Type I IFN production by <i>Lm</i>-infected DCs.

<p>(A) D1 cells were infected with <i>Lm</i> at an MOI of 40. Supernatants were collected at different time points and IFNβ and IFNα levels were quantified by ELISA. (B) D1 cells were infected with <i>Lm</i> at an MOI of 40. RNA was extracted at different time points and used for qRT-PCR analysis. The fold-increases, relative to β-actin, for IFNα4, IFNα9, IFNα5, IFNα2 are shown. (C) D1 cells were infected with <i>Lm</i>, and the RNA was extracted and analyzed by RT-PCR. IFNα6 and IFNα1 mRNA levels are shown. Data shown are representative of at least three independent experiments.</p

IFNγ production by NK cells cultured with BMDCs stimulated with <i>Lm</i> or <i>E. coli</i>.

<p>BMDCs were infected with <i>Lm</i> or <i>E. coli</i> at an MOI of 20 and cultured with syngeneic NK cells for 18 hr. Where indicated, recombinant IFNβ was added to the co-culture one hour after infection. Levels of IFNγ in the culture supernatants were quantified by ELISA. (A) Experimental design. (B) Co-culture experiments. The means ± SDs of three independent experiments are shown. p value <0.01.</p

Analysis of VOCs in the headspace of treated <i>G. biloba</i> leaves.

<p>Data are expressed as micrograms of VOCs per gram of leaf fresh weight (±SEM). Retention times (RT) and Kováts Index (KI) are indicated for each compound. For the same time point, boldface HW values indicate significant (P<0.05) differences between MD and HW. HW, herbivore wounding; MD, mechanical damage.</p

Time-course quantitative gene expression of some ROS scavenging genes in <i>G. biloba</i> upon herbivory.

<p>Gene expression of superoxide dismutase (SOD) and catalase (CAT) was up-regulated by herbivory at all times. Upon herbivory, peroxidase (POX) was significantly down-regulated after 4 h, whereas ascorbate peroxidase (APX) was down-regualted after 30 min. The dotted lines represent control values (mechanical damage), different letters indicate significant (P<0.05) differences, asterisk indicates significant (P<0.05) differences with respect to control.</p

Time-course quantitative gene expression of some <i>G. biloba</i> genes involved in phenylpropanoid and terpenoid metabolism upon herbivory.

<p>Phenylalanine ammonia lyase (<i>PAL</i>) and anthocyanidin reductase (<i>ANR</i>) were significantly up regulated by herbivory only after 4 h, whereas flavonol synthase (<i>FLS</i>) was down-regulated at 30 min and 4 h. Chalcone synthase (<i>CHS</i>) showed a constant up-regulation, whereas flavanone 3-hydroxylase (F3H) showed an increased up regulation after 4 h. Farnesyl diphosphate synthase (<i>FPPS</i>) was significantly upregulated only after 30 min whereas geranylgeranyl diphosphate synthase (<i>GGPP</i>) showed no regulation at all times. Levopimaradiene synthase (<i>PPS</i>) was significantly down-regulated at all times. The dotted lines represent control values (mechanical damage), different letters indicate significant (P<0.05) differences, asterisks indicate significant (P<0.05) differences with respect to control.</p

<i>In vivo</i> IFNβ production during <i>Lm</i> infection.

<p>Mice (n = 5/group) were injected with 1×10⁶ CFU of <i>Lm</i> and <i>E. coli</i>. RNA was extracted at 4 hr, 8 hr and 24 hr p.i. and the IFNβ mRNA was quantified. (A) IFNβ gene expression from total spleen at the time points indicated. (B) IFNβ gene expression in CD11c⁺ and CD11c⁻ cells purified from total spleen. (C) IFNβ gene expression in total spleen from mice infected with <i>E. coli</i> (1×10⁶ CFU) as a positive control. The housekeeping gene <i>PPIA</i> was used as a reference to normalize data. Data shown are representative of at least three independent experiments.</p

Induction of type I IFN genes.

<p>Samples derived from two biological replicates per time point were used for subsequent GeneChip probe arrays. (A) Heat map of the time course of IRG expression in DCs after exposure to <i>Lm</i>. (B) Heat map of the time courses of IFNβ and IFNα gene expression. RNAs were collected at different time points and the gene expression levels were measured by microarray analysis. Heat maps were generated with the GeneSpring hierarchical clustering algorithm. Data show mean fold-changes normalized to the pre-exposure 0 hr time point.</p

NK cell activation in the spleen of mice infected with <i>Lm</i>.

<p>Mice (n = 5/group) were infected with <i>Lm</i> (1×10⁶ CFU)± IFNβ. Spleens were removed five hours after infection and NK cell activation was evaluated. (A) Intracellular staining for IFNγ in DX5-positive cells. (B) Splenocytes were co-cultured with YAC-1 cells and the percentage of target cell lysis was determined after three hours; p-value <0.01. The mean of three independent experiments is shown.</p

Subcellular localization of [Ca²⁺]_cyt and H₂O₂ in <i>G. biloba</i> leaves upon herbivory.

<p><b>A</b>. False color images from confocal laser scanning microscopy shows that upon herbivory [Ca²⁺]_cyt was found mainly in the cytosol, indicated by the calcium orange dye as green patches not associated with any specific organelle. Metric bar = 10 µm. <b>B</b>. H₂O₂ localization by Amplex Red shows a clear associations with microbodies (probably peroxisomes) and/or mitochondria but not with chloroplasts. Metric bar = 20 µm. In both panels, single arrows indicate the dye, double arrows indicate chloroplasts.</p

H₂O₂ variations in <i>G. biloba</i> upon mechanical damage and herbivore wounding.

<p><b>A</b>. Mechanically-wounded <i>G. biloba</i> leaves, values (n = 5) are expressed as µM H₂O₂ calculated from a calibration curve. The same letter indicates not significant (P>0.05) variation. B. Herbivore-wounded <i>G. biloba</i> leaves, values (n = 5) are expressed as µM H₂O₂. Different letters indicate significant (P<0.05) differences, the asterisk indicate significant (P<0.05) differences with respect to mechanical damage. In both panels, amplex indicates the absence of pharmacological agents.</p