Treatment of plants with caterpillars and aphids was conducted in separate experiments. Sampling of plants occurred several hours after removal of insect herbivores so that plant gene expression results were not confounded by insect rna or by plant gene expression elicited by the physical movement of insect removal. The caterpillar treatment scheme was designed to capture early gene expression events and minimize variation due to leaf age and amount of insect damage. Six to 10 second and third instar S. exigua and P. rapae caterpillars were allowed to feed for 2-4 hours to generate 3 'middle-aged' leaves with 10-30% leaf area removed. Caterpillars were wrangled as needed with camel hair brushes (size 0) to concentrate their feeding on 3 leaves, and leaves of control plants were jiggled with a camel-hair brush to simulate the leaf movement caused by wrangling. Once sufficient damage was achieved, caterpillars were removed and the plants were returned to the growth chamber. The mechanical wounding treatment was designed to mimic insect damage to tissues. Mechanical damage was inflicted by running a sterile pattern wheel across either side of the midrib of 6 leaves on each plant, once at the beginning of the caterpillar wrangling and once half way through the caterpillar wrangling period. Control plants were jiggled with a camel-hair brush to simulate the leaf movement caused by mechanical wounding. Leaves were harvested for gene expression at 6 and 24 hr, with damaged (i.e. local) leaves harvested separately from size-matched undamaged (i.e. systemic) leaves. All leaves selected for treatment and harvest came from the fully-expanded 'middle-aged' leaf and leaves from 2-3 plants were pooled for each of the four bioreplicates. Aphids have much smaller effects on plants than caterpillars (Mewis et al 2005, 2006) and cannot be readily contained on individual leaves. Twenty sub-adult (final instar) and adult aphids were caged on plants and allowed to feed for 1 week. Controls were caged plants without aphids. All aphids were removed and caged controls were jiggled with a camel-hair brush to simulate the leaf movement caused by caterpillar removal. Whole plants were harvested for gene expression at 6 and 24 hr after aphid removal.
Growth protocol
Insect material: The dietary specialist aphid Brevicoryne brassicae (L.) and the dietary generalist aphid Myzus persicae (Sulzer) were maintained as plant virus free clones on pak-choi plants. For chewing insects we selected the dietary specialist caterpillar Pieris rapae L. and the dietary generalist caterpillar Spodoptera exigua Hübner. Eggs of S. exigua were obtained from Benzon Research (Carlisle, PA) and larvae were kept on commercially available artificial Spodoptera diet (Bioserv, Frenchtown, NJ, USA). Eggs of P. rapae were purchased form the Carolina Biological Supply Service (North Carolina) and reared on pak-choi. Both caterpillar species were transferred to Col-0 WT plants one day before the experiments. Plant material: Ecotype Columbia (Col-0) seeds were vernalized and sown into 6 x 5 cm pots filled with sterile Metromix 200 (Scotts company, Marysville, Ohio, USA; contains sphagnum, peat moss, and horticultural perlite). Plants were kept in growth chambers at 22 ± 1 °C, 65 ± 5 % relative humidity, at 250 µmol m-2 s-1 light intensity, and on a 10:14 (L:D) photoperiod. Approximately twice a week the plants were watered as needed and fertilized every two weeks (21-7-7, Miracle Gro).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using a modified TRIZOL extraction method as follows. Approximately 1 g of plant material was ground in liquid nitrogen using a mortar and pestle, resuspended in 15 ml TRIZOL reagent (Invitrogen, Carlsbad CA, USA), vortexed and incubated at 65°C for 5 min with regular mixing. Cell debris was pelleted by centrifugation for 30 min at 12 000 g and 4°C and the supernatant was extracted with 3 ml chloroform twice. After centrifugation for 20 min at 12 000 g, the aqueous phase was recovered and RNA was precipitated at room temperature for 5 min with 0.5 volumes of 0.8 m sodium citrate and 0.5 volumes of isopropanol. After centrifugation for 30 min at 12 000 g, the pellet was washed with 70% ethanol and re-centrifuged. The RNA pellet was air dried for 5 min and resuspended in 200 μl RNAse free water.
Label
Cy3
Label protocol
Total RNA (80 μg) was incubated with 0.27 μm T17VN primer, 0.15 mm dATP, dCTP, and dGTP, 0.05 mm dTTP (Invitrogen), 0.025 mm Cyanidin3- or Cyanidin5-conjugated dUTP (Amersham, Piscataway, NJ, USA), 40 U RNAseInh (Promega, San Luis Obispo, CA, USA), and 400 U SuperscriptII (Invitrogen) in 10 mm DTT and 1x first strand buffer in a total volume of 40 μl. In addition, 0.3 fmol human cRNAs complementary to the human negative control oligonucleotides were used in labeling reactions (HsD17B1, KRT1, and MB). Prior to the addition of enzymes the solution was heated to 65°C for 5 min and for primer annealing cooled to 42°C. Following an incubation at 42°C for 2.5 h, the RNA was degraded with 8 μl 1 M sodium hydroxide for 15 min at 65°C, neutralized with 8 μl 1 m hydrochloric acid and buffered with 4 μl 1 m Tris, pH 7.5. Subsequently, the labeled cDNA was purified using a PCR purification kit according to the manufacturer's protocol (Qiagen, Mississauga, ON, Canada). DNA was eluted in 100 μl 10 mm Tris, pH 8.5, the two labeling reactions were combined, and 1 μl Cyanidin5-labeled GFP was added. Following an ethanol/sodium acetate precipitation the air-dried cDNA pellet was resuspended in 3 μl water, denatured at 95°C for 3 min, added to 50 μl pre-warmed array hybridization buffer no. 1 (Ambion, Austin, TX, USA), and kept at 65°C until further use.
Treatment of plants with caterpillars and aphids was conducted in separate experiments. Sampling of plants occurred several hours after removal of insect herbivores so that plant gene expression results were not confounded by insect rna or by plant gene expression elicited by the physical movement of insect removal. The caterpillar treatment scheme was designed to capture early gene expression events and minimize variation due to leaf age and amount of insect damage. Six to 10 second and third instar S. exigua and P. rapae caterpillars were allowed to feed for 2-4 hours to generate 3 'middle-aged' leaves with 10-30% leaf area removed. Caterpillars were wrangled as needed with camel hair brushes (size 0) to concentrate their feeding on 3 leaves, and leaves of control plants were jiggled with a camel-hair brush to simulate the leaf movement caused by wrangling. Once sufficient damage was achieved, caterpillars were removed and the plants were returned to the growth chamber. The mechanical wounding treatment was designed to mimic insect damage to tissues. Mechanical damage was inflicted by running a sterile pattern wheel across either side of the midrib of 6 leaves on each plant, once at the beginning of the caterpillar wrangling and once half way through the caterpillar wrangling period. Control plants were jiggled with a camel-hair brush to simulate the leaf movement caused by mechanical wounding. Leaves were harvested for gene expression at 6 and 24 hr, with damaged (i.e. local) leaves harvested separately from size-matched undamaged (i.e. systemic) leaves. All leaves selected for treatment and harvest came from the fully-expanded 'middle-aged' leaf and leaves from 2-3 plants were pooled for each of the four bioreplicates. Aphids have much smaller effects on plants than caterpillars (Mewis et al 2005, 2006) and cannot be readily contained on individual leaves. Twenty sub-adult (final instar) and adult aphids were caged on plants and allowed to feed for 1 week. Controls were caged plants without aphids. All aphids were removed and caged controls were jiggled with a camel-hair brush to simulate the leaf movement caused by caterpillar removal. Whole plants were harvested for gene expression at 6 and 24 hr after aphid removal.
Growth protocol
Insect material: The dietary specialist aphid Brevicoryne brassicae (L.) and the dietary generalist aphid Myzus persicae (Sulzer) were maintained as plant virus free clones on pak-choi plants. For chewing insects we selected the dietary specialist caterpillar Pieris rapae L. and the dietary generalist caterpillar Spodoptera exigua Hübner. Eggs of S. exigua were obtained from Benzon Research (Carlisle, PA) and larvae were kept on commercially available artificial Spodoptera diet (Bioserv, Frenchtown, NJ, USA). Eggs of P. rapae were purchased form the Carolina Biological Supply Service (North Carolina) and reared on pak-choi. Both caterpillar species were transferred to Col-0 WT plants one day before the experiments. Plant material: Ecotype Columbia (Col-0) seeds were vernalized and sown into 6 x 5 cm pots filled with sterile Metromix 200 (Scotts company, Marysville, Ohio, USA; contains sphagnum, peat moss, and horticultural perlite). Plants were kept in growth chambers at 22 ± 1 °C, 65 ± 5 % relative humidity, at 250 µmol m-2 s-1 light intensity, and on a 10:14 (L:D) photoperiod. Approximately twice a week the plants were watered as needed and fertilized every two weeks (21-7-7, Miracle Gro).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using a modified TRIZOL extraction method as follows. Approximately 1 g of plant material was ground in liquid nitrogen using a mortar and pestle, resuspended in 15 ml TRIZOL reagent (Invitrogen, Carlsbad CA, USA), vortexed and incubated at 65°C for 5 min with regular mixing. Cell debris was pelleted by centrifugation for 30 min at 12 000 g and 4°C and the supernatant was extracted with 3 ml chloroform twice. After centrifugation for 20 min at 12 000 g, the aqueous phase was recovered and RNA was precipitated at room temperature for 5 min with 0.5 volumes of 0.8 m sodium citrate and 0.5 volumes of isopropanol. After centrifugation for 30 min at 12 000 g, the pellet was washed with 70% ethanol and re-centrifuged. The RNA pellet was air dried for 5 min and resuspended in 200 μl RNAse free water.
Label
Cy5
Label protocol
Total RNA (80 μg) was incubated with 0.27 μm T17VN primer, 0.15 mm dATP, dCTP, and dGTP, 0.05 mm dTTP (Invitrogen), 0.025 mm Cyanidin3- or Cyanidin5-conjugated dUTP (Amersham, Piscataway, NJ, USA), 40 U RNAseInh (Promega, San Luis Obispo, CA, USA), and 400 U SuperscriptII (Invitrogen) in 10 mm DTT and 1x first strand buffer in a total volume of 40 μl. In addition, 0.3 fmol human cRNAs complementary to the human negative control oligonucleotides were used in labeling reactions (HsD17B1, KRT1, and MB). Prior to the addition of enzymes the solution was heated to 65°C for 5 min and for primer annealing cooled to 42°C. Following an incubation at 42°C for 2.5 h, the RNA was degraded with 8 μl 1 M sodium hydroxide for 15 min at 65°C, neutralized with 8 μl 1 m hydrochloric acid and buffered with 4 μl 1 m Tris, pH 7.5. Subsequently, the labeled cDNA was purified using a PCR purification kit according to the manufacturer's protocol (Qiagen, Mississauga, ON, Canada). DNA was eluted in 100 μl 10 mm Tris, pH 8.5, the two labeling reactions were combined, and 1 μl Cyanidin5-labeled GFP was added. Following an ethanol/sodium acetate precipitation the air-dried cDNA pellet was resuspended in 3 μl water, denatured at 95°C for 3 min, added to 50 μl pre-warmed array hybridization buffer no. 1 (Ambion, Austin, TX, USA), and kept at 65°C until further use.
Hybridization protocol
We pre-hybridized microarray slides for 45 min at 48°C in 5x SSC, 0.1% SDS, 0.2% BSA. Slides were washed twice with water for 1 min, dipped five times in isopropanol, and spun dry in Falcon tubes at 100 g for 3 min. The hybridization solution was applied to the microarray slides and covered with untreated glass cover slips (Fisher Scientific, Nepean, ON, Canada). Arrays were incubated over night in CMT hybridization chambers (Corning, Corning, NY, USA) submerged in a water bath at 42°C with moderate vertical shaking. Hybridization chambers were disassembled and slides were washed for 15 min at 42°C in 2x SSC, 0.5% SDS, and for two times 15 min in 0.5x SSC, 0.5% SDS. Subsequently, arrays were dipped five times in 0.1x SSC and spun dry as described above.
Scan protocol
Microarrays were scanned with a ScanArray Express (Perkin-Elmer, Woodbridge, ON, Canada) scanner with laser power set to 95% and photo-multiplier tube set to 64–77%. We identified and quantified spots using the ImaGene software (BioDiscovery, Marina Del Rey, CA, USA). Grids were manually placed and spot finding was performed using the ‘Auto adjust’ spot function repeated for three times. Spot finding was subsequently verified by visual inspection and manually adjusted when necessary. Poor spots were manually flagged (flag 1) and were not used in further data analyses.
Description
Sp_S_24h_rep3_Cy5_064
Data processing
The median pixel intensities for each spot were used. Background was defined per subgrid as the mean signal intensities of the lowest 10% of all gene specific spots. This background was subtracted from the raw signal intensities of all spots in a given subgrid. All signal intensities that did not exceed background plus 3 standard deviations of the calculated background were excluded from further analysis (flooring). On average 19% of all spots (ranging from 12% to 27%) were thus excluded from further analyses as non-detectable. Background corrected signal intensities were used for Loess normalization (Yang et al., 2002), thereby generating normalized log2expression ratios comparing each treatment with the corresponding control sample.
