The following supplementary material is available for this article:
Figure S1. Transgene insertion in plants of three independently transformed antisense-lox lines. Genomic DNA (10 µg) from two individual plants of each as-lox line (A363, A211, A300) and WT Nicotiana attenuata plants was digested with SspI or EcoRI, respectively, and blotted onto nylon membranes (Winz and Baldwin, 2001). The blots were hybridized with a PCR fragment of the region of NaLOX3 used for the antisense construct. The digestion with SspI, having a recognition site inside the NaLOX3 coding region, and EcoRI (without a recognition site in NaLOX3), indicate a single copy of the transgene in as-lox lines A211 and A300 and potentially two insertions in the genome of A363, in addition to the endogenous NaLOX3 gene, which was also detected in the WT. Download here.
Figure S2. Real time PCR assay for analysis of endogenous NaLOX3 and antisense-lox (as-lox) transgene expression. Real time PCR analysis was performed on a ABI PRISMTM 7000 Sequence detection System (Applied Biosystems, Darmstadt, Germany). A) Specific amplicons (green box), a PCR primer pair and a double fluorescent dye-labeled probe, were designed for the detection of endogenous NaLOX3 and as-lox transgene transcripts . The NaLOX3 assay amplified the 5' region of NaLOX3 and spans the transition to the region of NaLOX3 used for the construction of the transformation vector, pNATLOX1. The as-lox assay amplifies the 3' region of the expressed transgene with a gene specific primer annealing to the antisense-lox sequence and a second transformation vector-specific primer and the probe annealing to the transcribed cauliflower mosaic virus 35S terminator (TCAMV) sequence. The specificity of the PCR reaction was verified by gel analysis of the reaction products and the quality of the assay was evaluated by analyzing previously characterized RNA samples, analyzed by Northern analysis, and comparing the results of the two independent methods. Expression levels of NaLOX3 in WT plants 45 (R45) and 60 (R60) minutes after wounding and application of Manduca sexta oral secretions and regurgitant were analyzed by real-time PCR and compared to Northern blot analysis (left panel, see Fig. 1B lanes 45 and 60 min). Expression of NaLOX3 and as-lox was analyzed in plants treated with lanolin or methyl jasmonate and attacked by a single M. sexta larva (right panel, see Fig. 4). n.d. represents a sample not determined due to lack of RNA. Download here.
Table S1. Description of all genes spotted on the DNA microarray. Download here.
Table S2. Mean ((SE) expression ratios of all genes on the cDNA microarray. Download here.
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