Multiple alignment of translated cloned sequences from Tuta absoluta and homologues from other insect species
For Vacuolar ATPase subunit-A: Aedes aegypti (XP_001659520.1); Drosophila melanogaster (NP_652004.2); Tribolium castaneum (XP_976188.1); Manduca sexta (P31400.1); Bombyx mori (NP_001091829.1); Tuta absoluta (KM591219). Arginine Kinase: Spodoptera litura (ADW94627.1); Helicoverpa armigera (ADD22718.1); Bombyx mori (NP_001037402.1); Tribolium castaneum (EFA11419.1); Homalodisca vitripennis (AAT01074.1); Drosophila melanogaster (AAA68172.1); Tuta absoluta (KM591220).
High magnification of the trajectory of Cy3-labeled dsRNA molecules through the tomato leaflets
Detail of various regions on the treated leaf, indicating the dsRNA distribution over leaf areas (bar = 200 μm).
Agroinfiltration experiments with Agrobacterium expressing eGFP or eGFP plus GFPi
(A) Expression of eGFP in tomato leaf infiltrated with Agrobacterium suspension containing plasmid with eGFP, visualized under a confocal fluorescent microscope. (B) Infiltration of tomato leaf with two Agrobacterium clones, expressing eGFP together with a construct expressing GFPi, indicating reduction in GFP expression visualized under confocal fluorescent microscope. (C) tomato leaf tissues at a distance away from the area infiltrated with Agrobacterium suspension containing plasmid with eGFP (bar = 100 μm).
RNAi effects on larval development.
Tuta absoluta individuals after feeding for 11 days in tomato leaflets, after absorbing 500 ng dsRNA of the GFP control (a) and the target genes V-ATPase (b) or AK (c). Total amount of individuals at pupal stage, resultant from the larvae feeding on GFP control (d) and the target genes V-ATPase (e) or AK (f)
Amplification detection of expressed sequences (cDNA; siRNA and microRNA) from various transgenic events
(A) Detection of amplification products from cDNA (RT-PCR) extracted from various transgenic ‘Micro-tom’ events and non-transformed control (WT) using primers specific for insect V-ATPase (top panel; 139 bp) or AK (bottom panel;190 bp), and tomato ubiquitin (108 bp). (B). Detection of amplification products derived from stem loop pulsed RT-PCR for potential siRNA derived from target genes (V-ATPase or AK, both 60 bp), plus the microRNA156 (MIR156; 60 bp) control ran at 3% agarose gel electrophoresis. Numbers represent events (1st number) or plants within events (2nd number).
List of degenerated primers
Degenerated primers used to amplify and clone respective candidate gene targets for RNAi, with expected amplicon size for each reaction. Numbers refer to primer order used in amplification reactions.
List of primers for Gateway construct
Primers used to amplify target gene fragments with Gateway recombination borders attL1 and attL2 (underlined), with expected amplicon size (bp).
Summary of three transformation experiments using construct containing repetitive and inverted gene fragments
Specific primers designed for transcriptional analysis of gene-targets for silencing, with expected amplicon size in base pairs
List of primers to detect siRNA in transgenic plants
Gene-specific and the universal primer sequences used to detect the predicted small interfering RNAs (siRNA) derived from the target genes V-ATPase (siRNA AATACATGCGCGCTCTAGATGAC) and AK (siRNA AAGTATCGTCCACACTGTCTGGC) and the control microRNA156 (UGACAGAAGAGAGUGAGCAC) in transgenic plants.