i A MECHANISTIC EXPLORATION OF SIGNALING CROSSTALK REGULATING LIGHT ...

94 Pages·2016·4.43 MB·English

by  Dan Leuchtman

A MECHANISTIC EXPLORATION OF SIGNALING CROSSTALK REGULATING
LIGHT RESPONSES, GROWTH AND IMMUNITY IN ARABIDOPSIS

A Dissertation
Presented to
The Faculty of the Graduate School
At the University of Missouri

In Partial Fulfillment
Of the Requirements for the Degree

Doctorate of Philosophy

By
DANIEL LOUIS LEUCHTMAN

Dr. Emmanuel Liscum, III, Doctoral Supervisor
JULY 2016
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The undersigned, appointed by the dean of the Graduate School, have examined the
Thesis entitled

A MECHANISTIC EXPLORATION OF SIGNALING CROSSTALK REGULATING LIGHT
RESPONSES, GROWTH AND IMMUNITY IN ARABIDOPSIS

Presented by Daniel L. Leuchtman
A candidate for the degree of
Doctor of Philosophy,
And hereby certify that, in their opinion, it is worthy of acceptance.

Dr. Emmanuel Liscum, III

Dr. Walter Gassmann

Dr. Paula McSteen

Dr. Michael L. Garcia
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ACKNOWLEDGEMENTS

I would like to start by thanking my advisor Dr. Mannie Liscum, co-advisor Dr.
Walter Gassmann, current committee members Drs. Paula McSteen and Michael L.
Garcia, and former committee member Dr. David Braun. Each and every one of you
has been a critical part in my education through thoughtful questions and constructive
criticism. I thank you all for turning me into a scientist.

To The Boss Man, Mannie: You have been a key part of my education since my
undergraduate career in 2007. My grades were nothing special, but you saw enough
potential in me to provide me with an opportunity to prove myself as a plant biologist in
a PhD program. Your guidance and advice has helped immensely in my development.
You have also given me the freedom and opportunity to explore other avenues of
growth and learning both scientifically and professionally.

To Walter: I thank you for always being willing to provide guidance or a look at
new data, even at the most random or haphazard of times. I also thank you for being
my guide to the world of plant-microbe interactions. Learning new subjects is one of my
biggest motivators and having an expert guide the way makes it that much easier and
enjoyable.

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To the knucklheads that make even the most difficult days in the lab tolerable
and distract me from my biggest pressures, all of my labmates in the Liscum and
Gassmann labs: thank you. Starting with my first graduate student mentor Brandon
Celaya, I thank you for introducing me to lab life and making me aware of my potential.
I thank Ullas Pedmale and Jen Holland for always being there when I had questions.
To Diana Roberts for dragging me through the process of learning quality experimental
design and lab practices, thank you for being patient. I thank Jo Morrow for being the
Cloning Queen and always being willing to chat. I thank Kyle Willenburg, Scott
Askinosie and Katelynn Koskie for being there to challenge my thoughts and ideas and
helping to make them better. I thank Chris Garner, Morgan Halane, Ben Spears, Sange
He Kim and Saikat Bhattacharjee for always being willing to help, with whatever random
questions I walk in with. And finally I would like to thank Anthony Shumate for allowing
me to practice the art of mentoring. You have turned into an exceptional young scientist
and I see no limit to your potential. There isn’t much more I have to offer and I now
consider you a peer and a close friend. Good luck and have fun in Portland.

I must also thank the wonderful communities I am fortunate enough to be a part
of. I thank the community in BLSC and 3-west for always being willing help or lend a
reagent I need on the spot. I thank the IPG for creating an amazing place to study plant
biology that is truly world class. I thank the DBS for caring for me as a scientist and as
an individual.

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To my amazing wife Celsi Cowan: Thank you for enduring this with me, keeping
me stable, and for making sure I remember to eat, exercise and take a break now and
then. The future may be uncertain, but I’m not afraid of anything because I’m with you,
and I cannot wait for our next big step.

Lastly, I want to thank my parents and sister, Rick, Libby and Allie Leuchtman.
You have all supported me my entire life and are always there for comfort and
guidance. I would not be the person I am today without your constant love and
encouragement.

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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ................................................................................................. ii
LIST OF FIGURES ......................................................................................................... viii
CHAPTER 1. INTRODUCTION AND BACKGROUND .................................................... 1
The phototropins ........................................................................................................... 1
Phototropism ................................................................................................................. 2
Plant immunity .............................................................................................................. 3
Self-regulation of immune activity ................................................................................. 5
Influences of light in immune signaling and pathogen defense .................................... 6
Phytochromes ........................................................................................................... 6
Cryptochromes .......................................................................................................... 7
Phototropins .............................................................................................................. 8
Influence of the circadian clock ................................................................................. 9
Variation in methods used to assess photoreceptor involvement in defense produce
disparate results ...................................................................................................... 11
Thesis synopsis .......................................................................................................... 14
CHAPTER 2. SHINING A LIGHT ON IMMUNITY: CROSSTALK BETWEEN LIGHT AND
DEFENSE SIGNALING .................................................................................................. 16
Abstract ....................................................................................................................... 16
Introduction ................................................................................................................. 17
Experimental Procedures ........................................................................................... 19
Results ........................................................................................................................ 23
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HopA1 mediated resistance does not show dependency on PHOT1, PHOT2 or
PIXL under normal or monochromatic light ............................................................. 23
Phototropin double mutants show reduced PR2 accumulation ............................... 27
Prolonged induction of PIXL-HA results in seedling death ...................................... 29
TNL mutants tested have altered phototropic phenotypes ...................................... 31
No detectible difference in chloroplast movements in select group of defense
mutants ................................................................................................................... 33
Discussion .................................................................................................................. 36
CHAPTER 3. WHEN IN ROME: CROSS KINGDOM SUBSTITUTION OF A KEY
IMMUNE REGULATOR IN PLANTS ............................................................................. 39
Abstract ....................................................................................................................... 39
Introduction ................................................................................................................. 40
Experimental Procedures ........................................................................................... 42
Results ........................................................................................................................ 44
srfr1-4 root growth is severely stunted in the presence of hygromcin, and this
phenotype is diminished in mutants transformed with MmSRFR1 .......................... 44
Select MmSRFR1 transgenic lines also exhibit an increase in shoot growth
compared to srfr1-4 srfrEV-1 control ....................................................................... 46
MmSRFR1 transgenic lines are indistinguishable from srfr1-4 and srfrEV-1 controls
on normal media, and the srfrEV-1 shortened root phenotype is hygromycin
dependent ............................................................................................................... 48
Non-lethal doses of paraquat induce root stunting .................................................. 52
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Hygromycin resistant Col plants show increased root growth compared to
hygromycin resistant srfr1-4 plants. ........................................................................ 54
Discussion .................................................................................................................. 57
CHAPTER 4. CONCLUSIONS ....................................................................................... 60
Resistance to Pst DC3000 hopA1 does not show a strong phototropin or PIXL
dependency ................................................................................................................ 62
Phototropins influence PR2 accumulation in response to Pst DC3000 hopA1 and
avrRpt2 ....................................................................................................................... 62
Extended induction of PIXL-HA results in seedling death .......................................... 63
Certain defense mutants exhibit increased phototropic curvature, but do not show
changes in chloroplast accumulation or avoidance .................................................... 64
Hygromycin resistant srfr1-4 exhibits a hygromycin dependent shortened root
phenotype ................................................................................................................... 65
Transgenic MmSRFR1 in the srfr1-4 background shows an increase in root and shoot
growth ......................................................................................................................... 66
LITERATURE CITED ..................................................................................................... 67
VITA................................................................................................................................ 84

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LIST OF FIGURES
CHAPTER 1. INTRODUCTION AND BACKGROUND .................................................... 1
CHAPTER 2. SHINING A LIGHT ON IMMUNITY: CROSSTALK BETWEEN LIGHT AND
DEFENSE SIGNALING .................................................................................................. 16
Figure 2.1 HopA1 mediated resistance does not show a dependency on PHOT1,
PHOT2 or PIXL. ................................................................................................... 25
Figure 2.2 HopA1 mediated resistance does not show a phototropin dependency
under BL or RL conditions. .................................................................................. 26
Figure 2.3 Phototropin double mutant shows decreased PR2 accumulation. ..... 28
Figure 2.4 Prolonged induction of PIXL expression results in seedling death. .... 30
Figure 2.5 Mutants lacking PIXL, RPS6 or RPS4 exhibit increased phototropic
curvature. ............................................................................................................. 32
Figure 2.6 No change in chloroplast movement was observed in mutants lacking
PIXL, RPS6, EDS1-2 or RPS2 ............................................................................ 34
Figure 2.7 Model summarizing the association between phot1 and defense
proteins ................................................................................................................ 35
CHAPTER 3. WHEN IN ROME: CROSS KINGDOM SUBSTITUTION OF A KEY
IMMUNE REGULATOR IN PLANTS ............................................................................. 39
Figure 3.1 In the presence of hygromycin, resistant srfr1-4 shows a stunted root
growth phenotype and MmSRFR1 transgenic lines show increased growth. ..... 45
Figure 3.2. Shoot growth of MmSRFR1 transgenic lines. ................................... 47
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Figure 3.3. In the absence of hygromycin, MmSRFR1 transgenic root growth is
indistinguishable from srfr1-4. ............................................................................. 50
Figure 3.4. srfrEV-1 stunted root phenotype is hygromycin dependent .............. 51
Figure 3.5 Root growth of Col, srfr1-4 and srfrEV-1 in the presence of paraquat.
............................................................................................................................. 53
Figure 3.6 Root growth analysis of hygromycin resistant Col and srfr1-4 ........... 55
Figure 3.7 Model summarizing the association between hygromycin, SRFR1 and
the HPT hygromycin resistance protein ............................................................... 55
CHAPTER 4. CONCLUSIONS ....................................................................................... 60

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