Huang et al. ased cloning of CHS2 on chromosome IV. Positions of the markers used for mapping are indi- cated above the line. The correspond ChromosomeⅣ ng nucleotide positions are numbered 9400 9450 9500 9550 9600k in kilobases below the line. the num- ber of recombinants is indicated in rentheses. Predicted genes are 45 kb shown by arrows indicating the direc- tion of transcription. B, A schematic a4g16845at4g16855 4g16900at4g16940at4g16960 CHS2 gene. Boxes and lines indicate Genes exons and introns. res ely. The nucleotide substitutions in chs2 and (RPP4) (SNC1 chs2-s1 are shown. C, Complementa- tion of the chs2 mutant. wild-type col B chs2 chs2, and Col transformed with a ge chs2-1(C-T) TAG nomic clone containing the mutat CHS2/RPP4 chs2( Col/CHS2: chs 2) were grown at 22 C for 2 weeks(left)and then treated chs2-S1 (G-A) at 4C for 10 d(right). D, Screenin of the chs2 suppressor chs2-s1. EMS. C 22°C D ed chs2 p at 22C for 2 weeks and then treated at chs2-s 4C for 10d. E, PRI gene expression in Col, chs 2, Col/CHS2: chs2, and chs2 si plants treated at 4C for 6 d by real- time RT-PCR. The data represen means of three replicates t SD. Similar results were observed in three inde. pendent experiments. F, Trypan blue staining of the leaves from chs2, Col CHS2: chs, and chs2-s1 plants Bar 32 100m. chs2 Col/CHS2: chs2 chs2-s1 Transgenic plants harboring a GUS plants(Fig. 6C). However, we found that RPP4 in the driven by the RPP4 promoter were d and wild-type Col background was induced by benzothia- analyzed. GUS staining revealed that RPP4 was ex- diazole(an Sa analog) and cold. Strikingly, cold stress pressed at low levels in leaves, stems, flowers, and dramatically enhanced the induction of the mutated siliques, and it was barely expressed in roots(Fig 6A). RPP4 in chs2(Fig. 6C; Supplemental Fig. S4A). Cold This result is in agreement with public data from induced overexpression could be a consequence of Genevestigator(https://www.genevestigator.com/gv/feedbackregulationuponRgeneactivationwhich index. jsp)and was validated by quantitative reverse might account for the phenotypes of chs2 mutants transcription(rTr-PCr analvsis under cold stress RPP4 was expressed at relatively low levels in the To test if overexpression of wild-type RPP4 would plants, consistent with the low steady-state expression recapitulate the chs2 phenotype, we generated trans- levels of R genes under normal conditions. However, genic lines expressing wild-type RPP4 driven either R genes can be induced by certain stimuli such as by its native promoter(RPP4: RPP4)or by the cauli athogens and SA. Therefore, we investigated whether flower mosaic virus 35S promoter(35S: RPP4), and we RPP4 expression was responsive to various stimuli. The lyzed their phenotypes under cold conditions. In expression of RPP4 was not induced by the oxidative terestingl ither the rPp RPP4 nor 35S: RPP4 ducer methyl viologen in either wild-type Col or chs2 transgenic line, in which RPP4 was indeed overex Plant Physiol. Vol. 154, 2010Transgenic plants harboring a GUS reporter gene driven by the RPP4 promoter were generated and analyzed. GUS staining revealed that RPP4 was ex￾pressed at low levels in leaves, stems, flowers, and siliques, and it was barely expressed in roots (Fig. 6A). This result is in agreement with public data from Genevestigator (https://www.genevestigator.com/gv/ index.jsp) and was validated by quantitative reverse transcription (RT)-PCR analysis (Fig. 6B). RPP4 was expressed at relatively low levels in the plants, consistent with the low steady-state expression levels of R genes under normal conditions. However, R genes can be induced by certain stimuli such as pathogens and SA. Therefore, we investigated whether RPP4 expression was responsive to various stimuli. The expression of RPP4 was not induced by the oxidative inducer methyl viologen in either wild-type Col or chs2 plants (Fig. 6C). However, we found that RPP4 in the wild-type Col background was induced by benzothia￾diazole (an SA analog) and cold. Strikingly, cold stress dramatically enhanced the induction of the mutated RPP4 in chs2 (Fig. 6C; Supplemental Fig. S4A). Cold￾induced overexpression could be a consequence of feedback regulation upon R gene activation, which might account for the phenotypes of chs2 mutants under cold stress. To test if overexpression of wild-type RPP4 would recapitulate the chs2 phenotype, we generated trans￾genic lines expressing wild-type RPP4 driven either by its native promoter (RPP4:RPP4) or by the cauli- flower mosaic virus 35S promoter (35S:RPP4), and we analyzed their phenotypes under cold conditions. In￾terestingly, neither the RPP4:RPP4 nor 35S:RPP4 transgenic line, in which RPP4 was indeed overex￾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 indi￾cated above the line. The correspond￾ing nucleotide positions are numbered in kilobases below the line. The num￾ber of recombinants is indicated in parentheses. Predicted genes are shown by arrows indicating the direc￾tion 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, Complementa￾tion of the chs2 mutant. Wild-type Col, chs2, and Col transformed with a ge￾nomic 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 6 SD. Similar results were observed in three inde￾pendent experiments. F, Trypan blue staining of the leaves from chs2, Col/ CHS2:chs2, and chs2-s1 plants. Bar = 100 mm. Huang et al. 802 Plant Physiol. Vol. 154, 2010