a defined, shortened length. For the majority of deletion mutants, FliK was secreted, but hook length was not controlled. On the other hand, for some deletion mutants FliK was not secreted, but the hook length was controlled, indicating that FliK secretion is not necessary for hook-length control. We conclude that FliK regulates hook length as an internal molecular ruler.     

Core protein domains involved in hepatitis C virus-like particle assembly and budding at the endoplasmic reticulum membrane

Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon, self-assembles into HCV-like particles (HCV-LPs) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV particle morphogenesis by electron microscopy. Various mutated HCV core proteins with engineered internal deletions were expressed with this system, to identify core domains required or dispensable for HCV-LP assembly. The HCV core protein sequence was compared with its counterpart in GB virus B (GBV-B), the virus most closely related to HCV, to identify conserved domains. GBV-B and HCV display similar tropism for liver hepatocytes and their core proteins are organized similarly into three main domains (I, II and III), although GBV-B core is smaller and lacks approximately 35 amino acids (aa) in domain I. The deletion of short hydrophobic domains (aa 133-152 and 153-167 in HCV core) that appear highly conserved in domain II of both GBV-B and HCV core proteins resulted in loss of HCV core ER anchoring and self-assembly into HCV-LPs. The deletion of short domains found within domain I of HCV core protein but not in the corresponding domain of GBV-B core according to sequence alignment had contrasting effects. Amino acids 15-28 and 60-66 were shown to be dispensable for HCV-LP assembly and morphogenesis, whereas aa 88-106 were required for this process. The production of GBV-B core protein from a recombinant SFV vector was associated with specific ER ultrastructural changes, but did not lead to the morphogenesis of GBV-B-LPs, suggesting that different budding mechanisms occur in members of the Flaviviridae family.     

Single crystals of V-amylose complexed with alpha-naphthol

Lamellar square single crystals of V-amylose were obtained by adding alpha-naphthol to metastable dilute aqueous solutions of synthetic amylose chains with an average degree of polymerization of 100. The morphology and structure of the crystals were studied using low-dose transmission electron microscopy including high-resolution imaging, as well as electron and X-ray diffraction. The crystals are crystallized in a tetragonal P4(1)2(1)2 or P4(3)2(1)2 space group with unit cell parameters, calculated from X-ray diffraction data, a = b = 2.2844 nm (+/-0.0005) and c = 0.7806 nm (+/-0.001), implying the presence of two amylose chains per unit cell. High-resolution lattice images of the crystals confirmed that the amylose chains were crystallized as 8-fold helices corresponding to the repeat of four maltosyl units.