Function of a Mutant RPP4 in Response to Chilling Genetic Analysis Supplemental Figure S5. Expression of RPP4 in edsI, pad4, rarl, and sgt1b uble mutants, c/s 2-2 1996), Pad+-1 rage et al, 1999), rar1-20 (Muskett et al., 2002), sgt1bfefa3(Gray Supplemental Table S1 Gene-specific primers used in this study. al., 2003), sid2-2(Wildermuth et al, 2001), and mpr1 Durrant and Dong 2004). The F2 progeny were specifically genotyped Homozygosity of the chs2 g dCAPS markers and the CHS2-2F and CHS2-2R primers( Supplemental Table S1). ACKNOWLEDGMENTS Ion Leakage and Pro Content Assays ng plasmids. We thank L. Dangl, Julia De The electrolyte leakage test was performed as described al., 2002). Three-week-old plants grown in soil under no seeds<in Li, Jane E. Parker, Brain J. Staskawicz, and the ABRC for mutant ere treated at 4C for different periods of time. The percen Received April 12, 2010; accepted August 4, 2010; Published August 10, 2010 ted as the autoclaving. Pro content was measured as described by Bates LITERATURE CITED SA Measurement Aarts N, Metz M, Holub E, Staskawicz B, Daniels M, Parker JE (1998) Free SA and total SA were extracted and measured from 3-week-old plant fferent requirements for EDSI and NDRI by disease resistance gene grown at 22C or treated at 4C for 6 d as described with some modification efine at least two R gene-mediated signaling path (Liet al, 1999). The last extracted residue was dissolved in acetonitrile and Proc Natl Acad sci uSA 95: 10306-10311 analyzed by HPLC using 5% acetate(PH 3.2) as the mobile phase Ade J, DeYoung B, Golstein C, Innes Rw(2007) Indirect activation of a plant nucleotide binding site-leucine-rich repeat protein by a bacterial Analysis of Chlorophyll, and Electron Microscopy protease. Proc Natl Acad Sci USA 104: 2531-2536 Alcazar R, Garcia AV, Parker JE, Reymond M (2009)Incremental steps Total chlorophylls were determined as described previously(Huang et al., toward incompatibility revealed by Arabidopsis epistatic interaction 2009). Sections of leaf tissue were prepared for electron microscopic analysis nodulating salicylic acid pathway activation. Proc Natl Acad Sci USA as described (Huang et al, 2009) arker JE Histochemical Staining Assay regulatory role of sGTl in early r gene-mediated plant defense Science295:2077-2080 blue staining and DAB staining were performed as described Bartsch M, Gobbato E, Bednarek P, Debey S, Schultze JL, Bautor J, Parker (Bowling et al., 1997; Thordal-Christensen et al., 1997). Histochem dent ENHANCED DISEASE SuSCept- on of GUs activity was performed as described previously (Yang BILITYI signaling in Arabidopsis immunity and ce is regulated by etal,2006) enase FMO1 and the Nudix hydrolase NUDT7. Plant Cell Bates LS, Waldren RP, Teare ID(1972) Rapid determination of free proline Quantitative RT-PCR Bendahmane A, Farnham G, Moffett P, Baulcombe DC(2002)Constitutive Total RNA was isolated from 10-d-old seedli n Ms or 21-d-old gain-of-function mutants in a nucleotide binding site-leucine rich repeat seedlings in soil using TRIzol (Invitrogen) followed by treatment with RNase protein encoded at the Rx locus of 1 Plant J32:195-204 ee DNase I(Takara). Two micrograms of RNA was subjected to first-stran Bowling SA, Clarke D, Liu Y, Klessig DE, Dong X(1997) The cpr5 mutant cDNA synthesis using Moloney murine leukemia virus reverse transcriptase IPRl-dependent and NPR1-independer (Promega)and an oligo(dT) primer. The primers used for real-time PCR are sistance. Plant Cell 9: 1573-1584 sted in Supplemental Table S1. Real-time PCR was performed using SYBI Cao H, Bowling SA, Gordon AS, Dong x(1994) Characterization of an Green PCR Master Mix(Takara) Analysis was performed using the Applied abidopsis mutant that is nonresponsive to inducers of systemic ac- systems PRISM 7500 real-time PCR system. The primer efficiencies were measured and the relative expression levels were calculated as described Cao H, gla JD, Volk S, Dong x previously (Miura et al, 2007) NPRI gene that controls systemic acquired resist997)The Arabidopsis rotein containing ankyrin repeats. Cell 88: 57-6 Sequence data from this article can be found in the Arabidopsis Genome RPP4/CHS2 Century KS, Holub EB, Staskawicz BJ(1995) NDRI, a locus of Arabidopsis thaliana that is required for disease resistance to both a bacterial and a 4g16860;PAD4,At3g52430;EDS1,A3g48090;NPR1,Atlg64280;S|D2 t3g20600; RARl, At5g51700: SGT1b, At4g11260; SNC Clarke SM, Mur LA, Wood JE, Scott IM (2004) Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for ac- At4g25470CBF3,A4g25480RD29A,A5g52310COR47,Atlg20440;ZAT12, rmotolerance in Arabidopsis thaliana Plant J 38: 432-447 A55g59820; APX1, Atlg07890: CAT1, Atlg20630; FER1, At5g01600, CSDI, Clough S], Bent AF (1998)Floral dip: a simplified method for Atlg0S830; ACT2, Atg18780 ated transformation of Arabidopsis thaliana. PlantJ16: 735- Davletova S, Schlauch K, Coutu J, Mittler R(2005)The zinc-finger protein Supplemental Data Zat12 plays a central role in reactive oxygen and abiotic stress signaling The following materials are available in the online version of this article. Diener AC, Li H, Zhou w, whoriskey w], Nes WD, Fink GR Supplemental Figure Sl. The effect of light on the chs2 phenotype under transferase 1 controls the level of cholesterol in plants. Plant Cell Dinesh-Kumar SP, Tham WH, Baker B](2000)Structure-function anal Supplemental Figure S2. Expression of ROs-associated gene ic virus resistance gene N. Proc Natl Acad Sci USA 97:14789-14794 Supplemental Figure S3. Relative mRNA levels of cold-responsive genes Doherty C), Van Buskirk HA, Myers S), Thomashow MF(2009)Roles for n and freezing tolerance. Plant Cell 21: 972-984 Supplemental Figure S4. Expression of RPP4 and SNCI in chs2 under cold Dong CH, Hu X, Tang w, Zheng x, Kim YS, Lee BH, Zhu JK(2006) is critical for rNa Plant Ph Vol.154,2010 807Genetic Analysis To generate double mutants, chs2-2 was crossed to eds1-1 (Parker et al., 1996), pad4-1 (Jirage et al., 1999), rar1-20 (Muskett et al., 2002), sgt1b/eta3 (Gray et al., 2003), sid2-2 (Wildermuth et al., 2001), and npr1 (Durrant and Dong, 2004). The F2 progeny were specifically genotyped. Homozygosity of the chs2 mutation was identified using dCAPS markers and the CHS2-2F and CHS2-2R primers (Supplemental Table S1). Ion Leakage and Pro Content Assays The electrolyte leakage test was performed as described previously (Lee et al., 2002). Three-week-old plants grown in soil under normal conditions were treated at 4
C for different periods of time. The percentage of electrolyte leakage was calculated as the percentage of conductivity before versus after autoclaving. Pro content was measured as described by Bates et al. (1972). SA Measurement Free SA and total SA were extracted and measured from 3-week-old plants grown at 22
C or treated at 4
C for 6 d as described with some modifications (Li et al., 1999). The last extracted residue was dissolved in acetonitrile and analyzed by HPLC using 5% acetate (pH 3.2) as the mobile phase. Analysis of Chlorophyll, and Electron Microscopy Total chlorophylls were determined as described previously (Huang et al., 2009). Sections of leaf tissue were prepared for electron microscopic analysis as described (Huang et al., 2009). Histochemical Staining Assay Trypan blue staining and DAB staining were performed as described previously (Bowling et al., 1997; Thordal-Christensen et al., 1997). Histochem￾ical detection of GUS activity was performed as described previously (Yang et al., 2006). Quantitative RT-PCR Total RNA was isolated from 10-d-old seedlings on MS plates or 21-d-old seedlings in soil using TRIzol (Invitrogen) followed by treatment with RNase￾free DNase I (Takara). Two micrograms of RNA was subjected to first-strand cDNA synthesis using Moloney murine leukemia virus reverse transcriptase (Promega) and an oligo(dT)18 primer. The primers used for real-time PCR are listed in Supplemental Table S1. Real-time PCR was performed using SYBR Green PCR Master Mix (Takara). Analysis was performed using the Applied Biosystems PRISM 7500 real-time PCR system. The primer efficiencies were measured and the relative expression levels were calculated as described previously (Miura et al., 2007). Sequence data from this article can be found in the Arabidopsis Genome Initiative database under the following accession numbers: RPP4/CHS2, At4g16860; PAD4, At3g52430; EDS1, At3g48090; NPR1, At1g64280; SID2, At1g74710; NDR1, At3g20600; RAR1, At5g51700; SGT1b, At4g11260; SNC1, At4g16890; PR1, At2g14610; PR2, At3g57260; CBF1, At4g25490; CBF2, At4g25470; CBF3, At4g25480; RD29A, At5g52310; COR47, At1g20440; ZAT12, At5g59820; APX1, At1g07890; CAT1, At1g20630; FER1, At5g01600; CSD1, At1g08830; ACT2, At3g18780. Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. The effect of light on the chs2 phenotype under cold stress. Supplemental Figure S2. Expression of ROS-associated genes in chs2 plants under cold stress. Supplemental Figure S3. Relative mRNA levels of cold-responsive genes in chs2. Supplemental Figure S4. Expression of RPP4 and SNC1 in chs2 under cold stress. Supplemental Figure S5. Expression of RPP4 in eds1, pad4, rar1, and sgt1b mutants. Supplemental Table S1. Gene-specific primers used in this study. ACKNOWLEDGMENTS We thank Jian Hua for her helpful discussion of the manuscript and providing plasmids. We thank Jeffery L. Dangl, Julia Dewdney, Xinnian Dong, Xin Li, Jane E. Parker, Brain J. Staskawicz, and the ABRC for mutant seeds. Received April 12, 2010; accepted August 4, 2010; published August 10, 2010. LITERATURE CITED Aarts N, Metz M, Holub E, Staskawicz BJ, Daniels MJ, Parker JE (1998) Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R gene-mediated signaling pathways in Arabidopsis. Proc Natl Acad Sci USA 95: 10306–10311 Ade J, DeYoung BJ, Golstein C, Innes RW (2007) Indirect activation of a plant nucleotide binding site-leucine-rich repeat protein by a bacterial protease. Proc Natl Acad Sci USA 104: 2531–2536 Alcazar R, Garcia AV, Parker JE, Reymond M (2009) Incremental steps toward incompatibility revealed by Arabidopsis epistatic interactions modulating salicylic acid pathway activation. Proc Natl Acad Sci USA 106: 334–339 Austin MJ, Muskett P, Kahn K, Feys BJ, Jones JD, Parker JE (2002) Regulatory role of SGT1 in early R gene-mediated plant defenses. Science 295: 2077–2080 Bartsch M, Gobbato E, Bednarek P, Debey S, Schultze JL, Bautor J, Parker JE (2006) Salicylic acid-independent ENHANCED DISEASE SUSCEPTI￾BILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenase FMO1 and the Nudix hydrolase NUDT7. Plant Cell 18: 1038–1051 Bates LS, Waldren RP, Teare ID (1972) Rapid determination of free proline for water-stress studies. Plant Soil 39: 205–207 Bendahmane A, Farnham G, Moffett P, Baulcombe DC (2002) Constitutive gain-of-function mutants in a nucleotide binding site-leucine rich repeat protein encoded at the Rx locus of potato. Plant J 32: 195–204 Bowling SA, Clarke JD, Liu Y, Klessig DF, Dong X (1997) The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance. Plant Cell 9: 1573–1584 Cao H, Bowling SA, Gordon AS, Dong X (1994) Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic ac￾quired resistance. Plant Cell 6: 1583–1592 Cao H, Glazebrook J, Clarke JD, Volko S, Dong X (1997) The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell 88: 57–63 Century KS, Holub EB, Staskawicz BJ (1995) NDR1, a locus of Arabidopsis thaliana that is required for disease resistance to both a bacterial and a fungal pathogen. Proc Natl Acad Sci USA 92: 6597–6601 Clarke SM, Mur LA, Wood JE, Scott IM (2004) Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for ac￾quired thermotolerance in Arabidopsis thaliana. Plant J 38: 432–447 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacte￾rium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743 Davletova S, Schlauch K, Coutu J, Mittler R (2005) The zinc-finger protein Zat12 plays a central role in reactive oxygen and abiotic stress signaling in Arabidopsis. Plant Physiol 139: 847–856 Diener AC, Li H, Zhou W, Whoriskey WJ, Nes WD, Fink GR (2000) Sterol methyltransferase 1 controls the level of cholesterol in plants. Plant Cell 12: 853–870 Dinesh-Kumar SP, Tham WH, Baker BJ (2000) Structure-function analysis of the tobacco mosaic virus resistance gene N. Proc Natl Acad Sci USA 97: 14789–14794 Doherty CJ, Van Buskirk HA, Myers SJ, Thomashow MF (2009) Roles for Arabidopsis CAMTA transcription factors in cold-regulated gene expres￾sion and freezing tolerance. Plant Cell 21: 972–984 Dong CH, Hu X, Tang W, Zheng X, Kim YS, Lee BH, Zhu JK (2006) A putative Arabidopsis nucleoporin, AtNUP160, is critical for RNA Function of a Mutant RPP4 in Response to Chilling Plant Physiol. Vol. 154, 2010 807