Huang et al. necessary for the chs2 phenotype by crossing chs2 with 8), indicating that NPRI is dispensable for the chs2- the SA-deficient mutant sid2-2(Wildermuth et al. con 2001 ). The chs2 sid2 double mutants exhibited chilling sensitivity and extensive cell death phenotypes similar chs2-Induced Chilling Sensitivity Requires Multiple to those of chs2(Fig. 7, A and C). As expected, the mutants were reduced to a wild-type level una double levels of sa and total sa in the chs2 sid2 Signaling Components r cold To assess whether defense signaling components stress(Fig. 8). Therefore, the chs2-conferred chilling-(including EDSl, PAD4, SGTIb, and RARI) are in- sensitive phenotype does not require SA volved in the chs2-mediated temperature signaling NPRI is a master regulator of SA signaling and plant pathway, we first examined RPP4 expression in eds1-2 immunity( Cao et al., 1994). To examine the require( Col; Bartsch et al. 2006), pad4-1 rage et al., 1999), ment for NPRI in chs2-mediated signaling, a chs2 npr1 rar1-20(Muskett et al. 2002), and sgt1b/eta3(Gray double mutant was generated and then characterized et aL., 2003)mutants. RPP4 expression was slightly he loss of NPRI function, while significantly reduc- down-regulated by edsI and pad4 but not by rarl or ing PRl expression, did not abrogate the chs2-medi t1b(Supplemental Fig. S5). We also generated dou- ated cold-sensitive morphology, cell death, or the ble mutants of chs2 with eds1-1(Parker et al., 1996), accumulation of SA at low temperature(Figs. 7 and pad4-1, rar1-20, and sgt1bleta3 for further analyses chs2 chs2 eds chs2 pad4B chs2 rar1 chs2 sgt1b chs2 sid2 chs2 npr1 c Col chs2 chs2 eds chs2 pad4 1500 chs2 rar1 chs2 sgt1b chs2 sid2 chs2 npr1 1200 3 chs2 chs2 eds chs2 pad4 chs2 rar1 chs2 sgt1b chs2 sid2 chs2 npr1 Figure 7. Phenotypes of the chs 2 double mutants under cold conditions. Three-week-old 22C-grown plants were treated at 4C for 6d(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 um. Note that the photographs of 4( reated Col and chs 2 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 um. E, PRi 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 Plant Physiol. Vol. 154, 2010necessary for the chs2 phenotype by crossing chs2 with the SA-deficient mutant sid2-2 (Wildermuth et al., 2001). The chs2 sid2 double mutants exhibited chilling sensitivity and extensive cell death phenotypes similar to those of chs2 (Fig. 7, A and C). As expected, the levels of SA and total SA in the chs2 sid2 double mutants were reduced to a wild-type level under cold stress (Fig. 8). Therefore, the chs2-conferred chilling￾sensitive phenotype does not require SA. NPR1 is a master regulator of SA signaling and plant immunity (Cao et al., 1994). To examine the require￾ment for NPR1 in chs2-mediated signaling, a chs2 npr1 double mutant was generated and then characterized. The loss of NPR1 function, while significantly reduc￾ing PR1 expression, did not abrogate the chs2-medi￾ated cold-sensitive morphology, cell death, or the accumulation of SA at low temperature (Figs. 7 and 8), indicating that NPR1 is dispensable for the chs2- conferred phenotype. chs2-Induced Chilling Sensitivity Requires Multiple Signaling Components To assess whether defense signaling components (including EDS1, PAD4, SGT1b, and RAR1) are in￾volved in the chs2-mediated temperature signaling pathway, we first examined RPP4 expression in eds1-2 (Col; Bartsch et al., 2006), pad4-1 (Jirage et al., 1999), rar1-20 (Muskett et al., 2002), and sgt1b/eta3 (Gray et al., 2003) mutants. RPP4 expression was slightly down-regulated by eds1 and pad4 but not by rar1 or sgt1b (Supplemental Fig. S5). We also generated dou￾ble mutants of chs2 with eds1-1 (Parker et al., 1996), pad4-1, rar1-20, and sgt1b/eta3 for further analyses. 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 mm. 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 mm. E, PR1 gene expression in the double mutants by real-time PCR. The data represent means of three replicates 6 SD. * P , 0.01 (t test), significant difference from chs2. All experiments were repeated three times with similar results. Huang et al. 804 Plant Physiol. Vol. 154, 2010