A Gain-of-Function Mutation in the Arabidopsis Disease Resistance gene rpp Confers Sensitivity to Low TemperaturelIwIIOAl Xiaozhen Huang, Jianyong Li, Fei Bao, Xiaoyan Zhang, and Shuhua Yang' State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China How plants adapt to low temperature is not well understood. To identify components involved in low-temperature signaling, we characterized the previously isolated chilling-sensitive2 mutant(chs2)of Arabidopsis(Arabidopsis thaliana). This mutant grey normally at 22.C but showed phenotypes similar to activation of defense responses when shifted to temperatures below 16C. hese phenotypes include yellowish and collapsed leaves, increased electrolyte leakage, up-regulation of PATHOGENESIS RELATEd genes, and accumulation of excess hydrogen peroxide and salicylic acid(SA). Moreover, the chs2 mutant was seedling lethal when germinated at or shifted for more than 3 d to low temperatures of 4C to 12C. Map-based cloning revealed that a single amino acid substitution occurred in the TIR-NB-LRR (for Toll /Interleukin-1 receptor-nucleotide-binding Leucine-rich repeat)-type resistance(R)protein RPP4(for Recognition of Peronospora parasitica), which causes a deregulation of the R protein in a temperature-dependent manner. The chs2 mutation led to an increase in the mutated RPP4 mRNA transcript, activation of defense responses, and an induction of cell death at low temperatures. In addition, a chs2 intragenic suppressor, in which the mutation occurs in the conserved NB domain, abolished defense responses at lower temperatures Genetic analyses of chs2 in combination with known SA pathway and immune ng mutants indicate that the chs2 onferred temperature sensitivity requires ENHANCED DISEASE SUSCEPTIBILITY UIREd FOR Mla12 RESIStance and SUPPRESSOR OF G2 ALLELE OF skpl but does not require PHYTOALEXIN CIENT4, NONEXPRESSOR OF PR GENESI, or SA. This study reveals that an activated TIR-NB-LRR protein has a large impact on temperature sensitivity in plant growt For optimal growth and survival, plants have evolved 1992, 2004), and DREB/CBF-independent pathways unique and sophisticated defense mechanisms against have been identified as important for cold responses as multiple stresses, both abiotic and biotic. Cold stress well (Xin and Browse, 1998; Dong et al., 2006; Lee et al has a significant limiting effect on the geographic 2006; Xin et al. 2007; Zhu et al., 2008) location of plants and on crop prodt luctivity (G Plants have evolved at least two layers of defense 1990). It can disrupt cellular homeostasis by altering mechanisms against pathogens. One of them is medi- the fatty acid composition of membrane lipids, which ated by resistance(r)proteins. Interaction of an R can deactivate membrane proteins and uncouple ma protein with a specific pathogen avirulence protein for physiological processes(Los and Murata, 200 triggers the hypersensitive response(HR), which is a Plants respond and adapt to cold stress in many form of programmed cell death that limits path- biochemical and physiological processes. A numb ogen growth and spread(Scheel, 1998). Most of the of genes are involved in the DREB/CBF(for DRE- characterized R proteins encode proteins with nucle- binding protein/C-repeat-binding factor)-dependent otide-binding Leu-rich repeat(NB-LRR) domains. A pathway to control cold acclimation(Gilmour et al well-conserved ARC (for After the Nb domain, f-1,R protein, and and these two domains are often referred to as the nb. Foundation of China (grant nos. 30670181, 3077202, and 90817007) ARC domain. The NB-LRR proteins can be grouped the National Key Basic Research Program of (grant no. into two main classes based on their N-terminal struc- cultural of China for trans- ture, which has either a Toll/Interleukin-l receptor 2003) The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy The Arabidopsis(Arabidopsis thaliana)RPP5(for Rec- describedintheiNstructionsforAuthors(www.plantphysiol.org)is: ognition of Peronospora parasitica) locus in Columbia ShuhuaYang(yangshuhua@cau.edu.cn) Col)is composed of seven TIR-NB-LRR class r genes, Iw The online version of this article contains Web-only data. includins PP4 and SNC1(for Suppressor of nprI IOA] Open Access articles can be viewed online without a sub- constitutive 1)genes(Noel et al., 1999). RPP4 plays an important role in resistance to Hyaloperonospora www.plantphysiol.org/cgi/doi/10.1104/pp.110.157610 parasitica through multiple signaling components, in- PlantPhysiologyOctober2010,Vol.154,Pp.796-809,www.plantphysiol.org@2010AmericanSocietyofPlantBiologistsA Gain-of-Function Mutation in the Arabidopsis Disease Resistance Gene RPP4 Confers Sensitivity to Low Temperature1[W][OA] Xiaozhen Huang, Jianyong Li, Fei Bao, Xiaoyan Zhang, and Shuhua Yang* State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China How plants adapt to low temperature is not well understood. To identify components involved in low-temperature signaling, we characterized the previously isolated chilling-sensitive2 mutant (chs2) of Arabidopsis (Arabidopsis thaliana). This mutant grew normally at 22
C but showed phenotypes similar to activation of defense responses when shifted to temperatures below 16
C. These phenotypes include yellowish and collapsed leaves, increased electrolyte leakage, up-regulation of PATHOGENESIS RELATED genes, and accumulation of excess hydrogen peroxide and salicylic acid (SA). Moreover, the chs2 mutant was seedling lethal when germinated at or shifted for more than 3 d to low temperatures of 4
C to 12
C. Map-based cloning revealed that a single amino acid substitution occurred in the TIR-NB-LRR (for Toll/Interleukin-1 receptor- nucleotide-binding Leucine-rich repeat)-type resistance (R) protein RPP4 (for Recognition of Peronospora parasitica4), which causes a deregulation of the R protein in a temperature-dependent manner. The chs2 mutation led to an increase in the mutated RPP4 mRNA transcript, activation of defense responses, and an induction of cell death at low temperatures. In addition, a chs2 intragenic suppressor, in which the mutation occurs in the conserved NB domain, abolished defense responses at lower temperatures. Genetic analyses of chs2 in combination with known SA pathway and immune signaling mutants indicate that the chs2- conferred temperature sensitivity requires ENHANCED DISEASE SUSCEPTIBILITY1, REQUIRED FOR Mla12 RESISTANCE, and SUPPRESSOR OF G2 ALLELE OF skp1 but does not require PHYTOALEXIN DEFICIENT4, NONEXPRESSOR OF PR GENES1, or SA. This study reveals that an activated TIR-NB-LRR protein has a large impact on temperature sensitivity in plant growth and survival. For optimal growth and survival, plants have evolved unique and sophisticated defense mechanisms against multiple stresses, both abiotic and biotic. Cold stress has a significant limiting effect on the geographic location of plants and on crop productivity (Guy, 1990). It can disrupt cellular homeostasis by altering the fatty acid composition of membrane lipids, which can deactivate membrane proteins and uncouple ma￾jor physiological processes (Los and Murata, 2004). Plants respond and adapt to cold stress in many biochemical and physiological processes. A number of genes are involved in the DREB/CBF (for DRE￾binding protein/C-repeat-binding factor)-dependent pathway to control cold acclimation (Gilmour et al., 1992, 2004), and DREB/CBF-independent pathways have been identified as important for cold responses as well (Xin and Browse, 1998; Dong et al., 2006; Lee et al., 2006; Xin et al., 2007; Zhu et al., 2008). Plants have evolved at least two layers of defense mechanisms against pathogens. One of them is medi￾ated by resistance (R) proteins. Interaction of an R protein with a specific pathogen avirulence protein triggers the hypersensitive response (HR), which is a form of programmed cell death that limits path￾ogen growth and spread (Scheel, 1998). Most of the characterized R proteins encode proteins with nucle￾otide-binding Leu-rich repeat (NB-LRR) domains. A well-conserved ARC (for Apaf-1, R protein, and CED4) domain is found just after the NB domain, and these two domains are often referred to as the NB￾ARC domain. The NB-LRR proteins can be grouped into two main classes based on their N-terminal struc￾ture, which has either a Toll/Interleukin-1 receptor (TIR) domain or a coiled-coil domain (Meyers et al., 2003). The Arabidopsis (Arabidopsis thaliana) RPP5 (for Rec￾ognition of Peronospora parasitica5) locus in Columbia-0 (Col) is composed of seven TIR-NB-LRR class R genes, including RPP4 and SNC1 (for Suppressor of npr1-1, constitutive 1) genes (Noel et al., 1999). RPP4 plays an important role in resistance to Hyaloperonospora parasitica through multiple signaling components, in- 1 This work was supported by the National Natural Science Foundation of China (grant nos. 30670181, 3077202, and 90817007), the National Key Basic Research Program of China (grant no. 2009CB119100), and the Ministry of Agricultural of China for trans￾genic research (grant no. 2008ZX08009–003). * Corresponding author; e-mail yangshuhua@cau.edu.cn. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Shuhua Yang (yangshuhua@cau.edu.cn). [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a sub￾scription. www.plantphysiol.org/cgi/doi/10.1104/pp.110.157610 796 Plant Physiology, October 2010, Vol. 154, pp. 796–809, www.plantphysiol.org  2010 American Society of Plant Biologists