Two days before the onset of the experiment, plants were transferred to a climate-controlled room (22ºC, 50-60% humidity, 12 hr photoperiod). As control, Arabidopsis plants were maintained under the same condition without exposure to DBM larvae. All rosette leaves were harvested at 4h after onset of the experiment and flash frozen in liquid nitrogen.
Growth protocol
Arabidopsis thaliana plants (ecotype Landsberg erecta) were grown in plastic pots (10cm wide x 8cm tall) containing Terra-lite Redi-earth (W.R. Grace and Co., Ajax, Ontario, Canada). Each pot contained four or five plants, which were grown in a growth chamber (Controlled Environment Ltd., Winnipeg, MB, Canada) at 20˚C constant temperature, 8 hr/16 hr Light/Dark photoperiod at 50-60% ambient humidity, for 8 to 9 weeks. Short day conditions prevented the onset of flowering and the plants were thus maintained in growth stage 1 (leaf production) with 13 to 15 rosette leaves larger than 1mm (stage 1.13 to 1.14).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using a modified TRIZOL extraction method as follows. Approximately 1g 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 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. Following a spectrophotometric determination of RNA concentration, the RNA was precipitated with 2.5 volumes of ethanol and a 1/10 volume of 3 M sodium acetate at –20°C overnight, and subsequently pelleted at 20,000 g for 30 min at 4°C. The precipitate was washed with 70% ethanol, re-centrifuged, air dried and resuspended in RNAse free water to an approximate concentration of 5 μg/μl. Actual concentration was determined spectrophotometrically, and RNA quality was determined using a 2100 Bioanalyzer (Agilent Technologies, Mississauga ON, Canada).
Label
Cy5
Label protocol
Total RNA was used for a direct labelling procedure. 80 μg total RNA 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 1 x first strand buffer in a total volume of 40 μl. In addition, 0.3 fmole human cRNAs complementary to the human negative control oligonucleotides were used in labelling reactions (HsD17B1, KRT1, and MB). Prior to 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 1M Tris-pH 7.5. Subsequently, the labelled 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 1μl Cyanidin5-labelled GFP was added. Following an ethanol/sodium acetate precipitation (Sambrook and Russel, 2001) 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 #1 (Ambion, Austin, TX, USA), and kept at 65°C until use.
Channel 2
Source name
whole rosette, treated with diamond back moth (Plutella xylostella), 4h, rep 4
Two days before exposing A. thaliana plants to herbivore treatment, plants were transferred to a climate-controlled room (22ºC, 50-60% humidity, 12 hr photoperiod). For insect treatment, seven Diamondback moth (DBM, Plutella xylostella) larvae (third to fifth instars) were placed on a group of four or five plants until time of harvest, for each time point separately. All rosette leaves were harvested at 4h after onset of continuous herbivory and flash frozen in liquid nitrogen.
Growth protocol
Arabidopsis thaliana plants (ecotype Landsberg erecta) were grown in plastic pots (10cm wide x 8cm tall) containing Terra-lite Redi-earth (W.R. Grace and Co., Ajax, Ontario, Canada). Each pot contained four or five plants, which were grown in a growth chamber (Controlled Environment Ltd., Winnipeg, MB, Canada) at 20˚C constant temperature, 8 hr/16 hr Light/Dark photoperiod at 50-60% ambient humidity, for 8 to 9 weeks. Short day conditions prevented the onset of flowering and the plants were thus maintained in growth stage 1 (leaf production) with 13 to 15 rosette leaves larger than 1mm (stage 1.13 to 1.14). Diamondback moth (DBM, Plutella xylostella) larvae were maintained on cabbage (Brassica oleracea) plants in a climate-controlled room at 25ºC, 12 hr photoperiod with 50%-60% relative humidity
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using a modified TRIZOL extraction method as follows. Approximately 1g 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 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. Following a spectrophotometric determination of RNA concentration, the RNA was precipitated with 2.5 volumes of ethanol and a 1/10 volume of 3 M sodium acetate at –20°C overnight, and subsequently pelleted at 20,000 g for 30 min at 4°C. The precipitate was washed with 70% ethanol, re-centrifuged, air dried and resuspended in RNAse free water to an approximate concentration of 5 μg/μl. Actual concentration was determined spectrophotometrically, and RNA quality was determined using a 2100 Bioanalyzer (Agilent Technologies, Mississauga ON, Canada).
Label
Cy3
Label protocol
Total RNA was used for a direct labelling procedure. 80 μg total RNA 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 1 x first strand buffer in a total volume of 40 μl. In addition, 0.3 fmole human cRNAs complementary to the human negative control oligonucleotides were used in labelling reactions (HsD17B1, KRT1, and MB). Prior to 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 1M Tris-pH 7.5. Subsequently, the labelled 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 1μl Cyanidin5-labelled GFP was added. Following an ethanol/sodium acetate precipitation (Sambrook and Russel, 2001) 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 #1 (Ambion, Austin, TX, USA), and kept at 65°C until use.
Hybridization protocol
We pre-hybridized microarray slides for 45 min at 48°C in 5 x SSC, 0.1 % SDS, 0.2 % BSA. Slides were washed twice with water for 1 min, dipped 5 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 2 x SSC, 0.5 % SDS, and for 2 times 15 min in 0.5 x SSC, 0.5 % SDS. Subsequently, arrays were dipped five times in 0.1 x 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 54 to 64. 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. For all analyses, the median pixel intensities for each spot were used.
Description
Arabidopsis thaliana Ler plants (growth stage 1) were exposed to Diamondback moth (DBM, Plutella xylostella) larvae for 1h (continuous feeding experiment). Control plants were harvested at the same time point. Whole rosettes were used for RNA isolation, labelling and array This file describes results for replicate 4 of 4 comparing DBM treatment for 4h (Channel 2, Cy3) with control rosettes (Channel 1, Cy5)
Data processing
For all analyses, the median pixel intensities for each spot were used. Analyses were performed using customized scripts for R and Bioconducter. For background correction, we defined the mean of the lowest 10% of spot intensities from a particular subgrid as the background for that subgrid. This mean was subtracted from each spot in the subgrid. Signal intensities that did not exceed the background plus 3 standard deviations thereof were defined as not detectable and were excluded from further analyses. We normalized using loess curves thus generating log2 transformed expression ratios comparing DBM treatment with control.
