PDQuest-generated master gel image showing the general spot pattern of matched protein spots from the total leaf proteome of healthy or Las-infected lemon plants.

<p>Labeled spots were differentially produced in response to Las-infection and described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067442#tab1" target="_blank">Table 1</a>. A sum of 200 µg of total protein was separated according to charge on a pH 4-7 IpG strip and according to mass on 8-16% gradient SDS-polyacrylamide Tris-HCl gels. Protein spots were visualized by staining with Coomassie Brilliant Blue (CBB). <i>M</i>_r, relative molecular mass; pI, isoelectric point.</p

The leaf-nutrient concentrations of healthy or Las-infected lemon plants.

<p> (A) Macronutrients: calcium (Ca), potassium (K) and magnesium (Mg); (B) Micronutrients: iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu). Two-year old healthy plants were either graft-inoculated or uninoculated with PCR-confirmed Las-infected bud sticks and leaf samples were analyzed 6 months post-inoculation. Bars within a plant group with the same lower case letter are not significantly different from each other (<i>P</i> > 0.05).</p

Categorization of differentially produced proteins in lemon plants in response to Las infection.

<p>(A) Venn diagram showing the number of protein spots that showed higher-accumulation (▲) or lower-accumulation (▼) in infected lemon plants compared to healthy plants. (B) Functional category distribution of differentially produced protein spots from comparing 2-DE gel images of the total leaf proteome of healthy or Las-infected lemon plants.</p

Differentially produced protein spots from 2-DE analysis of total leaf proteins from healthy or Las-infected lemon plants.

<p>Panels A-M show magnified views of protein spots in representative 2-DE gels containing separated total proteins from leaves of healthy or Las-infected lemon plants. Labeled spots showed significant changes and correspond to the spots presented in in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067442#pone-0067442-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067442#tab2" target="_blank">Tables 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067442#tab3" target="_blank">3</a>. Two-year old healthy plants were either graft-inoculated with side shoots from PCR-confirmed Las-infected bud sticks or uninoculated and leaf samples were analyzed at six months post-inoculation. A sum of 200 µg of total protein was separated according to charge on a pH 4-7 IpG strip and according to mass on 8-16% gradient SDS-polyacrylamide Tris-HCl gels. Protein spots were visualized by staining with Coomassie Brilliant Blue (CBB). <i>M</i>_r, relative molecular mass; pI, isoelectric point.</p

Mitochondrial localization of the Las autotransporter LasA_I.

<p><b>A-G1</b>: confocal laser scanning micrographs. GFP expression and MitoTracker labeling were detected in tobacco leaves infiltrated with pGDY and pGDY-<i>lasA</i>_I-TD plasmids, respectively. <b>A</b>, <b>E</b>: GFP detection with 505-530 nm BP filter; <b>B</b>, <b>F</b>: MitoTracker detection with 560 nm LP filter; <b>C</b>, <b>G</b>: merged scans; <b>D</b>, <b>H</b>: differential interference contrast (DIC) micrographs of tobacco cells with chloroplasts (red arrows). <b>E1, F1, G1</b>: magnifications of yellow boxes in panels E, F and G. Mitochondria (yellow arrows).</p

Surface location in <i>E. coli</i> of the autotransporter LasA_I as determined by immuno-dot blot analysis.

<p>Serial dilutions of <i>E. coli</i> cells were deposited onto a nitrocellulose membrane. The presence of LasA_I was detected with anti-LasA_I antibody. <b>A</b>: <i>E. coli</i> containing control plasmid pET102; <b>B</b>: IPTG-induced <i>E. coli</i> containing recombinant plasmid pET102-<i>lasA</i>_I; <b>C</b>: IPTG induced <i>E. coli</i> containing recombinant plasmid pET102-<i>lasA</i>_I treated with Proteinase K.</p

Surface localization in <i>E. coli</i> of GFP fusion proteins of LasA_I and LasA_II by immuno-dot blot.

<p>Serial dilutions of bacterial cells were deposited onto a nitrocellulose membrane; GFP was detected with anti-GFP antibody. <b>Lane 1, 2, 3</b>: IPTG induced <i>E. coli</i> containing plasmid pET102-<i>gfp</i>; <b>Lane 4, 5, 6</b>: IPTG-induced <i>E. coli</i> containing plasmid pET102-<i>gfp</i>-<i>lasA</i>_I-TD; <b>Lane 7, 8, 9</b>: IPTG induced <i>E. coli</i> containing plasmid pET102-gfp-<i>lasA</i>_II.</p

Polar localization of Las autotransporters LasA_I and LasA_II from ‘<i>Candidatus</i> Liberibacter asiaticus’ (Las) in <i><b>E. coli</b></i>.

<p><b>A</b>, <b>D</b>, <b>G</b>: GFP expression detected by confocal laser scanning microscopy (CLSM) in <i>E. coli</i> containing recombinant plasmids pET102-<i>gfp</i>, pET102-<i>gfp</i>-<i>lasA</i>_I-TD and pET102-<i>gfp</i>-<i>lasA</i>_II, respectively. <b>A</b>, <b>D</b>, <b>G</b>: 505 nm LP filter; <b>B, E, H</b>: differential interference contrast (DIC) of bacterial cells; <b>C, F, I</b>: FITC-DIC merged.</p

Mitochondria aggregation and morphology in plant cells.

<p><b>A1-A3</b>: confocal laser scanning micrographs. MitoTracker labeling was detected in tobacco leaves infiltrated with pGDY (A1), pGDY-<i>lasA</i>_I (A2) and pGDY-<i>lasA</i>_II (A3). Arrows indicate normal and aggregated mitochondria respectively. <b>B1-B4</b>: transmission electron microscopy micrographs. <b>B1</b>: normal mitochondria (red arrow), chloroplast (blue arrow) and cell wall (green arrow) from infiltrated pGDY. <b>B2</b>: enlarged mitochondria (red arrow), abnormal chloroplast (blue arrow) and detached cell wall (green arrow) from infiltrated pGDY-<i>lasA</i>_I. <b>B3</b>: normal mitochondria (arrow) from healthy periwinkle. <b>B4</b>: aggregated abnormal mitochondria (arrow) from Las-infected periwinkle.</p

Prophage-Mediated Dynamics of‘<i>Candidatus</i> Liberibacter asiaticus’ Populations, the Destructive Bacterial Pathogens of Citrus Huanglongbing

<div><p>Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. There are at least two prophages in the chromosomes of <i>Candidatus</i> Liberibacter asiaticus’ (Las) Floridian isolates. Las is both unculturable and the most prevalent species of Liberibacter pathogens that cause huanglongbing (HLB), a worldwide destructive disease of citrus. In this study, seven new prophage variants resulting from two hyper-variable regions were identified by screening clone libraries of infected citrus, periwinkle and psyllids. Among them, Types A and B share highly conserved sequences and localize within the two prophages, FP1 and FP2, respectively. Although Types B and C were abundant in all three libraries, Type A was much more abundant in the libraries from the Las-infected psyllids than from the Las-infected plants, and Type D was only identified in libraries from the infected host plants but not from the infected psyllids. Sequence analysis of these variants revealed that the variations may result from recombination and rearrangement events. Conventional PCR results using type-specific molecular markers indicated that A, B, C and D are the four most abundant types in Las-infected citrus and periwinkle. However, only three types, A, B and C are abundant in Las-infected psyllids. Typing results for Las-infected citrus field samples indicated that mixed populations of Las bacteria present in Floridian isolates, but only the Type D population was correlated with the blotchy mottle symptom. Extended cloning and sequencing of the Type D region revealed a third prophage/phage in the Las genome, which may derive from the recombination of FP1 and FP2. Dramatic variations in these prophage regions were also found among the global Las isolates. These results are the first to demonstrate the prophage/phage-mediated dynamics of Las populations in plant and insect hosts, and their correlation with insect transmission and disease development.</p></div