A gain-of-function mutation in the Arabidopsis disease resistance gene RPP4 confers sensitivity to low temperature

Abstract

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.

Figure 1.

Cold sensitivity of chs2 mutant plants. A, Phenotypes of wild-type Col and chs2 plants grown in soil at 22°C for 4 weeks (top row), cold treated at 4°C for 10 d (middle row), followed by 22°C for 3 d (bottom row). B, Phenotypes of wild-type Col and chs2 plants grown in soil at 22°C for 6 weeks (top) followed by cold treatment at 4°C for 10 d (bottom). C and D, Phenotypes of Col and chs2 plants directly geminated on MS plates and grown at 4°C for 3 months (C) or grown at 22°C for 3 weeks and then transferred to 4°C for an additional 10 d (D). E, Phenotypes of wild-type Col and chs2 plants grown in soil at 16°C for 7 weeks. F, Phenotypes of chs2 plants grown on MS plates at the indicated temperatures for 4 weeks. Images are of representative plants.

Figure 2.

Physiological analysis of chs2 mutant plants. A, Ion leakage assay in chs2 plants. Plants grown at 22°C for 3 weeks were then treated at 4°C for the indicated times. The data represent means of three replicates ± sd. Similar results were observed in three independent experiments. B, Pro content in chs2 plants. Plants grown at 22°C for 3 weeks were treated at 4°C for 6 d. The data represent means of four replicates ± sd. * P < 0.01 (t test), significant difference from Col. Similar results were observed in three independent experiments. FW, Fresh weight.

Figure 3.

The effect of the chs2 mutation on chloroplast development under cold stress. Wild-type Col and chs2 plants were grown at 22°C for 3 weeks and then treated at 4°C for 10 d. A, Chlorophyll content of Col and chs2 seedlings. The data represent means of four replicates ± sd. * P < 0.01 (t test), significant difference from Col. Similar results were observed in three independent experiments. B, Transmission electron microscopy of plastids from chs2 plants. Bar = 5 μm (top row) and 2 μm (bottom row). Images are of representative plants.

Figure 4.

chs2 constitutively activates defense responses to cold. Wild-type Col and chs2 plants were grown at 22°C for 3 weeks and then treated at 4°C for 6 d. For A, B, and E, 20 plants were tested for each genotype. Images are of representative plants from one of three independent experiments. A, H₂O₂ accumulation in chs2 plants stained by DAB. Bar = 20 μm. B, Cold-induced cell death in chs2 plants. Detached leaves were stained with trypan blue. Bar = 100 μm. Images are of representative plants. C, Expression of PR1 and PR2 in wild-type and chs2 plants by real-time RT-PCR. The data represent means of three replicates ± sd. Similar results were observed in three independent experiments. D, GUS analysis of PR1 in chs2 plants. PR1:GUS transgenic plants were crossed with chs2 plants. The F2 homozygous lines were used for GUS staining analysis. Images are of representative plants. E, SA accumulation in chs2 under cold conditions. Three-week-old 22°C-grown plants were treated at 4°C for 6 d. The data represent means of three replicates ± sd. Similar results were observed in three independent experiments. FW, Fresh weight.

Figure 5.

Map-based cloning of CHS2. A, A genetic map of the CHS2 locus on chromosome IV. Positions of the markers used for mapping are indicated above the line. The corresponding nucleotide positions are numbered in kilobases below the line. The number of recombinants is indicated in parentheses. Predicted genes are shown by arrows indicating the direction of transcription. B, A schematic diagram of the genomic structure of the CHS2 gene. Boxes and lines indicate exons and introns, respectively. The nucleotide substitutions in chs2 and chs2-s1 are shown. C, Complementation of the chs2 mutant. Wild-type Col, chs2, and Col transformed with a genomic clone containing the mutated chs2 (Col/CHS2:chs2) were grown at 22°C for 2 weeks (left) and then treated at 4°C for 10 d (right). D, Screening of the chs2 suppressor chs2-s1. EMS-mutagenized chs2 plants were grown at 22°C for 2 weeks and then treated at 4°C for 10 d. E, PR1 gene expression in Col, chs2, Col/CHS2:chs2, and chs2-s1 plants treated at 4°C for 6 d by real-time RT-PCR. The data represent means of three replicates ± sd. Similar results were observed in three independent experiments. F, Trypan blue staining of the leaves from chs2, Col/CHS2:chs2, and chs2-s1 plants. Bar = 100 μm.

Figure 6.

Expression of RPP4 and SNC1 in chs2. A and B, Expression of RPP4 by GUS staining (A) and by real-time PCR (B). Total RNA was extracted from various tissues. The data represent means of three replicates ± sd. C and D, Expression of RPP4 (C) and SNC1 (D) under various treatments. Total RNA was extracted from plants treated with cold (4°C), methyl viologen (MV; 5 μm), or benzothiadiazole (BTH; 0.5 mm) for 24 h. E and F, Expression of RPP4 (E) and SNC1 (F) in 2-week-old 22°C-grown plants (Col, chs2, RPP4:RPP4, 35S:RPP4, snc1-1, and bon1-1) by real-time PCR. The data represent means of three replicates ± sd. * P < 0.01 (t test), significant difference from Col. All experiments were repeated three times with similar results. G, Phenotypes of the plants (Col, chs2, RPP4:RPP4, 35S:RPP4, snc1-1,and bon1-1) grown in soil at 22°C for 4 weeks and then cold treated at 4°C for 10 d.

Figure 7.

Phenotypes of the chs2 double mutants under cold conditions. Three-week-old 22°C-grown plants were treated at 4°C for 6 d (C–E), 14 d (A), or 5 weeks (B). A and B, Growth phenotypes of the double mutants under cold conditions. Representative plants are shown. C, Trypan blue staining of the leaves from the double mutants. Bar = 100 μm. Note that the photographs of 4°C-treated Col and chs2 plants stained with trypan blue are identical to those shown in Figure 2B. D, DAB staining of the leaves from the double mutants. Bar = 100 μm. E, PR1 gene expression in the double mutants by real-time PCR. The data represent means of three replicates ± sd. * P < 0.01 (t test), significant difference from chs2. All experiments were repeated three times with similar results.

Figure 8.

SA accumulation in the double mutants under cold conditions. Three-week-old 22°C-grown plants were treated at 4°C for 6 d. Shown are mean values of free and total SA amount in different genotypes of three replicates ± sd. Similar results were observed in three independent experiments.