HopW1 from <i>Pseudomonas syringae</i> Disrupts the Actin Cytoskeleton to Promote Virulence in Arabidopsis

A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis.</p

MiR-9 Controls Chemotactic Activity of Cord Blood CD34⁺ Cells by Repressing CXCR4 Expression

Improved approaches for promoting umbilical cord blood (CB) hematopoietic stem cell (HSC) homing are clinically important to enhance engraftment of CB-HSCs. Clinical transplantation of CB-HSCs is used to treat a wide range of disorders. However, an improved understanding of HSC chemotaxis is needed for facilitation of the engraftment process. We found that ectopic overexpression of miR-9 and antisense-miR-9 respectively down- and up-regulated C-X-C chemokine receptor type 4 (CXCR4) expression in CB-CD34＋ cells as well as in 293T and TF-1 cell lines. Since CXCR4 is a specific receptor for the stromal cell derived factor-1 (SDF-1) chemotactic factor, we investigated whether sense miR-9 and antisense miR-9 influenced CXCR4-mediated chemotactic mobility of primary CB CD34＋ cells and TF-1 cells. Ectopic overexpression of sense miR-9 and antisense miR-9 respectively down- and up-regulated SDF-1-mediated chemotactic cell mobility. To our knowledge, this study is the first to report that miR-9 may play a role in regulating CXCR4 expression and SDF-1-mediated chemotactic activity of CB CD34＋ cells

Slow increase in cellular osmolality in the <i>gltI</i> mutant.

<p>The cellular osmolality levels in the wild-type strain (BGR1), the <i>gltI</i> mutant (BGLT1), and <i>gltI</i> mutant complementation with pGLT1 in LB medium. The levels of cellular osmolality were measured from <i>B</i>. <i>glumae</i> strains cultured for 6, 12, 24, and 36 h. All samples were normalized to weight of cells. Error bars indicate the SE ranges of three independent experiments. The asterisks (*) indicate a significant difference (p < 0.05) in osmolality among the <i>B</i>. <i>glumae</i> strains as determined by ANOVA/Tukey’s correction for multiple comparisons.</p

Effect of the addition of glycine betaine on the growth of the <i>gltI</i> mutant.

<p>Growth of the <i>B</i>. <i>glumae</i> wild-type strain BGR1, the <i>gltI</i> mutant (BGLT1), and <i>gltI</i> mutant complementation with pGLT1 [BGRT1(pBGLT1)] with various concentration of glycine betaine as a compatible solute in LB media. Error bars indicate the SE ranges of three independent experiments.</p

Glutamate uptake is important for osmoregulation and survival in the rice pathogen <i>Burkholderia glumae</i>

<div><p>Bacteria exhibit an optimal growth rate in culture media with sufficient nutrients at an optimal temperature and pH. In addition, the concentration of solutes plays a critical role in bacterial growth and survival. Glutamate is known to be a major anionic solute involved in osmoregulation and the bacterial cell’s response to changes in solute concentration. To determine how glutamate uptake is involved in osmoregulation in the rice bacterial pathogen <i>Burkholderia glumae</i> BGR1, we mutated the <i>gltI</i> gene encoding a periplasmic substrate binding protein of a glutamate transport system to abolish glutamate uptake, and monitored the growth of the <i>gltI</i> null mutant in Luria-Bertani medium. We found that the <i>gltI</i> null mutant showed a slower growth rate than the wild-type strain and experienced hyperosmotic stress resulting in water loss from the cytoplasm in stationary phase. When the incubation time was extended, the mutant population collapsed due to the hyperosmotic stress. The <i>gltI</i> null mutant exhibited loss of adaptability under both hypoosmotic and hyperosmotic stresses. The growth rate of the <i>gltI</i> null mutant was restored to the level of wild-type growth by exogenous addition of glycine betaine to the culture medium, indicating that glycine betaine is a compatible solute in <i>B</i>. <i>glumae</i>. These results indicate that glutamate uptake from the environment plays a key role in osmoregulation in <i>B</i>. <i>glumae</i>.</p></div

The loss of adaptability in the <i>gltI</i> mutant under hypoosmotic and hyperosmotic conditions.

<p>The <i>B</i>. <i>glumae</i> wild-type strain BGR1, the <i>gltI</i> mutant (BGLT1), and the <i>gltI</i> mutant complementation with pGLT1 were grown in LB medium with various concentrations of NaCl (0–5%). Error bars indicate the SE ranges of three independent experiments.</p

Genetic organization of the <i>gltI</i> gene in <i>B</i>. <i>glumae</i>.

<p>Lanes represent the restriction map of pGLT1 plasmid DNA. Colored arrows below the restriction map represent the schematic organization of the <i>gltI</i> locus. Vertical bars on the map indicate the positions of Tn<i>5</i> insertions. Genetic information and gene identities were obtained from the <i>B</i>. <i>glumae</i> BGR1 genome database (GenBank accession numbers: CP001503–CP001508). The restriction enzyme sites are indicated as follows: E, <i>Eco</i>RI; B, <i>Bam</i>HI.</p

Transmission electron microscopy (TEM) examination of the <i>gltI</i> mutant under hyperosmotic stress.

<p>TEM ultrathin section micrographs of <i>B</i>. <i>glumae</i> wild-type strain BGR1, the <i>gltI</i> mutant BGLT1, and the <i>gltI</i> mutant complementation with pGLT1 [BGRT1(BGLT1)] with exogenous addition of glycine betaine for 36, 48, and 60 h. All micrographs were picked from at least 100 pictures showing similar results. Scale bars indicate 0.2 μm.</p

Low cellular glutamate levels in the <i>gltI</i> mutant during growth.

<p>The intracellular amounts of glutamate in the <i>B</i>. <i>glumae</i> wild-type strain, <i>gltI</i> mutant, and <i>gltI</i> mutant complementation during growth. Cells of <i>B</i>. <i>glumae</i> strains were harvested at each sampling time (6, 12, 24, and 36 h). Glutamate pool sizes were assessed by integration of peaks generated by high performance liquid chromatography with fluorescence detection (HPLC-FLD) and normalized to colony forming units (CFU). Error bars indicate the SE ranges of three independent experiments. The asterisks (*) represent a significant difference (p < 0.05) in glutamate pools between the <i>gltI</i> mutant and the wild-type strain or the complement as determined by analysis of variance (ANOVA)/Tukey’s correction for multiple comparisons.</p

Growth of the <i>Burkholderia glumae gltI</i> null mutant in Luria-Bertani (LB) medium.

<p>Growth of the <i>B</i>. <i>glumae</i> wild-type strain BGR1, the <i>gltI</i> mutant (BGLT1), and the <i>gltI</i> mutant complementation with pGLT1 in LB medium. Error bars indicate the standard error (SE) ranges of three independent experiments.</p