Materials and Reagents

1. 3-4 weeks old Nicotiana benthamiana plants
   
2. Agrobacterium tumefaciens (A. tumefaciens) C58C1 strain or similar transformed with the binary vector pMOG800 harboring the protein coding sequence. In our case they were:
   1. pMGFP-p7B encoding GFP-fused Melon necrotic spot virus (MNSV) p7B
      
   2. pMGFP-p7B [D₇AP₁₀A] encoding GFP-fused MNSV p7B carrying D₇AP₁₀A mutation
      
   3. pMGFP-p7B [K₄₉A] encoding GFP-fused MNSV p7B carrying K₄₉A mutation
      
   4. pMGFP-KDEL encoding a GFP engineered to be targeted to the ER lumen
      
3. Yeast extract (Difco, catalog number: 212750 )
   
4. Tryptone (Difco, catalog number: 211705 )
   
5. Sodium chloride (NaCl) (Panreac Applichem, catalog number: 131659 )
   
6. Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
   
7. MES (Sigma-Aldrich, catalog number: M8250 )
   
8. Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M9272 )
   
9. Antibiotics
   1. Kanamycin (50 mg/ml) (Sigma-Aldrich, catalog number: K4000 )
      
   2. Rifampicin (50 mg/ml) (Duchefa Biochemie, catalog number: R0146 )
      
10. Mowiol^® 4-88 (Sigma-Aldrich, catalog number: 81381 )
    
11. Dabco^® 33-LV (Sigma-Aldrich, catalog number: 290734 )
    
12. Tris base (Roche Diagnostics, catalog number: 03118142001 )
    
13. Tris-HCl (1 M, pH 5.6)
    
14. Immersion oil Immersol 518 F (ZEISS, catalog number: 444960-0000-000 )
    
15. Latrunculin B (Sigma-Aldrich, catalog number: L5288 )
    
16. 25 µM Latrunculin B solution in 10 mM DMSO
    
17. LB medium (see Recipes)
    
18. Agrobacterium infiltration buffer (see Recipes)
    
19. Mowiol mounting medium (see Recipes)
    

Equipment

1. 28 °C growing chamber
   
2. 15 ml culture tubes
   
3. Swinging centrifuge rotor for 15 ml tubes
   
4. 1.5 ml tubes (standard Eppendorf tubes or similar)
   
5. BioPhotometer plus (Eppendorf)
   
6. 1 ml syringes without needle
   
7. Plant growing chamber
   
8. Fine paddle forceps
   
9. Petri culture dishes 35 x 10 mm
   
10. Microscope slides 76 x 26 mm (Menzel-Gläser, catalog number: AA00000112E )
    
11. Microscope cover slips 24 x 24 mm Nr. 1 (Menzel-Gläser, catalog number: BB024024A1 )
    
12. Adhesive one-sided tape
    
13. A cork borer (one centimeter diameter) or similar cutting device such as a scalpel blade.
    
14. An inverted confocal microscope (ZEISS, model: LSM 780)
    
15. Plan-Apochromat 63x/1.40 oil objective
    

Software

1. Graphpad prism software (http://www.graphpad.com)
   
2. MS Excel
   

Procedure