Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Positioning of TRN2. By using 18 dispersed co dominant AFLP markers from chromosome 5 on 46 F2 mutants, derived from a cross of trn2 (Col) x WT (Ler), TRN2 could be positioned in genetic segment 33 on the bottom half of chromosome 5 above the TRN1 locus. Subsequently, 145 F2 mutants were screened with nine co dominant AFLP markers from this genetic segment. In this way TRN2 was located to a 535 kb region flanked by the Col /Ler⁺ AFLP markers SM189_261,1 and SM136_157,9, which are allelic to the Col⁺/Ler AFLP markers, SM189_259,7 and SM136_156,9, respectively. The identified region still contained 10 recombinants. InDel polymorphisms and SNPs in the region of interest were identified from the Cereon Arabidopsis Polymorphism Collection (Cereon Genomics, Cambridge, MA) (http://www.arabidopsis.org/Cereon/index.html)and reduced the TRN2 interval to 18 kb, flanked by the SNP markers CER441660 and CER457484. The TRN2 interval was completely comprised in the BAC clone MZA15 and according to the annotated chromosome sequence (http://mips.gsf.de/proj/thal/db/index.html) contained five genes. Four genes code for putative proteins, and the fifth for a senescence associated protein 5 (SA5) like protein. The five different genes were sequenced for the two Col alleles trn2 1 and trn2 2 (Cnops et al., 2000). Both alleles revealed a single base change in exon 2 of the SA5 like gene indicating that it corresponded to TRN2. The TRN2 gene is a small gene that consisted of two exons of 497 and 311 bp. The TRN2 open reading frame encodes a polypeptide of 269 amino acids with a molecular mass of 30.3 kDa
• Supplemental Figure 2 - Alignment of the Arabidopsis tetraspanin TET proteins, functional domains and trn alleles. Comparison of the TRN2/TET1 sequence with other members of the Arabidopsis tetraspanin family. Protein sequences were aligned using the BioEdit sequence alignment program taking into account the blocks of homology in the transmembrane (TM) domains and the extracellular (ECL) loops. Identical residues are presented in black; similar residues in gray. The four TM domains are indicated by arrows and represent the maximum size for each TM (as predicted for all the domains of the 17 TM4 members with the TMHMM program). The dotted lines correspond with the ECL domains. A conserved C is indicated by a black triangle. The corresponding MIPS code for the different TM4 family members can be found in Figure 2. Improvement of the automatically annotated sequence is presented by an asterisk. The numbers in italics above the sequence refer to the changes found in the different trn2 alleles. 1, position of the stop codon introduced into the genomic sequence of trn2 1; 2, G to E change in trn2 2; 3, P to E change in trn2 3; 4, the 10 amino acids missing in trn2 4; 6, position of the 4 bp insertion in trn2 6. This changes the downstream amino acid into NYHLP followed by a premature stop of translation.
• Supplemental Figure 3 - Sequence alignment used for the phylogenetic tree of plant tetraspanins in NEXUS format. The alignment was based on 68 expresssed sequence tags homologous to TRN2.
• Supplemental Figure 4 - PDR5:GUS activity in a transverse section of a trn leaf. Ectopic PDR5:GUS activity in trn2 1 leaf section is indicated by double arrows. Leaves were stained with 5 bromo 4 chloro 3 indolyl β D glucuronide and visualized under dark field. X marks midvein. Bar = 300 μm.