Subcellular localization of AtFUC95A

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Figure 24: Acetylesterase‐activity assay with transiently expressed AtFXG1 and AtFuc95A proteins. 

A) The graph illustrates the difference in the activity between AtFXG1 and AtFUC95A, whose spectrum is in the  same range as the empty vector control ppT8. Substrate was PNP‐acetate which was mixed with N. benthamiana  leaf extract and incubated for different time points. B) Data table with average values ob measured absorbance at  405 nm. 

benthamiana leaf transiently expressing AtFUC95A. A signal of about 90 kDa was expected. It was not possible to detect a clear signal with A. thaliana samples but in the case of N. benthamiana leaves transiently expressing AtFUC95A, a specific signal of the expected size was visible (Figure 25 A and B, lane 2, arrows). No signal was detectable when N. benthamiana leaves were transformed with the empty vector control p21GT. The signal was optimised by testing different protein extraction buffers.

Extraction with 40 mM NaAc pH5.0 resulted in a stronger signal (Figure 25 A and B, lane 2) than extraction with 1xPBS pH7.0 (Figure 25 A and B, lane 3). As the antibody was obviously functional to recognize AtFUC95A when overproduced, we raised the hypothesis that its titre might be too low to allow the detection of this protein in its natural, rather low amounts, in A. thaliana. A purification and concentration of the antibody was therefore undertaken.

Figure 25: Testing of rabbit sera against AtFUC95A protein by Western Blot analysis on transiently expressed  AtFUC95A and empty vector control p21GT.  

Both sear 1713 and 1714 were collected from rabbits on different days (given in brackets). Secondary antibody  was Alkaline Phosphatase labelled goat‐antirabbit IgG (H+L) and detection was done using BCIP/NBT. Expected  signal was about 90 kDa (A, B lanes 2 + 3, arrows). Lane 1 – Fermentas Prestained protein ladder, 4 µL. Loading  volume of samples: 7.5 µL of proteins extracted with 40 mM NaAc pH5.0 mixed with equal volume of sample  buffer; 12.5 µL of proteins extracted with 1xPBS pH7.0 mixed with equal volume of sample buffer. 

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3.4.1.2 Purification of antibodies from rabbit sera and detection of AtFUC95A

The antibody purification has been performed using a column of Affi-Gel 15 from BioRad on which the AtFUC95A peptide had been coupled by anhydrous coupling. The advantage of this kind of coupling is its rapidity, the coupling being made via primary amino groups of the peptides or proteins within a few hours. A second advantage is its high stability at pH ranging from 2 to 11. Affi-Gel 15 is composed of a derivate of cross- linked agarose gel having N-hydroxysuccinimide esters. Ligand coupling occurs spontaneously in aqueous and non-aqueous solution. Coupling has been performed according to the instruction manual using water-free DMSO for equilibration and coupling. Aliquots before and after coupling have been taken to monitor the coupling efficiency by measurement of peptide concentration. The analysis of the monitoring aliquots showed that the coupling did not work under these conditions. A second try was performed using DMSO supplemented by a small concentration of triethylamine and 1.5 µmol of dried peptide mixed with the gel and subsequent incubation for several hours at room temperature under agitation. The gel was collected on the following day and free reactive sites were blocked with triethylamine and butylamine (possessing primary amine to block unbound gel sites). The gel was transferred into a small column for antibodies purification.

The purification of the antibodies against AtFUC95A started by the incubation of the rabbit serum with the modified Affi-Gel-15 gel, which was performed at 4°C over night under agitation. The gel was washed and antibodies were eluted in a tube containing a buffering solution by decreasing the pH. Each fraction collected was tested by Western Blot on AtFUC95A transiently produced in N. benthamiana as well as on A. thaliana Col-0 wildtype seedlings. N. benthamiana transiently transformed with the empty vector p21GT, as well as with a vector containing AtFUC95A. A strong and specific signal at about 90 kDa was detected with N. benthamiana overexpressing samples (Figure 26 A and B, lanes 4), but not with A. thaliana Col-0 wildtype (Figure 26 A and B, lanes 2).

Figure 26: Western Blot analysis of AtFUC95A and empty vector control p21GT.  

Both were transiently expressed in N. benthamiana as well as A. thaliana Col‐0 wildtype and Atfuc95A knockout to  test purified antibody  against  the fucosidase. Primary  antibody was a pool of all  10 fractions of affinity  chromatography‐purified serum 1713. Secondary antibody was: A) Alkaline Phosphatase labelled goat‐antirabbit  IgG (H+L) followed by colorimetric detection using BCIP/NBT; B) Anti‐Rabbit IgG (whole molecule) – Peroxidase  antibody produced in goat (Sigma) followed by chemiluminescent detection using Super  Signal  West  Pico  Chemiluminescent Substrate from Thermo Scientific. Expected signal of AtFUC95A was about 90 kDa which was  detected in the sample transiently expressed in N. benthamiana (A, lane 4; B, lane 2). Lane 1 – Fermentas  Prestained protein ladder, 4 µL. X – empty lane. 

The same experiment was performed using Anti-Rabbit IgG (whole molecule)-HRP- conjugated secondary antibody allowing a much more sensitive detection by chemiluminiscence, but no clear specific signal could be obtained with A. thaliana Col-0 wildtype. Therefore the antibody was considered as not being useful for usage in electron-microscopy in order to determine AtFUC95A subcellular localisation in A.

thaliana. As a consequence it was decided to perform the subcellular localisation studies by GFP-fusion and detection by confocal laser scanning microscopy (CLSM).

3.4.2 Subcellular localisation studies of AtFUC95A by GFP-fusion in N.

benthamiana show its presence in the apoplast

3.4.2.1 Vector modification and cloning of AtFUC95A-GFP

The coding sequence of AtFUC95A was cloned into the plant expression vector for GFP-fusion p20F and expressed in N. benthamiana before analysis by confocal laser scanning microscopy. As transient expression driven by the common CaMV35S promoter present in vector p20F was very low, it was decided to exchange this promoter

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by an enhanced version of the CaMV35S promoter, called eCaMV35S, as this promoter is composed of a longer promoter sequence and contains in addition the 5'-leader sequence (a 5’-untranslated region called Ω) of tobacco mosaic virus [Batoko et al, 2000; Gallie, 2002]. The encoding promoter sequence was excised from the vector p30GALT1_Fc-GFP using the restriction enzymes HindIII and XbaI. The short version of the CaMV35S promoter was excised from vector p20F in the same way and the new promoter was cloned into p20F to generate the vector p20F eCaMV35S. The extremities of the new promoter were sequenced to confirm its presence with the proper orientation (sequencing primers are given in Table 9).

The coding sequence of AtFUC95A was then transferred in front of the promoter.

Plasmid DNA was sequenced before transfer of the Plasmid-DNA into A. tumefaciens strain UIA143 by electroporation.

3.4.2.2 Transient expression in N. benthamiana and confocal laser scanning microscopy (CLSM)

Transient expression was performed in N. benthamiana leaves. AtFUC95A-GFP was infiltrated alone as well as coinfiltrated with ST-mRFP, a Golgi-marker which is also known to label the apoplast of plant cells at high expression level (Batoko et al., 2000;

Strasser et al., 2007). Agrobacteria cultures at optical density OD600 0.01, 0.03, 0.1 and 0.3 were carefully injected into the lower leaf epidermis of young leaves and incubated for 48 and 72 hours. A small piece of leaf was cut and analysed by confocal laser scanning microscopy. Agroinfiltration at an OD600 of 0.1 gave the best results.

The pictures obtained by confocal microscopy showed a fluorescence labelling surrounding the cell. In no cases other cell compartments could be visualised. To clearly exclude that this labelling could correspond to a labelling of the cytoplasm or the plasma membrane, the results obtained by the colocalisation studies with ST-mRFP were analysed. ST-mRFP is labelling the full apoplast (as well as Golgi bodies at lower level).

Overlay of AtFUC95A-GFP and ST-mRFP images presented staining of the same compartment (Figure 27) indicating that AtFUC95A accumulates in the apoplast.

This is in accordance with other experiments on glycosidases active on xyloglucan oligosaccharides which were found in apoplastic fluid of A. thaliana (Iglesias et al., 2006).

Figure 27: Analysis of the expression pattern of AtFUC95A‐GFP in N. benthamiana leaf epidermal cells by  confocal laser scanning microscopy.  

A), D) and G) FUC95A‐GFP at OD₆₀₀ 0.1; B), E) and H) Golgi‐ and apoplast‐marker ST‐mRFP at OD₆₀₀ 0.1; C), F) and I)  overlay. The labelling of the same compartment of both AtFUC95A‐GFP and ST‐mRFP indicates that AtFUC95A  accumulates in the apoplast. Bars = 10 µm. 

3.5 GUS-promoter studies reveal a strong expression of AtFUC95A