TESQAKKLADSPEVVHVMADSFYELATTRTWDYLGLSVANPNNLLNDTNMGDQVIIGFIDTGVWPESESFNDNGVGPI PSHWKGGCESGEKFISTNCNRKLIGAKYFINGFLAENEGFNTTESRDYISARDFIGHGTHTASIAGGSFVPNISYKGLAG GNLRGGAPRARIAIYKACWYVDQLGAVACSSSDILKAMDESMHDGVDVLSLSLGAQIPLYPETDLRDRIATGAFHAVAKG IIVVCAGGNSGPAAQTVLNTAPWIITVAATTLDRSFPTPITLGNRKVILGQALYTGQELGFTSLVYPENAGFTNETFSGV CERLNLNPNRTMAGKVVLCFTTNTLFTAVSRAASYVKAAGGLGVIIARNPGYNLTPCRDDFPCVAIDYELGTDVLLYIRS TRSPVVKIQPSRTLVGQPVGTKVATFSSRGPNSISPAILKPDIGAPGVSILAATSPDSNSSVGGFDILAGTSMAAPVVAG VVALLKALHPNWSPAAFRSAIVTTAWRTDPFGEQIFAEGSSRKVADPFDYGGGIVNPEKAADPGLIYDMGPRDYILYLCS AGYNDSSITQLVGNVTVCSTPKTSVLDVNLPSITIPDLKDEVTLTRTVTNVGTVDSVYKVVVEPPLGIQVVVAPETLVFN SKTKNVSFTVRVSTTHKINTGFYFGNLIWTDSMHNVTIPVSVRTQILQNYYDEN80 160 240 320 400 480 560 640 720F I

TESQAKKLADSPEVVHVMADSFYELATTRTWDYLGLSVANPNNLLNDTNMGDQVIIGFIDTGVWPESESFNDNGVGPI PSHWKGGCESGEKFISTNCNRKLIGAKYFINGFLAENEGFNTTESRDYISARDFIGHGTHTASIAGGSFVPNISYKGLAG GNLRGGAPRARIAIYKACWYVDQLGAVACSSSDILKAMDESMHDGVDVLSLSLGAQIPLYPETDLRDRIATGAFHAVAKG IIVVCAGGNSGPAAQTVLNTAPWIITVAATTLDRSFPTPITLGNRKVILGQALYTGQELGFTSLVYPENAGFTNETFSGV CERLNLNPNRTMAGKVVLCFTTNTLFTAVSRAASYVKAAGGLGVIIARNPGYNLTPCRDDFPCVAIDYELGTDVLLYIRS TRSPVVKIQPSRTLVGQPVGTKVATFSSRGPNSISPAILKPDIGAPGVSILAATSPDSNSSVGGFDILAGTSMAAPVVAG VVALLKALHPNWSPAAFRSAIVTTAWRTDPFGEQIFAEGSSRKVADPFDYGGGIVNPEKAADPGLIYDMGPRDYILYLCS AGYNDSSITQLVGNVTVCSTPKTSVLDVNLPSITIPDLKDEVTLTRTVTNVGTVDSVYKVVVEPPLGIQVVVAPETLVFN SKTKNVSFTVRVSTTHKINTGFYFGNLIWTDSMHNVTIPVSVRTQILQNYYDEN80 160 240 320 400 480 560 640 720F I G . three. PME17 and SBT3.5 proteins are identified in cell-wall-enriched 10-d-old root protein extracts. Structural domains (top) and amino acid sequences with peptides identified by MS (bottom) are shown for PME17 (A) and SBT3.five (B). On the structural domains, numbers indicate amino acids in the catalytic site, based on numbering of crystalized models, and domain boundaries. On the amino acid sequences, peptides identified by MS are underlined. Residues involved in catalysis are highlighted in yellow and putative N-glycosylation web pages are coloured in pink and dotted underlined. Within the PME17 sequence, the putative simple processing motif RKLL is highlighted in black.Kazal family members of serine protease inhibitors (Tian and Kamoun, 2005). Following apoplastic washes, equal amounts of extracted proteins were resolved by SDS AGE (Fig. 6B), transferred to nitrocellulose membrane and probed with anti-c-Myc antibodies (Fig. 6C). Within the absence of SBT3.five, two bands inside a molecular mass selection of 35?8 kDa were detected in the apoplasm (Fig. 6C). This suggests that, even though a single RKLL sequence was identified, two processing motifs might be present in thePME17 amino acid sequence, each of that are cleaved by an endogenous tobacco subtilase/protease. An additional band at a molecular mass close to 61 kDa most likely represents the nonprocessed kind of PME17. The recovery of this non-processed form in apoplastic washes is probably to become explained by a slight contamination (5 ) with cytosolic content, as measured by way of an a-mannosidase enzymatic assay (Supplementary Data Table S4). When SBT3.5 was co-infiltrated with PME17, the bigger bandA??Senechal et al. — PME and SBT expression in ArabidopsisFLAG_208G03 pme17-1 PME17-R Promoter PME17-F SAIL_400F09 sbt3.5-1 SBT3.5-R Promoter SBT3.5-F GABI_672C08 sbt3.5-77-SALK_059908 pme17-2 PME17-qRPME17-qFSBT3.Methyl 2-chloro-3-methylisonicotinate In stock 5-qRSBT3.5-qFBs pm WC10 five?1 2?ee2 ol -0 pm CPME17 F/R EF1aRelative expression of SBT3.5/PEX4 (log10)pme17-pme17–.BuyFmoc-N-Me-Phe-OH .PMID:24487575 -10 Relative expression of PME17/PEX4 (log10)-tolsbSBT3.5 F/R EF1asbCt2?six two?sbt3.5-sbt3.5-D six?* Length of 10-d-old roots (cm) five?five 5?0 5?5 five?0 4?five 4?0 **Wspme17-Col-pme17-sbt3.5-sbt3.5-F I G . 4. Characterization of T-DNA insertions lines for PME17 and SBT3.5. (A) Localization of T-DNA insertions in PME17 (leading) and SBT3.five (bottom) genomic DNA sequences. Promoter, 5 -UTR and 3 -UTR, and exons are represented in white, light grey and dark grey bars, respectively. Introns are represented as a black line. Primers F/R and qF/qR were utilised for semi-quantitative PCR and qPCR analyses, respectively. (B) Semi-quantitative PCR on cDNA from 10-d-old roots of wild-type and mutant plants are shown for PME17 (top rated) and SBT3.five (bottom). PME17F/R and SBT3.5F/R primers, flanking the inser.