Function of a Mutant RPP4 in Response to Chilling A 22c 4°C 4. chs2 constitutively activates Col chs2 Col chs2 nts were grown at 3 weeks and then treated at are of representative plants from one of three independent experiments. A H,O, accumulation in chs2 plants Cold-induced cell death in chs2 nts. Detached leaves were stained ages are of representative plant of Pri and Pr2 in wild- type and chs 2 plants by real-time Rt- PCR. The data represent means of three eplicates sD. Similar results were observed in three ind ments. D, GUS analysis of PRi in ch B plants. PR1: GUS transgenic plants were crossed with chs2 plants. The F2 homozygous lines were used for GUS chs2 under cold conditions. Thre treated at 4C for 6 d. The data repre nt means of three replicates +sD. Similar results were observed in three sosN PR1:GUS/Col 22°C223d22°c6d 2℃22c-3d22C6d PR1: GUS/chs2 E Free Sa Total SA 60 10 20 22°C 4C PRI gene expression and the cell death phenotype proteins, and mutations in or close to this conserved were significantly suppressed in chs2-s1(Fig 5, E and motif might abrogate the activity of NB-LRR proteins F). This mutation was mapped to the original RPP4( Bendahmane et al., 2002) locus. Sequencing analysis revealed a second point mutation of e to K at amino acid position 300 in chs 2- sl, which resides close to the walker b/Kinase 2 motif RPP4 Expression in chs2 at Different Conditions of the rPp4 nB domain(Fig. 5B). This motif was To elucidate the physiological function of RPP4, we shown to be important for the function of NB-LRR examined its organ-specific expression in Arabidopsis Plant Ph Vol.154,2010PR1 gene expression and the cell death phenotype were significantly suppressed in chs2-s1 (Fig. 5, E and F). This mutation was mapped to the original RPP4 locus. Sequencing analysis revealed a second point mutation of E to K at amino acid position 300 in chs2- s1, which resides close to the Walker B/Kinase 2 motif of the RPP4 NB domain (Fig. 5B). This motif was shown to be important for the function of NB-LRR proteins, and mutations in or close to this conserved motif might abrogate the activity of NB-LRR proteins (Bendahmane et al., 2002). RPP4 Expression in chs2 at Different Conditions To elucidate the physiological function of RPP4, we examined its organ-specific expression in Arabidopsis. 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 mm. B, Cold-induced cell death in chs2 plants. Detached leaves were stained with trypan blue. Bar = 100 mm. Im￾ages 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 6 SD. Similar results were observed in three independent experi￾ments. 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 repre￾sentative 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 repre￾sent means of three replicates 6 SD. Similar results were observed in three independent experiments. FW, Fresh weight. Function of a Mutant RPP4 in Response to Chilling Plant Physiol. Vol. 154, 2010 801