Defense activation triggers differential expression of phospholipase-C (PLC) genes and elevated temperature induces phosphatidic acid (PA) accumulation in tomato

Recently, we provided the first genetic evidence for the requirement of tomato PLC4 and PLC6 genes in defense activation and disease resistance. The encoded enzymes were catalytically active as they were able to degrade phosphatidylinositol (PI), thereby producing diacylglycerol (DG). Here we report differential PLC gene expression following the initiation of defense signaling by the interaction between Cladosporium fulvum resistance (R) protein Cf-4 and its matching effector Avr4 in tomato hybrid seedlings that express both Cf-4 and Avr4. Furthermore, we observed that PLC3 and PLC6 gene expression is upregulated by elevated temperature in the control seedlings. This upregulation coincides with an increase in the levels of phosphatidic acid (PA) and a decrease in the levels of PI and phosphatidylinositol phosphate (PIP). The decrease in PI and PIP levels matches with the activation of PLC. In addition, the levels of the structural phospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) declined transiently during recovery after the exposure to elevated temperature., Further studies will be required to explain the mechanism causing the sustained accumulation of PA during recovery, combined with a reduction in the levels of structural phospholipids.     

Structure and dynamics of pin1 during catalysis by NMR

The link between internal enzyme motions and catalysis is poorly understood. Correlated motions in the microsecond-to-millisecond timescale may be critical for enzyme function. We have characterized the backbone dynamics of the peptidylprolyl isomerase (Pin1) catalytic domain in the free state and during catalysis. Pin1 is a prolyl isomerase of the parvulin family and specifically catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds. Pin1 has been shown to be essential for cell-cycle progression and to interact with the neuronal tau protein inhibiting its aggregation into fibrillar tangles as found in Alzheimer's disease. (15)N relaxation dispersion measurements performed on Pin1 during catalysis reveal conformational exchange processes in the microsecond timescale. A subset of active site residues undergo kinetically similar exchange processes even in the absence of a substrate, suggesting that this area is already "primed" for catalysis. Furthermore, structural data of the turning-over enzyme were obtained through inter- and intramolecular nuclear Overhauser enhancements. This analysis together with a characterization of the substrate concentration dependence of the conformational exchange allowed the distinguishing of regions of the enzyme active site that are affected primarily by substrate binding versus substrate isomerization. Together these data suggest a model for the reaction trajectory of Pin1 catalysis.     

Adaptation is not required to explain the long-term response of axons to molecular gradients

It has been suggested that growth cones navigating through the developing nervous system might display adaptation, so that their response to gradient signals is conserved over wide variations in ligand concentration. Recently however, a new chemotaxis assay that allows the effect of gradient parameters on axonal trajectories to be finely varied has revealed a decline in gradient sensitivity on either side of an optimal concentration. We show that this behavior can be quantitatively reproduced with a computational model of axonal chemotaxis that does not employ explicit adaptation. Two crucial components of this model required to reproduce the observed sensitivity are spatial and temporal averaging. These can be interpreted as corresponding, respectively, to the spatial spread of signaling effects downstream from receptor binding, and to the finite time over which these signaling effects decay. For spatial averaging, the model predicts that an effective range of roughly one-third of the extent of the growth cone is optimal for detecting small gradient signals. For temporal decay, a timescale of about 3 minutes is required for the model to reproduce the experimentally observed sensitivity.     

Neuronal nitric oxide synthase immunopositive neurons in cat claustrum—a light and electron microscopic study

Nitric oxide is a unique neurotransmitter, which participates in many physiological and pathological processes in the organism. Nevertheless there are little data about the neuronal Nitric Oxide Synthase immunoreactive (nNOS-ir) neurons and fibers in the dorsal claustrum (DC) of a cat. In this respect the aims of this study were: (1) to demonstrate nNOS-ir in the neurons and fibers of the DC; (2) to describe their light microscopic morphology and distribution; (3) to investigate and analyze the ultrastructure of the nNOS-ir neurons, fibers and synaptic terminals; (4) to verify whether the nNOS-ir neurons consist a specific subpopulation of claustral neurons; (5) to verify whether the nNOS-ir neurons have a specific pattern of organization throughout the DC. For demonstration of the nNOS-ir the Avidin-Biotin-Peroxidase Complex method was applied. Immunopositive for nNOS neurons and fibers were present in all parts of DC. On the light microscope level nNOS-ir neurons were different in shape and size. According to the latter they were divided into three groups-small (with diameter under 15 mum), medium-sized (with diameter from 16 to 20 mum) and large (with diameter over 21 mum). Some of nNOS-ir neurons were lightly-stained while others were darkly-stained. On the electron microscope level the immunoproduct was observed in neurons, dendrites and terminal boutons. Different types of nNOS-ir neurons differ according to their ultrastructural features. Three types of nNOS-ir synaptic boutons were found. As a conclusion we hope that the present study will contribute to a better understanding of the functioning of the DC in cat and that some of the data presented could be extrapolated to other mammals, including human.     

A PCR fingerprinting technique to distinguish isolates of Lactococcus lactis

A fingerprinting technique similar to repetitive extragenic palindromic PCR was developed to identify strains of Lactococcus lactis. The method distinguishes closely related strains and discriminates among some with identical ldh sequences. The fingerprinting primer LL-Rep1 complements a moderately repeated sequence found in low G+C Gram-positive bacteria and may therefore prove useful for discriminating among strains of other low G+C Gram-positive species.     

Phosphorylation of the C subunit (p66) of human DNA polymerase delta

Of the four subunits constituting DNA polymerase δ, subunit C or p66 has been shown to mainly mediate polymerase interaction with PCNA, an auxiliary factor that greatly enhances DNA polymerase δ processivity on primed DNA templates. Here, we provide evidence that a highly conserved region located between amino acids 384 and 399 in the C-terminus of p66 is phosphorylated, most probably by Protein kinase CK2, and that another region, most probably located within the PCNA interacting domain in its extreme C-terminus, regulates its interaction with PCNA. Phosphorylation of p66 is associated with its co-localization with large subunit of DNA polymerase δ, p125, and PCNA, to the insoluble chromatin fraction at the beginning of S-phase. Taken together, the results provide evidence that concurrent phosphorylation events in p66 may positively and negatively regulate its activity and interactions with other components of the replisome during the cell cycle.     

Protein-protein interactions as a proxy to monitor conformational changes and activation states of the tomato resistance protein I-2

Plant resistance proteins (R) are involved in pathogen recognition and subsequent initiation of defence responses. Their activity is regulated by inter- and intramolecular interactions. In a yeast two-hybrid screen two clones (I2I-1 and I2I-2) specifically interacting with I-2, a Fusarium oxysporum f. sp. lycopersici resistance protein of the CC-NB-LRR family, were identified. Sequence analysis revealed that I2I-1 belongs to the Formin gene family (SlFormin) whereas I2I-2 has homology to translin-associated protein X (SlTrax). SlFormin required only the N-terminal CC I-2 domain for binding, whereas SlTrax required both I-2 CC and part of the NB-ARC domain. Tomato plants stably silenced for these interactors were not compromised in I-2-mediated disease resistance. When extended or mutated forms of I-2 were used as baits, distinct and often opposite, interaction patterns with the two interactors were observed. These interaction patterns correlated with the proposed activation state of I-2 implying that active and inactive R proteins adopt distinct conformations. It is concluded that the yeast two hybrid system can be used as a proxy to monitor these different conformational states.     

Time‐resolved dissection of the molecular crosstalk driving Fusarium head blight in wheat provides new insights into host susceptibility determinism

Fungal plant diseases are controlled by a complex molecular dialogue that involves pathogen effectors able to manipulate plant susceptibility factors at the earliest stages of the interaction. By probing the wheat–Fusarium graminearum pathosystem, we profiled the coregulations of the fungal and plant proteins shaping the molecular responses of a 96‐hr‐long infection's dynamics. Although no symptoms were yet detectable, fungal biomass swiftly increased along with an extremely diverse set of secreted proteins and candidate effectors supposed to target key plant organelles. Some showed to be early accumulated during the interaction or already present in spores, otherwise stored in germinating spores and detectable in an in vitro F. graminearum exudate. Wheat responses were swiftly set up and were evidenced before any visible symptom. Significant wheat protein abundance changes co‐occurred along with the accumulation of putative secreted fungal proteins and predicted effectors. Regulated wheat proteins were closely connected to basal cellular processes occurring during spikelet ontogeny, and particular coregulation patterns were evidenced between chloroplast proteins and fungal proteins harbouring a predicted chloroplast transit peptide. The described plant and fungal coordinated responses provide a resourceful set of data and expand our understanding of the wheat–F. graminearum interaction.     

The tomato R gene products I-2 and MI-1 are functional ATP binding proteins with ATPase activity

Most plant disease resistance (R) genes known today encode proteins with a central nucleotide binding site (NBS) and a C-terminal Leu-rich repeat (LRR) domain. The NBS contains three ATP/GTP binding motifs known as the kinase-1a or P-loop, kinase-2, and kinase-3a motifs. In this article, we show that the NBS of R proteins forms a functional nucleotide binding pocket. The N-terminal halves of two tomato R proteins, I-2 conferring resistance to Fusarium oxysporum and Mi-1 conferring resistance to root-knot nematodes and potato aphids, were produced as glutathione S-transferase fusions in Escherichia coli. In a filter binding assay, purified I-2 was found to bind ATP rather than other nucleoside triphosphates. ATP binding appeared to be fully dependent on the presence of a divalent cation. A mutant I-2 protein containing a mutation in the P-loop showed a strongly reduced ATP binding capacity. Thin layer chromatography revealed that both I-2 and Mi-1 exerted ATPase activity. Based on the strong conservation of NBS domains in R proteins of the NBS-LRR class, we propose that they all are capable of binding and hydrolyzing ATP.     

‘Good-genes’ and ‘compatible-genes’ effects in an Alpine whitefish and the information content of breeding tubercles over the course of the spawning season

Some models of sexual selection predict that individuals vary in their genetic quality and reveal some of this variation in their secondary sexual characteristics. Alpine whitefish (Coregonus sp.) develop breeding tubercles shortly before their spawning season. These tubercles are epidermal structures that are distributed regularly along the body sides of both males and females. There is still much unexplained variation in the size of breeding tubercles within both sexes and with much overlap between the sexes. It has been suggested that breeding tubercles function to maintain body contact between the mating partners during spawning, act as weapons for defence of spawning territories, or are sexual signals that reveal aspects of genetic quality. We took two samples of whitefish from their spawning place, one at the beginning and one around the peak of spawning season. We found that females have on average smaller breeding tubercles than males, and that tubercle size partly reveals the stage of gonad maturation. Two independent full-factorial breeding experiments revealed that embryo mortality was significantly influenced by male and female effects. This finding demonstrates that the males differed in their genetic quality (because offspring get nothing but genes from their fathers). Tubercle size was negatively linked to some aspects of embryo mortality in the first breeding experiment but not significantly so in the second. This lack of consistency adds to inconsistent results that were reported before and suggests that (i) some aspects of genetic quality are not revealed in breeding tubercles while others are, or (ii) individuals vary in their signaling strategies and the information content of breeding tubercles is not always reliable. Moreover, the fact that female whitefish have breeding tubercles of significant size while males seem to have few reasons to be choosy suggests that the tubercles might also serve some functions that are not linked to sexual signaling.