At 18 hpi. Expression of B. cinerea-induced or-repressed genes was quantitated relative

At 18 hpi. Expression of B. cinerea-induced or-repressed genes was quantitated relative to control conditions (no infection), and corrected for expression of the control -actin gene. Microarray expression data were obtained from Tables 1 and 2. Error bars for qRT-PCR values are the standard deviations (n 3). hpi, hours post inoculation; At Actin2, Arabidopsis Actin2 gene. doi:10.1371/journal.pone.0125666.gPLOS ONE | DOI:10.1371/journal.pone.0125666 May 1,10 /Microarray Analysis of Arabidopsis-Stressed PlantsTable 3. Phenotypic analysis of T-DNA insertion alleles of common-regulated genes in response to B. cinerea. AGI number (probe set) a At2g33380 (255795) At3g05030 (259081) At2g39250 (267010) At5g49450 (248606) At5g48430 (248703) At5g41080 (249337) At5g19120 (249923) At3g50560 (252167) At3g50060 (252193) At4g21870 (254384) At4g01250 (255568) At1g21910 (260856) At1g24530 (265028) At2g20670 (265387) At2g26980 (266313)a bProtein/gene RD20 NHX2 SNZ BZIP1 aspartyl protease/Pepsin A30 GDPD2 aspartyl protease/Pepsin A20 SDR MYB77 HSP26.5-P WRKY22 DREB26 transducin /WD-40 repeat Unknown CIPKInsertion site Exon Exon 5′-UTR Exon Promoter Promoter Exon Exon Exon Exon Intron NA 5′-UTR NA IntronSAIL/SALK ID (stock number) SAIL_737_G01 (N876376) SALK_039611 (N657915) SALK_030031 (N668027) SALK_069489 (660942) SALK_128791 (N684580) SALK_047427 (N653183) GABI_023B01 (N402125) SAIL_424_A04 (N819551) SALK_067655 (N662814) SAIL_1284_H05 (N879227) SALK_047120 (N664590) NA SALK_039180 (N674562) NA SALK_137779 (N402125)Phenotypeb S Wt Wt Wt Wt Wt Wt Wt Wt Wt Wt ND ND WtExpression of common up-/down-regulated genes data were obtained from Table 2 of this study and [20].Wt, disease response comparable to wild-type plants; S, susceptible. SAIL_737_G01 plants show increased local susceptibility to B. cinerea (Fig 4). T-DNA insertion mutants were assayed for their disease responses at least three times. doi:10.1371/journal.pone.0125666.tBreeding Research GABI-Kat [22]; obtained from the NASC. Lines with homozygous insertions corresponding to 13 genes were isolated. The T-DNA insertion mutant lines were then challenged with B. cinerea as described [10], and a summary of the disease assay results is SP600125 cancer presented in Table 3. Most of the T-DNA mutant alleles had no detectable effect on the resistance phenotype, including insertions in NHX2, SNZ, BZIP1, GDPD2, SDR, MYB77, WRKY77, CIPK3, At5g19120, At5g48430, and At4g21870 (Table 3).The RD20 gene contributes to the plant resistance to biotic and abiotic stressesThe RD20 gene was induced by B. cinerea in inoculated wild-type plants (Table 2). In order to check the function of the RD20 gene, we isolated homozygous lines for the T-DNA insertion allele of the RD20 gene designated rd20 (SAIL_737_G01) using PCR (S2 Fig). Plants homozygous for the rd20 allele display increased susceptibility to B. cinerea infection compared with heterozygous (RD20/rd20) or wild-type plants (Fig 4A). At early stages of disease, symptoms developed as local chlorosis and necrosis on inoculated leaves of the mutant rd20. Extending the period of inoculation to 4 days, disease symptoms developed order QVD-OPH beyond the inoculated tissues. We also determined the fungal growth in planta. At 5 and 10 days post-inoculation (dpi), rd20 mutant plants exhibited more fungal biomass than the other genotypes, as assessed by accumulation of B. cinerea ActinA relative to At Actin2 (Fig 4B). To characterize the performance of rd20 plants under drought stress, 3-week-old se.At 18 hpi. Expression of B. cinerea-induced or-repressed genes was quantitated relative to control conditions (no infection), and corrected for expression of the control -actin gene. Microarray expression data were obtained from Tables 1 and 2. Error bars for qRT-PCR values are the standard deviations (n 3). hpi, hours post inoculation; At Actin2, Arabidopsis Actin2 gene. doi:10.1371/journal.pone.0125666.gPLOS ONE | DOI:10.1371/journal.pone.0125666 May 1,10 /Microarray Analysis of Arabidopsis-Stressed PlantsTable 3. Phenotypic analysis of T-DNA insertion alleles of common-regulated genes in response to B. cinerea. AGI number (probe set) a At2g33380 (255795) At3g05030 (259081) At2g39250 (267010) At5g49450 (248606) At5g48430 (248703) At5g41080 (249337) At5g19120 (249923) At3g50560 (252167) At3g50060 (252193) At4g21870 (254384) At4g01250 (255568) At1g21910 (260856) At1g24530 (265028) At2g20670 (265387) At2g26980 (266313)a bProtein/gene RD20 NHX2 SNZ BZIP1 aspartyl protease/Pepsin A30 GDPD2 aspartyl protease/Pepsin A20 SDR MYB77 HSP26.5-P WRKY22 DREB26 transducin /WD-40 repeat Unknown CIPKInsertion site Exon Exon 5′-UTR Exon Promoter Promoter Exon Exon Exon Exon Intron NA 5′-UTR NA IntronSAIL/SALK ID (stock number) SAIL_737_G01 (N876376) SALK_039611 (N657915) SALK_030031 (N668027) SALK_069489 (660942) SALK_128791 (N684580) SALK_047427 (N653183) GABI_023B01 (N402125) SAIL_424_A04 (N819551) SALK_067655 (N662814) SAIL_1284_H05 (N879227) SALK_047120 (N664590) NA SALK_039180 (N674562) NA SALK_137779 (N402125)Phenotypeb S Wt Wt Wt Wt Wt Wt Wt Wt Wt Wt ND ND WtExpression of common up-/down-regulated genes data were obtained from Table 2 of this study and [20].Wt, disease response comparable to wild-type plants; S, susceptible. SAIL_737_G01 plants show increased local susceptibility to B. cinerea (Fig 4). T-DNA insertion mutants were assayed for their disease responses at least three times. doi:10.1371/journal.pone.0125666.tBreeding Research GABI-Kat [22]; obtained from the NASC. Lines with homozygous insertions corresponding to 13 genes were isolated. The T-DNA insertion mutant lines were then challenged with B. cinerea as described [10], and a summary of the disease assay results is presented in Table 3. Most of the T-DNA mutant alleles had no detectable effect on the resistance phenotype, including insertions in NHX2, SNZ, BZIP1, GDPD2, SDR, MYB77, WRKY77, CIPK3, At5g19120, At5g48430, and At4g21870 (Table 3).The RD20 gene contributes to the plant resistance to biotic and abiotic stressesThe RD20 gene was induced by B. cinerea in inoculated wild-type plants (Table 2). In order to check the function of the RD20 gene, we isolated homozygous lines for the T-DNA insertion allele of the RD20 gene designated rd20 (SAIL_737_G01) using PCR (S2 Fig). Plants homozygous for the rd20 allele display increased susceptibility to B. cinerea infection compared with heterozygous (RD20/rd20) or wild-type plants (Fig 4A). At early stages of disease, symptoms developed as local chlorosis and necrosis on inoculated leaves of the mutant rd20. Extending the period of inoculation to 4 days, disease symptoms developed beyond the inoculated tissues. We also determined the fungal growth in planta. At 5 and 10 days post-inoculation (dpi), rd20 mutant plants exhibited more fungal biomass than the other genotypes, as assessed by accumulation of B. cinerea ActinA relative to At Actin2 (Fig 4B). To characterize the performance of rd20 plants under drought stress, 3-week-old se.