The eukaryotic translation initiation factor 4E controls lettuce susceptibility to the Potyvirus Lettuce mosaic virus

The eIF4E and eIF(iso)4E cDNAs from several genotypes of lettuce (Lactuca sativa) that are susceptible, tolerant, or resistant to infection by Lettuce mosaic virus (LMV; genus Potyvirus) were cloned and sequenced. Although Ls-eIF(iso)4E was monomorphic in sequence, three types of Ls-eIF4E differed by point sequence variations, and a short in-frame deletion in one of them. The amino acid variations specific to Ls-eIF4E(1) and Ls-eIF4E(2) were predicted to be located near the cap recognition pocket in a homology-based tridimensional protein model. In 19 lettuce genotypes, including two near-isogenic pairs, there was a strict correlation between these three allelic types and the presence or absence of the recessive LMV resistance genes mo1(1) and mo1(2). Ls-eIF4E(1) and mo1(1) cosegregated in the progeny of two separate crosses between susceptible genotypes and an mo1(1) genotype. Finally, transient ectopic expression of Ls-eIF4E restored systemic accumulation of a green fluorescent protein-tagged LMV in LMV-resistant mo1(2) plants and a recombinant LMV expressing Ls-eIF4E degrees from its genome, but not Ls-eIF4E(1) or Ls-eIF(iso)4E, accumulated and produced symptoms in mo1(1) or mo1(2) genotypes. Therefore, sequence correlation, tight genetic linkage, and functional complementation strongly suggest that eIF4E plays a role in the LMV cycle in lettuce and that mo1(1) and mo1(2) are alleles coding for forms of eIF4E unable or less effective to fulfill this role. More generally, the isoforms of eIF4E appear to be host factors involved in the cycle of potyviruses in plants, probably through a general mechanism yet to be clarified.     

Analogs of 1alpha,25-dihydroxyvitamin D3 as pluripotent immunomodulators

The active form of vitamin D(3), 1,25(OH)(2)D(3), is known, besides its classical effects on calcium and bone, for its pronounced immunomodulatory effects that are exerted both on the antigen-presenting cell level as well as directly on the T lymphocyte level. In animal models, these immune effects of 1,25(OH)(2)D(3) are reflected by a strong potency to prevent onset and even recurrence of autoimmune diseases. A major limitation in using 1,25(OH)(2)D(3) in clinical immune therapy are the adverse side effects on calcium and on bone. TX527 (19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)) is a structural 1,25(OH)(2)D(3) analog showing reduced calcemic activity associated with enhanced in vitro and in vivo immunomodulating capacity compared to the mother-molecule. Indeed, in vitro TX527 is more potent that 1,25(OH)(2)D(3) in redirecting differentiation and maturation of dendritic cells and in inhibiting phytohemagglutinin-stimulated T lymphocyte proliferation. In vivo, this enhanced potency of TX527 is confirmed by a stronger potential to prevent type 1 diabetes in nonobese diabetic (NOD) mice and to prolong the survival of syngeneic islets grafts, both alone and in combination with cyclosporine A, in overtly diabetic NOD mice. Moreover, these in vivo effects of TX527 are obtained without the adverse side effects observed for 1,25(OH)(2)D(3) itself. We believe therefore that TX527 is a potentially interesting candidate to be considered for clinical intervention trails in autoimmune diseases.     

Meiosis resumption, calcium-sensitive period, and PLC-beta1 relocation into the nucleus in the mouse oocyte

We aimed to determine whether the breakdown of the germinal vesicle of the mouse oocyte and the nuclear import of phospholipase C-beta1 were calcium-dependent. We chelated Ca2+ ions with BAPTA-dextran at different times after the release of the oocyte from the ovarian follicle, i.e. after meiosis resumption has started, and we studied the effects on the kinetics of germinal vesicle breakdown, and on the migration of phospholipase C-beta1. We discriminate between two key-periods of calcium-sensitivity during the process of meiosis resumption. During the first hour, changes in the cytosolic Ca2+ especially promoted the migration of phospholipase C-beta1 into the nucleus, whereas changes in the nuclear concentration of Ca2+ were not implicated. Moreover, at this time, the cytosolic calcium pathway is PLC-beta1-dependent. By contrast, during the second hour following the onset of meiosis resumption, and thus just previous GVBD, the PLC-beta1-dependent Ca2+ signals in both cellular compartments were equally necessary for the resumption of meiosis. This particular period of the meiotic process corresponds to the moment when the phospholipase C-beta1 has strongly migrated into the nucleus. Our results highlight also the role played by the nucleus during the second key-period in the control of the GVBD via a Ca2+-dependent pathway.     

ERECTA, an LRR receptor-like kinase protein controlling development pleiotropically affects resistance to bacterial wilt

Bacterial wilt, one of the most devastating bacterial diseases of plants worldwide, is caused by Ralstonia solanacearum and affects many important crop species. We show that several strains isolated from solanaceous crops in Europe are pathogenic in different accessions of Arabidopsis thaliana. One of these strains, 14.25, causes wilting symptoms in A. thaliana accession Landsberg erecta (Ler) and no apparent symptoms in accession Columbia (Col-0). Disease development and bacterial multiplication in the susceptible Ler accession depend on functional hypersensitive response and pathogenicity (hrp) genes, key elements for bacterial pathogenicity. Genetic analysis using Ler x Col-0 recombinant inbred lines showed that resistance is governed by at least three loci: QRS1 (Quantitative Resistance to R. solanacearum) and QRS2 on chromosome 2, and QRS3 on chromosome 5. These loci explain about 90% of the resistance carried by the Col-0 accession. The ERECTA gene, which encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) and affects development of aerial organs, is dimorphic in our population and lies close to QRS1. Susceptible Ler plants transformed with a wild-type ERECTA gene, and the LER line showed increased disease resistance to R. solanacearum as indicated by reduced wilt symptoms and impaired bacterial growth, suggesting unexpected cross-talk between resistance and developmental pathways.     

High phylogenetic diversity of transconjugants carrying plasmid pJP4 in an activated sludge-derived microbial community

Transconjugants of plasmid pJP4, originating from an agar plate mating of a Pseudomonas putida donor with an activated sludge-derived microbial community, were isolated and identified by partial 16S rDNA sequencing. The transconjugant strains belonged to a variety of genera of the alpha-, beta-, gamabeta- and gamma-classes of the Proteobacteria, mostly to the families Rhizobiaceae and Comamonadaceae and the genus Stenotrophomonas. Only P. putida and Delftia spp. strains were able to grow on 2,4-D as the sole carbon source.     

Condensed lignins are synthesized in poplar leaves exposed to ozone

Poplar (Populus tremula x alba) trees (clone INRA 717-1-B4) were cultivated for 1 month in phytotronic chambers with two different levels of ozone (60 and 120 nL L(-1)). Foliar activities of shikimate dehydrogenase (EC 1.1.1.25), phenylalanine ammonia lyase (EC 4.3.1.5), and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) were compared with control levels. In addition, we examined lignin content and structure in control and ozone-fumigated leaves. Under ozone exposure, CAD activity and CAD RNA levels were found to be rapidly and strongly increased whatever the foliar developmental stage. In contrast, shikimate dehydrogenase and phenylalanine ammonia lyase activities were increased in old and midaged leaves but not in the youngest ones. The increased activities of these enzymes involved in the late or early steps of the metabolic pathway leading to lignins were associated with a higher Klason lignin content in extract-free leaves. In addition, stress lignins synthesized in response to ozone displayed a distinct structure, relative to constitutive lignins. They were found substantially enriched in carbon-carbon interunit bonds and in p-hydroxyphenylpropane units, which is reminiscent of lignins formed at early developmental stages, in compression wood, or in response to fungal elicitor. The highest changes in lignification and in enzyme activities were obtained with the highest ozone dose (120 nL L(-1)). These results suggest that ozone-induced lignins might contribute to the poplar tolerance to ozone because of their barrier or antioxidant effect toward reactive oxygen species.     

Identification of the required acyltransferase step in the biosynthesis of the phosphatidylinositol mannosides of mycobacterium species

Fatty acyl functions of the glycosylated phosphatidylinositol (GPI) anchors of the phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM) of mycobacteria play a critical role in both the physical properties and biological activities of these molecules. In a search for the acyltransferases that acylate the GPI anchors of PIM, LM, and LAM, we examined the function of the mycobacterial Rv2611c gene that encodes a putative acyltransferase involved in the early steps of phosphatidylinositol mannoside synthesis. A Rv2611c mutant of Mycobacterium smegmatis was constructed which exhibited severe growth defects and contained an increased amount of phosphatidylinositol mono- and di-mannosides and a decreased amount of acylated phosphatidylinositol di-mannosides compared with the wild-type parental strain. In cell-free assays, extracts from M. smegmatis overexpressing the M. tuberculosis Rv2611c gene incorporated [14C]palmitate into acylated phosphatidylinositol mono- and di-mannosides, and transferred cold endogenous fatty acids onto 14C-labeled phosphatidylinositol mono- and di-mannosides more efficiently than extracts from the wild-type strain. Cell-free extracts from the Rv2611c mutant of M. smegmatis were greatly impaired in these respects. This work provides evidence that Rv2611c is the acyltransferase that catalyzes the acylation of the 6-position of the mannose residue linked to position 2 of myo-inositol in phosphatidylinositol mono- and di-mannosides, with the mono-mannosylated lipid acceptor being the primary substrate of the enzyme. We also provide the first evidence that two distinct pathways lead to the formation of acylated PIM2 from PIM1 in mycobacteria.     

The F-actin cross-linking and focal adhesion protein filamin A is a ligand and in vivo substrate for protein kinase C alpha

Filamin A is an established structural component of cell-matrix adhesion sites. In addition, it serves as a scaffold for the subcellular targeting of different signaling molecules. Protein kinase C (PKC) has been found associated with filamin; however, details about this interaction and its significance for cell-matrix adhesion-dependent signaling have remained elusive. We performed a yeast two-hybrid analysis using protein kinase Calpha as a bait and identified filamin as a direct binding partner. The interaction was confirmed in transfected HeLa cells, and serial truncation fragments of filamin A were employed to identify two binding sites on filamin. In vitro ligand binding assays revealed a Ca2+ and phospholipid-dependent association of the regulatory domain of protein kinase C with these sites. Phosphorylation of filamin was found to be isoform-restricted, leading to phosphate incorporation in the C termini of filamin A and C, but not B. PKC-dependent phosphorylation of filamin was also detected in cells. Our data suggest an intimate interaction between filamin and PKC in cell signaling.