Journal: Plant Communications

Article Title: A faster killing effect of plastid-mediated RNA interference on a leaf beetle through induced dysbiosis of the gut bacteria

doi: 10.1016/j.xplc.2024.100974

Figure Lengend Snippet: Generation and molecular characterization of transplastomic and nuclear transgenic poplar plants. (A) Physical maps of transformation vectors for dsRNA expression from the plastid genome of poplar. The cassettes designed to produce dsRNAs are schematically depicted below the map, along with the expected structures and sizes. The selectable marker gene aadA is driven by the psbA promoter combined with the 5′ untranslated region (5′ UTR) from gene 10 of bacteriophage T7 ( Cr P psbA:T7g10 ) and the 3′ UTR of the rbcL gene from Chlamydomonas reinhardtii ( Cr T rbcL ). The location of the hybridization probe is shown as a black bar. (B and C) Southern blot analysis of transplastomic poplar lines Pt -pt-ds ACT (B) and Pt -pt-ds SRP54K (C) . DNA samples were digested with BglII and hybridized to a Digoxin (DIG)-labeled probe as indicated. Absence of the 3.50-kb hybridization signal for the wild-type genome indicates homoplasmy of transplastomic lines. (D and E) Quantification of ds ACT (D) and ds SRP54K (E) accumulation in transplastomic poplar plants by northern blot analysis. Five micrograms of total cellular RNA were loaded from transplastomic and wild-type poplar plants in each lane. For semi-quantitative analysis, a dilution series of in vitro -synthesized dsRNAs was loaded for comparison. The GelView-stained gel prior to blotting is shown below the blot as a loading control. (F and G) The relative expression of hp ACT (F) and hp SRP54K (G) in the nuclear transgenic poplar lines was determined by qRT–PCR. The poplar EF1β gene was used as an internal standard. Data are shown as means ± SEM ( n = 3).

Article Snippet: After blocking with 5% nonfat dry milk, the membrane was incubated with a 3000-fold diluted polyclonal antibody against β-actin (ABclonal, China) for 90 min and then incubated for 90 min with a 5000-fold diluted horseradish peroxidase (HRP)-conjugated secondary antibody (Bioswamp, China).

Techniques: Transgenic Assay, Transformation Assay, Expressing, Marker, Hybridization, Southern Blot, Labeling, Northern Blot, In Vitro, Synthesized, Comparison, Staining, Control, Quantitative RT-PCR

Journal: Plant Communications

Article Title: A faster killing effect of plastid-mediated RNA interference on a leaf beetle through induced dysbiosis of the gut bacteria

doi: 10.1016/j.xplc.2024.100974

Figure Lengend Snippet: Feeding assays of first-instar P. versicolora larvae on transplastomic and nuclear transgenic poplar lines expressing ds ACT . (A) Kaplan–Meier survival curves of first-instar larvae ( n = 30) fed on detached leaves of Pt -wt, nuclear transgenic ( Pt -nu-hp ACT #18 and Pt -nu-hp ACT #36), and transplastomic ( Pt -pt-ds ACT #1 and Pt -pt-ds ACT #2) poplar plants. The log-rank test was used to assess the significance of differences between two survival curves. The statistical significance ( p < 0.05) is denoted by different letters. The p values for each pair of plant lines are shown. (B) Mean weight of larvae after 3 days of feeding on detached poplar leaves. (C) Leaves damaged by larvae after 3 days of feeding. Data are means ± SE (three biological replicates; 10 insects per sample). The letters above the bars indicate the statistical significance of differences between the groups ( p < 0.05, ANOVA with Bonferroni multiple-comparison test). The damaged area was determined with Image J. (D) Representative example of bioassays with detached leaves of the indicated poplar plants. Leaves were exposed to first-instar larvae and replaced with fresh leaves every day; the photograph was taken at day 3. The diameter of the culture dish was 9 cm. Relative expression levels of β-Actin in larvae fed with the indicated poplar leaves at 3 (E) and 4 (F) days were determined by qRT–PCR. Gene expression levels in larvae fed on detached leaves of Pt -wt were set to 1. Data are means ± SE (three biological replicates; one insect per sample). Different letters above the bars indicate significant differences between groups ( p < 0.05, ANOVA with Bonferroni multiple-comparison test).

Article Snippet: After blocking with 5% nonfat dry milk, the membrane was incubated with a 3000-fold diluted polyclonal antibody against β-actin (ABclonal, China) for 90 min and then incubated for 90 min with a 5000-fold diluted horseradish peroxidase (HRP)-conjugated secondary antibody (Bioswamp, China).

Techniques: Transgenic Assay, Expressing, Comparison, Quantitative RT-PCR, Gene Expression

Journal: Plant Communications

Article Title: A faster killing effect of plastid-mediated RNA interference on a leaf beetle through induced dysbiosis of the gut bacteria

doi: 10.1016/j.xplc.2024.100974

Figure Lengend Snippet: Gut bacteria accelerate the mortality of P. versicolora larvae in PM–RNAi. (A) Kaplan–Meier survival curves of axenic and non-axenic first-instar larvae ( n = 30) after feeding on detached aseptic poplar leaves of Pt -wt and Pt -pt-ds ACT plants. The log-rank test was used to assess the significance of differences between two survival curves. ∗∗∗ p < 0.001; ∗ p < 0.05; NS, not significant. (B) Relative expression levels of β-Actin in axenic and non-axenic larvae fed with detached aseptic leaves of Pt -wt and Pt -pt-ds ACT plants after 2 and 3 days, as determined by qRT–PCR. Data are mean ± SE (three biological replicates; one insect per sample). Different letters indicate significant differences as determined by t -test ( p < 0.05). (C) Kaplan–Meier survival curves of axenic first-instar larvae ( n = 30) fed with detached aseptic leaves of Pt -wt and Pt -pt-ds ACT plants after reintroduction of P. putida by painting onto aseptic poplar leaves at a concentration of about 1 × 10 4 CFU/cm 2 . The log-rank test was used to assess the significance of differences between two survival curves. ∗∗∗ p < 0.001; ∗ p < 0.05; NS, not significant. (D) Relative expression levels of β-Actin in axenic larvae fed with detached aseptic poplar leaves of Pt -wt and Pt -pt-ds ACT plants after 2 and 3 days of reintroduction of P. putida , as determined by qRT–PCR. Data are means ± SE (three biological replicates; one insect per sample). Different letters indicate significant differences as determined by t -test ( p < 0.05).

Article Snippet: After blocking with 5% nonfat dry milk, the membrane was incubated with a 3000-fold diluted polyclonal antibody against β-actin (ABclonal, China) for 90 min and then incubated for 90 min with a 5000-fold diluted horseradish peroxidase (HRP)-conjugated secondary antibody (Bioswamp, China).

Techniques: Bacteria, Expressing, Quantitative RT-PCR, Concentration Assay

Journal: Plant Communications

Article Title: A faster killing effect of plastid-mediated RNA interference on a leaf beetle through induced dysbiosis of the gut bacteria

doi: 10.1016/j.xplc.2024.100974

Figure Lengend Snippet: Foliar application of gut bacteria enhances the resistance of transplastomic poplar plants to P. versicolora larvae. (A) Kaplan–Meier survival curves of first-instar non-axenic larvae ( n = 30) fed with detached aseptic poplar leaves of Pt -wt and Pt -pt-ds ACT plants coated with P. putida . The poplar leaves were painted with P. putida at a concentration of about 1 × 10 2 CFU/cm 2 . The log-rank test was used to assess the significance of differences between two survival curves. ∗∗∗ p < 0.001; NS, not significant. (B) Representative example of bioassays with detached leaves of the indicated poplar plants. Leaves were exposed to first-instar larvae and replaced with fresh leaves every day; the photographs were taken separately at 2 and 3 days. The diameter of the culture dish was 9 cm. (C) Leaves damaged by larvae after 2 and 3 days of feeding. Data are means ± SE (three biological replicates; 10 insects per sample). The letters above the bars indicate the significance of differences as determined by t -test ( p < 0.05). The damaged area was determined with ImageJ. (D) Relative expression levels of ACT in larvae fed with indicated poplar leaves at 2 and 3 days, as determined by qRT–PCR. Gene expression levels in larvae fed on detached leaves of Pt -wt were set to 1. Data are means ± SE (three biological replicates; one insect per sample). Different letters above the bars indicate significant differences, as determined by t -test ( p < 0.05).

Article Snippet: After blocking with 5% nonfat dry milk, the membrane was incubated with a 3000-fold diluted polyclonal antibody against β-actin (ABclonal, China) for 90 min and then incubated for 90 min with a 5000-fold diluted horseradish peroxidase (HRP)-conjugated secondary antibody (Bioswamp, China).

Techniques: Bacteria, Concentration Assay, Expressing, Quantitative RT-PCR, Gene Expression

Journal: Plant Communications

Article Title: A faster killing effect of plastid-mediated RNA interference on a leaf beetle through induced dysbiosis of the gut bacteria

doi: 10.1016/j.xplc.2024.100974

Figure Lengend Snippet: A schematic model illustrating the introduction of gut bacteria to enhance the resistance of PM–RNAi in P. versicolora . The ingestion of long dsRNAs expressed in plastids leads to a reduction in target mRNA levels and is highly toxic to P. versicolora larvae. In comparison to control non-axenic larvae fed with leaves of Pt -pt-ds ACT , P. putida- introduced non-axenic larvae exhibit lower production of ROS and reduced transcription of AMP-encoding genes, leading to immunodeficiency (IMD). Moreover, the expression of AMPs is regulated by transcription of Relish and Domeless through IMD and JAK/STAT pathways, in turn suppressing immune responses. This limited immune response hampers the ability of P. versicolora to control the overgrowth of gut bacteria.

Article Snippet: After blocking with 5% nonfat dry milk, the membrane was incubated with a 3000-fold diluted polyclonal antibody against β-actin (ABclonal, China) for 90 min and then incubated for 90 min with a 5000-fold diluted horseradish peroxidase (HRP)-conjugated secondary antibody (Bioswamp, China).

Techniques: Bacteria, Comparison, Control, Expressing

Journal: Arthroscopy Techniques

Article Title: The “Double Lasso-Loop” Technique Used for Arthroscopic Proximal Biceps Tenodesis

doi: 10.1016/j.eats.2018.11.012

Figure Lengend Snippet: Advantages and Limitations

Article Snippet: Next, while viewing through the lateral SA portal, an anchor is placed next to the articular margin of the humeral head, in line with the LHBT (Y-Knot RC all-suture anchor; ConMed, Largo, FL) ( A, ).

Techniques: Infection