Oculated (3 L) with B. cinerea spore suspension (3×105 spores mL-1) respectively, as described previously [10]. Control plants were sprayed with 1 Sabouraud maltose broth buffer using a Preval sprayer (Valve Corp., Yonkers, NY, USA). Plants were further kept under a sealed transparent cover to maintain high humidity in a growth chamber with 21 day/18 night temperature and a 12-h light/12-h dark photoperiod cycle. Responses to B. cinerea infection were assayed at 18 hpi of leaves, unless otherwise stated. The drought sensitivity assay was performed on 3-week-old well-watered plants that were planted in soil. Seedlings were kept in a growth chamber under the same conditions mentioned above without watering (drought stress) for 10 days. Survival rates were scored 3 days after rewatering. Control plants were well-watered and kept under the same conditions.Identification of T-DNA insertion linesT-DNA insertion lines were identified as described previously [22]. PCR primers were designed to the Quisinostat biological activity Arabidopsis genomic sequence flanking the T-DNA insertion site. These primers were used to analyze 12 sibling plants from each T-DNA line to confirm the T-DNA insertionPLOS ONE | DOI:10.1371/journal.pone.0125666 May 1,3 /Microarray Analysis of Arabidopsis-Stressed Plantscosegregated with the mutant phenotype. The primers were also used for genotyping individual lines within a segregating population to identify individuals homozygous for the insertion allele. A combination of one genomic primer plus a T-DNA insert primer was used to detect the insertion allele. Two genomic primers were used together to detect the wild-type allele. rd20 (SAIL_737_G01; stock number N876376) was obtained from the Nottingham Arabidopsis Stock Centre (NASC, Nottingham, UK). The T-DNA insertion in the rd20 mutant was confirmed by PCR using a T-DNA-specific primer (LB2, 50 -GCTTCCTATTATATCTTCCC AAATTACCAATACA-30 ) and an RD20-specific primer (RP, 50 -AAGTACGGAACGATTTG GAGG-30 ). Homozygous rd20 mutant plants were identified by PCR using a pair of primers corresponding to sequences flanking the T-DNA insertion (LP, 50 -TTAACCGTTAGCGCG TATTTG-30 ; RP).RNA ZM241385 manufacturer extraction and expression analysisRNA extraction and qRT-PCR expression analyses were performed as described previously [10]. The qRT-PCR was performed using gene-specific primers, with Arabidopsis Actin2 (AtActin2) as an endogenous reference for normalization. Expression levels were calculated by the comparative cycle threshold method, and normalization to the control was performed as described [23]. Primer sequences are found in S1 Table.Statistical analysisFor each sample, three technical replicates of the qRT-PCR assay were used with a minimum of three biological replicates. Results were expressed as means ?standard deviation (SD) of the number of experiments. A Student’s t-test for the values was performed at P < 0.05. Data of B. cinerea growth in inoculated plants represent the mean ?SD from a minimum of 16 plants. Data of drought sensitivity assay performed on plants represent the mean ?SD (n = 12). Analysis of variance and Duncan's multiple range test were performed to determine the statistical significance [24]. Mean values followed by an asterisk are significantly different from the corresponding control (P < 0.05). All experiments were carried out in triplicate with similar results.Heat, salinity and osmotic stress treatmentsWe analyzed data from a previous study on the responses of Arabidopsis to various stress c.Oculated (3 L) with B. cinerea spore suspension (3x105 spores mL-1) respectively, as described previously [10]. Control plants were sprayed with 1 Sabouraud maltose broth buffer using a Preval sprayer (Valve Corp., Yonkers, NY, USA). Plants were further kept under a sealed transparent cover to maintain high humidity in a growth chamber with 21 day/18 night temperature and a 12-h light/12-h dark photoperiod cycle. Responses to B. cinerea infection were assayed at 18 hpi of leaves, unless otherwise stated. The drought sensitivity assay was performed on 3-week-old well-watered plants that were planted in soil. Seedlings were kept in a growth chamber under the same conditions mentioned above without watering (drought stress) for 10 days. Survival rates were scored 3 days after rewatering. Control plants were well-watered and kept under the same conditions.Identification of T-DNA insertion linesT-DNA insertion lines were identified as described previously [22]. PCR primers were designed to the Arabidopsis genomic sequence flanking the T-DNA insertion site. These primers were used to analyze 12 sibling plants from each T-DNA line to confirm the T-DNA insertionPLOS ONE | DOI:10.1371/journal.pone.0125666 May 1,3 /Microarray Analysis of Arabidopsis-Stressed Plantscosegregated with the mutant phenotype. The primers were also used for genotyping individual lines within a segregating population to identify individuals homozygous for the insertion allele. A combination of one genomic primer plus a T-DNA insert primer was used to detect the insertion allele. Two genomic primers were used together to detect the wild-type allele. rd20 (SAIL_737_G01; stock number N876376) was obtained from the Nottingham Arabidopsis Stock Centre (NASC, Nottingham, UK). The T-DNA insertion in the rd20 mutant was confirmed by PCR using a T-DNA-specific primer (LB2, 50 -GCTTCCTATTATATCTTCCC AAATTACCAATACA-30 ) and an RD20-specific primer (RP, 50 -AAGTACGGAACGATTTG GAGG-30 ). Homozygous rd20 mutant plants were identified by PCR using a pair of primers corresponding to sequences flanking the T-DNA insertion (LP, 50 -TTAACCGTTAGCGCG TATTTG-30 ; RP).RNA extraction and expression analysisRNA extraction and qRT-PCR expression analyses were performed as described previously [10]. The qRT-PCR was performed using gene-specific primers, with Arabidopsis Actin2 (AtActin2) as an endogenous reference for normalization. Expression levels were calculated by the comparative cycle threshold method, and normalization to the control was performed as described [23]. Primer sequences are found in S1 Table.Statistical analysisFor each sample, three technical replicates of the qRT-PCR assay were used with a minimum of three biological replicates. Results were expressed as means ?standard deviation (SD) of the number of experiments. A Student's t-test for the values was performed at P < 0.05. Data of B. cinerea growth in inoculated plants represent the mean ?SD from a minimum of 16 plants. Data of drought sensitivity assay performed on plants represent the mean ?SD (n = 12). Analysis of variance and Duncan's multiple range test were performed to determine the statistical significance [24]. Mean values followed by an asterisk are significantly different from the corresponding control (P < 0.05). All experiments were carried out in triplicate with similar results.Heat, salinity and osmotic stress treatmentsWe analyzed data from a previous study on the responses of Arabidopsis to various stress c.