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Open Access Highly Accessed Research article

Induction of protein body formation in plant leaves by elastin-like polypeptide fusions

Andrew J Conley12, Jussi J Joensuu23, Rima Menassa2* and Jim E Brandle24

Author Affiliations

1 Department of Biology, University of Western Ontario, London, ON, Canada

2 Southern Crop Protection and Food Research Centre, Agriculture and Agri- Food Canada, London, ON, Canada

3 VTT Technical Research Centre of Finland, Espoo, Finland

4 Vineland Research and Innovation Centre, Vineland Station, ON, Canada

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BMC Biology 2009, 7:48  doi:10.1186/1741-7007-7-48

Published: 7 August 2009

Additional files

Additional file 1:

Movie S1. A three-dimensional rendering of a cluster of novel protein bodies. Sixty confocal images of a Nicotiana benthamiana epidermal cell expressing an endoplasmic reticulum- (ER-)targeted green fluorescent protein-elastin-like polypeptide (GFP-ELP) fusion protein (pPGEK) were taken from a 6.00 μm projection in the Z-direction and compiled together to construct the rotating 3-D image representing a collection of protein bodies.

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Additional file 2:

Movie S2. The variously-sized protein bodies are densely packed throughout the cytoplasm of the cell. Consecutive confocal images were taken and assembled together in a time-lapse movie as the confocal plane progressed through the PGEK-expressing cell. One hundred and ten image frames were taken through an 11.00 μm projection in the Z-direction.

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Additional file 3:

Movie S3. The protein bodies compactly gather in the cytoplasmic space surrounding the cell's nucleus. Time-lapse confocal imaging as the confocal plane progressed through the cell expressing pPGEK. Seventy image frames were taken through a 14.00 μm projection in the Z-direction.

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Additional file 4:

Movie S4. Movement of the protein bodies within Nicotiana benthamiana leaf epidermal cells. Time-lapse confocal imaging of cells expressing endoplasmic reticulum- (ER-)targeted green fluorescent protein-elastin-like polypeptide (pPGEK) was performed to demonstrate the mobility of the novel protein bodies (PBs). A variety of patterns of movement were observed for the PBs. The PBs did not generally move with a constant velocity; rather, they sporadically moved about in a stop-and-go, saltatory fashion. Trafficking of the PBs appeared to occur along the underlying cortical ER network in the leaf cells. Sixty image frames were taken over the course of 2 min 58 s.

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Additional file 5:

Movie S5. Directed trafficking of protein bodies within the cell. Consecutive confocal images of PGEK-expressing cells were taken to show that some protein bodies (PBs) remained relatively still and slowly oscillated in position for an extended period resembling Brownian motion, while other resting PBs would suddenly accelerate. Observing the center region of the movie demonstrated that large numbers of PBs moved in a relatively constant direction with a steady velocity towards a specific area of the cell, until moving out of the confocal plane. Two hundred image frames were taken over the course of 8 min 15 s.

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Additional file 6:

Movie S6. Mobility of novel protein bodies in numerous neighboring cells expressing pPGEK. The speed, amount and type of protein body (PB) movement is highly variable between neighboring cells. Moreover, particular PBs were observed to be carried away at high velocities when they jumped onto streaming cytoplasmic strands. One hundred and fifty image frames were taken over the course of 6 min 11 s.

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Additional file 7:

Movie S7. The trafficking of protein bodies is dependent on intact microfilaments. A 25-μM solution of latrunculin B, a drug responsible for inducing disintegration of the actin cytoskeleton, was infiltrated into the abaxial surface of Nicotiana benthamiana leaves that were transiently expressing pPGEK. After 1 h of treatment, the infiltrated area was observed by time-lapse imaging via confocal microscopy. Depolymerization of the cytoskeleton prevented all translational movement of the induced protein bodies (PBs), demonstrating that intact microfilaments are necessary for normal PB trafficking. Fifty image frames were taken over the course of 2 min 37 s.

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Additional file 8:

Movie S8. Overexpression of a myosin tail inhibits movement of the novel protein bodies. A dominant-negative mutant of myosin XI-K was co-agro-infiltrated with pPGEK into the leaves of Nicotiana benthamiana and visualized 3 days post-transfection by confocal microscopy. As a result, protein body (PB) trafficking was prevented with the PBs simply oscillating in place, suggesting that a functional actomyosin motility system is required for active PB movement, but is not necessary for the formation of PBs. Fifty image frames were taken over the course of 3 min 34 s.

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Additional file 9:

Movie S9. Fluorescence recovery after photobleaching analysis of an endoplasmic reticulum-targeted green fluorescent protein-elastin-like polypeptide fusion protein (pPGEK) present within the novel protein bodies. A small region of interest (white circle) within a single protein body (PB) was photobleached. After bleaching, fluorescence recovered in the PB. Eighty six image frames were taken over the course of 3 min 3 s.

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Additional file 10:

Movie S10. Movement of pPGEK protein into protein bodies following photobleaching. Selective photobleaching of six protein bodies in close proximity (white circle) showing fluorescence recovery of pPGEK inside the bleached area. One hundred and seventeen image frames were taken over the course of 4 min 15 s.

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Additional file 11:

Movie S11. The free exchange of proteins occurs rapidly within protein bodies. A particularly large protein body (PB) was continuously bleached (white circle) and imaged in an alternating fashion for a relatively long period of time. Although only a small region of the PB was selectively photobleached, the entire volume of the PB was homogeneously bleached with time, suggesting very rapid mobility of proteins within the confines of the PB. Twenty eight image frames were taken over the course of 2 min 18 s.

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