Optimizing callus induction and proliferation for Agrobacterium-mediated transformation of Brachypodium distachyon

Brachypodium distachyon has emerged as the model species for important temperate grass crops such as wheat and barley and the genome of the B. distachyon community inbred line Bd21 has been sequenced. Methods for tissue culture and Agrobacterium-mediated transformation have been developed for this model grass as a resource for reverse genetics and functional genomic analyses. In order to obtain a high quantity and quality of compact embryogenic callus (CEC) in B. distachyon, it is important to examine and optimize the optimal concentration of the auxin 2,4-D (dichlorophenoxyacetic acid) to use in both callus induction and callus proliferation media. Here, we investigated the effects of different concentrations of 2,4-D on callus induction and callus proliferation of B. distachyon Bd21. Our results showed that 2.5 mg l–1 2,4-D is an optimal concentration to use for both callus induction and proliferation, although 5.0 mg l–1 may also be used for callus proliferation. Additionally, the suitability of hygromycin or bialaphos as selectable markers was examined and results indicated that hygromycin is significantly more efficient than bialaphos when using the Agrobacterium-mediated transformation system

The wheat SnRK1α family and its contribution to Fusarium toxin tolerance.

Deoxynivalenol (DON) is a mycotoxin produced by phytopathogenic Fusarium fungi in cereal grain and plays a role as a disease virulence factor. TaFROG (Triticum aestivum Fusarium Resistance Orphan Gene) enhances wheat resistance to DON and it interacts with a sucrose non-fermenting-1 (SNF1)-related protein kinase 1 catalytic subunit α (SnRK1α). This protein kinase family is central integrator of stress and energy signaling, regulating plant metabolism and growth. Little is known regarding the role of SnRK1α in the biotic stress response, especially in wheat. In this study, 15 wheat (Triticum aestivum) SnRK1α genes (TaSnRK1αs) belonging to four homoeologous groups were identified in the wheat genome. TaSnRK1αs are expressed ubiquitously in all organs and developmental stages apart from two members predominantly detected in grain. While DON treatment had either no effect or downregulated the transcription of TaSnRK1αs, it increased both the kinase activity associated with SnRK1α and the level of active (phosphorylated) SnRK1α. Down-regulation of two TaSnRK1αs homoeolog groups using virus induced gene silencing (VIGS) increased the DON-induced damage of wheat spikelets. Thus, we demonstrate that TaSnRK1αs contribute positively to wheat tolerance of DON and conclude that this gene family may provide useful tools for the improvement of crop biotic stress resistance

A PSTOL-like gene, TaPSTOL, controls a number of agronomically important traits in wheat

Background Phosphorus (P) is an essential macronutrient for plant growth, and is required in large quantities by elite varieties of crops to maintain yields. Approximately 70% of global cultivated land suffers from P deficiency, and it has recently been estimated that worldwide P resources will be exhausted by the end of this century, increasing the demand for crops more efficient in their P usage. A greater understanding of how plants are able to maintain yield with lower P inputs is, therefore, highly desirable to both breeders and farmers. Here, we clone the wheat (Triticum aestivum L.) homologue of the rice PSTOL gene (OsPSTOL), and characterize its role in phosphate nutrition plus other agronomically important traits. Results TaPSTOL is a single copy gene located on the short arm of chromosome 5A, encoding a putative kinase protein, and shares a high level of sequence similarity to OsPSTOL. We re-sequenced TaPSTOL from 24 different wheat accessions and (3) three T. durum varieties. No sequence differences were detected in 26 of the accessions, whereas two indels were identified in the promoter region of one of the durum wheats. We characterised the expression of TaPSTOL under different P concentrations and demonstrated that the promoter was induced in root tips and hairs under P limiting conditions. Overexpression and RNAi silencing of TaPSTOL in transgenic wheat lines showed that there was a significant effect upon root biomass, flowering time independent of P treatment, tiller number and seed yield, correlating with the expression of TaPSTOL. However this did not increase PUE as elevated P concentration in the grain did not correspond to increased yields. Conclusions Manipulation of TaPSTOL expression in wheat shows it is responsible for many of the previously described phenotypic advantages as OsPSTOL except yield. Furthermore, we show TaPSTOL contributes to additional agronomically important traits including flowering time and grain size. Analysis of TaPSTOL sequences from a broad selection of wheat varieties, encompassing 91% of the genetic diversity in UK bread wheat, showed that there is very little genetic variation in this gene, which would suggest that this locus may have been under high selection pressure

Phthiocerol Dimycocerosates of M. tuberculosis Participate in Macrophage Invasion by Inducing Changes in the Organization of Plasma Membrane Lipids

Phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), in particular during the early step of infection when bacilli encounter their host macrophages. However, their cellular and molecular mechanisms of action remain unknown. Using Mtb mutants deleted for genes involved in DIM biosynthesis, we demonstrated that DIM participate both in the receptor-dependent phagocytosis of Mtb and the prevention of phagosomal acidification. The effects of DIM required a state of the membrane fluidity as demonstrated by experiments conducted with cholesterol-depleting drugs that abolished the differences in phagocytosis efficiency and phagosome acidification observed between wild-type and mutant strains. The insertion of a new cholesterol-pyrene probe in living cells demonstrated that the polarity of the membrane hydrophobic core changed upon contact with Mtb whereas the lateral diffusion of cholesterol was unaffected. This effect was dependent on DIM and was consistent with the effect observed following DIM insertion in model membrane. Therefore, we propose that DIM control the invasion of macrophages by Mtb by targeting lipid organisation in the host membrane, thereby modifying its biophysical properties. The DIM-induced changes in lipid ordering favour the efficiency of receptor-mediated phagocytosis of Mtb and contribute to the control of phagosomal pH driving bacilli in a protective niche

Aza-β3-Cyclotetrapeptides

J. Org. Chem., 2008, 73, 8579-858