A multicomponent, elicitor-inducible cystatin complex in tomato, Solanum lycopersicum

We assessed the ability of the fungal elicitor arachidonic acid to induce cystatin genes in tomato (Solanum lycopersicum), using a cDNA expression library from arachidonate-treated leaves. The cDNAs of two novel cystatins were isolated, coding for an approx. 11-kDa protein, SlCYS10; and for a 23.6-kDa protein, SlCYS9, bearing an N-terminal signal peptide and a long, 11.5-kDa extension at the C terminus. Both genes were induced by arachidonate but not by methyl jasmonate, an inducer of the 88-kDa eight-unit cystatin, multicystatin, accumulated in the cytosol of leaf cells upon herbivory. A truncated form of SlCYS9, tSlCYS9, was produced by deletion of the C-terminal extension to assess the influence of this structural element on the cystatin moiety. As shown by kinetic and stability assays with recombinant variants expressed in Escherichia coli, deleting the extension influenced both the overall stability and inhibitory potency of SlCYS9 against cysteine proteases of herbivorous organisms. These findings provide evidence for a multicomponent elicitor-inducible cystatin complex in tomato, including at least 10 cystatin units produced via two metabolic routes.     

Proteomic profiling of aphid Macrosiphum euphorbiae responses to host-plant-mediated stress induced by defoliation and water deficit

Abiotic and biotic host-plant stress, such as desiccation and herbivory, may strongly affect sap-sucking insects such as aphids via changes in plant chemicals of insect nutritional or plant defensive value. Here, we examined (i) water deprivation and (ii) defoliation by the beetle Leptinotarsa decemlineata as stresses indirectly affecting the aphid Macrosiphum euphorbiae via its host plant Solanum tuberosum. For plant-induced stress, aphids were reared on healthy vs. continuously stressed potato for 14 days (no watering; defoliation maintained at approximately 40%). Aphid performance under stress was correlated with metabolic responses monitored by profiling of the aphid proteome. M. euphorbiae was strongly affected by water stress, as adult survival, total aphid number and biomass were reduced by 67%, 64%, and 79%, respectively. Aphids performed normally on defoliated potato, indicating that they were unaffected or able to compensate any stress induced by plant defoliation. Stressed aphid proteomes revealed 419-453 protein spots, including 27 that were modulated specifically or jointly under each kind of host-plant stress. Reduced aphid fitness on water-stressed plants mostly correlated with modulation of proteins involved in energy metabolism, apparently to conserve energy in order to prioritize survival. Despite normal performance, several aphid proteins that are known to be implicated in cell communication were modulated on defoliated plants, possibly suggesting modified aphid behaviour. The GroEL protein (or symbionin) of the endosymbiont Buchnera aphidicola was predominant under all conditions in M. euphorbiae. Its expression level was not significantly affected by aphid host-plant stresses, which is consistent with the high priority of symbiosis in stressed aphids.     

Disruption of diacylglycerol kinase delta (DGKD) associated with seizures in humans and mice

We report a female patient with a de novo balanced translocation, 46,X,t(X;2)(p11.2;q37)dn, who exhibits seizures, capillary abnormality, developmental delay, infantile hypotonia, and obesity. The 2q37 breakpoint observed in association with the seizure phenotype is of particular interest, because it lies near loci implicated in epilepsy in humans and mice. Fluorescence in situ hybridization mapping of the translocation breakpoints showed that no known genes are disrupted at Xp11.2, whereas diacylglycerol kinase delta (DGKD) is disrupted at 2q37. Expression studies in Drosophila and mouse suggest that DGKD is involved in central nervous system development and function. Electroencephalographic assessment of Dgkd mutant mice revealed abnormal epileptic discharges and electrographic seizures in three of six homozygotes. These findings implicate DGKD disruption by the t(X;2)(p11.2;q37)dn in the observed phenotype and support a more general role for DGKD in the etiology of seizures.     

A critical role for cortactin phosphorylation by Abl-family kinases in PDGF-induced dorsal-wave formation

Proper regulation of cell morphogenesis and migration by adhesion and growth-factor receptors requires Abl-family tyrosine kinases [1-3]. Several substrates of Abl-family kinase have been identified, but they are unlikely to mediate all of the downstream actions of these kinases on cytoskeletal structure. We used a human protein microarray to identify the actin-regulatory protein cortactin as a novel substrate of the Abl and Abl-related gene (Arg) nonreceptor tyrosine kinases. Cortactin stimulates cell motility [4-6], and its upregulation in several cancers correlates with poor prognosis [7]. Even though cortactin can be tyrosine phosphorylated by Src-family kinases in vitro [8], we show that Abl and Arg are more adept at binding and phosphorylating cortactin. Importantly, we demonstrate that platelet-derived growth-factor (PDGF)-induced cortactin phosphorylation on three tyrosine residues requires Abl or Arg. Cortactin triggers F-actin-dependent dorsal waves in fibroblasts after PDGF treatment and thus results in actin reorganization and lamellipodial protrusion [9]. We provide evidence that Abl/Arg-mediated phosphorylation of cortactin is required for this PDGF-induced dorsal-wave response. Our results reveal that Abl-family kinases target cortactin as an effector of cytoskeletal rearrangements in response to PDGF.     

Proteomic profiling of a parasitic wasp exposed to constant and fluctuating cold exposure

When insects are exposed to fluctuating thermal regimes (FTRs) (i.e., cold exposure alternating with periodic short pulses to high temperature), in contrast to constant low temperature (CLT), mortality due to accumulation of chill injuries is markedly reduced. To investigate the physiological processes behind the positive impact of FTR, based on a holistic approach, two-dimensional electrophoresis (2-DE) analysis were performed with the parasitic wasp Aphidius colemani. Parasitoid proteomes revealed 369 well-distinguishable protein spots, where the overall response to cold exposure was clearly specific to treatments (CLT versus FTR). The reduced mortality under FTR was associated with up-regulation of several proteins playing key roles in energy metabolism (glycolysis, TCA cycle, synthesis and conversion of ATP), protein chaperoning (Hsp70/Hsp90), and protein degradation (proteasome). Our results also support the idea that cytoskeleton components, particularly actin arrangement, could play a role in the higher survival rates of insects under FTR.     

MsCYS1, a developmentally-regulated cystatin from alfalfa.

Several roles have been attributed to cystatins in plants, ranging from the regulation of host [endogenous] cysteine proteases to the inhibition of herbivorous pest [exogenous] proteases. We report here the cloning, expression and functional characterization of a novel cystatin from alfalfa, Medicago sativa L. The new sequence, isolated from a cDNA expression library prepared from young leaves, encodes a protein, MsCYS1, with the typical inhibitory motifs of cystatins, namely the central signature motif QxVxG, a GG doublet in the N-terminal trunk, and a W residue in the C-terminal region, about 30 amino acids distant from the central inhibitory motif. As shown by a protein-based phylogenetic reconstruction, MsCYS1 is a close relative of other cystatins from Fabaceae presumably involved in the regulation of endogenous proteases. This cystatin is developmentally regulated in stems and leaves, and not induced by stress signals including methyl jasmonate, known to activate cystatins involved in plant defense. A recombinant form of MsCYS1 expressed in Escherichia coli was shown to strongly inhibit alfalfa leaf cysteine proteases while showing weak affinity for the digestive cysteine proteases of different herbivorous pests. Overall, these observations suggest an endogenous protease regulatory role for MsCYS1, possibly associated with the early development of stems and leaves.     

Grazing-induced changes in cell wall silicification in a marine diatom

In aquatic environments, diatoms (Bacillariophyceae) constitute a central group of microalgae which contribute to about 40% of the oceanic primary production. Diatoms have an absolute requirement for silicon to build-up their silicified cell wall in the form of two shells (the frustule). To date, changes in diatom cell wall silicification have been only studied in response to changes in the growth environment, with consistent increase in diatom silica content when specific growth rates decrease under nutrient or light limitations. Here, we report the first evidence for grazing-induced changes in cell wall silicification in a marine diatom. Cells grown in preconditioned media that had contained both diatoms and herbivores are significantly more silicified than diatoms grown in media that have contained diatoms alone or starved herbivores. These observations suggest that grazing-induced increase in cell wall silicification can be viewed as an adaptive reaction in habitats with variable grazing pressure, and demonstrate that silicification in diatoms is not only a constitutive mechanical protection for the cell, but also a phenotypically plastic trait modulated by grazing. In turn, our results corroborate the idea that plant-herbivore interactions, beyond grazing sensu stricto, contribute to drive ecosystem structure and biogeochemical cycles in the ocean.     

Alpha6beta1 integrin expressed by sperm is determinant in mouse fertilization

Background  

Based on inhibition tests, the alpha6beta1 integrin was suggested to be a sperm receptor, but further experiments using gene deletion techniques have shown that neither oocyte alpha6, nor beta1 integrin subunits were essential for mouse fertilization.