Table_1_Metabolic changes and potential biomarkers in "Candidatus Liberibacter solanacearum"-infected potato psyllids: implications for psyllid-pathogen interactions.xlsx

Psyllid yellows, vein-greening (VG), and zebra chip (ZC) diseases, which are primarily transmitted by potato psyllid (PoP) carrying Candidatus Liberibacter solanacearum (CLso), have caused significant losses in solanaceous crop production worldwide. Pathogens interact with their vectors at the organic and cellular levels, while the potential changes that may occur at the biochemical level are less well reported. In this study, the impact of CLso on the metabolism of PoP and the identification of biomarkers from infected psyllids were examined. Using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analysis, metabolomic changes in CLso-infected psyllids were compared to uninfected ones. A total of 34 metabolites were identified as potential biomarkers of CLso infection, which were primarily related to amino acid, carbohydrate, and lipid metabolism. The significant increase in glycerophospholipids is thought to be associated with CLso evading the insect vector’s immune defense. Matrix-assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI) was used to map the spatial distribution of these biomarkers, revealing that 15-keto-Prostaglandin E2 and alpha-D-Glucose were highly expressed in the abdomen of uninfected psyllids but down-regulated in infected psyllids. It is speculated that this down-regulation may be due to CLso evading surveillance by immune suppression in the PoP midgut. Overall, valuable biochemical information was provided, a theoretical basis for a better understanding of psyllid-pathogen interactions was offered, and the findings may aid in breaking the transmission cycle of these diseases.</p

Table_3_Metabolic changes and potential biomarkers in "Candidatus Liberibacter solanacearum"-infected potato psyllids: implications for psyllid-pathogen interactions.xlsx

Psyllid yellows, vein-greening (VG), and zebra chip (ZC) diseases, which are primarily transmitted by potato psyllid (PoP) carrying Candidatus Liberibacter solanacearum (CLso), have caused significant losses in solanaceous crop production worldwide. Pathogens interact with their vectors at the organic and cellular levels, while the potential changes that may occur at the biochemical level are less well reported. In this study, the impact of CLso on the metabolism of PoP and the identification of biomarkers from infected psyllids were examined. Using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analysis, metabolomic changes in CLso-infected psyllids were compared to uninfected ones. A total of 34 metabolites were identified as potential biomarkers of CLso infection, which were primarily related to amino acid, carbohydrate, and lipid metabolism. The significant increase in glycerophospholipids is thought to be associated with CLso evading the insect vector’s immune defense. Matrix-assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI) was used to map the spatial distribution of these biomarkers, revealing that 15-keto-Prostaglandin E2 and alpha-D-Glucose were highly expressed in the abdomen of uninfected psyllids but down-regulated in infected psyllids. It is speculated that this down-regulation may be due to CLso evading surveillance by immune suppression in the PoP midgut. Overall, valuable biochemical information was provided, a theoretical basis for a better understanding of psyllid-pathogen interactions was offered, and the findings may aid in breaking the transmission cycle of these diseases.</p

Table_2_Metabolic changes and potential biomarkers in "Candidatus Liberibacter solanacearum"-infected potato psyllids: implications for psyllid-pathogen interactions.xlsx

Psyllid yellows, vein-greening (VG), and zebra chip (ZC) diseases, which are primarily transmitted by potato psyllid (PoP) carrying Candidatus Liberibacter solanacearum (CLso), have caused significant losses in solanaceous crop production worldwide. Pathogens interact with their vectors at the organic and cellular levels, while the potential changes that may occur at the biochemical level are less well reported. In this study, the impact of CLso on the metabolism of PoP and the identification of biomarkers from infected psyllids were examined. Using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analysis, metabolomic changes in CLso-infected psyllids were compared to uninfected ones. A total of 34 metabolites were identified as potential biomarkers of CLso infection, which were primarily related to amino acid, carbohydrate, and lipid metabolism. The significant increase in glycerophospholipids is thought to be associated with CLso evading the insect vector’s immune defense. Matrix-assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI) was used to map the spatial distribution of these biomarkers, revealing that 15-keto-Prostaglandin E2 and alpha-D-Glucose were highly expressed in the abdomen of uninfected psyllids but down-regulated in infected psyllids. It is speculated that this down-regulation may be due to CLso evading surveillance by immune suppression in the PoP midgut. Overall, valuable biochemical information was provided, a theoretical basis for a better understanding of psyllid-pathogen interactions was offered, and the findings may aid in breaking the transmission cycle of these diseases.</p

Scanning Electrochemical Microscopy of DNA Hybridization on DNA Microarrays Enhanced by HRP-Modified SiO₂ Nanoparticles

Imaging of localized hybridization of nucleic acids immobilized on a glass DNA microarray was performed by means of generation collection (GC) mode scanning electrochemical microscopy (SECM). Amine-tethered oligodeoxynucleotide probes, spotted on the glass surface, were hybridized with an unmodified target sequence and a biotinylated indicator probe <i>via</i> sandwich hybridization. Spots where sequence-specific hybridization had occurred were modified by streptavidin-horseradish-peroxidase-(HRP)-wrapped SiO₂ nanoparticles through the biotin–streptavidin interaction. In the presence of H₂O₂, hydroquinone (H₂Q) was oxidized to benzoquinone (BQ) at the modified spot surface through the HRP catalytic reaction, and the generated BQ corresponding to the amount of target DNA was reduced in solution by an SECM tip. With this DNA microarray, a number of genes could be detected simultaneously and selectively enough to discriminate between complementary sequences and those containing base mismatches. The DNA targets at prepared spots could be imaged in SECM GC mode over a wide concentration range (10^–7–10^–12 M). This technique may find applications in genomic sequencing

Silver staining of purified LPS.

<p>LPS was purified by hot phenol-water method, fractionated over SDS-PAGE, and stained by silver. Each lane contains 0.5 µg of LPS. Lane 1: Wide-type strain Yb2 LPS. Lane 2: Mutant strain RA2640 LPS. Lane 3: Complementation strain cRA2640 LPS.</p

Construction and characterization of <i>R. anatipestifer</i> mutant strain RA2640 and complementation strain cRA2640.

<p>(A) Slide agglutination test: Mutant strain RA2640 lacked serum agglutination ability to serotype 2 positive sera. (B) Southern blot analysis. Lane M: DM15000 DNA Marker (CWBIO, Beijing, China). Lane 1: A single insertion of Tn4351-transposon was identified in mutant strain RA2640. Lane 2: No hybridization band was detected in wild-type strain Yb2. Lane 3: <i>Xba</i>I fragment (10.4 kb) of pEP4351 was identified with the <i>ermF</i> gene probe. (C) Real-time RT-PCR analysis. The expression of upstream AS87_04040 gene and downstream AS87_04055 gene were measured. The changes of mRNAs were expressed as fold expression and calculated using the comparative C_T (2^-△△CT) method. The expression of AS87_04050 in mutant strain RA2640 couldn't be detected. Error bars represent SD from three replicates. (D) PCR analysis. Lane M: DM2000 DNA Marker (CWBIO, Beijing, China). Lane 1: The Erm and <i>R. anatipestifer</i> 16S rRNA were amplified from the mutant strain RA2640 using primer pairs Erm-F/Erm-R and RA 16S rRNA-F/RA 16S rRNA-R, showing an 833-bp fragment of Erm or a 496-bp fragment of RA 16S rRNA. Lane 2: A 496-bp fragment was amplified from wild-type strain Yb2 with primers RA 16S rRNA-F/RA 16S rRNA-R. Lane 3: The AS87_04050 gene and <i>R. anatipestifer</i> 16S rRNA were amplified from the complementation strain cRA2640 using primer pairs AS87_04050 comp-F/AS87_04050 comp-R and RA 16S rRNA-F/RA 16S rRNA-R, showing a 972-bp fragment of AS87_04050 or a 496-bp fragment of RA 16S rRNA.. Lane 4: The AS87_04050 gene and <i>R. anatipestifer</i> 16S rRNA were amplified from wild-type strain Yb2 using primer pairs AS87_04050 comp-F/AS87_04050 comp-R and RA 16S rRNA-F/RA 16S rRNA-R, showing a 972-bp fragment of AS87_04050 or a 496-bp fragment of RA 16S rRNA.</p

Sensitivity to disinfectants and normal duck sera.

<p>(A) Susceptibilities to hydrogen peroxide and Triton X–100, expressed as survival rate after incubation of bacteria with disinfectants at 37°C for 30 min as compared to PBS. Bars represent the means ± standard deviation of the results found in three experiments. (B) Susceptibilities to normal duck sera, expressed as survival rate after incubation of bacteria with normal duck sera at 37°C for 30 min at different dilutions as compared to PBS. ***, <i>p</i><0.001. Error bars represent the standard deviation of three independent experiments.</p

Bacterial LD₅₀ determination.

<p>Bacterial LD₅₀ determination.</p

Bacterial MICs determination.

a<p>The concentrations tested for ampicillin were 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 10 µg/ml respectively; for streptomycin sulfate were 5, 10, 15, 20, 25 and 30 µg/ml respectively; for gentamicin were 6.25, 12.5, 25, 50 and 60 µg/ml respectively; for spectinomycin sulfate were 25, 50, 75 and 100 µg/ml respectively; for chloramphenicol were 4, 6.25, 12.5 and 25 µg/ml respectively; for polymyxin B were 80, 100, 120 and 140 µg/ml respectively; and for nalidixic acid were 115, 125, 135 and 145 µg/ml, respectively.</p><p>Bacterial MICs determination.</p

Bacterial loads in blood of <i>R. anatipestifer</i> infected ducks.

<p>Six ducks were injected intramuscularly with bacterial culture at 1×10⁷ CFU. Blood samples were collected at 3, 6, 12 and 24 h.p.i. and serially diluted for bacterial counting. Each point represents the mean ± standard deviation.</p