<i>In planta</i> survival of <i>Pseudomonas syringae</i> pv. <i>actinidiae</i> strains in <i>Actinidia deliciosa</i> cv Hayward leaf.

<p>Histogram reporting <i>in planta</i> survival of <i>Psa</i> strains: The average bacterial count (log cfu/ml) of three independent experiments is reported with standard deviations for 3^rd and 7^th day after bacterial inoculation (1–2×10⁶ cfu/ml) in <i>Actinidia deliciosa</i> cv. Hayward leaf. Statistical significance with respect to <i>Psa</i> wild type is indicated with one asterisk (<i>P</i><0.01).</p

Expression and regulation of <i>psa-pip.</i>

<p>Expression of <i>psa-pip</i> was assessed in presence and absence of kiwi leaf extract for wild type (WT) and <i>psaR2</i> mutant (<i>Psa-</i>mR2). Statistical analyses (Student’s <i>t</i> test) were performed to compare the significant difference in promoter analysis between wild type <i>Psa</i> strain and <i>Psa</i>-mR2 mutant in the presence and absence of kiwi extract. a, significant difference to b at <i>P</i><0.01 and significant difference to c at <i>P</i><0.05. c, not significant difference to c at <i>P</i><0.05 but significant difference to a and b at <i>P</i><0.05.</p

Lipase secretion score of <i>Pseudomonas syringae</i> pv. <i>actinidiae</i> strains.

<p>Mean values and standard deviations were calculated for halo obtained from three replications of lipase secretion in LB Agar-tributyrin plates. Statistical analyses (Student’s <i>t</i> test) were performed to compare the significant difference in lipase secretion between wild type <i>Psa</i> strain and mutated and complemented strains. a, significant difference to WT at <i>P</i><0.05. b, significant difference to ‘a’ at <i>P</i><0.01.</p

Structure-based multiple sequence alignment of the regulatory domains of the three <i>Psa</i> solos with QS LuxRs and with the prototype of the PAB LuxR solos subfamily.

<p>The residues belonging to <b>Cluster 1,</b> to <b>Cluster 2</b> and <b>Cluster 3</b> are highlighted in green, cyan and in orange, respectively. The 3D architecture of the boundaries of the ligand-binding site is schematized by <b><i>r</i></b> (roof), <b><i>f</i></b> (floor), <b><i>p</i></b> (proximal wall) and <b><i>d</i></b> (distal wall) and its tripartite topology by <b><i>c</i></b> (conserved core), <b><i>s</i></b> (specificity patch) and <b><i>v</i></b> (variable patch).</p

The Kiwifruit Emerging Pathogen <i>Pseudomonas syringae</i> pv. <i>actinidiae</i> Does Not Produce AHLs but Possesses Three LuxR Solos

<div><p><i>Pseudomonas syringae</i> pv. <i>actinidiae</i> (<i>Psa</i>) is an emerging phytopathogen causing bacterial canker disease in kiwifruit plants worldwide. Quorum sensing (QS) gene regulation plays important roles in many different bacterial plant pathogens. In this study we analyzed the presence and possible role of <i>N</i>-acyl homoserine lactone (AHL) quorum sensing in <i>Psa</i>. It was established that <i>Psa</i> does not produce AHLs and that a typical complete LuxI/R QS system is absent in <i>Psa</i> strains. <i>Psa</i> however possesses three putative <i>luxR</i> solos designated here as PsaR1, PsaR2 and PsaR3. PsaR2 belongs to the sub-family of LuxR solos present in many plant associated bacteria (PAB) that binds and responds to yet unknown plant signal molecules. PsaR1 and PsaR3 are highly similar to LuxRs which bind AHLs and are part of the canonical LuxI/R AHL QS systems. Mutation in all the three <i>luxR</i> solos of <i>Psa</i> showed reduction of <i>in planta</i> survival and also showed additive effect if more than one solo was inactivated in double mutants. Gene promoter analysis revealed that the three solos are not auto-regulated and investigated their possible role in several bacterial phenotypes.</p></div

Swarming and swimming movement score of <i>Pseudomonas syringae</i> pv. <i>actinidiae</i> strains.

<p>Mean values and standard deviations were calculated for swarming and swimming bacterial movement obtained from three replications each on 0.8%, 0.6% and 0.3% KBA. Statistical analyses (Student’s <i>t</i> test) were performed to compare the significant difference in bacterial movement between wild type <i>Psa</i> strain and mutated and complemented strains. a, significant difference to WT at <i>P</i><0.0001.</p

Multiple sequence alignment of the regulatory domain of PsaR2 with the PAB LuxR solos subfamily.

<p>The residues belonging to <b>Cluster 1, Cluster 2</b> and <b>Cluster 3</b> are highlighted in green, cyan and in orange, respectively.</p

<i>In vivo</i> analgesic activity of parental mAb αD11 in inflammatory and neuropathic pain models.

<p>(a) Analgesic effects of αD11 antibody administration on the late phase (15–40 min) in the course of the formalin test. Treatment consisted in saline (negative control) or antibody injection (single doses: 12.5 µg of mock mouse mAb or two different molecular formats of αD11, <i>i.e.</i> mAb or Fab) performed (in the same paw as for formalin) 45 min before formalin injection and testing. Statistical analysis was performed on each phase (ANOVA and Fisher's Test for comparison of each couple of groups). Each experimental group included at least 8 animals. (b) Analgesic effects of αD11 antibody in the short lasting protocol of neuropathic pain model: mAb αD11 significantly increased the value of ipsilateral/contralateral index (ratio between the threshold forces measured for the two hind paws, the one ipsilateral to surgery and the contralateral one. Mean value ± s.e.), starting from day 4 to day 14, one week after the last antibody injection. Control mice were injected with either mock mouse IgG, (1.4 mg/Kg) or saline solution (sal). ANOVA test for repeated measures resulted in statistical significance for treatment (p<0.0001), time (p<0.0001) and the interaction between the two factors (treatment×time) (p<0.0001). (c) Analgesic efficacy of mAb αD11 (one dose: 2 mg/kg) in the long lasting protocol of neuropathic pain model. MAb αD11 increased the ipsilateral/contralateral index, starting either from day 5. The analgesic effect, which disappeared around days 17–19, increases again to reach a plateau between day 27 and day 31, identifying a late phase in the action of mAb αD11 (long-term effect). ANOVA test for repeated measures resulted in statistical significance for treatment (p<0.005), time (p<0.005) and the interaction between two factors (treatment×time) (p<0.005).</p

SPR analysis.

<p>Summary of the derived kinetic and equilibrium binding constants of parental IgG αD11 and Fab hum-αD11 towards hNGF.</p

Hum-αD11 retains the biological activity of the parental mAb αD11 <i>in vitro</i>.

<p>(a–d) mNGF induced differentiation of PC-12 cells. Neurite outgrowth inhibition assay: photomicrographs of PC-12 cells, treated with (a) mNGF 100 ng/ml alone or preincubated (b) with mAb αD11 (5 µg/ml) or (c) with IgG1 hum-αD11 (5 µg/ml), concentrated from stable cotransfected CHO cells supernatants. (d) Negative control: untreated PC-12 cells. (e) TrkA phosphorylation inhibition assay in 3T3 TrkA cells. 3T3 TrkA cells were incubated with the indicated combinations of mNGF, parental mAb αD11, IgG1 hum-αD11 (purified by Protein A-Sepharose) and the irrelevant mAb SV5 as a negative control. Cell lysates were separated on a 10% SDS gel and phosphorylated TrkA detected using anti-phospho-Y490 TrkA Ab. The ubiquitous band of tubulin served as gel loading control.</p