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QUANTITATIVE ANALYSIS OF THE MOVEMENT OF AGROBACTERIUM- DELIVERED VIRE2 RPOTEINS
INSIDE PLANT CELL
LI LI
NATIONAL UNIVERSITY OF SINGAPORE
2014
I
QUANTITATIVE ANALYSIS OF THE MOVEMENT OF AGROBACTERIUM- DELIVERED VIRE2 RPOTEINS
INSIDE PLANT CELL
LI LI
(M. Sc., Peking University)
A THESIS SUBMITTED
FOR THE DEGREE OF MASTER OF SCIENCE
DEPARTMENT OF CHEMISTRY
NATIONAL UNIVERSITY OF SINGAPORE
2014
II
DECLARATION
I hereby declare that this thesis is my original work and it has been written by
me in its entirety, under the supervision of Associate Professor Pan Shen Quan,
National University of Singapore, between 04/08/2013 and 22/08/2014. I have
duly acknowledged all the sources of information which have been used in the
thesis. This has also not been submitted for any degree in any university
previously.
Li Li 18/02/2015
Name Signature Date
III
ACKNOWLEDGMENTS
This dissertation would not have been possible without the guidance and the
help of several individuals who extended their valuable assistance in the
preparation and completion of this study. The 1 years of research study have
been a truly memorable learning journey.
First and foremost, I would like to express my sincere appreciation to my
supervisor Associate Professor Dr. Pan Shen Quan for giving me the
opportunity to work on the project in his research lab. His stimulating
suggestions, encouragement and immense knowledge helped me greatly
throughout the project. I also would like to express my sincere appreciation to
my co-supervisor Professor Dr. Li Fong Yau, Sam for providing me great help
on research work.
I specially thanks to Dr. Yang Qinghua for his untiring help and excellent
advice throughout the research and the writing of my thesis. My sincere thanks
also go to Madam Tong Yan from the Centre of BioImaging Sciences for all
her help she has rendered during my particle tracking work.
This thesis would not have been possible without the help and support from
my fellow lab mates: Dr. Yang Qinghua, Dr. Tu Haitao, Dr. Li Xiaoyang, Dr.
Lim Zijie, Dr. Wang Bingqing, Miss Peng Ling, Miss Zhang Chen and Miss
Guo Song.
I am really grateful to my friends for their faithful support and kindly help
throughout my tough time within this year. They are Miss Luo Yingling, Miss
Liu Lei, Miss Guo Xue, Miss Feng Ting, Miss Liang Qin, Miss Liang
Changhua, Miss Wu Ye, Miss Guo Hui, Mr. Jia Zhongchao, Mr. Zhen Naiwen,
IV
Mr. Chen Pengyu.
I am also grateful to my parents and my family for their unconditional love,
help and supporet. I would like to give my sincere thanks to my husband and
my child for believing in me and giving me the moral support this year.
Last but not least, I gratefully acknowledge t the National University of
Singapore for providing me with a valuable research scholarship and for
funding the project.
V
TABLE OF CONTENTS
PAGE
Declaration II
Acknowledgement III
Table of contents V
Summary VII
List of tables IX
List of figures X
PAGE
Chapter 1: Literature Review 1
1.1 Introduction of Agrobacterium-mediated VirE2
transformation
1
1.2 Direct visualization and tracking of Agrobacterium-
delivered VirE2
2
1.3 Quantitative analysis of the trafficking of delivered-
VirE2
4
1.4 Objective and Scope 5
VI
Chapter 2: Materials and Methods 6
2.1 Materials 6
2.2 Tracking of Agrobacterium-mediated VirE2 7
2.3 Quantitative analysis of Agrobacterium-mediated VirE2 11
Chapter 3: Results and Discussion 17
3.1 Tracking of the movement of Agrobacterium-delivered
VirE2 in plant cells
17
3.2 Correlation Analysis of the speeds of moving VirE2 and
the distances from analysis between the nucleus in
Agrobacterium-mediated plant cells
25
3.3 Correlation Analysis of the speeds of moving VirE2 and
the distances from the nuclease in drugs treated plant
cells
35
Chapter 4: Conclusion and Recommendations 39
References 40
VII
SUMMARY
Agrobacterium tumefaciens is a natural genetic engineer widely used to
deliver DNA and VirE2 into plant cells. A recently developed split-GFP
approach has been proved to be one of the considerable methods to directly
visualize the movement of Agrobacterium-delivered VirE2 protein (marked
with GFP) inside plant cell. In this report, the quantitative characteristics of
the movement of Agrobacterium-delivered VirE2 protein and the effects of
three kinds of protein transferring inhibitors(BDM, BFA and CytoD) on VirE2
movement were analyzed inside plant cell. Previous work with the split-GFP
approach had been done in my lab to get confocal time-lapse images of
Agrobacterium-delivered VirE2 movement in plant recipient cells, as well as
in protein transfer inhibited cells. Having tracked moving VirE2 and
quantitative analyzed VirE2 moving track in the time-lapse confocal images,
VirE2 were found to be directive delivered from cellar cytoplasm into nucleus
with average moving speed of 0.62 um/s, maximum speed of 5.76 um/s and
average moving displacement of 25.25 um. While after analysis of three
inhibitor(BDM, BFA and CytoD) treated cells, we found the movement of
Agrobacterium-delivered VirE2 was found to be obviously inhibited. Thus,
that myosin, the transport of protein in ER, actin filament all had a major role
in trafficking of Agrobacterium-delivered VirE2, where myosin, the transport
of protein in ER, actin filament might perform accessory functions in the
process.
VIII
LIST OF TABLES PAGE
Table 3.1 Table 3.1. Quantitative analysis of Agrobacterium-
delivered VirE2 tracks in normal leaf epidermal cells
of N. benthamiana.
18
Table 3.2 Average parameters of Agrobacterium-delivered
VirE2 tracked in normal and drug treated samples of
leaf epidermal cells of N. benthamiana.
19
Table 3.3 Statistics of moving speed of Agrobacterium-
delivered VirE2 in normal plant cells.
26
Table 3.4 Statistics of the distances between Agrobacterium-
delivered VirE2 and the nucleus in normal plant
cells.
27
Table 3.5 Pearson correlation between moving speed of
Agrobacterium-delivered VirE2 and their distances
from the nucleus in normal plant cells.
28
Table 3.6 Average statistics of the distribution of various Speed
and DistN in Agrobacterium-delivered VirE2
movement in different treatments cells.
36
Table 3.7 Pearson correlation between moving speed of
Agrobacterium-delivered VirE2 and their distances
from the nucleus in drugs treated cells.
37
IX
LIST OF FIGURES
Figure 3.1 Movement of Agrobacterium-delivered VirE2-GFP
in plant cells.
18
Figure 3.2 Tracks of the movement of Agrobacterium-delivered
VirE2 in plant cells.
19
Figure 3.3 Distribution bar charts of moving speed of
Agrobacterium-delivered VirE2 in normal plant cells
26
Figure 3.4 Distribution bar charts of the distances between
Agrobacterium-delivered VirE2 and the nucleus in
normal plant cells
27
Figure 3.5 Scatter diagram of moving speed of Agrobacterium-
delivered VirE2 and their distances from the nucleus
in normal plant cells
28
Figure 3.6 Scatter diagram of moving speed of Agrobacterium-
delivered VirE2 and their distances from the nucleus
in drugs treated plant cells.
36
1
Chapter 1. Literature Review
1.1 Introduction of Agrobacterium-mediated VirE2 transformation
The bacterium Agrobacterium tumefaciens is a kind of soil borne phytopathogen which
induces gall formation on plant tissue, i.e. crown gall disease. The normal recipients for
the infection of A. tumefaciens in biological researches include plants such as Nicotiana
benthamiana and Arabidopsis thaliana and so on. Under natural conditions, A.
tumefaciens can transfer its single-strand T-DNA, VirD2 and VirE2 proteins into host
cells and incorporate them into the plant cell chromosomes (Chilton et al. 1977). As a
result of the genetic distribution above, the harmful galls appear on plant tissue and
negatively affect the growing of plant cells. However, the bacterium A. tumefaciens is
also a useful tool in the gene engineering filed because the DNA transmission
capabilities of Agrobacterium can act as a means of inserting foreign genes into plants
(Schell et al. 1977).
During infection, VirE2 is the most abundant protein delivered into host cells and is also
one of the essential proteins participating in the transformation of T-DNA (Engstrom et
al. 1987). In A. tumefaciens cells, VirE2 presents binding activity to the non-specific
single-stranded DNA (T-DNA) and combines to the length of T-DNA in order to form a
compact helical structure (Citovsky et al. 1992). As a result, the compact structure
mediates T-DNA import across membranes of two cells (bacterial and host cells) and
then toward to host cells. In host cells, the compact structure of T-DNA and VirE2
protein will transfer into the nucleus and separated from each other because of the
incorporation of bacterial T-DNA into the plant cell chromosomes, and then VirE2 will
2
transfer out of the nucleus (Grange et al. 2008). Thus, the analysis of Agrobacterium-
mediated VirE2 transformation is meaningful to exploring the effects of VirE2 on
bacterial T-DNA translocation into the host cell nucleus and figuring out the transfer
mechanisms of VirE2 in host cells.
1.2 Direct visualization and tracking of Agrobacterium-delivered VirE2
In order to solve the difficulty of direct visualization of Agrobacterium-delivered VirE2,
a recently develop split-GFP system (Cabantous et al. 2005)were adapted and proved to
be successful in presenting the natural VirE2 movement in plant recipient cells, without
blocking the translocation activity(Li et al. 2014).
By the application of the split-GFP approach described in Li’s research (2014),
Agrobacterium-delivered VirE2 could be direct visualized in plant recipient cells. The
principle of split-GFP approach is as follows: VirE2-GFP11 fusion was created by
inserting a small GFP fragment (GFP11) into VirE2 at a permissive site; then, after the
expression of VirE2-GFP11 in A. tumefaciens and a large GFP fragment (GFP1-10) in
recipient plant cells, A. tumefaciens was infiltrated into the recipient cells. VirE2-
GFP11 would be delivered into the recipient cells, meet the large GFP fragment (GFP1-
10) and combine together. At that time, GFP fluorescence signals would become direct
visualized under a confocal microscopy. They found that VirE2-GFP11 fusion was
functional like VirE2 and indicated the trafficking of Agrobacterium-delivered VirE2.
Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA
into plant cells. The bacterium can transfer single-stranded DNA molecule (T-DNA)
and bacterial virulence proteins, including VirE2. A recently developed split-GFP
3
approach has been proved to be one of the considerable methods to directly visualize
the movement of Agrobacterium-delivered VirE2 protein (marked with GFP) inside
plant cell.
In Li’s(2014) research, they analyzed the quantitative characteristics of the movement
of Agrobacterium-delivered VirE2 protein in plant cells and explored the effects of
three kinds of protein transferring inhibitors(BDM, BFA and CytochalasinD) on VirE2
movement. Previous work with the split-GFP approach had done in my lab to get
confocal time-lapse images of Agrobacterium-delivered VirE2 movement in plant
recipient cells( Goodneret al., 1999). , as well as in protein transfer inhibited cells.
Referring to the time-lapse confocal images, I tracked VirE2 movement and found
VirE2 were efficient and directive delivered with average moving speed of 0.62um/s
and average moving distance of 25um in both cytoplasm and nucleus of plant cells, but
in protein transfer inhibited cells VirE2 delivery were obviously weaken with the
manner of swinging movement. In the following frequency and correlation analysis,
VirE2 moving speed and their distance from the nucleus in plant cells were described
and found to be significantly positive correlation with coefficients in 0.15-0.3 range, but
that in protein transport inhibited cells presented no significantly correlation. The
conclusions of the research include that VirE2 may be delivered by active and directive
transport and of a speed increasing as the increased distance from nucleus(Bell, 1990).
.In order to explore the characteristic of Agrobacterium-delivered VirE2 movement in
plant cells, through the application of the split-GFP approach and, leaf epidermal cells
of Nicotiana benthamiana infected by Agrobacterium tumefaciens were used to direct
4
observe the movement of Agrobacterium-delivered VirE2 under a confocal microscope
and then produce time-lapse image series. Under the confocal microscope, VirE2 were
labeled with green fluorescence protein (GFP) and the cellular locations were labeled by
Discosoma red fluorescent protein (DsRed). VirE2-GFP (green signal) appeared as
spots or filamentous structures in both cytoplasm and nucleus, while ucleus in one plant
cell presented as a big round structure labeled with DsRed and concluding some VirE2-
GFP particles(Smith and Townsend, 1907). Time-lapse image series including 59
pieces of images with intervals time of 3.5-4second were obtained from analyzed plant
cells. Movement of Agrobacterium-delivered VirE2 in plant cells could be presented by
the positional changes of the cancroids of VirE2 particles as time lapsing.
1.3 Quantitative analysis of Agrobacterium-delivered VirE2 movement
This analysis is used to determine the connection between variables.
In statistics, dependence is defined as the statistical relationship between two
continuous random variables. Correlation refers to a class of dependence.
The correlation coefficient is the key factor to demonstrate the class of linear correlation
between two randomvariables, and it can be obtained as formula (2). The correlation
coefficient betweenvariablesX and Y ,ρX,Yor corr(X,Y)is defined as:
(2)
where μX and μY are expected values of variables; σX and σY are their standard
deviations; E is the expected value operator; cov stands for the covariance.
The Pearson correlation is defined under the conditions of that only if both of the
standard deviations are finite and nonzero. It is a corollary of the Cauchy–Schwarz
5
inequality that the correlation cannot exceed 1 in absolute value. The correlation
coefficient is symmetric: corr(X,Y) = corr(Y,X). The degree of liner connection
between two variables is determined by the value of coefficient according to table1.1.
Table1.1. Thecriterion of linear correlation between variables depending on correlation
coefficient.
1.4 Objective and Scope
In order to quantitative of the characteristic of Agrobacterium-delivered VirE2
movement in plant cells, time-lapse image confocal series of the movement of
Agrobacterium-delivered VirE2 from leaf epidermal cells of Nicotiana benthamiana
infected by Agrobacterium tumefaciens were used to tracking and quantitative analyzed.
In order to explore the mechanisms of delivery, inhibitors treated cells were also
analyzed.
Correlationcoefficient The degree of liner connection
|corr(X,Y)| =0 Totally uncorrelative
0＜|corr(X,Y)|≤0.3 Weakly correlative
0.3＜|corr(X,Y)|≤0.5 Low correlative
0.5＜|corr(X,Y)|≤0.8 Obviously correlative
0.8＜|corr(X,Y)|≤1 High correlative
|corr(X,Y)| =1 Totally correlative
6
Chapter 2. Materials and Methods
2.1 Materials
Previous work with the split-GFP approach had done in my lab to get confocal video
images (time-lapse confocal images) of Agrobacterium-delivered VirE2 movement in
plant recipient cells. The plant recipient cells in this report were leaf epidermal cells of
N. benthamiana showing normal VirE2 movement, as well as cells treated with three
kinds of inhibitors. These inhibitors included a myosin ATPase inhibitor 2,3-
butanedione monoxime (BDM), a protein transport inhibitor in endoplasmic reticulum,
Brefeldin A(BFA), and a actin filament inhibitor Cytochalasin D (CytoD) , which were
added into samples with the concentrations of 50μM, 100μg/mL and 4μM separately.
The brief procedures of obtaining the confocal images of Agrobacterium-delivered
VirE2 movement in plant recipient cells conclude following steps.
1. Construction of A. tumefaciens mutants in which DNA sequence of (virE2) was
inserted with GFP11-coding sequence at a permissive site.
2. Construction of transgenic N. benthamiana expressing GFP1-10 and DsRed.
3. Agroinfiltration: infiltrating the A. tumefaciens mutant suspension into leaf
epidermal cells of N. benthamiana using a syringe.
4. Confocal Microscopy and obtaining the confocal viedo images.
A PerkinElmer Spinning Disk confocal microscopy with a UltraView system was used
to observe the movement of Agrobacterium-delivered VirE2. Under the combined
software (Volocity®
3D Image Analysis Software 6.2.1), video images (time-lapse
confocal images) of VirE2 movement were recorded with multiple focal planes (Z-
7
stacks). Time-lapse confocal images in before drugs treatment and after drugs treatment
were cropped into 59 images separately. The intervals between images are in the range
of 3.5-4s automatically determined by the confocal system for considerate scanning of
the plant cells.
2.2 Tracking of Agrobacterium-mediated VirE2
In order to explore the trafficking of Agrobacterium-delivered VirE2 in the leaf
epidermal cells of control N. benthamianacells and in trafficking prohibited treatment
cells, three control recipient leaf cells and one VirE2 trafficking prohibited cells by each
of the drugs(BDM, BFA, CytoD)were selected in the tracking analysis for delivered
VirE2.
The time-lapse confocal images were cropped into movies of 59 time points. Then they
were exported into the software Imaris for surface tracking.
Imaris provides a variety of standard procedures for surface tracking and exporting
tracks data into Excel format.
Briefly, a manual image segmentation and object tracking methods were used to analyze
image Swimming Agrobacterium delivered-VirE2 (markedwith GFP) and acquire
positions, surface area and moving time of VirE2 particles.Besides, we also measured
the positions of nucleuses (marked with RFP) for further analysis.The procedure of
surface tracking of delivered VirE2-GFP is shown as follows:
1) Identify the create surfaces.Find the Objects toolbar of the Surpass view, click
on the icon to add a new Surfaces item.You can the window shown as follows.
8
Select“Track Surfaces” in the algorithm setting option, and then click
on (Next). You can see the window below. In the source channel window below,
select GFP as Source Channel and check the Smooth option. Set the surface area detail
level as a suitable valueseparately, and then select the Absolute Intensity under the
Thresholding option.
Clickon (Next) and we can see the Threshold window below.
9
In the Threshold adjustment option, set the minimum value as threshold 10.000. Then
we can see VirE2 surface selected inthe viewing area followed.Clickon (Next) .
2) Filtering out unobviousVirE2 surface.
On the following Tab”Creation Wizard - 4/7 Classify Surfaces”, we sorted and filtered
the resulting Surfaces by various filter criteria (shown in talbe1). Click on (Next).
In the next step”Creation Wizard - 5/7 Edit Surfaces” , we can edit the resulting
Surfaces.In this research, an editing is not necessary. Click on (Next).
3) Establish a time dependantconnection between surfaces of delivered VirE2-GFP
(track them over time).
In the next step, we can see the Tracking window below. In order to edit the resulting
surfaces, select the tracking algorithm as autoregressive and set max distance as 20.000
10
um and max gap size as 3. Click on (Next).
Click on (Next) and we could see the following tab “Creation Wizard - 7/7 Classify
Tracks”. We could sort and filter the resulting Tracks by various filter criteria shown in
table1. At last, we clicked on the Next button. To complete Surface creation click on the
Finish button.
4) Exporting the tracks data into XLS. Format and measuring the position of
nucleuses.
We selectedthe Surfaces option in the selected Object List in the
Surpass Tree.We could see the statistical data by selecting the Statistics tab . There
were three statistical tabs Overall, Detailed, Selection.In order to export the output, we
chose to click on the button for the Overall statistics of tracks and click on the
button for all the specific statistical values in different tabs of an Excel file.The
tracks data included the coordinates, surface area, time points and velocity of tracked
moving VirE2 during time-lapse confocal images. And then we captured the tracks
images for each samples.
We used measuring tool to get the coordinates of the cancroids of nucleuses, which
were stable during time-lapse confocal images.
11
2.3 Quantitative analysis of Agrobacterium-mediated VirE2
Statistical data of moving GFP particles in time-lapse confocal images, such as the
coordinates, surface area, time points and velocity of tracked moving VirE2 and the
position of nucleus were imported into Excel software and then were filtered and
processed. As the velocity of VirE2 particles were obtained under the default that in the
software the time intervals of images is one second, the data was regenerated according
to themselves real intervals.
Then, with the coordinates of the moving VirE2 particles and the centroids of nucleuses,
the distance between each VirE2 particle and nucleus centralswere calculated according
to formula (1).
The distance between (x1, y1) and (x2, y2) is given by:
(1)
In order to explore the trafficking of Agrobacterium-delivered VirE2 in the leaf
epidermal cells of N. benthamiana, three recipient leaf cells were selected as parallel
analyzed control samples.Moreover, to analyze the effects of trafficking prohibited
drugs (BDM, BFA, CytochalasinD) treatment on the transformation of delivered-VirE2,
we selected BDM treated recipient cell(with the representative movements of the three
drug treated samples) for the further statistical analysis. For the purpose of exploring the
regularity of VirE2 movement in sample cells, we decided to figure out the distribution
of the values of VirE2 tracks’ speed and the distances between VirE2 and each nucleus,
as well as analysis the correlation between tracks’ speed and each distance, together
with the correlation between tracks’ acceleration and each distance.These data was
12
proceeded under the software SPSS (Statistical Product and Service Solutions; version
19.0) for StatisticsAnalysis( Frequencies analysis, Descriptive analysis and Correction
analysis).The statistics analysis steps of Control 1 are showed as an example.
This analysis is mainly used to figure out the values distribution of variables separately.
Firstly, after importing the tracks data of Control 1 VirE2 particles, such as speed,
acceleration and distances from the two nucleuses, select “analyze” in the menu list,
click the “Descriptive Statistics” then select the order “Frequencies”. Here is the
operation window of“Frequencies Analysis”.
Secondly, select analyzed variables(Speed, Dist1 and Dist2) and agree to “Display
frequency tables”. Then after clicking the “Statistics” option, we can see the statistics
windows below. After that, select the parameters as follows:
Percentile values: quartiles;
Central Tendency: mean, median
Dispersion: Std.deviation, minimum, variance, maximum
Distribution: skewness, kurtosis
Agree to “values are group midpoints”.
13
Then click “continue”.
Thirdly, after clicking the “Charts” option,we can see the Charts windows below. Then
set the chart type as histograms and tick “show normal curve on histograms.And then
click “continue”.
Fourthly, after clicking the “Format” option, we can see the Format windows below.
Then set “order by” as “ascending values” and “multiple variables” as “compare
variables”. And then click “continue”.
14
Lastly, click to “OK” in the Frequencies window. At this time, the output of frequencies
analysis would be the basic frequencies statistical data the basic descriptive statistics of
speed, Dist1( the distance between VirE2 particles and the first nucleus ) and Dist2( the
distance between VirE2 particles and the second nucleus ) and histogram with normal
curve.
This analysis is used to determine the connection between variables. If there are two
continuous random variables, the Pearson correlation coefficients in the “Bivariate
Correlation” are suitable; while if there are more than two variables, it is better to
control one variable, then analysis the correlation coefficients under the “Partial
correlation” method. Here we discuss the correlation between VirE2 particles and their
distances from two nucleuses.
At the beginning, the scatter diagram for the variables of Speed and Dist were made for
the visual observation of the connection of the speed of VirE2 particles and each of their
distances away from the controids of nucleuses.
The procedures is as follows: click on the “Graphs” option in the menu, and then choose
the “Scatter/Dot” option in the“Legacy Dialogs” list, and select “Simple Scatterplot”. At
that time, we could see the Scatter window below.
15
Select analyzed variables, speed and one of the distance, and then click on OK forming
the scatter diagram.
The second part of correlation analysis was to produce the quantitative results such as
the correlation coefficient between variables. The detailed steps are as follows.
Firstly, select “analyze” in the menu list, click the “Correlate” then select the order
“Partial Correlation”. Here is the operation window of “Partial Correlation”.
16
Secondly, select analyzed variables (Speed and Dist1) and set “Controlling for” as
“Dist1”. Then after clicking the “Options”button, we can see the statistics windows
below.
Thirdly, select statistics for “mean and standard deviations” and “zero-order correlations”
and missing values for “exclude cases listwise”. And then click “continue”.
Fourthly, after selecting test of significance as “Two-tailed” and agreeing to “display
actual significance level”, click “OK” to get results. At this time, the output of
correlation analysis would be the basic descriptive statistics (not shown because of
repetition) and correlations coefficients (seen in table 2.2).
17
Chapter 3. Results and Discussion
3.1 Tracking of the movement of Agrobacterium-delivered VirE2 in plant cells
In order to explore the characteristics of Agrobacterium-delivered VirE2 movement in
plant cells, the split-GFP approach was adopted; leaf epidermal cells of Nicotiana
benthamiana were infected by Agrobacterium tumefaciens and then used to directly
observe the movement of Agrobacterium-delivered VirE2 under a confocal microscope.
The movement was documented by capturing a series of time-lapse image. Additionally,
in order to analyze the transport mechanism of Agrobacterium-delivered VirE2
movement in plant cells, normal plant cells described above were added with three
kinds of drugs (BDM: a myosin ATPase inhibitor; BFA: a protein transport inhibitor in
endoplasmic reticulum; CytoD: a actin filament inhibitor) and then VirE2 movement in
the drug treated plant cells were also observed and recorded as 59 pieces time-lapse
images series.
Under the confocal microscope, VirE2 were labelled with green fluorescence protein
(GFP) and the cellular locations were labelled by Discosoma red fluorescent protein
(DsRed). VirE2-GFP (green signal) appeared as spots or filamentous structures in both
cytoplasm and nucleus, while nucleus in one plant cell presented as a big round
structure labelled with DsRed and including some VirE2-GFP particles. Time-lapse
image series including 59 pieces of images with intervals time of 3.5-4second were
obtained from the plant cells analyzed. The movement of Agrobacterium-delivered
VirE2 in plant cells could be presented by the positional changes of the cancroids of
VirE2 particles as time lapsing.
18
Figure 3.1. Movement of Agrobacterium-delivered VirE2-GFP in plant cells. Different rows show different cells: top row, normal leaf
epidermal cells of N. benthamiana; Second row, normal cells treated with inhibitors. Arrows or triangles were used to point out the
movement of VirE2-GFP (Green) by presenting the positional changes of VirE2-GFP in time-lapse images. The value above time bar
shows the current no. of time point with a total of 59 in time-lapse images series. Bars are 10um.
19
Figure 3.2.Tracks of the movement of Agrobacterium-delivered VirE2 in plant cells. Each column corresponds to tracks in different cells
marked at the bottom: from left to right, three control(-BDM) cells having obvious difference among cells and one representative BDM
treated (+BDM) cells. Top row, Representative confocal images; Middle row, Dots and connecting lines show tracks of moving VirE2 in
time-lapse images, the black dots present moving VirE2 tracked in the current time confocal images. Bottom row, tracks of moving VirE2
showed on the confocal images. The color change in time bar at top right indicated time lapse. Bars are10um.
20
Table 3.1. Quantitative analysis of Agrobacterium-delivered VirE2 tracks in normal leaf
epidermal cells of N. benthamiana.
Items Normal
Cell 1 Cell 2 Cell 3
Average
and
standard
deviations
of three
cells
No. of Movable VirE2Particles 45 69 35 50±7
Mean
value
of
items
among
VirE2
tracks
in the
cell
Moving time rate (%) of
one VirE2 track
0.51 0.48 0.68 0.56±0.12
Average moving speed of
one VirE2 track (um/s) 0.49 0.76 0.69 0.62±0.14
Track displacement length
(um) 15.24 38.02 22.48 25.25±5.11
1. Moving time rate (%) is presented by the rate of moving time to the
overall time of images
2. Track displacement length(um) is presented by the distance between the
first and last VirE2 position in the track.
21
Table 3.2. Average parameters of Agrobacterium-delivered VirE2 tracked in normal
and drug treated samples of leaf epidermal cells of N. benthamiana.
Average in cells with
different treatment
Normal
Drug treated
BDM BFA CytoD
No. of Moving
Particles 50 28 39 41
Moving time rate (%) 0.56 0.40 0.25 0.37
Average moving speed
(um/s) 0.62 0.30 0.27 0.44
Track displacement
length (um) 25.25 5.21 3.45 4.02
As shown in the top row of Figure 3.1, one VirE2-GFP (green) filament pointed by
arrows was moving in time-lapse image series. The value presented in time bar showed
the sequence of current image in 59 pieces time-lapse images series. VirE2 moved with
a distance of ~5um from the 3rd
image to the 11th
image and with a distance of ~10um
from the 11th
image to the 15th
image, implicating that the mean speed of the VirE2
moved among 5 images (from 11th
to the 15th
image) was 3.6 times of that in previous 9
images (from 3rd
to 11th
image). In other words, in the previous 9 pieces time-lapse
images, the VirE2 moved with a tiny speed but in the following 4 pieces time-lapse
images, it moved with a greatly increased speed. After the observation of overall VirE2
movement in plant cells, we found that the characteristic of VirE2 movement in plant
cells are as follows: as time lapsing, most Agrobacterium-delivered VirE2 were obvious
and directive moving in plant cells; in most of the time, VirE2 moved with small speed
22
but sometimes they moved with a large speed. We could get the conclusion that
Agrobacterium-delivered VirE2 were efficient delivered in both cytoplasm and nucleus
of plant cells.
As shown in the second row of Figure 3.1, two VirE2-GFP (green) filament pointed by
arrows and triangles presented unobvious movement as time lapsing. Similarly, the two
VirE2 all moved with a distance of 1-2um from the 2nd
to the 6th
image, as well as from
the 6th
to 11th
image. The phenomenon indicated that the two VirE2 all moved with a
tiny speed in the whole 11 pieces time-lapse images. After the observation of overall
VirE2 movement in drugs treated plant cells, we found that the characteristic of VirE2
movement in drugs treated plant cells are as follows: as time lapsing, most
Agrobacterium-delivered VirE2 were nearly swinging around their original positions
with small speed. We could get the conclusion that the movement of Agrobacterium-
delivered VirE2 in plant cells was obviously inhibited under the conditions of myosin
ATPase inhibited(BDM treated cells) or protein transport inhibited in endoplasmic
reticulum(BFA treated cells) or actin filament inhibited (CytoD treated cells) .
In order to quantitative analyze the characteristic of Agrobacterium-delivered VirE2
movement in plant cells, the tracking of VirE2 movements in one cell as time lapsing
and the collection of quantitative data in VirE2 tracks were processed under the
software Imaris. Firstly, during 59 pieces time lapse confocal image series, the
movements of one VirE2 were tracked as one track. Secondly, dozens or hundreds of
tracks of moveable VirE2 in one cell were combined, forming a drawing of the overall
VirE2 tracks in the cell. With the changing positional data of moveable VirE2 as time
23
lapsing, together with the current time among 59 pieces time-lapse images series
automatically recorded and calculated under the Imaris software, quantitative data such
as the number of movable VirE2, moving time and mean moving speed and track
displacement length averaged among different tracks could be obtained.
In general, according to the tracks of VirE2 (Figure 3.2), VirE2 movements in normal
cells presented more vigorous and directive movement, indicating the efficient delivery
of VirE2 particles, while VirE2 movements in all of the drugs treated cells were found
unobvious and presented as swinging around the original position, indicating the
inhibited effects of drugs treatment on the delivery of VirE2. However, the efficient
VirE2 delivery among plant cells (three normal cells) presented some considerable
differences. As can be seen in tracks data of three normal cells shown in Figure 3.2 and
Table 3.1, VirE2 movement in normal Cell 1 presented as the lest vigorous moving of
VirE2 with less moveable VirE2 and less moving speed and less track displacement
length, but VirE2 movement in normal Cell 3 presented as most vigorous movement
because of presenting largest values on the three parameters. Moreover, the efficient
delivery of VirE2 was strongly inhibited could be further supported by the quantitative
analysis of VirE2 tracks (Table 3.2). Firstly, the average number of moving VirE2
particles in normal cells was 50 and that in drugs treated cells were reduced to 28-41.
Secondly, the average moving time rate was reduced from 0.56 to 0.25-0.37. Thirdly,
the average moving speed of was reduced from 0.62 um/s to 0.27- 0.44 um/s. Lastly, the
average moving distance of VirE2 was reduced from 25um to 3-5 um, indicating the
reduced delivery distance of VirE2 in drugs treated cells.
24
To sum up, in normal plant cells, Agrobacterium-delivered VirE2 were efficient
transport with mostly tiny speed and sometimes large speed, with a large track distance
during 59 time-lapse images. while in drugs treated cells, the efficient transport of
VirE2 were greatly inhibited.The negative effects of inhibitors (BDM: myosin ATPase
interaction inhibitor; BFA: protein transport in endoplasmic reticulum inhibitor; CytoD:
actin filament interaction inhibitor) on the delivery of VirE2 could be concluded as the
reduction of the number of movable particles, moving time, moving distance and
moving speed.
25
3.2 Correlation Analysis of the speed of Agrobacterium-delivered
VirE2 and the distance from the nucleus in plant cells
Studies of the trafficking of Agrobacterium-delivered VirE2 in plant cells
found that, in plant cells infected by Agrobacterium VirE2 proteins were
delivered from the cellular membrane into nucleuses and then delivered out of
nucleuss. Referring to this directive and active transport of VirE2, I supposed
that among the movement of obvious delivered VirE2 in cellular cytoplasm,
there may be obvious correlation among moving speed of VirE2 proteins and
their distance from the position of nucleus.
In order to testify the hypothesis, moving speed of Agrobacterium-delivered
VirE2 proteins and their distance from the position of nucleus in plant cells
were considered as variables (Speed and DistN) and processed with Frequency
Analysis and Pearson Correlation Analysis under the software SPSS. After
tracking all moving VirE2 proteins among time-lapse confocal images series
within one plant cell under the software Imaris, the positional changes as time
lapsing and the stable position of nucleus in the cell could be obtained.
According to those data in normal plant cells, Speed and DistN were calculated.
In order to analyze the characteristics of obvious delivered VirE2 movement,
movement with speed less than 0.3um/s (the average speed in drugs treatment
cells) were filtered out, as well as movements with DistN less than 3um
(normal radius in sample cells).
26
Figure 3.3. Distribution bar charts of moving speed of Agrobacterium-
delivered VirE2 in normal plant cells: A, Cell 1; B, Cell 2 ; C, Cell 3.
27
Table3.3. Statistics of moving speed of Agrobacterium-delivered VirE2 in
normal plant cells.
Items of speed Cell1 Cell2 Cell3 Average
No. of VirE2 movements 131 193 188 171
Speed of
VirE2(um/s)
Mean value 0.73 1.57 1.14 1.15
Variance value 0.40 0.57 0.50 0.49
Range 4.03 5.42 5.23 4.89
Minimum 0.30 0.30 0.30 0.30
Maximum 4.27 5.76 5.52 5.18
28
Figure 3.4. Distribution bar charts of the distances between Agrobacterium-
delivered VirE2 and the nucleus in normal plant cells: A, Cell 1; B, Cell 2; C,
Cell 3.
29
Table3.4. Statistics of the distances between Agrobacterium-delivered VirE2
and the nucleus in normal plant cells.
Items of DistN (um) Cell1 Cell2 Cell3 Average
No. of VirE2 movements 131 193 188 171
DistN (um)
Mean
value 17.2 27.78 26.72 23.90
Variance
value 24.82 44.95 30.28 33.35
Range 41.24 35.72 37.78 38.25
Minimum 3.00 3.00 3.00 3.00
Maximum 44.24 38.72 40.78 41.25
30
Figure 3.5. Scatter diagram of moving speed of Agrobacterium-delivered
VirE2 and their distances from the nucleus in normal plant cells: A, Cell 1; B,
Cell 2; C, Cell 3.
31
Table 3.5. Pearson correlation between moving speed of Agrobacterium-
delivered VirE2 and their distances from the nucleus in normal plant cells.
Normal plant cells Cell 1 Cell 2 Cell 3
Correlation coefficient 0.254**
0.198
* 0.150
*
Sig. (2-tailed) 0.003 0.015 0.039
N 131 193 188
*. Correlation is significant at the 0.05 level (2-tailed).
**. Correlation is significant at the 0.01 level (2-tailed).
32
Figure 3.3 and Table3.3 shows the Distribution of moving speed (Speed) of
Agrobacterium-delivered VirE2 in normal plant cells. In normal cell 1, 131
VirE2 movements with a maximum speed of 4.27 and a mean speed of
0.73µm/s were analyzed. The percentiles mean that within 131 VirE2
movements, there were 25% having a speed less than 0.45µm/s, 50% having a
speed less than 0.56µm/s and 75% having a speed less than 0.87µm/s. In
normal cell 2, 193 VirE2 movements with a maximum speed of 5.76 and a
mean speed of 1.57µm/s were analyzed. The percentiles mean that within 193
VirE2 movements, there were 25% having a speed less than 0.80µm/s, 50%
having a speed less than 1.02µm/s and 75% having a speed less than 1.93µm/s.
In normal cell 3, 188 VirE2 movements with a maximum speed of 5.52 and a
mean speed of 1.14um/s were analyzed. The percentiles mean that within 188
VirE2 movements, there were 25% having a speed less than 0.48µm/s, 50%
having a speed less than 0.72µm/s and 75% having a speed less than 1.67µm/s.
Moreover, compared with previous VirE2 average speed of 0.62µm/s, an
obvious increased average speed of 1.15 µm/s were obtained through filtering
out of speed less than 0.3µm/s. We could easily get the conclusions that even
with an average range of 4.89µm/s, more than 75% VirE2 speed were
concentrative distributed at low speed(less than 2µm/s).
Figure 3.4 and Table3.4 shows the Distribution of the distance (DistN)
between Agrobacterium-delivered VirE2 and the nucleus in normal plant cells.
In normal cell 1, among the 131 VirE2 movements, their distance (DistN) from
the nucleus was presented a maximum of 44.24µm and a mean value of
17.20µm. The percentiles mean that there were 25% VirE2 presenting a DistN
less than 13.29µm, 50% presenting a DistN less than 15.70µm and 75%
33
presenting a DistN less than 22.34µm. In normal cell 2, among the 193 VirE2
movements, their distance (DistN) from the nucleus was presented a maximum
of 38.72µm and a mean value of 27.78µm. The percentiles mean that there
were 25% VirE2 presenting a DistN less than 16.94µm, 50% presenting a
DistN less than 22.51µm and 75% presenting a DistN less than 28.08µm. In
normal cell 3, among the 137 VirE2 movements, their distance (DistN) from
the nucleus was presented a maximum of 40.78µm and a mean value of
26.72µm. The percentiles mean that there were 25% VirE2 presenting a DistN
less than 18.52µm, 50% presenting a DistN less than 23.24µm and 75%
presenting a DistN less than 33.79um. Moreover, the similarly presented
maximum DistN among cells presented an average of 41.25, which is
coincided with normal radius of analyzed plant cells. Having compared with
mean DistN among different normal cells, we found that mean DistN in Cell 1
is 17.2, far below the average mean of 23.90, but mean DistN in the other two
cells presented a bit more than average value. Moving VirE2 in Cell 1was
distributed closer with the position of nucleus but farther in the other two.
Figure 3.5 and Table3.5 shows the results of Pearson correlation analysis
between variables Speed and DistN. In scatter diagram (Figure 3.5), the values
shown in horizontal and vertical ordinate stands for the distribution of Speed
and DistN. Dots stand for VirE2 movement with corresponding Speed and
DistN. Thus, it could be also used to describe the distribution of Speed and
DistN. It could be easily find that in normal plant Cell 1, VirE2 movement
having a speed less than 1.00um/s were nearly averaged distributed on the
range of DistN, indicating the broadly delivered VirE2 in the cell. However,
VirE2 movement having a larger speed, more than 1.00um/s, presented a
34
probably correlation with variable DistN. Similarly, the different distribution of
VirE2 movement could also be find in Cell 2 and 3, but separated by the speed
of 2.00um/s.
Table3.5 shows the Pearson correlation coefficient between variable Speed
and DistN and the responding significance level. The correlation coefficient in
normal cell 1 presented positive correlation of 0.254 at the 0.01level,
indicating that Speed is positively related to DistN in cell 1 with a probability
of 99%. The correlation coefficient in normal cell 2 presented positive
correlation of 0.198 at the 0.05level, indicating that Speed is positively related
to DistN in cell 2 with a probability of 95%. The correlation coefficient in
normal cell 3 presented positive correlation of 0.150 at the 0.05level,
indicating that Speed is positively related to DistN in cell 2 with a probability
of 95%. Moreover, the correlation coefficient in these cells is distributed on
the range of 0.15-0.25, indicating that the two variables (Speed and DistN) are
weakly correlative.
Thus, we could get the conclusion that among obvious Agrobacterium-
delivered VirE2 movement (Speed more than 0.3um/s) in cytoplasm of normal
cells (DistN of more than 3um), the speed of VirE2 is significantly positive to
the distance between VirE2 and nucleus. However, the weakly correlation
between Speed and DistN is considerable, because of the complex mechanisms
of Agrobacterium-delivered VirE2. There may exist a different mechanism for
VirE2 transport depending on the type of carriers.
35
3.3 Correlation Analysis of the speed of Agrobacterium-delivered
VirE2 and the distance from the nuclease in drugs treated plant cells
In order to analysis the inhibited effects of the three kinds of drugs(BDM,
BFA and CytoD) on VirE2 trafficking , variables Speed and DistN of VirE2
movement in three cells from each drug treated samples were processed for
Frequency Analysis and Pearson Correlation Analysis. Movement with speed
less than 0.1um/s were filtered out, as well as movements with DistN less than
3um (normal radius in sample cells).
Figure 3.6 and Table3.6 shows the Distribution of moving speed (Speed) of
Agrobacterium-delivered VirE2 and the distance（DistN） between nucleuses
in drugs treated plant cells. We found that VirE2 movement in planted cells
treated by three drugs, where myosin ATPase interaction inhibited (BDM
treated) or protein transport in endoplasmic reticulum inhibited (BFA treated)
or actin filament interaction inhibited (CytoD) presented highly similarity as
follows: mean speed and maximum speed were reduced by a half and 70%,
respectively, of that in normal cells; moreover，DistN were distributed in one
or two small range , implicating that the all cell distributed VirE2 movement
in normal cells were inhibited strongly: VirE2 movement was totally ceased in
some area and strongly weaken in some area.Table3.7 shows the Pearson
correlation between moving speed of Agrobacterium-delivered VirE2 and
their distances from the nucleus in drugs treated cells. We found that there
were no correlation between Speed and DistN. Taken together, these data
strongly suggested that myosin, the transport of protein in ER, actin filament
all have a major role in trafficking of Agrobacterium-delivered VirE2,where
36
myosin, the transport of protein in ER, actin filament might perform accessory
functions in the process.
Figure 3.6. Scatter diagram of moving speed of Agrobacterium-delivered
VirE2 and their distances from the nucleus in drugs treated plant cells. A,
BDM treated cell; B, BFA treated cell ; C, CytoD treated cell.
37
Table 3.6. Average statistics of the distribution of various Speed and DistN in
Agrobacterium-delivered VirE2 movement in different treatments cells.
Items Normal BDM
treated
BFA
treated
CytoD
treated
No. of VirE2 movements 171 102 127 132
Speed of
VirE2(um/s)
Mean 1.15 0.53 0.62 0.58
Minimum 0.30 0.10 0.10 0.10
Maximum 5.18 1.53 1.67 1.72
DistN (um)
Mean 23.90 23.72 18.99 30.32
Minimum 3.00 3.00 3.00 3.00
Maximum 41.25 38.24 43.56 42.13
38
Table 3.7. Pearson correlation between moving speed of Agrobacterium-
delivered VirE2 and their distances from the nucleus in drugs treated cells.
Drugs treated cells BDM treated BFA treated CytoD treated
Pearson Correlation -0.178 0.214 0.342
Sig. (2-tailed) 0.139 0.524 0.256
N 68 89 76
*. Correlation is significant at the 0.05 level (2-tailed).
**. Correlation is significant at the 0.01 level (2-tailed).
No star shows no obvious significant correlation relationship.
39
Chapter 4. Conclusion and Recommendations
Through tracking and quantitative analysis of VirE2 under time-lapse confocal
images, VirE2 were found to be directive delivered from cellar cytoplasm into
nucleus with average moving speed of 0.62 um/s, maximum speed of 5.76
um/s and average moving displacement of 25.25 um. While after analysis of
three inhibitor(BDM, BFA and CytoD) treated cells, we found that the
movement of Agrobacterium-delivered VirE2 was obviously inhibited. Thus,
that myosin, the transport of protein in ER, actin filament all had a major role
in trafficking of Agrobacterium-delivered VirE2, where myosin, the transport
of protein in ER, actin filament might perform accessory functions in the
process.
The research is an exploration of methods by using tracking and statistical
analysis for the movement of Agrobacterium-delivered VirE2 proteins inside
plant cell. It could be processed with more parameters such as acceleration for
deep study of the mechanisms of Agrobacterium-delivered VirE2.
40
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