A missense mutation in CHS1, a TIR-NB protein, induces chilling sensitivity in Arabidopsis

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1 The Plant Journal (2013) 75, doi: /tpj A missense mutation in CHS1, a TIR-NB protein, induces chilling sensitivity in Arabidopsis Yuancong Wang 1, Yao Zhang 1, Zheng Wang 1, Xiaoyan Zhang 1 and Shuhua Yang 1,2,3, * 1 State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing , China, 2 Coordinated Research Center for Crop Biology, China Agricultural University, Beijing , China, and 3 National Plant Gene Research Center, Beijing , China Received 6 March 2013; revised 28 April 2013; accepted 2 May 2013; published online 8 May *For correspondence ( yangshuhua@cau.edu.cn). SUMMARY Low temperature is an environmental factor that affects plant growth and development and plant pathogen interactions. How temperature regulates plant defense responses is not well understood. In this study, we characterized chilling-sensitive mutant 1 (chs1), and functionally analyzed the role of the CHS1 gene in plant responses to chilling stress. The chs1 mutant displayed a chilling-sensitive phenotype, and also displayed defense-associated phenotypes, including extensive cell death, the accumulation of hydrogen peroxide and salicylic acid, and an increased expression of PR genes: these phenotypes indicated that the mutation in chs1 activates the defense responses under chilling stress. A map-based cloning analysis revealed that CHS1 encodes a TIR-NB-type protein. The chilling sensitivity of chs1 was fully rescued by pad4 and eds1, but not by ndr1. The overexpression of the TIR and NB domains can suppress the chs1 conferred phenotypes. Interestingly, the stability of the CHS1 protein was positively regulated by low temperatures independently of the 26S proteasome pathway. This study revealed the role of a TIR-NB-type gene in plant growth and cell death under chilling stress, and suggests that temperature modulates the stability of the TIR-NB protein in Arabidopsis. Keywords: CHS1, TIR-NB-type protein, chilling stress, defense responses, Arabidopsis. INTRODUCTION To survive in complex environments, plants have evolved a sophisticated mechanism to deal with the different stresses, including biotic and abiotic stresses. Plants must keep pathogens out during their whole lives. Previous studies have identified that there are at least two layers of inducible defense responses beside physical barriers (Chisholm et al., 2006; Jones and Dangl, 2006). The first is called microbial- or pathogen-associated molecular patterns (MAMPs or PAMPs)-triggered immunity (PTI). Plants trigger general but relatively weak resistance by using transmembrane pattern recognition receptors (PRRs) to sense the slowly evolving molecular patterns, called MAMPs or PAMPs. The second is termed effector-triggered immunity (ETI), which is triggered when host disease resistance (R) proteins recognize pathogen effectors. These effectors function to help the pathogens overcome the PTI and invade the hosts. Unlike PTI, ETI is specific for the pathogens whose effectors are recognized by the hosts, and triggers a much stronger resistance than is triggered The Plant Journal 2013 John Wiley & Sons Ltd by PTI (Jones and Dangl, 2006). ETI is often accompanied by: the accumulation of the defense hormone salicylic acid (SA); the induction of PATHOGENESIS-RELATED (PR) genes; the production of reactive oxygen species (ROS); and programmed cell death, termed the hypersensitive response (Hammond-Kosack and Jones, 1996). The largest class of R proteins consists of proteins containing a nucleotide-binding leucine-rich repeat (NB-LRR) domain at the C terminus and either a coiled-coil (CC) domain or Toll/Interleukin 1 receptor (TIR) domain at the N terminus (Dangl and Jones, 2001). Besides these R proteins, there are an additional 57 proteins that lack LRR domains but are considered to be related to R proteins; 49 of them contain a TIR domain at the N-terminus. These proteins are divided into two subclasses. Proteins harboring an NB domain are called TIR-NB types, and the others, which lack both an LRR and an NB domain, are called TIR X types (Meyers et al., 2002). Several key factors have been identified to be crucial to the functions of these R genes in hosts. 553

2 554 Yuancong Wang et al. R proteins belonging to the TIR class require the function of ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and PHY- TOALEXIN-DEFICIENT 4 (PAD4) (Glazebrook et al., 1996; Aarts et al., 1998), and those belonging to the CC class depend on the action of NON-RACE SPECIFIC DISEASE RESISTANCE 1 (NDR1) (Aarts et al., 1998; Glazebrook, 2001). It has been reported that the subcellular localization of some R proteins is critical for disease resistance. For instance, in order to trigger plant resistance responses, some R proteins need to accumulate in the nucleus, including N, MILDEWA 10 (MLA 10), RESISTANCE TO PSEUDO- MONAS SYRINGAE 4 (RPS4) and SUPPRESSOR OF npr1 1 CONSTITUTIVE 1 (SNC1) (Burch-Smith et al., 2007; Shen et al., 2007; Wirthmueller et al., 2007; Cheng et al., 2009). Emerging evidence has demonstrated a close link between biotic and cold stress signaling (Alcazar and Parker, 2011). Recent studies have shown that temperatures that are higher than the normal growth range can cause reduced PTI against virulent pathogens and NB-LRRconditioned ETI against avirulent pathogens (Yang and Hua, 2004; Wang et al., 2009). Enhanced disease resistance and inhibited plant growth, caused by the transcriptional accumulation of EDS1 and PAD4 in an auto-active immune background, can be suppressed by high temperatures (Yang and Hua, 2004; Rietz et al., 2011). Plant pathogen interactions have been demonstrated to be regulated differently at different temperatures (Garrett et al., 2006). The R gene Mi can confer nematode resistance to tomatoes (Solanum lycopersicum), and this resistance is lost when the temperature is above 32 C (Hwang et al., 2000). Other evidence supporting this notion includes N in tobacco (Nicotiana tabacum; Someya et al., 2004), and SSI4, RPW8 and the RPP1-like TIR-NB-LRR cluster in Arabidopsis (Xiao et al., 2003; Bomblies et al., 2007; Zhou et al., 2008; Alcazar et al., 2009). Some auto-active resistance mutants are also typically temperature-dependent. Different mutations in SNC1, chilling sensitive 2 (CHS2) and CHS3 show variant auto-active immune phenotypes in a temperature-dependent manner (Zhang et al., 2003; Yang and Hua, 2004; Huang et al., 2010; Yang et al., 2010; Zhu et al., 2010). A recent study showed that ABA-deficient mutations enhance resistance mediated by SNC1 and RPS4 through promoting their nuclear accumulation, thus suggesting an intersection between ABA and disease resistance through the localization of R proteins (Mang et al., 2012). Previous studies reported that one chs1 mutant grew normally at 22 C; however, when it was transferred to the chilling temperature the chs1 mutant showed leaf chlorosis, and did not survive (Hugly et al., 1990). Analyses of microarray data and comparative protein and lipid profiling indicate that the chs1 mutation also disrupts the chloroplast metabolism and lipid trafficking (Hugly et al., 1990; Patterson et al., 1993; Provart et al., 2003). In this report, we show that a mutated TIR-NB-type gene (CHS1) confers sensitivity to chilling temperatures. The function of the CHS1 gene in plant growth and cell death under chilling stress was investigated, and our results revealed that the accumulation of the TIR-NB protein is regulated by low temperatures in Arabidopsis. RESULTS Characterization of the chilling-sensitive mutant chs1 To better understand the molecular regulation of the plant response to chilling stress, we investigated the chs1 1 and chs1 2 mutants, which were isolated previously (Hugly et al., 1990). Because these two alleles showed similar phenotypes, we chose to examine chs1 2 in this study. The chs1 2 mutant was indistinguishable from wild-type Col plants when grown at a normal temperature (22 C; Hugly et al., 1990). However, chs1 2 showed chillingsensitive phenotypes when germinated and grown at chilling temperatures (16 C or below), or when moved from 22 to 16 C (Hugly et al., 1990) (Figure 1a,b). To better analyze the speed of death of chs1 2, compared with the wild type, the levels of chlorophyll a and b were measured in Col and chs1 2. Two-week-old chs1 2 seedlings grown at 22 C were transferred to 16 C and maintained for the indicated time. After 3 days at 16 C, the levels of chlorophyll a and b were dramatically decreased in chs1 2 (which did not show any visible defect phenotype), compared with the wild type; the levels decreased continually with the chilling treatment (Figure 1c). Because chs1 2 grown at 16 C showed a chlorotic phenotype, an ion leakage assay was performed on 2 week-old chs1 2 seedlings before and after the chilling treatment. No obvious changes in ion leakage were observed in the wild type before the chilling treatment. In contrast, ion leakage in the chs1 2 seedlings was significantly increased after 3 days at 16 C, and the leakage continued to increase after that (Figure 1d). We then examined whether chs1 2 conferred cell death at 16 C by performing trypan blue staining. At 22 C, chs1 2 resembled the wild-type Col in morphology, and neither leaves of chs1 2 nor leaves of Col displayed visible trypan blue staining. However, after transferring the plants to 16 C for 7 days, cell death was evident in chs1 2 but not in Col (Figure 1e). Electron transport chains can be perturbed by low temperatures, thus inducing ROS production (Fryer et al., 2002; Hideg et al., 2002; Pfannschmidt et al., 2003). Therefore, we tested the accumulation of hydrogen peroxide (H 2 O 2 ) by using 3,3 diaminobenzidine (DAB) staining. Strong staining was detected in chs1 2, but not in the wild type, following the chilling treatment at 16 C (Figure 1e), indicating that the chs1 2 mutant accumulates a higher level of H 2 O 2 than the wild type under chilling stress. We next determined whether the expression of defenseassociated genes was altered in chs1 2 under chilling

3 chs1 mediated chilling sensitivity 555 (a) (b) (e) (c) (f) (g) Figure 1. Phenotype of the chs1 2 mutant. (a, b) Chilling sensitivity of the chs1 2 mutant. (a) Wild-type Col and chs1 2 mutant plants were grown on MS medium at 16 C for 3 weeks. (b) Col and chs1 2 were grown in soil at 22 C for 2 weeks before transfer to 16 C for 1 week. (c) Measurement of chlorophyll a and b in Col and chs1 2 after they were grown at 22 C for 2 weeks and treated at 16 C for the indicated time. Ion leakage of seedlings described in (c). (e) Two-week-old wild-type Col and chs1 2 plants were grown at 22 C before transfer to 16 C for 7 days; detached leaves were stained with trypan blue (upper panel) and 3,3 diaminobenzidine (DAB) (lower panel). Scale bar: 1 mm. (f) The expression of PR1 and PR2 in Col and chs1 2 grown at 22 C for 2 weeks and transferred to 16 C for the indicated time, as measured by qrt-pcr. The data represent the means of four replicates SDs. (g) Free and total salicylic acid (SA) accumulation in Col and chs1 2. The data represent the means of four replicates SDs. Similar results were observed in three independent experiments. conditions. Total RNA was extracted from 2 week-old chs1 2 and Col plants grown at 22 C at 0 and 3 days after transfer to 16 C, and PR1 and PR2 expression was analyzed by quantitative real-time PCR (qrt-pcr). The expression levels of both PR1 and PR2 were dramatically increased in chs1 2 after the transfer to 16 C, whereas no obvious change in the PR1 or PR2 transcript levels was detected in Col plants under the same conditions (Figure 1f). Because induced PR gene expression is often associated with SA signaling, the endogenous levels of SA in chs1 2 and Col were measured. The levels of both total and free SA were similar to those of the wild type at 22 C. In contrast, the chilling-treated chs1 2 plants accumulated 10- and eight-fold higher levels of total and free SA than the wild type (Figure 1g). These results demonstrated that the mutation in CHS1 results in the activation of the defense response under chilling stress. ROS-associated gene expression in chs1 2 under chilling conditions The above data showed that chs1 2 experienced cell death and H 2 O 2 accumulation at 16 C (Figure 1e). Excess H 2 O 2 induces the expression of genes involved in oxidative stress (Iba, 2002; Mittler et al., 2004; Rizhsky et al., 2004). We next used qrt-pcr to examine the expression of ROS-associated genes, including genes encoding the ROS-producing enzyme NADPH oxidase (RbohD) and ROS-scavenging enzymes, such as ascorbate peroxidase (APX1), catalase (CAT1) and glutathione reductase (GR1), after a 16 C treatment for 3 days. No obvious differences

4 556 Yuancong Wang et al. were detected in the expression of these genes between Col and chs1 2 plants grown at 22 C; however, all four of these ROS-associated genes were upregulated between two- and threefold in chs1 2 compared with the wild type following the chilling treatment (Figure 2). ZAT12 is a C 2 H 2 zinc finger-type transcription factor gene that plays an important role in regulating several genes involved in plant responses to oxidative stress (Davletova et al., 2005). ZAT12 was upregulated (approximately 50 fold) in chs1 2 following the 16 C treatment (Figure 2). The FER1 gene encoding ferritin, which is pivotal for protecting cells against oxidative damage (Ravet et al., 2009), was also highly expressed in the chs1 2 plants subjected to chilling stress (Figure 2). Taken together, these results suggest that the chs1 conferred chilling-sensitive phenotype links the over-activation of ROS production and detoxification with the oxidative signaling pathways caused by the mutation of CHS1. CHS1 encodes a TIR-NB-type protein The chs1 2 mutant was previously reported to be a recessive mutant in a single nuclear locus (Hugly et al., 1990). To identify the CHS1 gene, we generated a segregating population by crossing chs1 2 with Landsberg erecta (Ler). A total of 2500 chilling-sensitive mutant plants were selected for mapping from the segregating F 2 population. The CHS1 locus was mapped to a 275 kb region between markers F28G4 and F11A6 on chromosome I (Figure 3a). A DNA sequencing analysis in this region showed that that a G? A substitution at nucleotide 28 was present in the At1 g17610 gene; this substitution results in an alanine (Ala)? threonine (Thr) substitution (Figures 3b and S1). The same nucleotide mutation was found in the chs1 1 mutant. CHS1 encodes a TIR-NB-type protein. A construct harboring the CHS1 coding region, driven by its native promoter into chs1 2 plants (approximately 2 kb upstream of CHS1), can fully rescue the chilling-sensitive phenotype of chs1 2, cell death and PR expression (Figure 3c f). These results indicate that the chs1 2 mutant phenotype is caused by the mutation observed in At1g To dissect the nature of the chs1 2 mutation, we transformed Col plants with the CHS1 coding region fused with the green fluorescent protein (GFP) gene under the control of the Super promoter (CHS1-GFP). Of the eight independent transgenic lines generated, six showed overexpression of CHS1 (Figure 4a), and they exhibited similar phenotypes, so we chose two lines for further study (lines 3 and 4). Intriguingly, the other two lines (1 and 2) displayed underexpression of CHS1 compared with the wild type Col (Figure 4a), which may be caused by a genesilencing effect. After growing at 16 C for 3 weeks, underexpressing lines showed smaller and weaker stature, whereas overexpressing lines tended to grow better and bigger than the wild type (Figure 4b). These results implied that chs1 2 is a recessive, negative-interfering mutation. Next, transgenic plants overexpressing the mutated chs1 GFP in Col were generated (Figure 4c). These plants showed higher expression of PR1, small stature, obvious lesions and cell-death phenotypes (Figure 4c e). Furthermore, the overexpression of CHS1-GFP in the chs1 2 background at least partially restored the chilling-sensitive phenotype of chs1 2 (Figure 4f, g). F 1 progeny of chs1 2 and Ler showed semi-chilling sensitivity, intermediate between that of Col/Ler and chs1 2 plants (Figure 4h). Taken together, these results suggest that the chilling sensitivity of chs1 2 is dosage dependent. A genetic screen for suppressors of chs1 2 was performed to identify mutants that restore the chs1 2 phenotype to wild-type morphology. Two suppressors of chs1 2, named chs1 r1 and chs1 r2, were mapped to the original CHS1 locus. A sequencing analysis revealed that the mutations of G152R and A138V in chs1 r1 and chs1 r2 were both located in the TIR domain of CHS1 (Figure S2). Both chs1 r1 and chs1 r2 fully restored the chs1 2 chillingsensitive phenotype (Figure S3a). Moreover, the cell death, H 2 O 2 accumulation and expression of PR genes in chs1 r1 and chs1 r2 all recovered to wild-type levels (Figure S3b,c). Figure 2. Expression of reactive oxygen species (ROS)-associated genes in Col and chs1 2 plants under chilling stress, as measured by qrt-pcr. Plants were grown at 22 C for 2 weeks and transferred to 16 C for the indicated time. The data represent the means of three replicates SDs. All of the experiments were repeated three times with similar results.

5 chs1 mediated chilling sensitivity 557 (a) (c) (b) (e) (f) Figure 3. Map-based cloning of CHS1. (a) Genetic map of the CHS1 locus. The markers used for mapping are shown with the number of recombinants. Predicted genes are indicated by arrows, indicating the direction of transcription. (b) The genomic structure of the CHS1 gene. The filled box and unfilled box present the coding region and untranslated region of CHS1 exon, respectively. The positions of the chs1 mutations are shown. (c, d) Complementation test of the chs1 2 mutant. A genomic fragment of CHS1 driven by its native promoter was transformed into chs1 2 plants. (c) Twoweek-old plants grown at 22 C were transferred to 16 C for 1 week. Seedlings grown on MS medium for 3 weeks at 16 C. (e) Trypan blue staining of true leaves from plants that were described in. Scale bar: 100 lm. (f). The PR1 and PR2 transcript levels in the plants grown at 22 C for 2 weeks and transferred to 16 C for 3 days, as measured by qrt-pcr. The data represent the means of three replicates SDs. Similar results were observed in three independent experiments. These results imply that the second mutations in chs1 r1 and chs1 r2 abolish or weaken the negative interference of the chs1 2 mutation. The expression pattern of CHS1 To examine the expression pattern of CHS1, we transformed Col plants with a CHS1:GUS (b glucuronidase) construct carrying the GUS reporter gene driven by a 2.0 kb fragment of the CHS1 promoter. In these transgenic lines, strong GUS staining was primarily observed in cauline leaves, flowers (mainly in sepals) and old rosette leaves (Figure 5b e). GUS signals were also observed in the stems, but not in the siliques or roots (Figure 5a,d f). In addition, semiquantitative RT-PCR demonstrated that CHS1 transcripts were primarily detected in the leaves and flowers (Figure 5g), which is consistent with the GUS staining results. The subcellular localization of CHS1 To investigate the function of CHS1, we examined the subcellular localization of the CHS1-GFP protein in Arabidopsis protoplasts. As a control, a nuclear-localized marker, AT-hook motif nuclear-localized protein 22 (AHL22), fused with red fluorescent protein (RFP), was co-expressed in Arabidopsis protoplasts (Figure 5h). CHS1-GFP was detected in both the cytoplasm and the nucleus (Figure 5h). In some cases, the activation of the TIR-NB-LRR protein that is caused by point substitutions is known to alter its subcellular localization. For example, the accumulation of mutated SNC1 in the nucleus results in the constitutive defense response and dwarf phenotype (Zhu et al., 2010). To determine whether the mutation of CHS1 also affects its subcellular localization, we transiently co-expressed chs1 GFP and AHL22-RFP in Arabidopsis protoplasts. chs1 GFP

6 558 Yuancong Wang et al. (a) (b) (c) (e) (h) (f) (g) Figure 4. Phenotypes of Super:CHS1-GFP and Super:chs1 GFP plants in Col or chs1 2 backgrounds at 16 C. (a) Expression of CHS1 in Col, chs1 2 and four independent transgenic plants of Super:CHS1-GFP grown at 16 C for 2 weeks, as measured by qrt-pcr. (b) Phenotypes of 4 week-old plants described in (a). (c) Expression of CHS1 and PR1 in Col, chs1 2 and three independent transgenic plants of Super:chs1 GFP grown at 16 C for 2 weeks, as measured by qrt-pcr. Phenotypes of the 4 week-old plants described in (c). (e) Trypan blue staining of true leaves from the plants described in. Scale bar: 100 lm. (f) RT-PCR analysis of CHS1 in Col and two independent transgenic lines of chs1/super:gfp and chs1/super:chs1-gfp. EF1a was used as a control. (g) Phenotypes of the 4 week-old plants described in (f). (h) Phenotypes of Col, Ler, chs1 2 and F 1 progeny of chs1 2 crossed with Ler grown at 22 C for 4 weeks before transfer to 16 C for an additional 8 days. accumulated in the cytoplasm and nucleus (Figure 5h). The mutated chs1 did not obviously change the subcellular localization of this protein. Overexpression of CHS1-TIR and CHS1-NB domains can suppress the phenotype of chs1 2 A BLAST analysis showed that CHS1 contains TIR and NB domains. To gain insight into the function of each domain, we generated two constructs carrying the coding region that encodes the TIR or NB domain fused with GFP under the control of the Super promoter, and we transformed them into chs1 2. The transgenic lines indeed overexpressed the TIR and NB domains by RT-PCR (Figure 6a). The transgenic lines carrying TIR-GFP could partially rescue the chilling-sensitive phenotype of chs1 2, whereas the transgenic lines carrying NB-GFP could completely restore the sensitive phenotype of chs1 2 following a 16 C treatment (Figure 6b). Trypan blue and DAB staining showed that the cell death and H 2 O 2 accumulation phenotypes in chs1 2 were partially compromised by the overexpression

7 chs1 mediated chilling sensitivity 559 (a) (b) (c) (g) (h) (e) (f) crossed with plants expressing the nahg gene, which encodes an enzyme that degrades SA to catechol. Col, chs1 2 and chs1 2 nahg plants were grown at 16 C for 3 weeks, and nahg almost completely rescued the chillingsensitive phenotype of chs1 2 (Figure 7a). Consistent with the phenotype, cell death and H 2 O 2 accumulation were clearly suppressed (Figure 7b,c), and PR1 expression also reverted to the wild-type level in the chs1 2 nahg plants (Figure 7d). To further determine the role of SA in the chs1 2 phenotype, chs1 2 was crossed with sid2 1, a mutant with defects in SA biosynthesis (Wildermuth et al., 2001), and npr1 1, a mutant defective in SA signaling (Durrant and Dong, 2004). The chilling-sensitive phenotype and PR1 expression in chs1 2 were partially suppressed by sid2 1 and npr1 1, and the rate of cell death in chs1 2 sid1 2 and chs1 2 npr1 1 was slightly slower than in chs1 2; however, the eventual cell death phenotype was not substantially affected (Figure 7a). These results indicate that the chilling sensitivity and defense activation in chs1 2 partially requires SA. The chs1 2 phenotype is dependent on EDS1 and PAD4 Figure 5. Expression pattern and subcellular localization of CHS1. (a f) GUS expression in the CHS1:GUS transgenic lines. b glucuronidase (GUS) activity was analyzed in the different seedling stages as follows: the two-leaf (a) and eight-leaf (b) stages; (c) the leaves in (b); inflorescences and flowers; (e) a zoom in of the area designated by the arrow in ; and (f) siliques. (g) RT-PCR analysis of CHS1 in the roots (RT), stems (ST), mature rosette leaves (RL), cauline leaves (CL), flowers (FL) and siliques (SL). (h) Subcellular localization of CHS1 and mutated chs1 in Arabidopsis protoplasts. CHS1-GFP or chs1 GFP was co-transformed with a nuclear marker gene (AHL22-RFP) into protoplast prepared from 15 day-old seedlings grown on MS medium. Scale bars: 20 lm. of TIR-GFP, and were completely suppressed by the overexpression of NB-GFP (Figure 6c). We also tested the mrna levels of PR1 and PR2 by qrt-pcr. The transcript level of PR1 in the chs1/nb-gfp lines showed no visible differences from the wild type, and the mrna level of PR1 in the chs1/tir-gfp line was between those of the wild type and chs1 2. The PR2 transcript level in the chs1/tir-gfp lines was partially or completely restored to the wild-type level (Figure 6d). These data indicate that overexpression of the TIR or NB domain, respectively, can partially or completely rescue the chillingsensitive phenotype of chs1 2. The chs1 2 phenotype is partially dependent upon SA To determine whether an increased SA level in chs1 2 is required for the chilling-sensitive phenotype, chs1 2 was Previous studies have demonstrated that the function of traditional TIR-NB-LRR R proteins requires EDS1 and PAD4 (Glazebrook et al., 1996; Aarts et al., 1998). Because CHS1 contains TIR-NB domains, we tested whether the phenotype of chs1-2 also requires EDS1 and PAD4 by crossing chs1 2 with pad4 1 (Jirage et al., 1999) and eds1 2 (in the Col background) (Bartsch et al., 2006). We found that the eds1 and pad4 mutations completely suppressed the phenotype of chs1 2 at 16 C (Figure 7a). The double mutants displayed no obvious cell-death phenotype, H 2 O 2 accumulation or constitutive PR1 gene expression (Figure 7b d). However, the chs1 2 ndr1 1 double mutant behaved similarly to chs1 2 with respect to both morphological and cell-death phenotypes (Figure 7a d). Therefore, the chs1 conferred phenotypes under chilling stress require EDS1 and PAD4, but not NDR1. The CHS1 protein accumulates under chilling stress Because the phenotype of chs1 2 was dependent upon a low temperature, we wondered whether the CHS1 transcript level was temperature-regulated. A semiquantitative RT-PCR analysis showed that CHS1 expression in wild-type plants grown at 22 C and at 16 C remained at the same levels (Figure 8a), indicating that the CHS1 transcript level is not regulated by temperature. We then investigated whether the CHS1 protein level is influenced by temperature. The transgenic lines overexpressing GFP, CHS1-GFP, or chs1 GFP were grown at 16 and 22 C for 7 days, and the GFP signal was detected under a confocal microscope. The GFP signals from the roots of CHS1-GFP and chs1-gfp seedlings grown at 16 C were stronger than in those grown at 22 C (Figure 8b). As

8 560 Yuancong Wang et al. (a) (b) (c) Figure 6. Overexpression of the TIR and NB domains of CHS1 suppresses the chs1 2 phenotype. Wild-type Col, chs1 2 and transgenic plants overexpressing the TIR and NB domains of CHS1 driven by the Super promoter in chs1 2 (chs1/tir-gfp and chs1/nb-gfp) were grown at 16 C. (a) RT-PCR analysis of TIR and NB domains in 2 week-old Col and chs1 2 plants, and in two independent transgenic lines of chs1/tir-gfp and chs1/nb-gfp, respectively. EF1a was used as a control. (b) Phenotypes of the plants described in (a) grown at 16 C for 3 weeks. (c) Trypan blue (upper panel) and DAB (lower panel) staining of true leaves from the plants described in (a) grown at 16 C for 3 weeks. Scale bar: 100 lm. Expression of PR1 and PR2 genes in the plants described in (a) grown at 16 C for 2 weeks, as measured by qrt-pcr. The data represent the means of three replicates SDs. Similar results were observed in three independent experiments. a control, the GFP signals in the GFP seedlings grown at 22 C were similar to those grown at 16 C (Figure 8c). To further determine which domain of CHS1 was sensitive to temperature, we observed the GFP signals in the TIR-GFP and NB-GFP seedlings grown at 22 and 16 C, respectively. TIR-GFP showed reduced expression at 22 C compared with 16 C, whereas no obvious difference in the NB-GFP signals was observed at 16 or 22 C (Figure 8b). Consistently, an immunoblot analysis revealed that the protein levels of CHS1, chs1 and TIR were lower at 22 C than at 16 C, whereas the NB-GFP and GFP protein levels remained unchanged at 16 and 22 C (Figure 8c). These results indicate that the CHS1 protein is low-temperature regulated at the post-transcriptional level. To further study the kinetics of temperature-dependent CHS1-GFP degradation, 14 day-old seedlings of GFP, CHS1-GFP, TIR-GFP and NB-GFP grown at 16 C were transferred to higher temperature (28 C) conditions, which can accelerate CHS1 degradation. Total protein was extracted from these seedlings at 0, 1, 3 and 6 h after treatment at 28 C. The CHS1-GFP and TIR-GFP proteins were indeed degraded following the treatment at 28 C, whereas the GFP and NB-GFP proteins displayed no notable changes under the same treatment (Figure 8d). Notably, the TIR-GFP protein degraded much faster than the CHS1- GFP protein (Figure 8d), suggesting that the NB domain of CHS1 may stabilize the CHS1 protein. To determine whether the TIR domain of CHS1 protein degradation is dependent on the 26S ubiquitin-proteasome pathway, we treated TIR1-GFP seedlings with the proteasome inhibitor MG132 (Smalle and Vierstra, 2004). MG132 treatment did not suppress high temperature-induced degradation of the TIR domain (Figure 8e). These results suggest that the temperature-dependent degradation of the CHS1 protein and its TIR domain is not mediated by the 26S proteasome pathway.

9 chs1 mediated chilling sensitivity 561 (a) (b) (c) Figure 7. Phenotypes of Col, chs1 2 and double mutants grown at 16 C. (a) The phenotypes of Col, chs1 2 and double mutants were grown at 16 C for 3 weeks. (b, c) Trypan blue (b) and DAB (c) staining of the plants described in (a). Scale bars: 100 lm. Expression of PR1 in the plants described in (a) grown at 16 C for 2 weeks, as measured by qrt-pcr. The data represent the means of three replicates SDs. Similar results were observed in three independent experiments. DISCUSSION In this study we characterized the chilling-sensitive mutant chs1, which was previously reported upon by Hugly et al. (1990). This mutant exhibits growth arrest and a seedlinglethal phenotype when grown at chilling temperatures of 16 C or below. CHS1 encodes a TIR-NB-type protein. The overexpression of the TIR and NB domains can suppress the chs1-confered phenotypes. Furthermore, the accumulation of CHS1 protein was positively regulated by low temperatures in a 26S proteasome pathway-independent manner. Recent studies reported that the nuclear accumulation of SNC1 and N may contribute to the resistance mediated by them. The mutated snc1 promotes its nuclear accumulation and triggers the auto-defense response in Arabidopsis (Zhu et al., 2010). In this study, however, CHS1 and mutated chs1 are both localized in the nucleus and cytoplasm, suggesting that other factors affect CHS1 function rather than change its localization. One previous study showed that the LRR domain of CC-NB-LRR-type potato R protein Rx1 can promote localization in the cytoplasm (Slootweg et al., 2010). One mutation of SNC1 that changes the ratio of nuclear to cytoplasm distribution also occurs in the LRR domain of SNC1 (Zhu et al., 2010). Thus, the lack of an LRR domain of CHS1 may contribute to the stable subcellular localization of CHS1. Previous studies have demonstrated that the functions of TIR-NB-LRR and CC-TIR-LRR R proteins are dependent upon EDS1 and NDR1, respectively (Glazebrook et al., 1996; Aarts et al., 1998; Glazebrook, 2001). Our genetic analysis demonstrated that the CHS1 conferred chilling sensitive phenotype at 16 C is dependent upon EDS1 and PAD4, but not upon NDR1, which is consistent with the characteristics of most of the TIR-type R proteins. Therefore, although CHS1 does not contain an LRR domain like traditional R proteins, it can activate similar pathways like a TIR-NB-LRR-type R protein. It is possible that unknown TIR-NB-LRR-type protein(s) may participate in the pathway that causes the phenotype of chs1. Several lines of evidence support the hypothesis that chs1 2 appears to be a recessive, negative-interfering mutation in a dosage-dependent manner. First, chs1 2 is a recessive mutation (Hugly et al., 1990). Second, the growth of seedlings is promoted in transgenic plants overexpressing CHS1, and inhibited in plants underexpressing CHS1. Third, the wild-type plants become chilling-sensitive when the mutated chs1 GFP is overexpressed, whereas the chilling sensitivity of chs1 mutants is recovered when CHS1- GFP is overexpressed. Furthermore, F 1 progeny of chs1 2 and Ler showed semi-chilling sensitivity that was intermediate between that of Col/Ler and chs1 2. In traditional NB-LRR R proteins, the NB domain has been shown to represent a switch that can turn the R

10 562 Yuancong Wang et al. (a) (c) (b) (e) Figure 8. CHS1 is degraded at high temperatures. (a) Expression of CHS1 in response to low temperature by semiquantitative RT-PCR. Two-week-old seedlings grown at 22 C were transferred to 16 C for the indicated times. (b) GFP signals in roots from 7 day-old transgenic seedlings grown at 22 and 16 C were visualized with a confocal microscope. Scale bar: 50 lm. (c) Immunoblot analysis of CHS1 protein and different domains of CHS1 in seedlings described in (a). Total protein was extracted from 7 day-old transgenic lines grown at 22 and 16 C, and analyzed by immunoblotting with antibodies against GFP. Immunoblot analysis of CHS1 and different domains of CHS1 under temperature-shifted conditions. Seven-day-old seedlings grown at 16 C were transferred to 28 C for the indicated times. Total protein was extracted and analyzed by immunoblotting with antibodies against GFP. (e) Immunoblot analysis of the TIR domain of CHS1 in the presence of MG132 under temperature-shifted conditions. Seven-day-old seedlings grown at 16 C were transferred to 28 C in the presence of MG132 or DMSO (as a control) for the indicated time. Total protein was extracted and analyzed by immunoblotting with antibodies against GFP. proteins on or off by binding ATP or ADP, respectively (Lukasik and Takken, 2009). The mutation of CHS2 (an active form of RPP4) is in the NB domain and may constitutively activate RPP4 at low temperatures (Huang et al., 2010). However, in CHS1 we found that the mutation causing the functional change of CHS1 is close to the N terminus of the TIR domain. The TIR domain has been identified as an intracellular part of Toll-like receptors (TLRs) in animals (Tapping, 2009). The TIR domain also occurs at an N terminal location in a major subclass of cytoplasmic NB-LRR-plant R proteins. These R proteins can perceive the pathogen effectors and trigger defense

11 chs1 mediated chilling sensitivity 563 responses to protect the plants from pathogens (Chisholm et al., 2006; Jones and Dangl, 2006; Rafiqi et al., 2009; Dodds and Rathjen, 2010). Evidence suggests that the TIR domain is dispensable for the effector recognition essential for defense signaling (Bernoux et al., 2011). Effector-independent cell death can be triggered by the overexpression of the TIR domain plus amino acids from some TIR-NB-LRR proteins of Arabidopsis, tobacco and flax (Linum usitatissimum; Frost et al., 2004; Michael Weaver et al., 2006; Swiderski et al., 2009; Krasileva et al., 2010). Based on our results, we speculate that both wildtype CHS1 and mutant chs1 can form a heterodimer with a TIR-NB-LRR-type protein through its TIR domain, but whereas the heterodimer containing mutated chs1 may activate this TIR-NB-LRR protein, the wild-type CHS1 does not. When overexpressed TIR domain of wild-type CHS1 in chs1-2, it competes with the mutated chs1 in forming heterodimer with one or more unknown TIR-NB-LRR proteins, and thereby reducing the ratio of the dimer containing mutate chs1. Since the dimer containing wild-type TIR domain cannot activate the TIR-NB-LRR proteins, it can partially restore the phenotype of chs1-2. It has been reported that the overexpression of other domains can suppress the activation of R or R-like proteins. For instance, the phenotype of chs3 1 is suppressed by overexpressing the LIM domain of CHS3, suggesting that the LIM domain might repress N terminal R like activity (Yang et al., 2010). Overexpressing the NB-ARC domain can also inhibit the auto-activity and self-association of the L6 TIR domain (Bernoux et al., 2011). Consistently, the overexpression of the NB ARC domain of CHS1 may also repress the dimerization, thus totally suppressing the chilling-sensitive phenotype of chs1 2. Furthermore, we tested the transcription level of CHS1 in chs1/super:tir- GFP and chs1/super:nb-gfp plants, and found that CHS1 expression in all transgenic lines remained the same compared with the wild type (Figure S3). These results exclude the possibility that the gene silencing of CHS1 contributes to the suppression of the chs1 phenotype. Although the chs1 mutant displays extensive cell death at low temperatures (16 C or below), it grows normally at normal temperatures (22 C). This phenomenon prompted us to test the effect of temperature on CHS1 protein stability. Previous study reported both the transcriptional and protein levels of SNC1 were higher at 22 C than 28 C (Yang and Hua, 2004; Zhu et al., 2010). But for CHS1, unlike SNC1, the mrna level remained unchanged at 16 C; however, CHS1-GFP and TIR-GFP proteins accumulated to a greater extent at 16 C than at 22 C, whereas NB-GFP was maintained at a consistent level at 22 and 16 C. Furthermore, the CHS1 protein was degraded several hours after the plants were transferred from 22 to 28 C. Intriguingly, we noticed that the TIR domain of CHS1 degraded much faster than that of the full-length protein. In contrast, the NB domain was not responsive to the temperature change. These results imply that one function of the NB domain is to stabilize CHS1 at high temperatures. EXPERIMENTAL PROCEDURES Plant materials and growth conditions Arabidopsis thaliana plants of the Columbia (Col) and Landsberg erecta (Ler) ecotypes were used in this study. The chs1 1 and chs1 2 mutants (Hugly et al., 1990) were obtained from the Arabidopsis Biological Resource Center (ABRC, The mutants sid2 1 (Wildermuth et al., 2001), npr1 1 (Durrant and Dong, 2004), pad4 1 (Jirage et al., 1999), eds1 2 (in Col background) (Bartsch et al., 2006) and ndr1 1 (Century et al., 1995) are in the Col background. The Arabidopsis plants used in this study were grown at 22 or 16 C under a 16 h light/8 h dark photoperiod (100 lmol m 2 sec 1 ), with 50 70% relative humidity. The Arabidopsis seeds were planted on MS medium (Sigma-Aldrich, supplemented with 2% sucrose and 0.8% agar before they were transferred to soil. Map-based cloning of the CHS1 gene To map the chs1 2 mutation, chs1 2 (Col background) was crossed with Ler. The F 1 plants from the cross were self-fertilized, and the resulting F 2 seeds were collected. A total of 2500 chs1 2 mutant plants were chosen from the segregating F 2 population based on their hypersensitivity at 16 C. Genomic DNA was used to perform PCR-based mapping with simple sequence length polymorphism (SSLP) and cleaved amplified polymorphic sequence (CAPS), and derived CAPS (dcaps) markers. Plasmid construction and plant transformation A 3.55 kb genomic fragment containing the CHS1 promoter, coding region and 3 untranslated region (3 UTR) region was amplified from Col genomic DNA using the primers CHS1-1-1F and CHS1-1-3R; the fragment was cloned into the pcambia1300 vector (CAMBIA) to generate the CHS1:CHS1 construct. To generate the CHS1:GUS construct, a 2.07 kb genomic fragment upstream of the CHS1 ATG was amplified using primers CHS1-1-1F and CHS1-1-2R, and was cloned into the pzpgus2 vector (Diener et al., 2000). To construct Super:TIR-GFP, Super:NB-GFP and Super:CHS1-GFP, the DNA fragments were amplified from Col by PCR using the primers CHS1-2-TIR-F and CHS1-2-TIR-R, CHS1-2-NB-F and CHS1-2-NB-R, and CHS1-2-TIR-F and CHS1-2-NB-R, respectively; the fragments were cloned into the pcambia1300 vector containing the Super promoter (Ni et al., 1995). The mutated chs1 DNA fragment used to construct Super:chs1 GFP was amplified from the chs1 2 mutant. The primers are listed in Table S1. All of the constructs were transformed into the designated genotypes via floral-dip transformation (Clough and Bent, 1998). Electrolyte leakage assays and chlorophyll measurement Two-week-old seedlings grown at 22 C were transferred to 16 C for 0, 3, 6 and 9 d. The electrolyte leakage assay was performed as described previously (Lee et al., 2002). The contents of chlorophyll a and b were extracted and measured as previously described (Huang et al., 2009).

12 564 Yuancong Wang et al. Salicylic acid (SA) measurement Free and total SA were extracted and measured from 2 week-old seedlings grown at 22 C and then transferred to 16 C for 3 days, as previously described (Li et al., 1999). Histochemistry The histochemical detection of GUS activity was performed as previously described (Yang et al., 2006). Trypan blue and DAB staining was also performed as previously described (Bowling et al., 1997; Thordal-Christensen et al., 1997). Quantitative real-time PCR (qrt-pcr) Two-week-old seedlings grown at 22 C were transferred to 16 C for 3 days. Total RNA was prepared using the TRIzol reagent (Invitrogen, and was reverse transcribed using M MLV reverse transcriptase (Promega, qrt-pcr was performed using a SYBR Green PCR Master Mix kit (TaKaRa, The relative expression levels were calculated as previously described (Huang et al., 2010). The primers used are listed in Table S1. Protoplast transformation Protoplast isolation and transformation were performed as previously described (Zhai et al., 2009). Briefly, protoplasts were prepared from 10 day-old wild-type seedlings grown on MS medium. After transformation, the protoplasts were incubated at 16 C, and the GFP signals were observed after h of incubation. Confocal laser microscopy Transgenic plants expressing Super:CHS1-GFP, Super:CHS1-TIR- GFP, Super:CHS1-NB-GFP or Super:chs1 GFP were grown at 22 or 16 C for 7 days. GFP fluorescence in the roots of the transgenic plants or in transgenic protoplasts was visualized using a confocal laser scanning microscope (LSM510; Zeiss, Immunoblot analysis Total protein was prepared from 7 day-old transgenic plants that were grown at 22 or 16 C, or were transferred from 16 to 22 C for different time periods, as previously described (Huang et al., 2009). The CHS1-GFP fusion proteins were visualized on immunoblots using an anti-gfp antibody (Sigma-Aldrich). Rubisco was stained with Ponceau S as a control. ACKNOWLEDGEMENTS We thank Jane E. Parker and ABRC for mutant seeds. This work was supported by the National Key Basic Research Program of China (2011CB915401), China National Funds for Distinguished Young Scientists ( ), the National Natural Science Foundation of China ( ), and the Ministry of Agriculture of China for transgenic research (2011ZX ). SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. Amino-acid sequence of CHS1. Figure S2. Phenotypes of Col, chs1 2 chs1 r1 and chs1 r2. Figure S3. Expression of CHS1 in Super:TIR-GFP and Super: NB-GFP plants grown at 16 C for 2 weeks by semiquantitative RT-PCR. Table S1. Gene-specific primers used in this study. REFERENCES Aarts, N., Metz, M., Holub, E., Staskawicz, B. J., Daniels, M. J. and Parker, J. E. 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