α-mannosidase II (HGL1/MANII) β1,2-n-acetyl-glucosaminyltransferase II (GNTII)

Transcription

1 Oligosaccharyltransferase (OST) (STT3a, STT3b, DD1, DD2, DGL1) Glucosidase I, Glucosidase II ER-Mannosidase α-mannosidase I (MNI) β1,2-n-acetyl-glucosaminyltransferase I (CGL1/GNTI) α-mannosidase II (HGL1/MNII) β1,2-n-acetyl-glucosaminyltransferase II (GNTII) α1,3-fucosyltransferase (FUCTa, FUCTb) β1,2-xylosyltransferase (XYLT) β1,3-galactosyltransferase (GLT) α1,4-fucosyltransferase (FUCTc) β1,2-hexosaminidase (HEX) F X GlcNc Man Glc Gal Fuc Xyl X F F X X P F F F ER Golgi apparatus PM / cell wall STT3a (t5g19690) STT3b (t1g34130) DGL1 (t5g66680) dgl1-1 (-62) dad1 stt3b-1 stt3a-1 stt3a & stt3b Koiwa et al. (2003) dgl1-1 Lerouxel et al. (2005) DD1 (t1g32210) DD2 (t2g35520) dad2 mania-1 dad1 & dad2 (P. Gallois, pers. commun.) MNIa (t1g51590) MNIb (t3g21160) HGL1 (MNII) (t5g14950) CGL1 (GNTI) (t4g38240) fucta manib-2 cgl1-3 hgl1-1 mania-1 manib-2 (H. Koiwa, Kajiura et al.) hgl1-1 Strasser et al. (2006) cgl1-3 Frank et al. ( 2008) FUCTa (t3g19280) FUCTb (t1g49710) xylt fuctb fucta fuctb xylt Strasser et al. (2004), Kang et al. (2008) XYLT (t5g55500) GLT1 (t1g26810) galt1-1 galt1-1 Strasser et al. (2007) (bp) Suppl. Fig. 1. ) Overview of N-glycosylation and N-glycan modification steps in the plant secretory pathway. ER, Endoplasmic Reticulum; PM, plasma membrane. ) Mutant alleles used in this study.

2 250 +elf18 luminescence (RLU) efr time (min) 600 +flg22 luminescence (RLU) fls time (min) Suppl. Fig. 2. Early MMP responses in. Cut leaf pieces of wild type, efr, fls2 and mutant plants were elicited with elf18 or flg22, respectively. ROS production was monitored over time. ars indicate means ± SD (n = 3).

3 120 + elf flg22 growth (% of control) efr C24 stt3a-1 growth (% of control) fls2 C24 stt3a-1 elf18 C24 stt3a-1 efr C efr fls2 C24 stt3a-1 elf26-tyr- 125 I flg22 fls2 ~150 ~100 EFR FLS2 RPL4 125 I-Tyr-flg22 ~175 ~155 Suppl. Fig. 3. MMP responses, ligand binding, and PRR gene expression in the stt3a-1 allele. ) Seedling growth arrest of stt3a-1 in the C24 background. ) Cross-linking of radio-labeled elf26 and flg22 peptides by indicated lines. C) ccumulation of EFR and FLS2 transcripts revealed by RT-PCR of wild type (, C24) and indicated lines. RPL4 was used as a control.

4 elf cgl1-3 fucta fuctb xylt cgl1-3 fucta fuctb xylt elf26-tyr- 125 I ~150 ~100 Suppl. Fig. 4. Elf26 binding in and mutant combinations. Equal amounts of ground seedling material of the indicated lines were incubated with elf26-tyr-125i in the absence (-) or presence (+) of 10 µm elf18. Similar results were obtained in at least three independent experiments.

5 Häweker et al. SiaT-mRFP SiaT-mRFP EFRN143Q-YFP EFRN288Q-YFP stt3b-1 D E EFRN143Q-YFP EFRN288Q-YFP efr +Tu C +Tu Suppl. Fig. 5. Subcellular localization of and its glycosylation variants. Indicated constructs were transiently expressed in protoplasts and observed by confocal microscopy. -D) Representative images of and its variants (green) in wild type, without or with 10 µm tunicamycin (+Tu) incubation or in glycosylation mutants ( or stt3b-1) are shown upon co-expression with ER marker (Frank et al., 2008) or Golgi marker SiaT-mRFP (magenta). Co-localization appears white in d channels. Similar results were obtained in at least two independent experiments. E) Transient expression of and its variants in protoplasts of the efr mutant. Scale bars = 5 µm.

6 Häweker et al. N288 N328 N366 N239 EFR N191 N439 N342 N180 N478 N143 N127 N55 N590 N571 N95 T30 N28 I664 N432 FLS2 N406 N466 N371 N361 N631 N525 N262 N588 N453 N388 N217 N347 N684 N179 N704 N720 N733 N772 N744 N94 T780 K1 I30 N26 N79 N103 N114 N127 N149 N183 K198 Suppl. Fig. 6. Prediction of likely N-glycosylation sites in the LRR domains of EFR, FLS2, and K1. First and last amino acids of the 3D models are shown in black. Less conserved glycosylation sites in EFR (compared with EFR-like proteins) are marked in green, conserved sites in blue, and absolutely conserved sites in red. sparagine residues (N) that were changed to glutamine (Q) by site-directed mutagenesis are highlighted by a red frame.

7 +Tu K N. benth. α-gfp ~175 ~140 FLS2-YFP +Tu K N. benth. α-gfp ~200 ~155 Suppl. Fig. 7. and FLS2-YFP accumulation in the presence of tunicamycin. EFR- and FLS2-YFP constructs were expressed in N. benthamiana by transient transformation. Tunicamycin (10 µm) was infiltrated 3 days later and incubated for the indicated time points (min). Controls, silencing suppressor strain (19K) and non-transformed leaves (N. benth.). Immunoblots were revealed with GFP antibodies.

8 dgl1-1 elf18 galt1-1 dad1 dad2 α-fls2 ~175 ~70 elf26-tyr- 125 I ~150 ~100 flg22 Coomassie 125 I-Tyr-flg22 ~175 ~155 Suppl. Fig. 8. Other glycosylation mutants and OST subunits. ) FLS2 immunoblot. Seedlings were grown in vitro up to 12 days. Mutant seedlings were selected based on their obvious growth phenotype. ll material obtained from the dgl1-1 mutant was loaded, whereas 80 mg fresh weight of seedlings were used. Coomassie staining is shown as loading reference. The presented immunoblots were exposed as follows: for 2 min and dgl1-1 for 30 min. ) Crosslinking experiments with radio-labeled elf26 and flg22 peptides using ground material of the indicated seedlings.

9 elf18 FLS2-YFP EFR N143Q -YFP EFR N288Q -YFP EFR Y702S -YFP - + FLS2-YFP EFR N143Q -YFP EFR N288Q -YFP EFR Y702S -YFP elf26-tyr- 125 I α-gfp Coomassie C EFR GFP actin D FLS2-YFP EFR N143Q -YFP EFR N288Q -YFP EFR Y702S -YFP control EFR N143Q -YFP EFR N288Q -YFP EFR Y702S -YFP Suppl. Fig. 9. nalyses of variants. YFP-fusion constructs were expressed in N. benthamiana by transient transformation. ) Cross-linking of radio-labeled elf26. ) ccumulation of YFP-fusion proteins was detected by immunoblotting with GFP antibodies. Coomassie staining is shown for equal loading. C) Transcript accumulation of the transgenes was controlled by RT-PCR. D) Subcellular localization was observed by confocal microscopy. Represen-tative patterns are shown for and its mutant variants. FLS2-YFP is shown for comparison. Control, not transformed. Scale bar = 20 µm. Similar results were obtained in two independent experiments.