Subcellular localization of DCL-TP-mVirD2-58-EGFP, RbcS-TP-mVirD2-58-EGFP,

DCL-TP and RbcS-TP proved to be strong chloroplast-targeting signals in both Arabidopsis and

tobacco leaves. Thus, these signals should be functional in fusion proteins of DCL-TP or RbcS-TP fused to

mVirD2-58 carrying the modified N-terminal NLS and a broad deletion of the C-terminal NLS and

mVirD2-80 carrying the modified N-terminal NLS and a precise deletion of the C-terminal NLS.

DCL-TP-mVirD2-58-EGFP was detected exclusively in the nucleus of Arabidopsis and tobacco

leaf cells (Figure 3.4.3.2a). There was no transmission between EGFP and autofluorescence channels in the

Arabidopsis leaf, whereas EGFP channel transmitted to autofluorescence detector in the tobacco leaf (* mark in Figure 3.4.3.2a). However, since the DAPI stained nucleus merged with EGFP, DCL-TP-mVirD2-

58-EGFP was localized in the nucleus.

In the Arabidopsis and tobacco leaves, RbcS-TP-mVirD2-58-EGFP was localized mainly in the

nucleus and very little in chloroplasts (arrows in Figure 3.4.3.2b). Also in the tobacco leaf, chloroplasts

containing the recombinant protein were not detected in most areas (Figure 3.4.3.2b); thus, most of the

recombinant protein was not imported into chloroplasts. Instead, it clearly localized in the tobacco leaf

nuclei. Some nuclei did not show transmission from the EGFP to autofluorescence channel in the

Arabidopsis leaf (** mark in Figure 3.4.3.2b). There was some transmission between EGFP and autofluorescence channels in tobacco leaves (* mark in Figure 3.4.3.2b). Despite transmission between the

channels, overlap of the DAPI stained nuclei and EGFP suggested that RbcS-TP-mVirD2-58-EGFP was

mainly localized in the nucleus and very little in chloroplasts of the Arabidopsis and tobacco leaves.

The expression of DCL-TP-mVirD2-80-EGFP in the Arabidopsisleaf was low. The recombinant

protein was detected in the nucleus (Figure 3.4.3.2c); also very low intensity signals of EGFP were

overlapping with the nuclei (open arrow in Figure 3.4.3.2c). There was no transmission between EGFP and

autofluorescence channels in the Arabidopsis leaf. The recombinant protein was also detected mostly in the

nucleus and little in the cytoplasm of the tobacco leaf cell (Figure 3.4.3.2c). Transmission occurred between

EGFP and autofluorescence channels in the tobacco leaf (* mark in Figure 3.4.3.2c). Although slightly

different localization of DCL-TP-mVirD2-80-EGFP was observed in the Arabidopsis and tobacco leaves,

116

RbcS-TP-mVirD2-80-EGFP was localized largely in the nucleus and slightly in chloroplasts in the

Arabidopsis leaf (arrows in Figure 3.4.3.2d). However, there was a larger amount of the recombinant protein localized in the chloroplast as compared to RbcS-TP-mVirD2-58 protein in Arabidopsis. In the

agro-infiltrated tobacco leaf, RbcS-TP-mVirD2-80-EGFP was also localized mainly in the nucleus and

slightly in the chloroplasts (arrows in Figure 3.4.3.2d). DAPI was unable to stain the same cell as the

observed one before the DAPI staining in Figure 3.4.3.2d, yet EGFP fluorescence was detected strongly in

the nucleus as the DAPI staining merged with EGFP in both before and after DAPI stained cells (Figure

3.4.3.2d). EGFP did not transmit to the autofluorescence channel in tobacco before DAPI staining, but there

was slight transmission between EGFP and autofluorescence channels in the Arabidopsis and tobacco

leaves after the DAPI staining (* mark in Figure 3.4.3.2d). Regardless of the slight transmission between

two channels, overlay of DAPI stained nuclei and EGFP suggested that RbcS-TP-mVirD2-EGFP was

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DAPI EGFP Autofluorescence DIC Merge

Figure 3.4.3.2a. DCL-TP-mVirD2-58-EGFP subcellular localization in transgenic Arabidopsis leaf (Top row) and in the tobacco leaves 48 h after agro- infiltration (Middle row: before DAP staining; Bottom row: after DAPI staining) examined under the confocal microscope.

DAPI: DAPI stained nuclei; EGFP: recombinant protein fused to EGFP; Autofluorescence: chloroplast autofluorescence; DIC: differential interference contrast; Merge: merged photo of DAPI, EGFP, Autofluorescence and DIC; * mark: transmission from EGFP. Scale bar = 10 µm.

Arabidopsis Tobacco: Before DAPI staining Tobacco: After DAPI staining

*

118

DAPI EGFP Autofluorescence DIC Merge

Figure 3.4.3.2b. RbcS-TP-mVirD2-58-EGFP subcellular localization in transgenic Arabidopsis leaf (Top row) and in the tobacco leaves 48 h after agro- infiltration (Middle row: before DAP staining; Bottom row: after DAPI staining) examined under the confocal microscope.

DAPI: DAPI stained nuclei; EGFP: recombinant protein fused to EGFP; Autofluorescence: chloroplast autofluorescence; DIC: differential interference contrast; Merge: merged photo of DAPI; EGFP, Autofluorescence and DIC, arrows: chloroplasts; * mark: transmission from EGFP;

** mark: no transmission to Autofluorescence; A: auto corrected. Scale bar = 10 µm.

*

**

Arabidopsis Tobacco: Before DAPI staining Tobacco: After DAPI staining A A

*

*

**

**

119

DAPI EGFP Autofluorescence DIC Merge

Figure 3.4.3.2c. DCL-TP-mVirD2-80-EGFP subcellular localization in transgenic Arabidopsis leaf (Top row) and in the tobacco leaves 48 h after agro- infiltration (Middle row: before DAP staining; Bottom row: after DAPI staining) examined under the confocal microscope.

DAPI: DAPI stained nuclei; EGFP: recombinant protein fused to EGFP; Autofluorescence: chloroplast autofluorescence; DIC: differential interference contrast; Merge: merged photo of DAPI, EGFP, Autofluorescence and DIC; open arrows: nuclei; * mark: transmission from EGFP; A: auto corrected. Scale bar = 10 µm.

*

A

*

Arabidopsis Tobacco: Before DAPI staining Tobacco: After DAPI staining

120

DAPI EGFP Autofluorescence DIC Merge

Figure 3.4.3.2d. RbcS-TP-mVirD2-80-EGFP subcellular localization in transgenic Arabidopsis leaf (Top row) and in the tobacco leaves 48 h after agro- infiltration (Middle row: before DAP staining; Bottom row: after DAPI staining) examined under the confocal microscope.

DAPI: DAPI stained nuclei; EGFP: recombinant protein fused to EGFP; Autofluorescence: chloroplast autofluorescence; DIC: differential interference contrast; Merge: merged photo of DAPI, EGFP, Autofluorescence and DIC; arrows in Arabidopsis EGFP: EGFP in chloroplasts; * mark: transmission from EGFP;

A: auto corrected. Scale bar = 10 µm.

*

A

*

A Arabidopsis Tobacco: Before DAPI staining Tobacco: After DAPI staining

*

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Original Autocorrected

Figure 3.4.3.2e. Original and autocorrected pictures indicating subcellular localization of recombinant proteins.

Autocorrection was done to enhance only green colour to make the subcellular localization of the recombinant proteins more explicit.

RbcS-TPmVirD2-58-EGFP in the transgenic Arabidopsis leaf

RbcS-TP-mVirD2-58-EGFP in the agro-infiltrated tobacco leaf

DCL-TP-mVirD2-80-EGFP in the transgenic Arabidopsis leaf

RbcS-TP-mVirD2-80-EGFP in the transgenic Arabidopsis leaf

RbcS-TP-mVirD2-80-EGFP in the agro-infiltrated tobacco leaf

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In document Genetic engineering of Agrobacterium tumefaciens to target chloroplasts and identification of a novel nuclear localization signal of VirD2 protein (Page 132-139)