Multiple stress analysis

2.2.1.1. Plant growth

Arabidopsis seeds (wildtype Col-0) were stratied in 0.1% w/v agarose at 4ºC for 72 hours in the dark. Stratied seeds were sown in pre-watered Arabidopsis soil mix (6:1:1 ratio of Levington F2S compost:sand:vermiculate) in 4 cm pots (P24, Plank- pak). Pots were covered with cling lm and placed in a growth chamber to germinate. The covers were removed 7 days after sowing (DAS) and the seedlings thinned out so that there was one plant per pot. Plants were grown in 16 hour light conditions (120mmol photons.m−2_.s−1_{) at 20ºC, 60% humidity, and 350 ppm CO}

2. 2.2.1.2. Botrytis inoculation

Two weeks prior to use, the Botrytis cinerea pepper strain (Denby et al., 2004) spores were germinated and cultured on sterile tinned apricot halves (Tesco) in deep Petri dishes, and incubated at 25ºC in complete darkness. Prior to infection, the spores were harvested and suspended in sterile water, and ltered through glass wool cloth. The inoculum was prepared nally in half-strength sterile grape juice (Tesco) and the concentration adjusted to 100 000 spores/ml. The spore concentration was measured using a hemocytometer.

Harvested leaves were placed on 0.8% w/v plant agar (Duchefa Biochemie) in propag- ator trays. Several 0.6 ml droplets of the above inoculum were placed on each leaf (between 3-6 spots, depending on the size of the leaf) ensuring coverage of the leaf. The propagator trays were covered with lids, and incubated under the same conditions as plant growth (Section 2.2.1.1), except increasing the relative humidity to 90%. Mock

inoculations were performed by applying droplets of half-strength grape juice to the leaves.

2.2.1.3. RNA extraction

RNA extractions were performed by homogenising the leaf tissue (approximately 1 g) with 1ml TRIzol (Invitrogen) in a pre-chilled Dremel drill. Samples were incubated at room temperature for 5 minutes to allow for the dissociation of nucleoprotein complexes before the addition of 200 ml chloroform. The samples were shaken vigorously by hand for 15 seconds and incubated for a further 3 minutes at room temperature. The samples were then centrifuged at 8 000 xg for 15 minutes at 4ºC. The upper aqueous phase was transferred to a fresh 1.5 ml Eppendorf tube, followed by the addition of 500 ml isop- ropanol, and incubated for 2 hours at -20ºC. The samples were centrifuged at 8 000 xg for 20 minutes at 4ºC. The RNA pellets were washed with 1 ml 70% v/v ethanol (made up with diethylpyrocarbonate-treated [DEPC] water), followed by centrifugation at 8 000 xg for 10 minutes at 4ºC. The supernatant was completely removed and the pellets allowed to air dry for 5 minutes before re-suspension in 100ml DEPC-treated water. Each sample was puried using the RNAeasy purication kit (QIAgen), according to manufacturer's instructions. The samples were eluted in 50 mL DEPC-treated water. Total RNA concentrations were determined using a Nanodrop ND-1000 spectrophoto- meter (Thermo Scientic) using a 1 ml sample. Total RNA integrity was determined using a 2100 BioAnalyzer with the RNA 6000 Nano LabChip kit (Agilent), according to manufacturer's instructions. RNA samples were stored at -80ºC.

2.2.1.4. cDNA synthesis

Using the RNA concentrations determined from the Nanodrop above, the appropriate volume of RNA solution was used to ensure a starting quantity of 1000 ng RNA. Prior to cDNA synthesis, any DNA in the sample was removed by treating the RNA sample with 1 ml RQ1 DNaseI (Promega), 1 ml 10xRQ1 DNaseI buer, and enough water to make up the solution to 10 ml. This sample was incubated at 37ºC for 30 minutes, before the addition of 1 ml RQ1 DNase Stop Solution (Promega). The sample was incubated at 65°C for 10 minutes to inactivate the DNase.

The rst strand synthesis was performed by the addition of 1 ml 50 uM oligo(dT)18 primers and 1 ml 10 mM dNTPs to the above solution. The sample was incubated at 65ºC for 5 minutes to allow the primers to anneal to the RNA. Following the incuba- tion, 4 ml 5X First Strand Buer (Invitrogen), 2 ml 0.1 M DTT (Invitrogen) and 1 ml RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) was added to the sample, and incubated at 42ºC for 2 minutes. Finally 1 ml SuperScript II Reverse Transcriptase (Invitrogen) was added, and allowed to incubate at 42ºC for 50 minutes, followed by 70ºC for 15 minutes to inactivate the enzyme. cDNA samples were stored at -20ºC.

2.2.1.5. qPCR analysis

Primers for the qPCR analysis were designed using Primer3Plus (Untergasser et al., 2007), using the qPCR setting. The cDNA samples were 10x diluted prior to the qPCR analysis and the qPCR was performed on a CFX384 Touch Real-Time PCR Detection platform (Bio-Rad). One 384 well qPCR plate was used for each gene being analysed. Each sample was made up of 1 ml cDNA template, 0.5 ml each of the forward and re- verse primers (5 mM) for the gene of interest, 5 ml SsoAdvanced SYBR Green Supermix (Bio-Rad), and 3 ml water. Each sample was performed in triplicate as technical rep- licates. For each biological replicate, the position of each harvest on the qPCR plate was randomised, and within each of the harvests, the position of the treatments were randomised. A no-template control was included for each replicate to ensure no con- tamination was present.

The qPCR run was set at 95ºC for 3 minutes, 45 cycles of 95ºC for 10 seconds, and 55ºC for 30 seconds. After each of these cycles, the uorescence in each well was de- termined. At the end of the run, a melt curve analysis was performed by applying 95ºC for 10 seconds, and then running a temperature gradient from 65ºC to 95ºC in 0.5ºC increments every 5 seconds. The uorescence was measured after each temperature change.

2.2.1.6. Analysis of qPCR data

Following the analysis of the plates, the threshold cycle number (Ct) was calculated using the qpcR package in R (Ritz and Spiess, 2008). This package allowed for the dynamic calculation of these values, instead of a simple threshold. In this package, a four-parameter logistic curve was tted to the uorescence curves, and any reactions which failed this tting process were discarded as poor or incomplete reactions. Using these tted curves, it was possible to calculate the Ct values by determining the max- imum of the second derivative of the tted curve (Luu-The et al., 2005). The three technical replicates were tted simultaneously to provide an overall level of condence of the biological replicate. In addition, the package was used to calculate the eciencies of the primers for each reaction.

The relative dierence in gene expression was calculated as the dierence between the Ct value of the gene of interest, and the Ct of the reference gene. Thus

∆Ct=Ctgene−Ctref erence.