Tomato TFT1 Is Required for PAMP-Triggered Immunity and Mutations that Prevent T3S Effector XopN from Binding to TFT1 Attenuate Xanthomonas Virulence

XopN is a type III effector protein from Xanthomonas campestris pathovar vesicatoria that suppresses PAMP-triggered immunity (PTI) in tomato. Previous work reported that XopN interacts with the tomato 14-3-3 isoform TFT1; however, TFT1''s role in PTI and/or XopN virulence was not determined. Here we show that TFT1 functions in PTI and is a XopN virulence target. Virus-induced gene silencing of TFT1 mRNA in tomato leaves resulted in increased growth of Xcv ΔxopN and Xcv ΔhrpF demonstrating that TFT1 is required to inhibit Xcv multiplication. TFT1 expression was required for Xcv-induced accumulation of PTI5, GRAS4, WRKY28, and LRR22 mRNAs, four PTI marker genes in tomato. Deletion analysis revealed that the XopN C-terminal domain (amino acids 344–733) is sufficient to bind TFT1. Removal of amino acids 605–733 disrupts XopN binding to TFT1 in plant extracts and inhibits XopN-dependent virulence in tomato, demonstrating that these residues are necessary for the XopN/TFT1 interaction. Phos-tag gel analysis and mass spectrometry showed that XopN is phosphorylated in plant extracts at serine 688 in a putative 14-3-3 recognition motif. Mutation of S688 reduced XopN''s phosphorylation state but was not sufficient to inhibit binding to TFT1 or reduce XopN virulence. Mutation of S688 and two leucines (L64,L65) in XopN, however, eliminated XopN binding to TFT1 in plant extracts and XopN virulence. L64 and L65 are required for XopN to bind TARK1, a tomato atypical receptor kinase required for PTI. This suggested that TFT1 binding to XopN''s C-terminal domain might be stabilized via TARK1/XopN interaction. Pull-down and BiFC analyses show that XopN promotes TARK1/TFT1 complex formation in vitro and in planta by functioning as a molecular scaffold. This is the first report showing that a type III effector targets a host 14-3-3 involved in PTI to promote bacterial pathogenesis.     

Phylogeny and systematics of the bee genus Osmia (Hymenoptera: Megachilidae) with emphasis on North American Melanosmia: subgenera,synonymies and nesting biology revisited

The predominantly Holarctic bee genus Osmia Panzer is species‐rich and behaviourally diverse. A robust phylogeny of this genus is important for understanding the evolution of the immense variety of morphological and behavioural traits exhibited by this group. We infer a phylogeny of Osmia using DNA sequence data obtained from three nuclear genes (elongation factor 1‐α, LW‐rhodopsin and CAD) and the mitochondrial gene COI. Our taxon sampling places special attention on North American members of the subgenus Melanosmia Schmiedeknecht; we discuss the novel placement of a number of species traditionally assigned to O. (Melanosmia) and examine the relative support for alternative classifications of this species‐rich subgenus. We use this new phylogeny to guide a reassessment of morphological and behavioural characters within Osmia. Our results provide support for the recognition of Osmia (Hapsidosmia), subgen.n ., a monotypic subgenus containing Osmia iridis Cockerell & Titus. We synonymize Osmia (Mystacosmia) Snelling under O. (Melanosmia), syn.n . We synonymize Osmia (Acanthosmioides) Ashmead under O. (Melanosmia), syn.n ., propose ‘odontogaster species group’ as a replacement for the subgeneric name Acanthosmioides, and refine the morphological characters that serve to diagnose the species group. We additionally propose ‘nigrifrons species group’ for a clade within O. (Melanosmia) containing most species formerly placed in Osmia (Centrosmia) Robertson. We demonstrate more cohesive patterns of nest substrate use in the nigrifrons and odontogaster species groups than was previously believed to occur, reconsider character polarity of aspects of the female mandible, and show that a large number of morphological characters have evolved convergently within the genus. In order to facilitate discussion of relevant taxa, we propose the following 15 new synonymies: O. bakeri Sandhouse under O. melanopleura Cockerell; O. crenulaticornis Michener under O. pinorum Cockerell; O. claremontensis Michener under O. sedula Sandhouse; O. cockerelli Sandhouse under O. dakotensis Michener; O. francisconis White under O. enixa Sandhouse; O. hurdi White under O. austromaritima Michener; O. sladeni Sandhouse under O. nifoata Cockerell; O. titusi Cockerell under O. phenax Cockerell; O. subtrevoris Cockerell, O. physariae Cockerell, and O. erecta Michener under O. giliarum Cockerell; and O. universitatis Cockerell, O. integrella Cockerell, O. amala Cockerell, and O. metitia Cockerell under O. nigrifrons Cresson, syn.n . We remove O. wyomingensis Michener from synonymy with O. nifoata Cockerell, stat.n ., and O. pinorum Cockerell from synonymy with O. physariae Cockerell, stat.n . This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:A3E7D63B‐5C4C‐4ACF‐BF33‐48E5C5DD1B0D .     

Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result from simultaneous open/shut events of two or more channel units.     

Electrical and adaptive properties of rod photoreceptors in bufo marinus. I. Effects of altered extracellular Ca(2+) levels

The effects of altering extracellular Ca(2+) levels on the electrical and adaptive properties of toad rods have been examined. The retina was continually superfused in control (1.6 mM Ca(2+)) or test ringer’s solutions, and rod electrical activity was recorded intracellularly. Low-calcium ringer’s (10(-9)M Ca(2+)) superfused for up to 6 min caused a substantial depolarization of the resting membrane potential, an increase in light-evoked response amplitudes, and a change in the waveform of the light-evoked responses. High Ca(2+) ringer’s (3.2 mM) hyperpolarized the cell membrane and decreased response amplitudes. However, under conditions of either low or high Ca(2+) superfusion for up to 6 min, in both dark-adapted and partially light-adapted states, receptor sensitivity was virtually unaffected; i.e., the V-log I curve for the receptor potential was always located on the intensity scale at a position predicted by the prevailing light level, not by Ca(2+) concentration. Thus, we speculate that cytosol Ca(2+) concentration is capable of regulating membrane potential levels and light-evoked response amplitudes, but not the major component of rod sensitivity. Low Ca(2+) ringer’s also shortened the period of receptor response saturation after a bright but nonbleaching light flash, hence accelerating the onset of both membrane potential and sensitivity recovery during dark adaptation.

Exposure of the retina to low Ca(2+) (10(-9)M) ringer’s for long periods (7-15 min) caused dark-adapted rods to lose responsiveness. Response amplitudes gradually decreased, and the rods became desensitized. These severe conditions of low Ca(2+) caused changes in the dark-adapted rod that mimic those observed in rods during light adaptation. We suggest that loss of receptor sensitivity during prolonged exposure to low Ca(2+) ringer’s results from a decrease of intracellular (intradisk) stores of Ca(2+); i.e., less Ca(2+) is thereby released per quantum catch.

     

Construction of a cyclin D1-Cdk2 fusion protein to model the biological functions of cyclin D1-Cdk2 complexes

Cyclin D1 is frequently overexpressed in human breast cancers, and cyclin D1 overexpression correlates with poor prognosis. Cyclin D1-Cdk2 complexes were previously observed in human breast cancer cell lines, but their role in cell cycle regulation and transformation was not investigated. This report demonstrates that Cdk2 in cyclin D1-Cdk2 complexes from mammary epithelial cells is phosphorylated on the activating phosphorylation site, Thr(160). Furthermore, cyclin D1-Cdk2 complexes catalyze Rb phosphorylation on multiple sites in vitro. As a model to investigate the biological and biochemical functions of cyclin D1-Cdk2 complexes, and the mechanisms by which cyclin D1 activates Cdk2, a cyclin D1-Cdk2 fusion gene was constructed. The cyclin D1-Cdk2 fusion protein expressed in epithelial cells was phosphorylated on Thr(160) and catalyzed the phosphorylation of Rb on multiple sites in vitro and in vivo. Kinase activity was not observed if either the cyclin D1 or Cdk2 domain was mutationally inactivated. Mutational inactivation of the cyclin D1 domain prevented activating phosphorylation of the Cdk2 domain on Thr(160). These results indicate that the cyclin D1 domain of the fusion protein activated the Cdk2 domain through an intramolecular mechanism. Cells stably expressing the cyclin D1-Cdk2 fusion protein exhibited several hallmarks of transformation including hyperphosphorylation of Rb, resistance to TGFbeta-induced growth arrest, and anchorage-independent proliferation in soft agar. We propose that cyclin D1-Cdk2 complexes mediate some of the transforming effects of cyclin D1 and demonstrate that the cyclin D1-Cdk2 fusion protein is a useful model to investigate the biological functions of cyclin D1-Cdk2 complexes.     

Antibodies to Aeromonas salmonicida Lipopolysaccharide and cell wall antigens in rabbit and salmon immune sera

Atlantic salmon (Salmo salar L.) immunised with A-layer positive or A-layer negative strains ofAeromonas salmonicida did not produce antibodies reactive with proteinase K-digested LPS in the low molecular weight area corresponding to the core-region of LPS. The salmon produced antibody titres as high as those produced by rabbit when assayed against whole bacteria or LPS in ELISA. The salmon antibodies against the A-layer positive strain of A. salmonicida lysed rabbit erythrocytes sensitised with LPS from the A-layer positive strain of A. salmonicida. This was in contrast to the non-haemolytic activity of the salmon antibodies against the A-layer negative strain, indicating differences in epitopes between the two strains.     

Emergence of Complex Haplotypes from Microevolutionary Variation in Sequence and Structure of <Emphasis Type="Italic">Colias</Emphasis> Phosphoglucose Isomerase

A molecular evolutionary explanation of natural genetic variation requires analysis of specific variants’ evolutionary dynamics. To pursue this for phosphoglucose isomerase (PGI) of Colias butterflies, whose polymorphism is maintained by strong natural selection, we assembled a large data set of wild haplotypes, highly variable at the amino acid and DNA levels. The most common electrophoretic, i.e., charge macrostate, allele class, 3, is conserved in its pattern of charged amino acid residues. The next most common macrostate, 4, has multiple patterns of charge, i.e., microstates, while less common (1, 2, 5, 6) macrostates are very diverse. Macrostate 4 shows significant linkage disequilibrium (LD) among its variants, especially for two groups of five haplotypes each. We find extensive intragenic recombination among all haplotypes except the two high-LD groups of macrostate 4, which display none. Phyletic relations among haplotypes are largely reticulate, again except for the high-LD groups of macrostate 4, which form clades with strong bootstrap support. Charge-changing and linked charge-neutral amino acid variants occur in diverse parts of PGI’s sequence. Homology-based modeling of PGI’s structure shows that these regions are related spatially in ways suggesting functional interaction. The high-LD groups of macrostate 4 display parallel amino acid variation in these regions. This pattern of haplotype clades with high LD among multiple varying sites, emerging from chaotically recombining variation, may be a “signature” of refinement of complex adaptive sequences by recombination and selection. It should be tested further in this study system and others as a possibly general feature of the evolution of living complexity.     

Homology modeling, simulation and molecular docking studies of catechol-2, 3-Dioxygenase from Burkholderia cepacia: Involved in degradation of Petroleum hydrocarbons

Catechol 2, 3-dioxygenase is present in several types of bacteria and undergoes degradation of environmental pollutants through an important key biochemical pathways. Specifically, this enzyme cleaves aromatic rings of several environmental pollutants such as toluene, xylene, naphthalene and biphenyl derivatives. Hence, the importance of Catechol 2, 3-dioxygenase and its role in the degradation of environmental pollutants made us to predict the three-dimensional structure of Catechol 2, 3-dioxygenase from Burkholderia cepacia. The 10ns molecular dynamics simulation was carried out to check the stability of the modeled Catechol 2, 3- dioxygenase. The results show that the model was energetically stable, and it attains their equilibrium within 2000 ps of production MD run. The docking of various petroleum hydrocarbons into the Catechol 2,3-dioxygenase reveals that the benzene, O-xylene, Toluene, Fluorene, Naphthalene, Carbazol, Pyrene, Dibenzothiophene, Anthracene, Phenanthrene, Biphenyl makes strong hydrogen bond and Van der waals interaction with the active site residues of H150, L152, W198, H206, H220, H252, I254, T255, Y261, E271, L276 and F309. Free energy of binding and estimated inhibition constant of these compounds demonstrates that they are energetically stable in their binding cavity. Chrysene shows positive energy of binding in the active site atom of Fe. Except Pyrene all the substrates made close contact with Fe atom by the distance ranges from 1.67 to 2.43 Å. In addition to that, the above mentioned substrate except pyrene all other made π-π stacking interaction with H252 by the distance ranges from 3.40 to 3.90 Å. All these docking results reveal that, except Chrysene all other substrate has good free energy of binding to hold enough in the active site and makes strong VdW interaction with Catechol-2,3-dioxygenase. These results suggest that, the enzyme is capable of catalyzing the above-mentioned substrate.     

Asymmetric Cell Division: A Persistent Issue?

Heterogeneity within a clonal population of cells can increase survival in the face of environmental stress. In a recent issue of Science, Aldridge et al. (2012) demonstrate that cell division in mycobacteria is asymmetric, producing daughter cells that differ in size, growth rate, and susceptibility to antibiotics.     

藏药七十味珍珠丸改善APP/PS1小鼠学习记忆能力

藏药七十味珍珠丸（ratanasampil,RNSP）可改善大脑氧化应激水平,改善大脑功能,有安神和促进学习记忆的功效,然而RNSP是否可改善阿尔茨海默症（AD）小鼠的学习记忆功能,尚缺乏系统研究。本研究采用APP/PS 1转基因小鼠为研究对象,并随机将其分为实验组和对照组。对实验组进行为期12周的RNSP灌胃给药,对照组进行12周的蒸馏水灌胃,采用Morris水迷宫与开场实验评价小鼠学习记忆能力,比较小鼠体重与相关器官质量,并比较器官质量指数,通过分子生物学检测指标评价小鼠脑内老年斑数量,Aβ生成量及BACE1表达水平。本研究证实,与对照组相比,给药组小鼠定位航行潜伏期明显缩短（22.60±13.26 vs. 46.44±8.41, P<0.01, day 5）,穿越平台次数明显增加（1.29±0.37 vs. 0.54±0.29, P<0.01）,探洞次数明显增加（32.11±9.85 vs. 20.89±8.78, P<0.05）,表明RNSP提高了APP/PS 1小鼠的学习记忆能力和空间探索能力。与对照组相比,给药组小鼠大脑重量及脑质量指数均增高（0.4135±0.0102 vs. 0.3833±0.0254, P<0.05;2.04±0.08 vs. 1.84±0.15, P<0.05）,脑内老年斑数量减少（18.70±7.88 vs. 38.83±6.15, P<0.05）,Aβ1- 42水平及BACE1表达均显著降低（0.19±0.08 vs. 0.41±0.12, P<0.05; 0.136±0.04 vs. 0.206±0.02, P<0.05）,表明RNSP延缓了APP/PS 1小鼠的脑萎缩进程,降低脑内老年斑的形成,下调脑内Aβ1-42水平和BACE1裂解酶的蛋白质表达量。本研究提示,RNSP可改善APP/PS 1小鼠的学习记忆能力,其机制可能和RNSP抑制脑萎缩,降低BACE1蛋白表达以及减少脑内Aβ沉积有关。