Huang et al. 2008); in contrast, reduced ATP hydrolysis with nor- the signaling components required for the tempera- mal atP binding can result in constitutive activation ture sensitivity of chs2 mutants show similarities and of some R proteins( Takken et al. 2006; Ade et al., 2007, differences with those required for RPP4 function in van Ooij jen et al., 2008). We hypothesize that the chs pathogen resistance. The different genetic requirement mutation might interfere with ATP hydrolysis, thus of chs2 and RPP4 might be due to the nature of the causing a gain-of-function activity. It is possible that mutation in the CHS2 protein. The molecular mecha- low temperature induces a conformational change nism by which chs2 regulates temperature-dependent within chs2, resulting in an active signaling state (on cell death is still unknown, and the subcellular local- state) under cold conditions. In accordance with this ization of rPp4 or CHS2 remains unclear. Further study, a number of mutants with deregulated R-like study on the protein localization, protein activities, proteins have been shown to have temperature- and suppressors of chs2 will shed more light on the dependent autoimmune responses(Xiao et al. 2003; function of RPP4 in the regulation of temperature- Yang and Hua, 2004; Zhou et al., 2008; Alcazar et al., dependent cell death and the interconnected mecha 2009) nisms of cold stress and defense signalin Temperature Sensitivity and R Genes MATERIALS AND METHODS In-O enes con Plant Material and Growth Conditions temperature sensitivity. However, their temperature- sensitive ranges can be different. RPP4 and SNCI are lewskija were used in this study. The chs 2-1 and chs2-2(Schneider et al, 1995) highly homologous in their predicted amino acid mutants were obtained from the Arabidopsis Biological Resource Center equences; in addition, their gene structures are ver imilar,including their position at the rPP5 locus and under a long-day (16 h of light/8 h of dark)photoperiod at 100 umol their numbers of exons and introns (van der biezen with 50% to 70% relative humidity in soil or on Ms medium (Sigma et aL, 2002 ). A gain-of-function snc1-1 mutant shows a containing 2%o Suc and 0. 8% agar growth-defective phenotype and activated defense responses at 22 C but not at 28C(Yang and Hua, Genetic Mapping and Cloning of the CHS2 Gene 2004). Nevertheless, snc1-1 can survive and set seeds even at temperatures of 4C to 22 C. In contrast, chs The chts2-2 seeds were treated with 0.3% EMS for 8 h. Approximately 20,000 M2 Plants(derived from 5,000 M1 seeds) were screened at 4C for chs2 shows obvious defense activation at 16C to 18 C and mutants with a wild-type phenotype is lethal at temperatures below 12%C. As these R or To map the chs2-2 mutation, a homozygous chs2-2 mutant(Col back R-like genes share downstream signaling components round) was crossed with LeT The FI plants from the cross were self-fertilized such as EDSI (Li et al., 2001), the temperature sensi- and the resulting F2 seeds were collected. The segregating F2 populati seedlings with a wild-type phenotype were used for mapping. A total of 3,000 tivity likely comes from R genes, as different mutants F2 Plants were selected. Genomic DNA from these F2 plants was extracted have different ranges of temperature sensitivity. This d used for PCR-based was demonstrated recently by altering the tempera- Additional ture sensitivity of defense responses through manip- tions/deletions identified from the Cereon Arabidopsis polymorphism and ulatingRgenesSpecificmissensemutationsinSncILersequencecollection(www.arabidopsis.org).genomicDnacorresponding and N genes could retain defense responses normally to candidate genes was PCR amplified from the mutant and sequenced to inhibited at elevated temperatures, and additional missense mutations in the SNCl protein reverse the To map chs2-s1 mutations, the F2 populations were derived from genetic rossing between the mutants(in Col) and Ler. Bulked segregation analysis temperature sensitivity of defense responses(Zhu was performed with simple sequ length polymorphism, cleaved-ampli et al. 2010). Thus, differences in temperature sensitiv- fied polymorphic sequence, and dCAPS markers. ity and sensitivity range are most likely due to varying temperature sensitivity in r protein, and different Plasmid Construction and plant Transformation forms of NB-LRR proteins mediate temperature sen- tivity in plant s by conformation- A 12-kb Pstl genomic fragment containing the RPP4 promoter and cod ally transitioning between off and on states( (Zhu et al,mmwa出和mk厘1 artificial chromosome clone F5D3(A 2010) construct.A 1.0-kb EcoRV-EcoRI genomic fragment containing the ca e RPP4 Regulates Cold Response and Defense Responses Da was amplified by PCR using CHS2-IF and CHS2-1R from the ger of chs2 plants and used to replace the wild-type fragment in RPP4: RPP4 via Both Common and Distinct Signaling Mediators to generate the An 8.3-kb genomic fragment containin Previous studies show that rPp confers resistance translated region from RPP4: RPP4 was cloned into the binary vector to H parasitica, which requires the action of multiple PGreen-0229(Hellens et al. 2000)to generate the 35S: RPP4 construct. For the CHS2 Gus fusion, a 1.46-kb genomic fragment upstream of the NPRI NDRI PAL, PBS2, PBS3, SGT1b, RARI RPS5. PIR primers(Supplemental Table SI)and fused with the GUS reporter gene in SIDI, SID2, and SA. In this study, we found that chs 2 is S2(Diener et al., 2000) dependent on EDSI, SGTI, and RARI but is indepen- used r trachesform tumefaciems strain Gvs3101 carrying different constructs was Plant Physiol. Vol. 154, 20102008); in contrast, reduced ATP hydrolysis with nor￾mal ATP binding can result in constitutive activation of some R proteins (Takken et al., 2006; Ade et al., 2007; van Ooijen et al., 2008). We hypothesize that the chs2 mutation might interfere with ATP hydrolysis, thus causing a gain-of-function activity. It is possible that low temperature induces a conformational change within chs2, resulting in an active signaling state (on state) under cold conditions. In accordance with this study, a number of mutants with deregulated R-like proteins have been shown to have temperature￾dependent autoimmune responses (Xiao et al., 2003; Yang and Hua, 2004; Zhou et al., 2008; Alcazar et al., 2009). Temperature Sensitivity and R Genes Many gain-of-function mutations of R genes confer temperature sensitivity. However, their temperature￾sensitive ranges can be different. RPP4 and SNC1 are highly homologous in their predicted amino acid sequences; in addition, their gene structures are very similar, including their position at the RPP5 locus and their numbers of exons and introns (van der Biezen et al., 2002). A gain-of-function snc1-1 mutant shows a growth-defective phenotype and activated defense responses at 22
C but not at 28
C (Yang and Hua, 2004). Nevertheless, snc1-1 can survive and set seeds even at temperatures of 4
C to 22
C. In contrast, chs2 shows obvious defense activation at 16
C to 18
C and is lethal at temperatures below 12
C. As these R or R-like genes share downstream signaling components such as EDS1 (Li et al., 2001), the temperature sensi￾tivity likely comes from R genes, as different mutants have different ranges of temperature sensitivity. This was demonstrated recently by altering the tempera￾ture sensitivity of defense responses through manip￾ulating R genes. Specific missense mutations in SNC1 and N genes could retain defense responses normally inhibited at elevated temperatures, and additional missense mutations in the SNC1 protein reverse the temperature sensitivity of defense responses (Zhu et al., 2010). Thus, differences in temperature sensitiv￾ity and sensitivity range are most likely due to varying temperature sensitivity in R protein, and different forms of NB-LRR proteins mediate temperature sen￾sitivity in plant immune responses by conformation￾ally transitioning between off and on states (Zhu et al., 2010). RPP4 Regulates Cold Response and Defense Responses via Both Common and Distinct Signaling Mediators Previous studies show that RPP4 confers resistance to H. parasitica, which requires the action of multiple signaling components including DTH9, EDS1, PAD4, NPR1, NDR1, PAL, PBS2, PBS3, SGT1b, RAR1, RPS5, SID1, SID2, and SA. In this study, we found that chs2 is dependent on EDS1, SGT1, and RAR1 but is indepen￾dent of PAD4, NPR1, and SA. This result indicates that the signaling components required for the tempera￾ture sensitivity of chs2 mutants show similarities and differences with those required for RPP4 function in pathogen resistance. The different genetic requirement of chs2 and RPP4 might be due to the nature of the mutation in the CHS2 protein. The molecular mecha￾nism by which chs2 regulates temperature-dependent cell death is still unknown, and the subcellular local￾ization of RPP4 or CHS2 remains unclear. Further study on the protein localization, protein activities, and suppressors of chs2 will shed more light on the function of RPP4 in the regulation of temperature￾dependent cell death and the interconnected mecha￾nisms of cold stress and defense signaling. MATERIALS AND METHODS Plant Material and Growth Conditions Arabidopsis (Arabidopsis thaliana) plants of the accessions Col and Wassi￾lewskija were used in this study. The chs2-1 and chs2-2 (Schneider et al., 1995) mutants were obtained from the Arabidopsis Biological Resource Center (ABRC; stock nos. CS6298 and CS6299). Plants were grown at 22
C or 4
C under a long-day (16 h of light/8 h of dark) photoperiod at 100 mmol m22 s 21 with 50% to 70% relative humidity in soil or on MS medium (Sigma) containing 2% Suc and 0.8% agar. Genetic Mapping and Cloning of the CHS2 Gene The chs2-2 seeds were treated with 0.3% EMS for 8 h. Approximately 20,000 M2 plants (derived from 5,000 M1 seeds) were screened at 4
C for chs2-s mutants with a wild-type phenotype. To map the chs2-2 mutation, a homozygous chs2-2 mutant (Col back￾ground) was crossed with Ler. The F1 plants from the cross were self-fertilized, and the resulting F2 seeds were collected. The segregating F2 population seedlings with a wild-type phenotype were used for mapping. A total of 3,000 F2 plants were selected. Genomic DNA from these F2 plants was extracted and used for PCR-based mapping with simple sequence length polymor￾phism and derived cleaved-amplified polymorphic sequence (dCAPS) markers. Additional mapping markers were developed based on inser￾tions/deletions identified from the Cereon Arabidopsis polymorphism and Ler sequence collection (www.arabidopsis.org). Genomic DNA corresponding to candidate genes was PCR amplified from the mutant and sequenced to identify the mutation. To map chs2-s1 mutations, the F2 populations were derived from genetic crossing between the mutants (in Col) and Ler. Bulked segregation analysis was performed with simple sequence length polymorphism, cleaved-ampli- fied polymorphic sequence, and dCAPS markers. Plasmid Construction and Plant Transformation A 12-kb PstI genomic fragment containing the RPP4 promoter and coding region was cloned from bacterial artificial chromosome clone F5D3 (ABRC) into the binary vector pCAMBIA1300 (CAMBIA) to generate the RPP4:RPP4 construct. A 1.0-kb EcoRV-EcoRI genomic fragment containing the chs2 mu￾tation was amplified by PCR using CHS2-1F and CHS2-1R from the genomic DNA of chs2 plants and used to replace the wild-type fragment in RPP4:RPP4 to generate the CHS2:chs2 construct. An 8.3-kb genomic fragment containing the RPP4 coding region and 3# untranslated region from RPP4:RPP4 was cloned into the binary vector pGreen-0229 (Hellens et al., 2000) to generate the 35S:RPP4 construct. For the CHS2:GUS fusion, a 1.46-kb genomic fragment upstream of the CHS2 ATG start codon was amplified by PCR using the CHS2-p1F and CHS2- p1R primers (Supplemental Table S1) and fused with the GUS reporter gene in the binary vector pZPGUS2 (Diener et al., 2000). Agrobacterium tumefaciens strain GV3101 carrying different constructs was used to transform wild-type (Col) plants via floral dip transformation (Clough and Bent, 1998). Huang et al. 806 Plant Physiol. Vol. 154, 2010