Localization studies with CLR1 fusion proteins transiently expressed in N. benthamiana

benthamiana and stably expressed in A. thaliana

CLR1 is phosphorylated in a chitin- and CERK1-dependent manner in vivo (section 3.3.1).

Microsomal preparations of plants expressing pCLR1::CLR1-eCFP from the endogenous promoter revealed that the majority of the protein is found in the soluble protein fraction, whereas a small portion also localized to the microsomal fraction (Figure 22). To get more insight into the in planta subcellular localization of CLR1, pCLR1::CLR1-GFP was expressed transiently in N. benthamiana as well as stably in A. thaliana and analysed by confocal laser scanning microscopy (CLSM). To assess the potential effect of myristoylation on subcellular localization, pUBQ10::CLR1-M1-GFP and pUBQ10::CLR1-M2-GFP (compare section 3.4) were also transformed and included in the subsequent analyses. Figure 23 shows the localization of the different CLR1 fusion proteins transiently expressed in N. benthamiana by Agrobacterium-mediated transformation. As positive control free GFP was expressed and as a negative control, leaves infiltrated with the empty Agrobacterium strain (GV3101 pMP90RK) were used.

R e s u l t s| 91

Figure 23. Subcellular localization of CLR1-GFP fusion proteins transiently expressed in N. benthamiana. The genomic pCLR1::CLR1-GFP construct, as wells as pUBQ10::CLR1-M1-GFP and pUBQ10::CLR1-M2-GFP were transformed into N. benthamiana by Agrobacterium-mediated transformation. Agrobacteria containing a vector for expression of free GFP were used as positive control. The empty bacteria strain GV3101 pMP90RK was used as negative control (mock). Pictures were taken 3 dpi using the Leica TCS SP5 CLSM. Pictures show overlays of GFP fluorescence in green and chloroplast autofluorescence in red. The plasmolysis images also contain the bright field channel. First column: Empty arrows indicate localization at a reticulate structure which is likely the endoplasmic reticulum (ER), arrows indicate localization at the cell boundary. Second column: Striped arrows indicate cytoplasmic strands, asterisks mark the nuclei. Third column: Plasmolysis, full arrow heads indicate Hechtian strands, empty arrow heads mark plasmolysis events without visible Hechtian strands. Size markers are given in µm.

92 |R e s u l t s

Confocal laser scanning microscopy (CLSM) revealed that the three tested CLR1 fusion proteins (expressed from the pCLR1::CLR1-GFP, pUBQ10::CLR1-M1-GFP and pUBQ10::CLR1-M2-GFP constructs) all showed a very similar subcellular localization pattern.

CLR1-GFP signal can be observed at the cell periphery, but CLR1-GFP is also present in the cytoplasm, indicated by fluorescence signal in cytoplasmic strands (Figure 23, striped arrows). All three CLR1 fusion proteins were also found in a reticulate structure which likely represents the endoplasmic reticulum (ER) (empty arrows, first column) and in nuclei (asterisk) as well as nucleoli. The free GFP control was used to distinguish the true CLR1 subcellular localization from that of possibly unspecific signal due to free GFP. The free GFP control is found at the cell boundary, in the cytoplasmic strands and nuclei, but not in the nucleoli or ER. To investigate if the signal at the cell periphery corresponds to the plasma membrane, plasmolysis experiments with 1 M NaCl were performed (Figure 23, third column). With both CLR1-GFP and free GFP the fluorescence signal detached from the cell wall, but while CLR1-GFP was clearly present in Hechtian strands, no fluorescence signal associated with Hechtian strands was visible with free GFP. These findings indicate plasma membrane localization of CLR1-GFP.

Next, the subcellular localization of fusion proteins expressed from the pCLR1::CLR1-GFP, pUBQ10::CLR1-M1-GFP and pUBQ10::CLR1-M2-GFP constructs was analysed in stably transformed Arabidopsis plants by CLSM. In comparison to the expression in N. benthamiana, CLR1-GFP signals in the stable transgenic Arabidopsis plants were much weaker (Figure 24).

R e s u l t s| 93

Figure 24. Subcellular localization of CLR1-M1-GFP and CLR1-M2-GFP stably expressed from the Ubiquitin10 promoter in Arabidopsis thaliana Col-0. Col-0 expressing pCERK1::CERK1-GFP was used as positive control, whereas untransformed wild type Col-0 plants were used as negative control. Pictures were taken using the Leica TCS SP5 CLSM. Pictures in the first and second column represent overlays of GFP fluorescence shown in green and chloroplast autofluorescence shown in red. The overlays in the third and fourth columns also contain the bright field image. First column: Striped arrow heads indicate cytoplasmic strands (cyt). Asterisks mark nuclei (nuc). Second column: Arrows indicate the cell periphery. Third and fourth column depict the subcellular localization upon 1 M NaCl treatment leading to plasmolysis. Third column: Arrows indicate the plasmolysed plasma membrane and empty arrow heads mark circular invaginations. Fourth column: Arrows show plasma membrane detachment from the cell wall and arrow heads mark Hechtian strands. Size markers are given in µm.

With pCLR1::CLR1-GFP no specific signal could be detected at all and thus this construct could not be included in the CLSM analysis. For the two CLR1 constructs driven by the Ubiquitin10 promoter fluorescence could be detected in the cell periphery (Figure 24). Both

94 |R e s u l t s

fusion proteins are also present in the cytoplasm, indicated by fluorescence localized to cytoplasmic strands. pUBQ10::CLR1-M2-GFP but not pUBQ10::CLR1-M1-GFP was also visible in nuclei (Figure 24, asterisk). After treatment with 1M NaCl to induce plasmolysis, pUBQ10::CLR1-M2-GFP showed Hechtian strands but pUBQ10::CLR1-M1-GFP did not.

Another difference between the two Ubiquitin10 promoter-driven CLR1 constructs was the formation of circular structures within the cell after NaCl treatment of pUBQ10::CLR1-M2-GFP (Figure 24, empty arrow heads). Since fluorescence of free GFP would have been too strong, pCERK1::CERK1-GFP was used as a control. As expected, pCERK1::CERK1-GFP localized to the cell periphery and also shows a fluorescence signal in the Hechtian strands after NaCl treatment.

Taken together, these results indicate that the native expression levels of the pCLR1::CLR1-GFP fusion protein were too low to be detected by CLSM in the present experimental setup. However, both CLR1-GFP fusion proteins driven by the Ubiquitin10 promoter were stably expressed in Arabidopsis to expression levels detectable by CLSM. The expression levels of pUBQ10::CLR1-M2-GFP seemed to be higher than those of pUBQ10::CLR1-M1-GFP. The fact that CLR1-M2-GFP but not CLR1-M1-GFP could be observed in nuclei and Hechtian strands, might be explained by the different fluorescence intensities of the fusion proteins.

3.5.3 Expression of β-glucuronidase under the native CLR1 promoter reveals expression