FLS2 Molecular Cell Volume 5, Issue 6, Pages 1003-1011 (June 2000) Lourdes Gómez-Gómez, Thomas Boller  Molecular Cell  Volume 5, Issue 6, Pages 1003-1011 (June 2000) DOI: 10.1016/S1097-2765(00)80265-8

Figure 1 Phenotypes of Wild-Type and fls2 Seedlings Untreated or Treated with Flg22 Flg22 treatment caused growth inhibition in wild-type plants, while it did not induce this response in mutant plants. Molecular Cell 2000 5, 1003-1011DOI: (10.1016/S1097-2765(00)80265-8)

Figure 2 Positional Cloning of the FLS2 Gene and Functional Complementation (A) Map of the fls2 locus on chromosome 5. The FLS2 gene was mapped at the bottom of chromosome 5 between marker LFY3 and DFR. Six additional PCR-based genetic markers (TRX3, MLN [mi83], MCL19.9, MPL12.1, MPL12.10, CIC10G6) developed from EST sequences, YAC end sequences, and from genes already mapped in this region were used to further delimit the FLS2 gene. For marker TRX3, located on the telomeric side of FLS2 gene, a total of 58 heterozygous were identified from 548 chromosomes. Of these 58 heterozygous for TRX3, 26 were homozygous La-er for marker MNL1. From the 26 detected at the MNL marker, 12 heterozygous chromosomes were detected for marker MCL19–9. For marker MPL12.1, located in the MPL12 cosmid, six heterozygous were detected. For marker Kn44/45 (Niyogi et al. 1993), located on the telomeric side of the FLS2 gene, 44 heterozygous chromosomes were detected from 582 total chromosomes. Eight of these chromosomes were heterozygous for marker CIC10G6. More F2 flg22-insensitive plants were analyzed for recombinants in this marker, and 12 recombinants out of 606 chromosomes were identified. No recombinants were identified for marker MPL12.10. The position of the FLS2 gene was finally restricted to the cosmid clone MPL12 (GenBank accession number AB010698). The predicted gene organization of MPL12 is displayed; the boxes represent predicted genes. E and B represent restriction sites for EcoRI and BamHI, respectively. The diagram below represents the FLS2 transcript, with exons indicated by straight horizontal lines and the intron indicated by lines angled downward. The initiator (ATG) and termination (TAG) codons are indicated. The upward pointing arrows indicate the locations and amino acid changes of the three sequenced mutations. (B) Complementation of fls2 mutants in response to flg22 treatment. The pBBFLS2 construct provides suppression of the fls2-24 and fls2-17 flagellin insensitive phenotype. Fresh weight of seedlings was determined 15 days after treatment. Pictures of representative T2 generation plants were taken 15 days after flg22 treatment. Molecular Cell 2000 5, 1003-1011DOI: (10.1016/S1097-2765(00)80265-8)

Figure 3 Deduced Structural Features of FLS2 The amino acid sequence predicted from the DNA sequence of FLS2 is shown divided into nine domains (A–I) indicated as follows: (A), potential signal sequence; (B), unknown domain; (C), LRR domain (a 23–amino acid island that fit the consensus sequence of leucine zipper motifs was buried between the 16th and 17th LRR and is shown underlined); (D and F), charged; (E), transmembrane; (G), juxtamembrane; (H), serine-threonine kinase; and (I), COOH-terminal tail. Molecular Cell 2000 5, 1003-1011DOI: (10.1016/S1097-2765(00)80265-8)

Figure 4 FLS2 Is a Single Copy Gene Southern blot analysis of 10 μg of digested gDNA isolated from wild-type (La-er) Arabidopsis. NdeI made no cut, EcoRI cut twice, and HindIII cut seven times in the gDNA sequence of FLS2. Molecular Cell 2000 5, 1003-1011DOI: (10.1016/S1097-2765(00)80265-8)

Figure 5 FLS2 mRNA Is Present in All the Analyzed Organs (A) FLS2 RNA levels in roots, stems, flowers, and leaves of La-er plants were detected by RT-PCR. (B) RT-PCR analysis of 7-day-old La-er and fls2-24 seedlings treated or untreated with flg22 during 24 hr. Molecular Cell 2000 5, 1003-1011DOI: (10.1016/S1097-2765(00)80265-8)

Figure 6 Elevated FLS2 Transcript Levels Enhance and Reduced FLS2 Transcript Levels Reduce Flagellin Responses (A) RT-PCR analysis of FLS2 transcript levels in wild type (wt) and in several T1 35S::FLS2 Arabidopsis plants. (B) Callose deposition in Arabidopsis leaves 12 hr after flg22 treatment. (C) Oxidative burst in the leaf tissues of Col-0 wild type, 35S::FLS2-Col2, and 35S::FLS2-Col4, after treatment with 10 nM flg22. (D) Relative fresh weight measurements of flg22 untreated or treated T2 generation seedlings from both transgenic parental lines, 35S::FLS2-Col2 and 35S::FLS2-Col4, and of wild-type Col-0 seedlings untreated or treated with 10 μM flg22 for 7 days. (Lower panel) Phenotype of 35S::FLS2 transgenic and Col-0 plants treated or untreated with 10 μM flg22 for 7 days. Shown are representative hygromycin resistant T2 generation seedlings from 35S::FLS2-Col2 and 35S::FLS2-Col4 parental lines. The photographs were taken at the same magnification. Molecular Cell 2000 5, 1003-1011DOI: (10.1016/S1097-2765(00)80265-8)