Biscutella environmental stress experiment RNAseq methods

Plant material One individual (termed “original individual” henceforth) of Biscutella laevigata subsp. austriaca (Brassicacea) (2n = 2x = 18) was grown under standardized greenhouse conditions (16h/8h light/dark, 22-26°C/16-18°C, 65% relative air humidity, 20-40kLux) for over a year. This individual stems from the alpine population of Schneeberg in Austria (ca. 1800m a.s.l.; GPS: 47.697209, 15.609734). From this individual a number of cuttings, including root and several leaves, where made and regenerated for four weeks at 12h/12h light/dark, 24°C day and 18°C night temp. and 65% relative air humidity. These clones were then repotted into larger pots, transferred back to the greenhouse described above and kept there until they formed new leaves after an additional 6 weeks. This way one genotype can be cloned and multiplied with a survival rate of 50%-75% of all cuttings. All plants involved in the stress experiment were such 10-week old clones. Experiment design The time-frame of the experiment can be divided into three phases, acclimation, treatment and harvest. Acclimation started when all clones involved in the experiments were relocated to the growth chamber under control conditions. This phase lasted for seven days (until day 7 of the experiment). The treatment phase started after seven days of acclimation, when the watering for the drought treated clones was stopped. 10 days later (day 17), the drought treated clones started to show signs of drought stress (wilting leaves on at least three clones). The same day at around noon (day 17), after first signs of drought stress had been observed, the second longest treatment (herbivory, 30h) was started. By placing eight Plutella xylostella third- to fifth-instar larvae (6-9mm in length, after Ehlting et al. 20081) onto one mature leaf per clone. The cold treatment was initiated at the same day (day 17) at 6pm, by relocating clones to the cold room onto a lamp shelf. The Cold treatment was ended at 6pm of day 18 at the onset of the harvesting phase. The Heat treatment was started at 9am the next day (day 18), by relocating the clones to a Percival initially set to control conditions. Over 3h the temperature was raised to 42°C, where the clones remained for 6h until 6pm of day 18 when the harvest phase began. Harvest phase started at 6pm of day 18, when plants were harvested within half an hour. Harvesting comprised of sampling stressed leaves (wilting and fed-on leaves for drought and herbivory respectively, fully expanded leaves for control, cold and heat) and snap freezing them in liquid nitrogen, still under the corresponding treatment conditions. The experiment was terminated by relocating all clones back to the growth chamber under control conditions, where they were regenerated. Control treatment After stabilizing the plants at standard greenhouse conditions (16h/8h light/dark, 22-26°C/16-18°C, 65% relative air humidity, 20-40kLux), they were relocated to a growth chamber, where they acclimatized to control conditions for 7 days. Conditions in this growth chamber were 16h/8h, 22°C, 45% relative air humidity, 100-120uM PAR and daily watering, no fertilizer and no pesticides were applied. Cold treatment The cold treatment consisted of relocating five clones to a 4°C-room for 24h (starting at 6pm of day 17), where they rested on a lamp-shelf and experienced a lower amount of light (70-90uM PAR) as compared to the control condition. The light/dark-cycle was maintained at 16h/8h, as in the control conditions. It is noteworthy that for the alpine B. laevigata subsp. austriaca genotype used in these treatments, 24h of 4°C treatment represents only mild cold stress. Drought treatment The drought treatment was conducted in the same growth chamber as the control and herbivory treatments, with the only difference being the watering regime. To achieve a drought stress, we stopped watering for a total of 10 days, which was when the first signs of wilting leaves appeared on at least three of the five drought-treated clones. Leaves were harvested an additional 1.5 days later, at the same time as the other treatments were terminated. Heat treatment For the heat treatment, five clones were relocated to a growth chamber with initially the same temperature and light settings of 22°C and 100-120µM, as in the growth chamber under control conditions. In order to not apply a direct heat shock, the temperature was raised gradually for 3h until a maximum of 42°C had been reached, where the plants remained for 6h. Larkindale and colleagues have shown that a gradual increase in temperature, as compared to heat shock, or large (>8°C) step-wise increments of temperature, leads to higher-fold transcriptome changes (Larkindale et al., 20082). Herbivory treatment The herbivory treatment was conducted in the growth chamber under control conditions. We applied eight larva of the Diamondback Moth (Plutella xylostella) onto one mature leaf of each of the five clones, for 30h before onset of the harvesting phase. The herbivory treated leaf of each clone was encapsulated with a small transparent plastic cage, wherein the eight larvae were held. At the onset of the harvesting phase, visible feeding damage had occurred on all the treated leaves for about 10-20% of the encapsulated leaf area. RNA extractions and sequencing RNA was extracted from 50-100mg of snap frozen leaf tissue, using the RNAeasy mini kit (QIAGEN, cat. no. 74104) following the manufacturers protocol. RNA was quantified with the Qubit 4 fluorometer (Invitrogen) and then treated with DNAse 1 (Thermo Fisher, cat. no. EN0525). Subsequently RNA integrity was assessed using the Bioanalyzer 2100 system (Agilent), whereupon RNA extracts with a ribosomal RNA integrity number (RIN) over 7.0 or higher were considered for library preparation. Library preparation and RNA-sequencing was outsourced to the next-generation sequencing platform of the University of Bern (Bern, Switzerland). For leave tissue from the environmental stress experiment, libraries were prepared using the "TruSeq Stranded Total RNA with Ribo-Zero Plant"-kit (Illumina, Cat. No. 20020610), including ribosomal RNA depletion and a size selection step for 300bp fragments. 50bp paired-end (PE) RNA sequencing was conducted for 17 libraries (4x control, 4x cold, 3x heat, 3x drought and 3x herbivory) on two lanes of an S2 flow cell of the NovaSeq6000 system yielding approximately 1.1 billion raw PE-reads. References1. Ehlting, J. et al. Comparative transcriptome analysis of Arabidopsis thaliana infested by diamond back moth (Plutella xylostella) larvae reveals signatures of stress response, secondary metabolism, and signalling. BMC genomics 9, 154; 10.1186/1471-2164-9-154 (2008).