addgene plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: The MBW complex regulates volatiles in petunia flowers: EOBV interacts with AN1 to suppress biosynthesis of phenylpropenes

doi: 10.64898/2026.03.01.708793

Figure Lengend Snippet: a) Developmental expression patterns of AN1 and AN11 . RT-qPCR analysis was performed on flower tissues collected at different developmental stages. DPA, days postanthesis. Data are means ± SEM (n = 3). Significance of differences was calculated by Tukey’s multiple comparison test following one-way ANOVA. Values with different letters are significantly different at P ≤ 0.05. (b and c) Petunia cv. Mitchell flowers were inoculated at anthesis with agrobacteria carrying pTRV2-AN1, pTRV2-AN11 or pTRV2-CHS as a control. Flowers were harvested 2 days after inoculation and subjected to localized headspace sampling followed by GC–MS analysis (b) or RNA extraction followed by RT-qPCR analysis (c) . Data are means ± SEM ( n = 12). Significance of differences was calculated using Dunnett’s test with pTRV2-CHS as the control following one-way ANOVA (b) or two-tailed unpaired Student’s t -test (c) (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). (a and c) RT-qPCR data were normalized to ACTIN , and the presented data were normalized to the sample with the highest expression level. Standard errors are indicated by vertical lines.

Article Snippet: Tobacco rattle virus RNA2 (pTRV2) vectors pTRV2-sgRNA1-sgRNA2 and pTRV2-sgRNA1-sgRNA3, were synthesized by Twist Bioscience (San Francisco, CA, USA), based on the pTRV2 vector with GenBank accession AF406991 .

Techniques: Expressing, Quantitative RT-PCR, Comparison, Control, Sampling, Gas Chromatography-Mass Spectrometry, RNA Extraction, Two Tailed Test

Journal: bioRxiv

Article Title: The MBW complex regulates volatiles in petunia flowers: EOBV interacts with AN1 to suppress biosynthesis of phenylpropenes

doi: 10.64898/2026.03.01.708793

Figure Lengend Snippet: a) Phylogenetic tree displaying the similarity between EOBV and subgroup 4 R2R3-MYBs from other plants. Protein sequences were aligned by ClustalW, followed by neighbor-joining method with 1000 bootstraps using MEGA version 11. b) Nuclear localization of EOBV in petal epidermal cells by confocal microscopy. Petunia petals were inoculated with agrobacteria carrying EOBV-GFP or GFP-EOBV and RFP - NLS, or only RFP - NLS as a control. Bars = 10 μm. BF = bright field. NLS, nuclear localization signal. c) Expression of EOBV in different plant organs. Tissues from different organs were collected at 1100 h. RT-qPCR data were normalized to ACTIN , and the presented data were normalized to the sample with the highest expression level. Significance of differences was calculated by Tukey’s multiple comparison test following one-way ANOVA. Values with different letters are significantly different at P ≤ 0.05. d) EOBV transcripts are enriched in the petal adaxial epidermis. Normalized counts in the petal adaxial epidermis (purple bars) vs. whole petal (gray bars) based on the transcriptome data from Skaliter et al . (2024) . Significance of differences was calculated by two-tailed unpaired Student’s t -test: * P ≤ 0.05. EVER, EPIDERMIS VOLATILE EMISSION REGULATOR. CAB, CHLOROPHYLL A/B BINDING PROTEIN. e) Transient suppression of EOBV in petunia cv. Mitchell flowers inoculated with agrobacteria carrying pTRV2-CHS as a control or pTRV2-EOBV. Flowers were harvested 2 days after inoculation and subjected to localized headspace sampling followed by GC–MS analysis or RNA extraction followed by RT-qPCR analysis (inset). Data are means ± SEM ( n = 11–12). RT-qPCR data were normalized to ACTIN , and the presented data were normalized to those from pTRV2-CHS. Significance of differences between treatments was calculated by two-tailed unpaired Student’s t -test: (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). Standard errors are indicated by vertical lines.

Article Snippet: Tobacco rattle virus RNA2 (pTRV2) vectors pTRV2-sgRNA1-sgRNA2 and pTRV2-sgRNA1-sgRNA3, were synthesized by Twist Bioscience (San Francisco, CA, USA), based on the pTRV2 vector with GenBank accession AF406991 .

Techniques: Confocal Microscopy, Control, Expressing, Quantitative RT-PCR, Comparison, Two Tailed Test, Binding Assay, Sampling, Gas Chromatography-Mass Spectrometry, RNA Extraction

Journal: bioRxiv

Article Title: The MBW complex regulates volatiles in petunia flowers: EOBV interacts with AN1 to suppress biosynthesis of phenylpropenes

doi: 10.64898/2026.03.01.708793

Figure Lengend Snippet: a) Schematic representation of the genomic sequence of EOBV and location of the three Cas9 target sites. b) Top: schematic representation of the T-DNA carrying tobacco rattle virus RNA2 (pTRV2) vectors used used for gene editing. SGP, subgenomic promoter; LB, left border; 35S-P, cauliflower mosaic virus 35S promoter; SGP, subgenomic promoter; sgRNA, single guide RNA; NOS-T, nopaline synthase terminator; RB, right border; Cas9, human co-don-optimized Cas9 gene. Bottom: targeted genomic sequences and Sanger sequencing of eobv -knockout lines. Green nucleotides, spacer sequences; red nucleotides, protospacer adjacent motif (PAM). Black arrows mark the predicted cleavage site of Cas9. Magenta box, EOBV start codon. c) Predicted EOBV protein product in eobv -knockout and wild-type (WT) lines. Functional domains are indicated by colored rectangles. Numbers denote amino acid positions. d) Representative petunia cv. Mitchell flowers 2 days postanthesis from control Cas9 and eobv lines. Bar = 1 cm.

Article Snippet: Tobacco rattle virus RNA2 (pTRV2) vectors pTRV2-sgRNA1-sgRNA2 and pTRV2-sgRNA1-sgRNA3, were synthesized by Twist Bioscience (San Francisco, CA, USA), based on the pTRV2 vector with GenBank accession AF406991 .

Techniques: Sequencing, Virus, Genomic Sequencing, Knock-Out, Functional Assay, Control