Additional file 1 of Development and application of a virus-induced gene silencing protocol for the study of gene function in narrow-leafed lupin
Creators: name: Mancinotti, Davide
name: Rodriguez, Maria Cecilia
name: Frick, Karen Michiko
name: Dueholm, Bjørn
name: Jepsen, Ditte Goldschmidt
name: Agerbirk, Niels
name: Geu-Flores, Fernando
Publisher: name: figshare
Year: 2021
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Fig. S1. RT-PCR-based amplification of ALSV fragments from systemic N. benthamiana leaves infected with ALSV-0 or ALSV-PDS. Expected PCR fragment sizes are indicated above the respective fragments. M, molecular weight marker. The sizes of selected fragments of the molecular weight marker are indicated to their left side (bp). Fig. S2. Normalized relative abundance of the ten most abundant QAs in leaves of plants infected with ALSV-GFP or ALSV-PDS. Box plots represent data for 18 or 12 biological replicates for plants infected with ALSV-GFP or ALSV-PDS, respectively. Whiskers represent values within 1.5 times the interquartile range over or under the 75th and 25th percentiles, respectively. Dots and triangles represent the individual data points, the dashed line represents the mean of ALSV-GFP and significant differences are represented by one or two asterisks (two-sided Wilcoxon test; P ≤ 0.05 or P ≤ 0.01, respectively). Fig. S3. Normalized relative expression of LaPDS in leaves of plants infected with ALSV–GFP, ALSV–PDS or ALSV–PDS-GFP. Box plots represent data for 5 (ALSV–GFP and ALSV–PDS) or 7 (ALSV–PDS-GFP) biological replicates. Whiskers represent values within 1.5 times the interquartile range over or under the 75th and 25th percentiles, respectively. Dots represent the individual data points, the dashed line represents the mean of ALSV-GFP and the asterisks represent significant differences between leaves of plants infected with ALSV–GFP and ALSV-PDS or ALSV–PDS-GFP (two-sided Wilcoxon test; P ≤ 0.01). Fig. S4. Representative chromatograms from the LC–MS analysis of leaf extracts from PDS co-silenced plants. (A) Extract of a bleached leaf from an ALSV-PDS-GFP plant (44 mg). (B) Extract of a bleached leaf from an ALSV-PDS-LDC plant (45 mg). The colored, solid traces represent the extracted ion chromatograms (EICs) of the QAs [M + H]+ ions ± 0.005 Da. The dashed, light grey traces represent the full-scan base peak chromatograms, superimposed on the EICs. The m/z ranges of the EIC traces are: red, 249.191–249.201; blue, 235.175–235.185; green, 235.175–235.185; violet, 347.228–347.238; light orange, 411.223–411.233; azure, 369.212–369.222; dark grey, 395.228–395.238. The marked peaks are: 1, 13-hydroxy(iso)lupanine; 2, isolupanine; 3, lupanine; 4, isoangustifoline; 5, angustifoline; 6, 13-(2-methylbut-2-enoyl)oxylupanine (angeloyl or tigloyl); 7, 13-coumaroyloxylupanine (cis or trans); 8, 13-benzoyloxylupanine; 9, 13-cis-cinnamoyloxylupanine; 10, 13-trans-cinnamoyloxylupanine; and 11, caffeine (internal standard). Sequence S1. Coding sequence of LaPDS from NLL cv. Oskar. The VIGS fragment is highlighted in cyan, and the qPCR amplicon is highlighted in yellow. Individual residues highlighted in grey are located at exon-exon junctions. Sequence S2. Coding sequence of LaLDC from NLL cv. Oskar. The VIGS fragment is highlighted in cyan, and the qPCR amplicon is highlighted in yellow. LaLDC is a single-exon gene. Sequence S3. Coding sequence of LaCAO from NLL cv. Oskar. The qPCR amplicon is highlighted in yellow. Individual residues highlighted in grey are located at exon-exon junctions. Sequence S4. Coding sequence of GFP (U87973). The fragment cloned as a negative control into pALSV-RNA2u is highlighted in cyan. Table S1. DNA oligo sequences used in this study.