Function of a Mutant RPP4 in Response to Chilling A C Figure 6. Expression of RPP4 and SNCI o Col A and B, Expression of RPP4 by GUS stai and by real-time PCR(B). Total RNA wa from various tissues. The data represent means of hree replicates sD C and D, Expression of RPP4 (C)and SNC1( D)under various treatments. Total RNA was extracted from plants treated 4C), methyl viologen(MV; 5 AM), or be Mock MV BTH Cold diazole(BTH: 0.5 mM) for 24 h. E and F D sion of RPP4(E)and SNCI( F)in 2-week-old 22.grown plants(Col, chs2, RPP4: RPP4, 355. RPP4, snc]-1, and bon1-1)by real esent means of three replicates +sD P<0.01 (t test), significant difference from n nI CoL. All experiments were repeated three times with similar results. G. Phenot ants(Col, chs 2, RPP4: RPP4, 3.5S: RPP4, snc1-1 Mock MV BTH Cold and bon]-1)grown in soil at 22C for 4 weeks and then cold treated at 4C for 10 d E F RPP4: RPP4 35S: RPP4 pressed(Fig. 6E), exhibited chs2-like phenotypes at transcript than did cold-treated Col plants(Fig. 6D 4oC(Fig. 6G). Therefore, the chs2-conferred pheno- Supplemental Fig $4B) types are not simply caused by constitutive expression To determine whether up-regulation of SNCl also of RPP4 but rather by the amino acid substitution in contributes to the chs2 phenotype, we tested the cold chs2. All of these data indicate that chs2 is a gain-of- sensitivity of sncl-I and bonl-1 plants, in which SNCI function mutant and that cold-induced overexpression is activated or derepressed(Yang and Hua, 2004; L of the mutated RPP4 gene is required for the chs2 et al., 2007; Fig 6F). Neither of them showed a chs2-like phenotype lethal phenotype at cold stress(Fig. 6G). In addition, we transformed the CHS2 chs2 clone into sncl-1l loss- of-function mutant plants. All 10 independent trans- chs2-Induced Chilling Sensitivity Is Independent genic lines displayed a chs2-like chilling-sensitive of SNcl phenotype(data not shown), indicating that the chs2 mutation confers a chs2 phenotype independent of Since the RPP5 locus R genes are coordinately SNCI regulated (Yi and Richards, 2007), we examined the expression of SNC1, a close homolog of RPP4, in the chs2-Induced Chilling Sensitivity Is Independent of SA chs2 mutant. Similar expression patterns of SNC1 induction were found in wild-type Col and chs2 plants (Fig. 6D). SNCI expression was induced by benzothia Because chs2 plants accumulated high levels of free diazole and cold stress in both genotypes. Moreover, SA and total SA after cold treatment(Fig. 4E), we then chs2 plants accumulated higher levels of the SNC1 determined whether activation of the Sa pathway is Plant Ph Vol.154,2010pressed (Fig. 6E), exhibited chs2-like phenotypes at 4
C (Fig. 6G). Therefore, the chs2-conferred pheno￾types are not simply caused by constitutive expression of RPP4 but rather by the amino acid substitution in chs2. All of these data indicate that chs2 is a gain-of￾function mutant and that cold-induced overexpression of the mutated RPP4 gene is required for the chs2 phenotype. chs2-Induced Chilling Sensitivity Is Independent of SNC1 Since the RPP5 locus R genes are coordinately regulated (Yi and Richards, 2007), we examined the expression of SNC1, a close homolog of RPP4, in the chs2 mutant. Similar expression patterns of SNC1 induction were found in wild-type Col and chs2 plants (Fig. 6D). SNC1 expression was induced by benzothia￾diazole and cold stress in both genotypes. Moreover, chs2 plants accumulated higher levels of the SNC1 transcript than did cold-treated Col plants (Fig. 6D; Supplemental Fig. S4B). To determine whether up-regulation of SNC1 also contributes to the chs2 phenotype, we tested the cold sensitivity of snc1-1 and bon1-1 plants, in which SNC1 is activated or derepressed (Yang and Hua, 2004; Li et al., 2007; Fig. 6F). Neither of them showed a chs2-like lethal phenotype at cold stress (Fig. 6G). In addition, we transformed the CHS2:chs2 clone into snc1-11 loss￾of-function mutant plants. All 10 independent trans￾genic lines displayed a chs2-like chilling-sensitive phenotype (data not shown), indicating that the chs2 mutation confers a chs2 phenotype independent of SNC1. chs2-Induced Chilling Sensitivity Is Independent of SA and NPR1 Because chs2 plants accumulated high levels of free SA and total SA after cold treatment (Fig. 4E), we then determined whether activation of the SA pathway is 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 6 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 mM), or benzothia￾diazole (BTH; 0.5 mM) for 24 h. E and F, Expres￾sion 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 6 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. Function of a Mutant RPP4 in Response to Chilling Plant Physiol. Vol. 154, 2010 803