The Arabidopsis V-ATPase is localized to the TGN/EE via a seed plant-specific motif
- Department of Cell Biology, Centre for Organismal Studies, Heidelberg University, Germany
- Electron Microscopy Core Facility, Heidelberg University, Germany
- Department of Biodiversity and Plant Systematics, Centre for Organismal Studies, Heidelberg University, Germany
- Division of Cellular Dynamics, National Institute for Basic Biology, Japan
- The Department of Basic Biology, SOKENDAI (The Graduate University for Advanced Studies), Japan
Figures
Figure 1 with 3 supplements
The targeting signal of VHA‐a1 is located between L132 and E179 and the a1-TD is sufficient for targeting of VHA‐a3 to the TGN/EE.
Root tips of 6-day-old Arabidopsis seedlings were analyzed by confocal laser scanning microscopy (CLSM) (A) The chimeric constructs a1NT179a3-GFP and a1NT228a3-GFP show dual localization at the …
Figure 1—figure supplement 1
The tri-peptide motif that is responsible for the targeting of Stv1p is absent in VHA-a1.
Amino acid sequence alignment of the yeast subunit a isoforms Vph1p and Stv1p with the Arabidopsis isoforms VHA-a1 and VHA-a3. Residues similar between all proteins at the same position are shown …
Figure 1—figure supplement 2
The targeting signal of VHA‐a1 is not located in the first 85 amino acids.
(A) Chimeric proteins were made which consisted of increasing lengths of the VHA‐a1 N‐terminus fused to decreasing lengths of the C‐terminal domain of VHA‐a3. All constructs were fused to GFP. (B) …
Figure 1—figure supplement 3
Three-dimensional models of the VHA-a1 and VHA-a3 N-termini.
Homology modeling of the N- termini of VHA-a1 and VHA-a3 was done using cryo-EM models of Stv1p (PDB607U) and Vph1p (PDB607T) respectively as templates. The models are color ramped.
Figure 2 with 1 supplement
Site-directed mutagenesis reveals the importance of conserved amino acids in the targeting of VHA‐a1 to the TGN/EE.
(A) The VHA-a1-clade consensus sequence for the a1-TD region was made on the weblogo platform (Crooks et al., 2004). Sequence numbers are based on Arabidopsis VHA-a1. Conserved amino acids were …
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Figure 2—source data 1
Source data for Figure 2K.
- https://cdn.elifesciences.org/articles/60568/elife-60568-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
The VHA-a1-TD is conserved in Angiosperms.
Amino acid sequence alignment of representative sequences from the VHA-a1 and VHA-a3 clades. The sequence numbers are in reference to the A. thaliana VHA-a1 sequence. Residues highlighted in black, …
Figure 3 with 1 supplement
VHA-a1 is retained at the ER after AtSar1b-GTP-CFP expression.
After 6 hr of induction with 60 µM DEX, AtSar1b-GTP-CFP is expressed in 6-day-old Arabidopsis root tip cells. (A) VHA-a1-GFP is retained in the ER and also agglomerates to produce bright punctae …
Figure 3—figure supplement 1
AtSar1b-GTP-CFP expression blocks the ER exit of secretory pathway proteins.
(A) EM of high-pressure frozen Arabidopsis root tips. After 6 hr of induction with 60 µM DEX, AtSar1b-GTP-CFP expression causes bloating of the ER, aggregation of Golgi stacks and leads to an …
Figure 4
The mutations in the a1-TD affect an ER-exit motif.
The localization and fluorescence intensity of mutated VHA-a1-GFP proteins was analyzed in the presence (+DEX) and absence (DMSO) of AtSar1b-GTP-CFP. Root tips of 6-day-old Arabidopsis seedlings …
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Figure 4—source data 1
Source data for Figure 4B.
- https://cdn.elifesciences.org/articles/60568/elife-60568-fig4-data1-v2.xlsx
Figure 5 with 1 supplement
Tonoplast localization of mutated VHA-a1 increases in the absence of VHA-a2 and VHA-a3.
(A) The localization of mutated VHA-a1 proteins tagged to GFP were analyzed in the vha-a2 vha-a3 double mutant background after 20 min staining with FM4-64. Root tips of 6-day-old Arabidopsis …
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Figure 5—source data 1
Source data for Figure 5B.
- https://cdn.elifesciences.org/articles/60568/elife-60568-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
A competition exists to enter provacuoles in wild type Arabidopsis root tip cells.
VHA-a3-R729N-GFP is retained in the wild type background and localizes to the tonoplast in the vha-a2 vha-a3 double mutant background. Scale bars = 10 µm.
Figure 6 with 1 supplement
Spermatophyte VHA-a isoforms cluster into two distinct clades, the a1-TD is conserved in the VHA-a1 clade and originates in the gymnosperm sequences.
(A) Phylogenetic analysis of the N-terminal sequences of VHA-a proteins was done. A graphical summary of the tree is depicted. VHA-a isoforms from seed plants including the gymnosperm Pinus taeda …
Figure 6—figure supplement 1
Phylogenetic analysis of the N-terminal sequences of VHA-a related proteins.
VHA-a related protein sequences for selected species are shown. Branch support is calculated on the basis of 500 bootstraps.
Figure 7 with 2 supplements
The mutated VHA-a1 subunits complement the vha-a2 vha-a3 double mutant to varying degrees.
Plants were grown in short day conditions (22 °C and 10 hr light) for 6 weeks. (A) All mutant variants of VHA-a1-GFP displayed bigger rosette size than the vha-a2 vha-a3 double mutant. E161S which …
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Figure 7—source data 1
Source data for Figure 7B and C.
- https://cdn.elifesciences.org/articles/60568/elife-60568-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
The mutated VHA-a1-GFP proteins complement the vha-a2 vha-a3 double mutant to varying degrees in long day conditions.
Plants were grown in long day conditions (22 °C and 16 hr of light) for 4 weeks. All mutant variants of VHA-a1-GFP displayed bigger rosette size than the vha-a2 vha-a3 double mutant. VHA-a1 with the …
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Figure 7—figure supplement 1—source data 1
Source data for Figure 7—figure supplement 1B.
- https://cdn.elifesciences.org/articles/60568/elife-60568-fig7-figsupp1-data1-v2.xlsx
Figure 7—figure supplement 2
Protein levels of the mutated VHA-a proteins at the tonoplast.
Abundance of the GFP tagged proteins was determined via western blot. Tonoplast membrane proteins were separated by SDS-PAGE and subsequently immunoblotted with an anti-VHA-a1, VHA-a3 and VHA-B …
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Figure 7—figure supplement 2—source data 1
Source data for Figure 7—figure supplement 2.
- https://cdn.elifesciences.org/articles/60568/elife-60568-fig7-figsupp2-data1-v2.zip
Figure 8 with 2 supplements
MpVHA-a containing complexes are functional in Arabidopsis and can replace VHA-a3 and VHA-a2 at the tonoplast.
(A) MpVHA-a-mVenus is dual localized at the tonoplast and TGN/EE in the vha-a2 vha-a3 background. The TGN/EE localization was confirmed by treatment of root cells with 50 µm BFA for 3 hr followed by …
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Figure 8—source data 1
Source data for Figure 8C.
- https://cdn.elifesciences.org/articles/60568/elife-60568-fig8-data1-v2.xlsx
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Figure 8—source data 2
Source data for Figure 8D.
- https://cdn.elifesciences.org/articles/60568/elife-60568-fig8-data2-v2.zip
Figure 8—figure supplement 1
Subcellular localization of MpVHA-a-mVenus in M. polymorpha.
(A and B) Single confocal images of M. polymorpha dorsal thallus cells co-expressing mRFP-MpSYP6A and MpVHA-a-mVenus driven by the CaMV35S (A) or MpEF1α (B) promoter. Green, magenta, and blue pseudo …
Figure 8—figure supplement 2
MpVHA-a-mVenus is dual localized at the TGN/EE and tonoplast in Arabidopsis wildtype root cells.
UBQ10:MpVHA-a-mVenus was co-expressed with UBQ10:VHA-a3-pmScarlet-I and VHA-a1:VHA-a1-mRFP in Arabidopsis wildtype. Confocal analysis was done on 6-day-old Arabidopsis roots. (A) MpVHA-a-mvenus …
Figure 9 with 2 supplements
VHA-a1 is essential for pollen development but not for vegetative growth.
(A) The exon-intron structure of the first seven exons of VHA-a1 shows the sites which were targeted in independent CRISPR approaches. vha-a1-1, vha-a1-2, vha-a1-3 and vha-a1-4 are examples for vha–a…
Figure 9—figure supplement 1
vha-a1-1 can be distinguished from the wildtype allele without sequencing and mutations in VHA-a1-GFP lead to absence of GFP signal.
(A) Mutations in VHA-a1 were distinguished from mutations in VHA-a1-GFP by use of specific primers. Shown are the exon-intron structures of gene and transgene. Primer one is VHA-a1 specific, while …
Figure 9—figure supplement 2
MpVHA-a-mVenus is dual localized in vha-a1.
(A) vha-a1 (Cas9+) was crossed with UBQ10:MpVHA-a-mVenus in wildtype background and F1 seedlings were analyzed by CLSM. Root tips of 6-day-old seedlings were analyzed. MpVHA-a-mVenus was dual …
Figure 10 with 2 supplements
VHA-a2 and VHA-a3 replace VHA-a1 at the TGN/EE in vha-a1 during vegetative growth.
(A) vha-a1 vha-a1-GFP hypocotyls are hypersensitive to 125 nM ConcA, in contrast to wildtype hypocotyls indicating that a target of ConcA is present at the TGN/EE. vha-a1 hypocotyls expressing UBQ10:…
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Figure 10—source data 1
Source data for Figure 10A.
- https://cdn.elifesciences.org/articles/60568/elife-60568-fig10-data1-v2.xlsx
Figure 10—figure supplement 1
vha-a1 is hypersensitive to Concanamycin-A (ConcA).
Root morphology of 4-day-old etiolated seedlings. vha-a1 roots are hypersensitive to 125 nM ConcA, in contrast to wild type roots and roots of plants expressing vha-a1 VHA-a1-GFP. Scale bars = 75 µm.
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Figure 10—figure supplement 1—source data 1
Source data for Figure 10—figure supplement 1A.
- https://cdn.elifesciences.org/articles/60568/elife-60568-fig10-figsupp1-data1-v2.xlsx
Figure 10—figure supplement 2
vha-a1 vha-a2/+ and vha-a1 vha-a3/+ have reduced rosette sizes.
(A) vha-a1 (Cas9+) was crossed with the vha-a2 vha-a3 double mutant. Analysis of F1 plants revealed that vha-a1 vha-a2/+ vha-a3/+ is reduced in growth. Rosette areas of 3.5-week-old plants were …
Figure 11
A competition exists to enter COPII vesicles in Arabidopsis.
Upper panel: COPII-mediated ER-export is the only way to exit the ER in Marchantia. Marchantia VHA-a (MpVHA-a) does not contain a TGN/EE-retention signal. MpVHA-a complexes acidify the TGN/EE en …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background Arabidopsis thaliana | Col-0 | Nottingham Arabidopsis Stock center (NASC) | NASC: N37008 | |
| Strain, strain background (Marchantia polymorpha) | Tak-1 | Ishizaki et al., 2008 PMID:18535011 | ||
| Genetic reagent Arabidopsis thaliana | vha-a2 vha-a3 | (Krebs et al., 2010) PMID:20133698 | At2g21410,At4g39080 | SALK_142642 SALK_29786 |
| Genetic reagent Arabidopsis thaliana | vha-a1-1 | this study | At2g28520 | See Materials and methods, Construct design and plant transformation |
| Genetic reagent Arabidopsis thaliana | vha-a1-2 | this study | At2g28520 | See Materials and methods, Construct design and plant transformation |
| Genetic reagent Arabidopsis thaliana | vha-a1-3 | this study | At2g28520 | See Materials and methods, Construct design and plant transformation |
| Genetic reagent Arabidopsis thaliana | vha-a1-4 | this study | At2g28520 | See Materials and methods, Construct design and plant transformation |
| Genetic reagent Arabidopsis thaliana | BRI1: BRI-GFP | (Geldner et al., 2007) PMID:17578906 | ||
| Genetic reagent (Marchantia polymorpha) | CaMV35S:mRFPMpSyp6A | (Kanazawa et al., 2016) PMID:26019268 | ||
| Gene Arabidopsis thaliana | VHA-a1 | arabidopsis.org | At2g28520 | |
| Gene Arabidopsis thaliana | VHA-a3 | arabidopsis.org | At4g39080 | |
| Gene Arabidopsis thaliana | Sar1B | arabidopsis.org | At1g56330 | |
| Gene (Rattus norvegicus) | Beta-galactoside alpha-2,6-sialyltransferase 1 (ST) | RGD | 3676 | |
| Gene Arabidopsis thaliana | Brassinosteroid insensitive 1 (BRI1) | arabidopsis.org | At4g39400 | |
| Gene (Marchantia polymorpha) | MpVHA-a | Marchantia genome database | Mp3g15140 | |
| Gene Arabidopsis thaliana | MpSYP6A | Marchantia genome database | Mp3g18380 | |
| Gene (Amborella trichopoda) | Amborella trichopoda VHA-a | Phytozome | evm_27.TU.AmTr _v1.0_scaffold00080.37 | |
| Gene (Selaginella moellendorffii) | Selaginella moellendorffii VHA-a | Phytozome | 182335 | |
| Gene (Pinus taeda) | Pinus taeda VHA-a | PineRefSeq, Tree genes | 5A_I15_VO_L_1_ T_29156/41278 | |
| Transfected construct Arabidopsis thaliana | CRISPR VHA-a1 U6-26p: gRNA one and U6-29p:gRNA two in pHEE401E | this study | Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | CRISPR VHA-a1 U6-26p: gRNA three in pHEE401E | this study | Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | CRISPR VHA-a1 U6-26p: gRNA four in pHEE401E | this study | Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10: VHA-a1 NT 35 aa-VHA-a3 | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10: VHA-a1 NT 85 aa-VHA-a3 | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10: VHA-a1 NT 131 aa-VHA-a3 | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10: VHA-a1 NT 179 aa-VHA-a3 | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10: VHA-a1 NT 228 aa-VHA-a3 | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10:VHA-a1-intron10-GFP | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ:VHA-a3-pmScarlet-I | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10:VHA-a1-intron10 E156Q-GFP | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10:VHA-a1-intron10 E161S-GFP | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10:VHA-a1-intron10 F134Y-GFP | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10:VHA-a1-intron10 L159T-GFP | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10:VHA-a1-intron10 E156Q + L159T-GFP | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10:VHA-a1-intron10 L159T + E161S -GFP | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10:VHA-a1- intron10 ELE-GFP | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10:VHA-a1-intron10 ΔEEI-GFP | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ:VHA-a3R729N-GFP | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10:VHA-a3-a1-TD-GFP | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | Dex:Sar1BH74L-CFP | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ10:MpVHA-a-mVenus | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct (Marchantia polymorpha) | CaMV35S:MpVHA-a-mVenus | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct (Marchantia polymorpha) | MpEF1α:MpVHA-a-mVenus | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ: A.trichopoda -VHA-a-NT-VHA-a1-mCherry | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ:P.taeda-VHA- a-NT-VHA-a1-mVenus | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ:S.moellendorffii -VHA-a-NT-VHA-a1-mCherry | this study | See Materials and methods, Construct design and plant transformation | |
| Transfected construct Arabidopsis thaliana | UBQ:ST-GFP | this study | See Materials and methods, Construct design and plant transformation | |
| Recombinant DNA reagent (plasmid) | UBQ10 promoter | Lampropoulos et al., 2013 PMID:24376629 | pGGA006 | |
| Recombinant DNA reagent (plasmid) | pOp6 | Schürholz et al., 2018 PMID:30026289 | pGGA016 | |
| Recombinant DNA reagent (plasmid) | B-Dummy | Lampropoulos et al., 2013 PMID:24376629 | pGGB003 | |
| Recombinant DNA reagent (plasmid) | entry module C | Lampropoulos et al., 2013 PMID:24376629 | pGGC000 | |
| Recombinant DNA reagent (plasmid) | pGGC-VHA-a3 | this study | pKSC003 | See Materials and methods, Construct design and plant transformation |
| Recombinant DNA reagent (plasmid) | pGGC-VHA-a1-intron 10 | this study | pKSC012 | See Materials and methods, Construct design and plant transformation |
| Recombinant DNA reagent (plasmid) | pGGC-ST | this study | pKSC013 | See Materials and methods, Construct design and plant transformation |
| Recombinant DNA reagent (plasmid) | GR-LhG4 | Schürholz et al., 2018 PMID:30026289 | pGGC018 | |
| Recombinant DNA reagent (plasmid) | linker GFP | Lampropoulos et al., 2013 PMID:24376629 | pGGD001 | |
| Recombinant DNA reagent (plasmid) | linker-CFP | Lampropoulos et al., 2013 PMID:24376629 | pGGD004 | |
| Recombinant DNA reagent (plasmid) | GFP (A206K) no linker | Lampropoulos et al., 2013 PMID:24376629 | pGGD011 | |
| Recombinant DNA reagent (plasmid) | GSL-mVenus | this study | p2456 | See Materials and methods, Construct design and plant transformation |
| Recombinant DNA reagent(plasmid) | GSL-pmScarlet-I | this study | p1324 | See Materials and methods, Construct design and plant transformation |
| Recombinant DNA reagent (plasmid) | GSL-mCherry | Waadt et al., 2017 PMID:28850185 | p2897 | |
| Recombinant DNA reagent (plasmid) | rbcS terminator | Lampropoulos et al., 2013 PMID:24376629 | pGGE001 | |
| Recombinant DNA reagent (plasmid) | HSP18.2M terminator | Waadt et al., 2017 PMID:28850185 | p1296 | |
| Recombinant DNA reagent (plasmid) | BastaR | Lampropoulos et al., 2013 PMID:24376629 | pGGF001 | |
| Recombinant DNA reagent (plasmid) | SulfR | Lampropoulos et al., 2013 PMID:24376629 | pGGF012 | |
| Recombinant DNA reagent (plasmid) | KanR | Lampropoulos et al., 2013 PMID:24376629 | pGGF007 | |
| Recombinant DNA reagent (plasmid) | HygR_pNos | Waadt et al., 2017 PMID:28850185 | p1317 | |
| Recombinant DNA reagent (plasmid) | HygR_pUbq10 | Lampropoulos et al., 2013 PMID:24376629 | pGGF005 | |
| Recombinant DNA reagent (plasmid) | F-H adapter | Lampropoulos et al., 2013 PMID:24376629 | pGGG001 | |
| Recombinant DNA reagent (plasmid) | H-A adapter | Lampropoulos et al., 2013 PMID:24376629 | pGGG002 | |
| Recombinant DNA reagent (plasmid) | intermediate vector M | Lampropoulos et al., 2013 PMID:24376629 | pGGM000 | |
| Recombinant DNA reagent (plasmid) | intermediate vector N | Lampropoulos et al., 2013 PMID:24376629 | pGGN000 | |
| Recombinant DNA reagent (plasmid) | UBQ10:GR-LHG4:trbcS | this study | pKSM002 | See Materials and methods, Construct design and plant transformation |
| Recombinant DNA reagent (plasmid) | pOP6:Sar1BH74L-CFP | this study | pKSN009 | See Materials and methods, Construct design and plant transformation |
| Recombinant DNA reagent (plasmid) | pGGZ001 | Lampropoulos et al., 2013 PMID:24376629 | ||
| Recombinant DNA reagent (plasmid) | pGGZ003 | Lampropoulos et al., 2013 PMID:24376629 | ||
| Recombinant DNA reagent (plasmid) | pGGZ004 | this study | See Materials and methods, Construct design and plant transformation | |
| Recombinant DNA reagent (plasmid) | pHEE401E | Wang et al., 2015 PMID:26193878 | See Materials and methods, Construct design and plant transformation | |
| Antibody | VHA-a1 (rabbit polyclonal) | Agrisera | AS142822 | (1:1000) |
| Antibody | VHA-a3 (rabbit polyclonal) | Agrisera | AS204369 | (1:1000) |
| Antibody | VHA-B (mouse polyclonal) | Ward et al., 1992 PMID:16668845 | (1:100) | |
| Antibody | anti-GFP (rabbit polyclonal) | Roth et al., 2018 PMID:30410018 | (1:5000) | |
| Chemical compound, drug | Concanamycin-A (ConcA) | Santa Cruz | sc-202111A | |
| Chemical compound, drug | Brefeldin A | LC Laboratories | B-8500 | |
| Chemical compound, drug | FM4-64 | Thermo Fisher Scientific | T13320 | |
| Chemical compound, drug | Dexamethasone (DEX) | Sigma-Aldrich | D4902 | |
| Chemical compound, drug | Malachite Green, Acid Fuchsin, Orange G | Thermo Fisher Scientific | AC413490250, AC400210250, AC229820250 | Alexander stain |
| Commercial assay or kit | CloneJET PCR Cloning kit | Thermo Fisher Scientific | K1231 | |
| Strain, strain background (E. coli) | NEBα | In-house facility | COS Heidelberg | |
| Strain, strain background (A. tumefaciens) | GV3101 | In-house facility | COS Heidelberg | |
| Strain, strain background (A. tumefaciens) | ASE1 | In-house facility | COS Heidelberg | |
| Software, algorithm | Adobe Illustrator 2020 | Adobe Inc | Figure assembly | |
| Software, algorithm | Zen Software | Carl Zeiss | Microscopy | |
| Software, algorithm | Leica LSF | Leica | Microscopy | |
| Software, algorithm | Originpro 2020 | Origin | Statistics and graph plotting | |
| Software, algorithm | Intas Imager | Intas | Western blot | |
| Software, algorithm | Image J | NIH | Image quantification |
Additional files
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Supplementary file 1
Supplementary tables.
- https://cdn.elifesciences.org/articles/60568/elife-60568-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/60568/elife-60568-transrepform-v2.docx
