*4.2. Constructs*

The identity and source of protein constructs was as follows: 1S:GFP and 1SGFPm, N-terminal fragment 1-178 from *Arabidopsis* HMGR1S (accession AAF16652) fused, respectively, to GFP or monomeric GFP [19]. ER:GFP, GFP sequence with appended ER signal peptide at the N-terminus and ER retention signal at the C-terminus, encoded by plasmid pVKH-GFP-HDEL [65].

#### *4.3. Transient Expression in Nicotiana Benthamiana Leaves*

Transient expression of 1S:GFP and 1S:GFPm in *N. benthamiana* leaves was achieved by agroinfiltration as previously described [66]. 1S:GFP and 1S:GFPm was previously cloned into plasmid pPCV002 under control of the cauliflower mosaic virus 35S promoter, which confers high constitutive expression [19]. The pPCV002 derivatives were transformed into *Agrobacterium tumefaciens* strain GV3101 pMP90RK [67]. The transformed bacteria were grown in YEB medium (per litre: 5 g beef extract, 1 g yeas<sup>t</sup> extract, 5 g bacteriological peptone, 5 g sucrose, 2 mmol MgSO4) containing 100 μg/mL each of kanamycin, rifampicin and carbenicillin at an OD600 of 0.5 to 1.0. Cells were harvested by centrifugation and suspended in 10 mM *<sup>N</sup>*-morpholino ethanesulfonic acid (MES, pH 5.7), 10 mM MgCl2 and 0.2 mM acetosyringone to an OD600 of 1.0. Bacteria were infiltrated into leaves with a 1-mL disposable syringe without a needle. Expression was examined daily, until day 6 after agroinfiltration.

#### *4.4. Source and Use of Antibodies*

The catalytic domain of *Arabidopsis* HMGR1S (CD1) produced in *Escherichia coli* was used as immunogen to produce a polyclonal antibody in rabbit and the resulting serum was immunosubstracted to remove IgG reacting against the bacterial proteins [48]. The immunopurified serum (Ab-CD1-i) was used as primary antibody at 1:500 for whole mount and 1:1000 for transmission EM. Anti-rabbit IgG secondary antibodies for HMGR detection were code Ab150066 (Abcam, Toronto, ON, Canada) coupled to Alexa Fluor-555 at 1:1000 for whole mount (Figure 1a,b), code Ab150068 (Abcam, Toronto, ON, Canada) coupled to Alexa Fluor-594 at 1:1000 for whole mount (Figure 1g), code 111-215-144 (Jackson Immunoresearch, Cambridge, UK) coupled to an 18-nm gold particle at 1:30 for transmission EM (Figure 1d) and code 111-205-144 (Jackson Immunoresearch, Cambridge, UK) coupled to a 12-nm gold particle at 1:30 for transmission EM (Figure 1e,f,l,m).

GFP was detected with Ab-5450 (Abcam, Toronto, ON, Canada) as the primary antibody at 1:1000 for whole mount and transmission EM. Anti-goat IgG secondary antibodies for GFP detection were code Ab150133 (Abcam, Toronto, ON, Canada) coupled to Alexa Fluor-488 at 1:1000 for whole mount, and code 705-215-147 (Jackson Immunoresearch, Cambridge, UK) coupled with an 18-nm gold particle at 1:15 for transmission EM.

#### *4.5. Immunolocalization in Whole Mount*

Whole-mount in situ immunolocalization was done as described [68] with modifications. After fixation in 4% ( *w*/*v*) paraformaldehyde, seedlings were incubated in methanol to remove chlorophylls. Five cycles of seedling freezing and thawing on glass slides were performed to permeate tissue, followed by incubation with 2% ( *w*/*v*) Driselase (D8037; Sigma Aldrich-Merk, Madrid, Spain) to allow for the penetration of antibodies through the plant cell wall. After blocking with 3% ( *w*/*v*) bovine serum albumin (BSA) solution, simultaneous incubation with one or two primary antibodies (Ab-CD1-i at 1:500, Ab-5450 at 1:1000) was performed directly on the slides. Samples were then washed with phosphatebuffered saline (PBS) and incubated with the corresponding secondary antibody at 1:1000. The secondary antibody to detect HMGR was Abcam Ab150066 (Alexa Fluor-555) or Ab150068 (Alexa Fluor-594). The secondary antibody to detect GFP was Abcam Ab150133 (Alexa Fluor-488). After washing with PBS, samples were sealed for observation at the confocal microscope.

## *4.6. Confocal Microscopy*

Confocal laser microscopy was performed with spectral microscope Olympus FV1000 (objectives UPLSAPO 60x O, numerical aperture: 1.35 and UPLSAPO 60x W, numerical aperture: 1.20) or Leica TCS SP5 (objectives HCX PL APO CS 40x/1.25 Oil UV, HCX PL APO 63x/1.20 W corrected for UV, and HCX PL APO CS 63x/1.20 water UV) at room temperature. Fluorophores were detected with the following excitation and emission wavelengths: GFP (excitation = 488 nm, emission = 500–545 nm), Alexa Fluor-555 (excitation = 559 nm, emission = 570–610 nm) and Alexa Fluor-594 (excitation = 559 nm, emission = 575–620 nm) and chlorophyll (excitation = 559 nm, emission = 640–680 nm). Images were acquired with the software FV10-ASW 4.1 (Olympus, Hamburg, Germany) and LAS AF 2.7.3.9723 (Leica Microsystems, Wetzlar, Germany) and processed with the software ImageJ 1.50i (http://imagej.nih.gov/ij, accessed on 28 May 2020).

#### *4.7. Chemical Fixation for Ultrastructural Studies*

Explants from *Nicotiana* leaves or *Arabidopsis* seedlings were excised under a stereomicroscope and transferred to glass vials filled with 1.5% (*v*/*v*) paraformaldehyde and 1.5% (*v*/*v*) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) containing 2 mM CaCl2. The vials were degassed briefly to allow penetration of the fixative into tissue and incubated at 4 ◦C for 24 h. After washing with the cacodylate-CaCl2 buffer without fixative, samples were post-fixed for 3 h at 4 ◦C with 1% ( *w*/*v*) osmium tetroxide and 0.8% ( *w*/*v*) K3Fe(CN)6 in the same buffer. Samples were subsequently dehydrated in acetone, infiltrated with Spurr resin for 2 d, embedded in the same resin and polymerized at 60 ◦C for 48 h.

#### *4.8. High-Presure Freezing (HPF) for Ultrastructural Studies*

Explants from *Nicotiana* leaves or *Arabidopsis* seedlings were excised under a stereomicroscope and transferred to aluminium planchette with a 200 μm-deep cavity that was subsequently filled with yeas<sup>t</sup> (*Saccharomyces cerevisiae*) paste and cryoimmobilized immediately with a high pressure freezer (EM Pact (Leica)). Freeze substitution of frozen samples was performed in an automatic freeze substitution system (EM AFS (Leica)) with acetone containing 2% ( *w*/*v*) osmium tetroxide and 0.1% ( *w*/*v*) uranyl acetate, for 3 d at

−90 ◦C. On the fourth day, the temperature was raised by 5 ◦C per hour to room temperature. At this temperature, samples were rinsed in acetone, infiltrated with Epon (*Nicotiana* samples, Figure 4) or Spurr (*Arabidopsis* samples, Figure 5) resin for 2 d, embedded in a thin layer of the same resin and polymerized at 60 ◦C for 48 h.
