i

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 andSaikat 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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CHAPTER 1

INTRODUCTION AND BACKGROUND

1.1 The phototropins

Optimizing photocapture is paramount to plant health. Plants evolved complex

response mechanisms to seek and optimize utilization of the best available light.

Examples of these responses include entrainment of the circadian clock, shade

avoidance syndrome and phototropism (Hsu and Harmer, 2014; de Wit et al., 2016;

Kutschera and Briggs, 2016). Phototropins are blue light photoreceptors that mediate a

variety of physiological responses including chloroplast movements, stomatal opening,

cotyledon/leaf expansion and the aforementioned phototropism, which is the process of

oriented growth towards directional light (Goyal et al., 2013; Okajima, 2016).

Phototropism has been studied for centuries and is one of the most well characterized

of these responses. In the path from light perception to oriented growth, many areas of

active research remain. This list includes determining structure, biochemical function,

sub-cellular localization, intracellular movement, interaction partners, and downstream

signaling targets of key proteins (Hohm et al., 2014; Liscum et al., 2014; Okajima,

2016). However, investigation of signal integration and cross-communication are more

recent areas of study.

The phototropins, phot1 and phot2, were originally identified in a screen for

mutants that failed to exhibit phototropism (Liscum and Briggs, 1995). Protein structure

is similar between the two phototropins, with two conserved amino-terminal FMN (Flavin

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MonoNucleotide)-binding LOV (Light, Oxygen, and Voltage) domains and a carboxyl-

terminal PKD (Ser/Thr Protein Kinase Domain). Following perception of UV-A/blue light

by the FMN chromophore, a covalent adduct is formed at the LOV domain and results in

a conformational change (Okajima, 2016). Change in protein shape allows for

autophosphorylation, which has been shown to be essential for phototropism (Inoue et

al., 2008). phot1 and phot2 exhibit partially overlapping function in intermediate light,

with phot1 being predominantly responsible for the responses to low light intensities

(Christie et al., 2015).

1.2 Phototropism

Asymmetric growth between the shaded and irradiated sides of the stem/shoot is

what enables the canonical bending response. This difference in growth is due to

redistribution of the plant growth hormone auxin, IAA (indole-3-acetic acid). While

several phot1 interacting partners and downstream signaling components have been

identified, the exact mechanism by which active phototropins induce lateral auxin

gradients remains elusive. The most detailed models involve modulation of auxin efflux

carriers, via direct or indirect regulation, with some also implicating intracellular Ca2+ flux

(Hohm et al., 2013).

Attempts to resolve precise mechanisms leading from light recognition to

physiological response has resulted in many attempts to identify new phot1 interacting

partners, with some unexpected outcomes. One such example is the identification of

PIXL (phot1 interacting cross-linked), which is a previously uncharacterized R

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(resistance)-like protein belonging to the TNL (Toll/Interleukin-1 receptor, nucleotide-

binding site, leucine-rich repeat) sub-family.

1.3 Plant immunity

The plant immune system is conceptualized as a two-tier system (Asai and

Shirasu, 2015; Cui et al., 2015). The first system is involved in recognizing invading

pathogens outside the cells via extracellular receptors known as PRRs (Pattern

Recognition Receptors). These receptors recognize conserved microbial molecular

patterns such as bacteria flagellin, chitin, and other structural carbohydrates commonly

found in various microbes known as MAMPs (Microbial Associated Molecular Patterns),

PAMPs (Pathogen Associated Molecular Patterns), or DAMPs (Damage Associated

Molecular Patterns) from molecules found in the apoplast following damage to the plant

due to pathogens or herbivores (Zipfel, 2014). Upon recognition of the pathogen, a

cascade of defense responses is elicited known as PTI (PAMP Triggered Immunity).

Examples of such responses include closure of stomates, synthesis of antimicrobial

compounds such as phytoalexins, strengthening of the cell wall by mechanisms such as

deposition of callose, and translation of defense related proteins and peptides (Zipfel,

2014; Asai and Shirasu, 2015; Bigeard et al., 2015; Yu et al., 2016). Pathogens,

however, have evolved to circumvent these responses by injecting molecules known as

effectors directly into the host cell in order to disrupt PTI. Such strategies include re-

opening of stomates, suppressing defense related gene expression, or manipulating

hormonally regulated defense responses (Nomura et al., 2006; Jelenska et al., 2007;

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Jelenska et al., 2010; Anderson et al., 2012; Lozano-Duran et al., 2014). To counter

these measures, plants have in turn evolved the means to then recognize these foreign

molecules in order to activate the second tier of the immune system known as ETI

(Effector Triggered Immunity). ETI restores and amplifies PTI basal transcription and is

typically characterized by a rapid release of ROS (Reactive Oxygen Species) that serve

as both antimicrobial compounds as well as signaling molecules to neighboring cells

(Cui et al., 2015; Lehmann et al., 2015). ROS accumulation results in a form of

programmed cell death known as a HR (Hypersensitive Response) and can be seen in

leaves as local lesions of necrotic tissue surrounded by healthy tissue. It is important to

note that a detectible HR is not always found upon R protein mediated recognition of

effectors and ETI (Gassmann, 2005).

In response to primarily biotrophic and hemi-biotrophic pathogens, signals are

sent throughout the rest of the plant where defenses are then upregulated resulting in

SAR (Systemic Acquired Resistance) (Fu and Dong, 2013). SAR is characterized by

accumulation of the defense hormone SA (Salicylic Acid), massive transcriptional

reprogramming and chromatin modifications that prime systemic tissues for defense

related gene expression (Gruner et al., 2013; Kachroo and Robin, 2013). Other

hormones that play decisive roles in defense include ET (Ethylene) and JA (Jasmonic

Acid) with these two playing more pivotal roles in defense against necrotrophic

pathogens and herbivores (Kachroo and Kachroo, 2012; Verma et al., 2016).

Additionally, SA and JA have been found as mutually antagonistic with respect to the

defense mechanisms each induces, with increased resistance to biotrophic pathogens

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increasing susceptibility to necrotrophic pathogens and vice versa (Robert-Seilaniantz

et al., 2011). Some pathogens have even evolved to exploit this disparity by stimulating

the opposite hormone, an example being biotrophic pathogens stimulating JA mediated

responses (Jiang et al., 2013; Gimenez-Ibanez et al., 2014).

1.4 Self-regulation of immune activity

The plant immune system must be under strict control to ensure that defense is only

activated when necessary in order to prevent energy loss and deleterious side effects.

Mechanisms by which plants control activation and suppression of the immune system

are still being elucidated. SRFR1 is a gene originally identified in a screen for negative

regulators of immunity by assaying for reactivation of resistance to Pst avrRps4 in the

normally susceptible Arabidopsis accession RLD (Kwon et al., 2004). Investigation of

the amino acid sequence reveals several key features with implications in transcriptional

regulation, protein-protein interaction and complex formation. Cross kingdom homology

is seen across the length of the protein with the most notable region being a N-terminal

tetratricopeptide repeat (TPR) domain (Kwon et al., 2009). TPR domain containing

proteins in animal systems are also associated with immune system regulation (Zeytuni

and Zarivach, 2012). In certain genetic backgrounds, plants lacking SRFR1 have

markedly different morphology, reduced viability, appear stunted and constitutively

express several defense related genes (Kim et al., 2010; Li et al., 2010).

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1.5 Influences of light in immune signaling and pathogen defense

Light has been shown as a necessary component of an effective immune

response. Using the model pathogen Pseudomonas syringae pv. maculicola in a series

of experiments with varying light conditions Zeier and colleagues (2004) showed an

uncoupling of certain defense responses, namely PR-1 expression, free SA, camalexin,

HR, SAR and apoplastic bacterial growth. The authors suggest that light directly

functions as a signal in immune signaling rather that contributing via indirect effects

through photosynthesis and energetic needs. Additionally, this study highlighted the

importance of light in SA mediated resistance as certain JA inducible genes show no

light depend expression patterns.

1.5.a.1 Phytochromes

The phytochromes phyA and phyB are thought to have a synergistic effect on

HR, PR1 expression and apoplastic growth of incompatible pathogen Pseudomonas

syringae pv. tomato DC3000 avrRpt2 with a strong influence of phytochrome on SA

mediated PR1 expression (Genoud et al., 2002). When studying a variegation mutant,

it was found that white areas (those with low chloroplast counts) showed a significant

reduction in HR, assayed via conductivity, while PR1 expression was comparable to

green areas with typical chloroplast abundance. The authors concluded that

phytochrome signaling modulates SA perception in Arabidopsis, and speculated that

plastids may be an alternative source of ROS in addition to a plasmalemma-associated

NADPH oxidase. However, PR expression is still light-dependent presenting a crucial

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role for light, independent of the photosynthetic machinery. Calcium is noted as a

potential point of crosstalk due to the fact that calcium signaling has been implicated in

both light and defense signaling responses (Genoud et al., 2002).

1.5.a.2 Cryptochromes

To test for additional influences of photoreceptors in defense responses,

infections using Psm avrRpm1 and photoreceptor double mutants phyA phyB, cry1 cry2

and phot1 phot2 were performed. The cryptochromes CRY1 and CRY2 are blue light

photoreceptors that entrain the circadian clock and mediate certain developmental

responses such as seedling de-etiolation (Galvao and Fankhauser, 2015). SA

accumulation was significantly higher in the cry1cry2 plants and significantly lower in

phyA phyB plants. HR was no different among the lines tested. SA and PR1

expression was detectible in phyA phyB mutant plants, but at levels lower than Col.

This was consistent with results from Genoud and colleagues, though results testing for

HR using trypan blue staining showed no significance. It is important to note that

Genoud and colleagues did see significant differences in HR between wild type plants

and photoreceptor mutants but investigated this using an electrolyte leakage assay.

This experiment quantifies the release of ions from infected leaves and can thus be

used as a quantitative indicator of HR.

In inoculations with incompatible bacteria, no significant differences were seen in

the photoreceptor mutants. In inoculations with compatible bacteria, however, the phyA

phyB double mutant showed a 3-fold higher growth than wild type. When the authors

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investigated systemic tissues (or re stimulated with inoculum) they found that cry1 cry2

and phot1 phot2 were able to mount an effective immune response against an

incompatible pathogen compared to the same assay preformed with a compatible

pathogen. Similar leaves were taken prior to infection to see if this result could be

attributed to presence of SA. Indeed, phyA phyB mutants accumulated significantly less

SA, and there was no noticeable difference between bacteria present from either the

incompatible or compatible interactions. Together these data suggest that SAR is

abolished in the phyA phyB mutant due to an inability to spread systemic signal post

infection. The authors proposed two mechanisms by which light may be influencing the

system. (1) Energetic status and metabolic environment of the cell through

photosynthesis. (2) Cross talk between photoreceptor and defense associated

signaling components. Based on these studies, photoreceptors alone are not

responsible for the critical dependency on light the immune system has. Work on

phytochrome has demonstrated an important role in SA mediated defenses soon after

infection. However, evidence such as expression of certain defense markers post

infection and the presence of a HR response in the phyA phyB mutant show that

phytochromes have a limited influence on SA mediated defenses at sites of infection.

1.5.a.3 Phototropins

Studies performed by Jeong and colleagues (2010) studying Arabidopsis

resistance to TCV (Turnip Crinkle Virus) found that stability of R protein, HRT, is light

and photoreceptor dependent (Jeong et al., 2010). By investigating possible influences

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of photoreceptors in light dependent stability of HRT, specifically phyC, phyD, phyE,

cry1, cry2, phot1, phot2, phyA and phyB, it was found that mutations in phytochromes

did not alter HRT mediated resistance but instead phot1, phot2, cry1 and cry2 showed

altered susceptibility, suggesting that blue light photoreceptors are necessary for

resistance to TCV through HRT. Although transcript levels of HRT were comparable to

wild type in all genetic backgrounds, protein levels were greatly reduced in phot2 and

cry2 mutant backgrounds. While phot1 and CRY1 were not needed for the stability of

the HRT protein, susceptibility data suggests they still play a role elsewhere in the HRT

mediated resistance. The authors found that although altered levels of HRT were found

between cry1 and cry2, phot1 and phot2, SA accumulation was significantly lower in

both phot1 and phot2 mutant despite the fact that these mutants still showed HR and

PR-1 expression at levels similar to wild type. It was hypothesized that blue light

photoreceptors act redundantly, possibly in conjunction with factors other than the

photoreceptors themselves. These data demonstrate a particularly important role for

CRY2 in HRT mediated resistance to TCV. In addition, the study found that HRT

interacted with COP1, an E3 ubiquitin ligase known to negatively regulate phot2

mediated activity (Mao et al., 2005).

1.5.a.4 Influence of the circadian clock

The circadian clock allows plants to anticipate pattern changes in the

environment as well as biotic threats. According to Ingle and colleagues (2015),

Arabidopsis has altered susceptibility to necrotrophic pathogen Botrytis cinerea based

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on inoculation time. Plants infected at subjective dawn were more resistant than those

infected at subjective dusk under constant light treatments and this was abolished in

clock mutants elf3-1 and cca1 lhy. Microarray analysis revealed that a core set of clock

target genes related to defense were induced faster in plants infected at subjective

dawn. In addition, enhanced susceptibility seen in plants infected at subjective night

was lost in jaz6 mutants, suggesting a role for jasmonates in circadian mediated

defense regulation (Ingle et al., 2015).

Transcriptome data has been used to investigate circadian and light dependent

changes to Pst DC3000 interactions. In tomato, Yang and colleagues (2015) observed

that Pst DC3000 susceptibility is highest before midnight. Light treatment, especially

red light treatment, given at this time point enhances resistance. This phenomenon is

correlated with increased SA accumulation and expression of defense related genes.

Analysis of the transcriptome revealed that red light induces expression of genes

implicated in maintaining circadian rhythms as well as phytochrome and SA mediated

defenses, further highlighting the ability of red light to influence SA mediated defenses

against Pst DC3000. Additionally, genes involved in redox homeostasis, calcium

signaling, transcriptional regulation and cellulose synthesis were differentially expressed

in response to red light and pathogen challenge (Yang et al., 2015).

Within a set photocycle, the specific time a plant is exposed to a pathogen can

have a profound influence on defense output. When challenged with incompatible

bacteria Psm avrRpm1, HR, PR expression and SA accumulation were higher in tissues

inoculated early in the day and significantly lower in plants infected in the evening or at

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night. Testing for circadian regulation of SA mediated defenses, Griebel and Zeier

(2008) performed infections in the subjective morning and subjective evening with

plants that had been acclimated to continuous light or continuous darkness. Under

continuous darkness SA was indistinguishable between the subjective day and

subjective night time points. In the continuous light treatment, SA was higher at

subjective night compared to subjective day. Circadian regulation of SA would predict

lower levels at subjective night, but this result showed the opposite. This highlights how

the duration of light exposure immediately following infection can influence SA

production.

1.5.b Variation in methods used to assess photoreceptor involvement in defense

produce disparate results

Results from various studies of photoreceptor activity in plant immunity may vary

however, as Jeong and colleagues (2010) did not detect a notable difference in

bacterial proliferation in phot and cry single mutants compared to wildtype when

challenged with Pst avrRpt2 or Pst avrRps4. It is important however, to keep in mind

the redundancies between photoreceptor paralogs when interpreting these results,

especially under routine lighting conditions. In contrast, We and Yang (2010) noticed a

dependency on CRY1 for AvrRpt2 mediated resistance specifically under continuous

light. In these conditions, Pst avrRpt2 showed increased proliferation in cry1 mutants

but decreased growth in CRY1 overexpressing lines. These authors also performed

experiments under blue and red light conditions to test for dependency on

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active/inactive CRY1. Blue-light treated plants displayed the same pattern as those

grown under white-light with CRY1-overexpressing plants showing less bacterial growth

than wild type and cry1 mutant plants showing increased growth. However, this pattern

was abolished in plants grown in red-light following infection, suggesting that active

CRY1 influences resistance to Pst avrRpt2 under these conditions. As compatible

pathogen Pst DC3000 grew to normal levels in all backgrounds tested, the authors

conclude that basal resistance is not influenced by CRY1 but rather R-protein mediated

resistance.

Genoud and colleagues (2002) found light dependency for PR-1 when treated

with SA, and HR when challenged with Pst avrRpt2. There was also light dependency

of defense against Pst avrRpt2, as well as phytochrome dependency for all of the

aforementioned results. Zeier and colleagues (2004) used Psm avrRpm1 and also

found a light dependency on HR and PR-1 expression, as well as SA accumulation.

Griebel and Zeier (2008), again, found light dependency of SA accumulation and PR-1

expression and HR using Psm avrRpm1. These authors also found, to a lesser extent,

phytochrome dependency on SA accumulation and PR-1 expression qualitatively in

agreement with results from Genoud and colleagues (2002), however they did not find

HR to be phytochrome dependent. Two different methods were used to come to these

conclusions, Genoud and colleagues (2002) assessed HR measuring electrolyte

leakage and trypan blue staining was used by Griebel and Zeier (2008).

Additionally, defense during incompatible interactions in the phyA phyB double

mutant were not found to be significantly impaired by Griebel and Zeier (2008), but

13

instead defense during compatible interactions. Genoud and colleagues (2002),

however, did see a significant increase in incompatible pathogen growth between phyA

phyB double mutants and wild type seedlings. Differences between the two studies

could be attributed to the different strains of bacteria used (Pst avrRpt2 vs Psm

avrRpm1), or growth conditions. Genoud and colleagues (2002) used a growth

chamber with 30µmol m-2 s-1 white light, a 9h L: 15h D photocycle, 20ºC temperature,

and 60% humidity. Seedlings used were approximately 3 weeks old. Griebel and Zeier

(2008) used growth conditions set to 70µmol m-2 s-1, 9h L: 15h D, 18ºC and used 6

week old plants. Age of the plants, specific pathogen used and intensity of light in the

growth space could attribute to the differences seen.

In the same conditions mentioned above, Griebel and Zeier (2008) found no

difference between cry1 cry2 or phot1 phot2 and their respective wild type backgrounds.

However, Wu and Yang (2010) saw increased growth in cry1 when challenged with Pst

avrRpt2 when grown in continuous light at 18-22ºC and light at 120µmol m-2 s-1 using 4-

5 week old seedlings. In comparison, Jeong and colleagues (2010) found no significant

difference between phot or cry single mutants when challenged with Pst avrRpt2 or Pst

avrRps4, consistent with the study conducted by Griebel and Zeier (2008) using Psm

avrRpm1 on phot and cry double mutants. Jeong and colleagues (2010) used growth

conditions as follows 14h L: 10h D, 22ºC, 65% relative humidity, 106.7µmol m-2 s-1. To

summarize, these results highlight the variation in environments, pathogens used to

challenge plants, and subtle influence photoreceptors have on pathogen defense. On

the contrary, the results demonstrate the clear influence light has on HR and SA

14

mediated defenses in Arabidopsis. Interestingly, light dependency on HR has also been

demonstrated in Arabidopsis challenged with viruses (Chandra-Shekara et al., 2006;

Jeong et al., 2010).

1.6 Thesis synopsis

The goal of my thesis was to begin bridging the molecular signaling mechanisms

behind light mediated growth and development and plant immunity. Both of these

processes have been studied for decades, but typically in isolation, and for very good

reason since only under strict growth environments have findings thus far been

possible. However, now that major players in most principle signaling pathways have

been identified, we may begin studying these systems in synergy. Additionally, while in

silico methods to study network organization are quite powerful, only through tandem

use with mechanistic examination and validation may concise and concrete conclusions

be made.

The PIXL-phot1 interaction in Arabidopsis represented a potential mechanistic

starting point to connect the well characterized systems of light/phototropin mediated

developmental physiology and immune function. Balancing adaptations to varied

lighting environments with defense responses is an excellent example of the challenges

behind resource allocation and utilization in plants. For instance, studies on the

influence of phytochrome B and UV-B receptor, UVR8, have highlighted a fulcrum that

either tips the system in the direction of increased defense potential in the way of

increased R:FR and high UV-B, or a focus on growth and elongation in low R:FR and

15

UV-B (Mazza and Ballare, 2015). A better understanding of these turning points help

explicate energetic commitments made by plants and could result in improved

agricultural management strategies.

Furthermore, while study of light dependent physiology, plant defense and the

respective signaling components have excellent potential to reveal possible crosstalk,

our studies have also led us to investigate the balance between immunity and normal

growth and development, specifically through influences of SRFR1. While functions as

a negative regulator of defense remains clear, other possible functions present an open

question. TPR domain containing proteins can be found across all kingdoms, and while

many also participate in immunity others have diverse roles ranging from protein

trafficking, vesicle formation and organelle function (Zeytuni and Zarivach, 2012). The

sequence of SRFR1 shows marked similarity across kingdoms and presence of the

conserved TPR domain, but portions of conserved sequence are still of unknown

function. It is quite possible that the roles of SRFR1 extend beyond simply regulating

defense signaling and these possibilities provide another exceptional model for

signaling crosstalk. Substitution of SRFR1 with MmSRFR1 is a unique opportunity to

investigate this possibility.

16

CHAPTER 2

SHINING A LIGHT ON IMMUNITY: CORSSTALK BETWEEN LIGHT AND DEFENSE

SIGNALING

Authors: Daniel L. Leuchtman, Anthony Shumate, Ullas V. Pedmale, Walter Gassmann

and Emmanuel Liscum

2.1 Abstract

For plants, light serves not only as a source of energy, but also as a source of

key environmental information. Recent studies have found that light also plays a critical

role for the plant immune system, and this requirement has been found to be

independent of the energy which light provides. Photoreceptors are molecules that

equip plants with a means to sense the quantity and quality of surrounding light and

allow anticipation of important changes throughout the growing season. Phototropins

are blue-light photoreceptors that arbitrate several physiological processes such as

stomatal opening, chloroplast movements and phototropism. In our research, we are

investigating the potential crosstalk between light signaling and pathogen defense using

Arabidopsis as a model. PIXL is a putative resistance protein found to be an interactor

of phototropin1. Mutants lacking PIXL have altered light responsiveness, suggesting

the wild-type protein could act as an intermediary between light and defense signaling.

Further characterization of this system will provide a mechanism to connect these two

seemingly unrelated molecular networks.

17

2.2 Introduction

While several studies have highlighted the importance of light environments

during pathogen defense, research on precise mechanistic bases for this phenomenon

is still new and quite active. Many hypotheses exist including photosynthetic

implications on ROS accumulation, biosynthesis of defense related compounds,

chloroplast modifications, influences of the circadian clock and light signaling events

mediated by photoreceptors (Hua, 2013; Karpinski et al., 2013; Ballare, 2014;

Kangasjarvi et al., 2014; Trotta et al., 2014). Associations between proteins traditionally

studied for their roles in light signaling and R proteins have been demonstrated (Jeong

et al., 2010). Further analysis of R proteins in the context of light signaling could reveal

further signaling overlap between these two systems.

Most R proteins, as well as RGAs (Resistance Gene Analogs), are intracellular

NB-LRR (Nucleotide Binding – Leucine Rich Repeat) proteins that can be grouped

according to an amino-terminal CC (Coiled Coil) domain, known as the CNLs, or TIR

(Toll/Interleukin 1 Receptor-like) domain, known as the TNLs. In the off state, NB-LRR

proteins bind ADP. Stimulation by an elicitor, which may be through direct or indirect

recognition of a pathogen effector, results in a conformational change allowing for the

exchange of ADP for ATP resulting in the active state of the molecule. ATP is then

hydrolyzed to return to the off state. EDS1 (Enhanced Disease Susceptibility 1) has

been found necessary for elicitation of resistance via TNLs, while NDR1 (Non-Race

specific Disease Resistance 1) is essential for CNL mediated resistance (Wu et al.,

2014; Sukarta et al., 2016). Recognition of effector molecules is typically associated

18

with cell death and a HR, but these phenomena are not absolute as resistance initiated

through these proteins can be uncoupled from this response by the host cell (Oh et al.,

2006; Senthil-Kumar and Mysore, 2013). Recent insights have highlighted the

mechanisms behind protein function and how multiple NB-LRR proteins may work in

concert to initiate defense. For example, TNL resistance proteins RRS1 and RPS4

have been shown to elicit defense responses to several effector proteins coordinately

(Williams et al., 2014).

Most characterized resistance proteins have been discovered from screens

testing for resistance or susceptibility to certain effector molecules or pathogens.

Several other roles for these proteins have been identified. Some NB-LRRs, known as

‘helper NB-LRRs’, have demonstrated the ability to initiate resistance through atypical

mechanisms relative to what their structure would suggest. Helper NB-LRRs cannot

initiate resistance independently and must do so in conjunction with multiple immune

receptors, even those normally involved in basal defense (Bonardi et al., 2011; Jacob et

al., 2013; Roberts et al., 2013). While lacking the LRR domain, CHS1 (Chilling

Sensitive 1) and CHL1 (CHS Like 1) are TIR-NB genes involved in acclimation to

temperature stress (Zbierzak et al., 2013). Several other NB-LRRs, including RPP4,

have roles in coordinating acclimation to temperature stress with defense (Huang et al.,

2010; Zhu et al., 2010; Heidrich et al., 2013). These examples show that NB-LRR

proteins can play key roles intersecting biotic and abiotic stress.

PIXL is a RGA (Resistance Gene Analog), or resistance-like gene, belonging to

the TNL sub-group that was identified as a phot1-interacting protein. BLAST analysis

19

shows PIXL has closest similarity with R-protein RPS6 (Meyers et al., 2003). RPS6

was identified by screening for mutants susceptible to Pst DC3000 hopA1, where

HopA1 normally acts as an avirulence effector that signals through EDS1 (Kim et al.,

2009). Another proposed R-protein-phototropin interactor is RPS2, a CNL R protein.

phot1 and phot2 were identified in a screen for RPS2 interacting partners with the phot2

interaction being confirmed (Qi and Katagiri, 2009; Jeong et al., 2010). However, no

implications in RPS2 mediated resistance have been identified for phot1 or phot2 thus

far. The PIXL-phot1 interaction presented us with an opportunity to explore mechanistic

explanations associating light and defense signaling using reverse genetics to search

for physiological relevance.

2.3 Experimental Procedures

Plant material: Arabidopsis thaliana seedlings used in this study were all in the

Columbia (Col) accession. Phototropin and defense mutants have been described

previously: phot1-5 (Huala et al., 1997), phot2-1 (Kagawa et al., 2001), phot1-5/2-1

(Kinoshita et al., 2001), rps4-2 and srfr1-4 (Kim et al., 2010), eds1-2 (Bartsch et al.,

2006), ndr1-1 (Century et al., 1995), rps2-201 (Kunkel et al., 1993), and fls2

(SALK_093905) (Heese et al., 2007). The rps6-3 allele (SALK_029541) is from the

Salk Arabidopsis insertion library and was obtained from the Arabidopsis Biological

Resource Center. The second exon was determined to be the site of T-DNA insertion

using PCR and is consistent with the suggested position of insertion from the T-DNA

Express website (http://signal.salk.edu/cgi-bin/tdnaexpress). The pixl-1 allele

20

(SAIL_259_B09) is from the Syngenta Arabidopsis insertion library (Sessions et al.,

2002), and was obtained from the Arabidopsis Biological Resource Center. The first

exon was determined to be the site of T-DNA insertion using sequencing. This is

consistent with the suggested position of insertion determined by raw flanking sequence

from the T-DNA Express website (http://signal.salk.edu/cgi-bin/tdnaexpress).

Syringe inoculation assay: Arabidopsis plants were grown in a reach-in growth chamber

under an 8 h light/16 h dark cycle at 24°C, 70% relative humidity, and a light intensity of

90 to 140 µmol m-2 s-1. Four week old plants were infiltrated with plate grown bacteria

isolated and diluted to a suspension of 5 x 104 cfu/mL in 10mM MgCl2 using a 1mL

needleless syringe. Leaf discs with a total area of 0.5 cm2 were ground in 10mM MgCl2

and serial dilutions were plated on selective medium in triplicate at the time points

indicated.

Flood inoculation assay: Two week old seedlings were grown in high wall plates

containing 1/2x MS, 1x Gamborg vitamins, 1% sucrose and 0.3% phytagel kept in a

walk-in growth chamber under 12 h light/ 12 h dark cycle at 22ºC and light intensity of

50 to 70 µmol m-2 s-1. Plates were flooded with 40 ml of bacterial suspension made in

sterile water containing 0.025% silwet L-77 resulting in a final concentration of 1 x 105

cfu/mL and incubated at room temperature for 2-3 minutes. Bacterial growth was

assessed immediately after bacterial treatment and at day 4 post infection. Internal

bacterial populations were evaluated from three biological replicates and each replicate

represented a pooled sample of four independent rosette seedlings from the same petri

plate. The total weight of inoculated seedlings was measured and used to normalize

21

bacterial growth. After weighing, seedlings were surface-sterilized with 5% H2O2 and

rinsed three times with sterile distilled water. Each pooled biological replicate was then

separately ground in 10mM MgCl2 and serial dilutions were plated on selective medium.

Western blotting analysis: 2-3 week old Arabidopsis seedlings, grown in a walk-in

growth chamber under 16 h light/ 8 h dark cycle at 22ºC and light at an intensity of 50 to

70 µmol m-2 s-1, were infected with bacteria suspended in 10 mM MgCl2 at a

concentration of 2.5 x 108 CFU/mL via the dip method. Approximately 50mg of tissue

was ground in 6M urea, centrifuged at 16,000 RCF for 5 minutes to separate debris and

then supernatants were separated and quantified using a Bradford assay. After diluting

to 20µg of protein per lane, samples were subjected to SDS-PAGE and transferred to

nitrocellulose membrane. After blocking with 5% milk in Tris buffered saline containing

0.1% Tween-20 (TBST), the membrane was incubated overnight at 4°C with the

indicated primary α-PR2 antibody (Agrisera, Vännäs, Sweden) followed by HRP

conjugated secondary antibodies (Santa Cruz, Dallas, TX). Membranes were then

washed three times in TBST, exposed to HRP substrate and visualized using

autoradiography film. Ponceau stain is included as a loading control.

Transgenic constructs: PIXL was amplified from genomic DNA prepared from Col leaf

tissue using a cTAB extraction protocol. Using approximately 100ng of template DNA,

10µM forward (5’ CACC ATG GCT TCT TCG TCC TCC TCT TCT 3’) and reverse (5’

CGT GTC TTC TTC TGC CTC TAA ATT A 3’) primers, Phusion High-Fidelity DNA

Polymerase (New England Biolabs, Ipswich, MA) with provided GC buffer, dNTPs and

recommended thermocycler settings. Products in the range of 3.9 kb were isolated via

22

agarose gel electrophoresis, excised, purified and used in a TOPO reaction for insertion

into the pENTR entry cloning vector. PIXL was then inserted into the binary destination

vector pLN604, which is derived from pER8, using Gateway LR Clonase II Enzyme Mix

(ThermoFisher, Waltham, MA) (Zuo et al., 2000; Nicaise et al., 2013). ß-Estradiol

inducible PIXL-HA was then stably transformed into pixl-1 mutants using the floral dip

method. Transgenic plants were screened to homozygosity on media containing

Hygromycin B.

ß-Estradiol growth assay: Eight individual seeds were sown per genotype and per

treatment on a plate containing 1/2x MS, 2% sucrose, 1x Gamborg Vitamins and either

2 µM ß-Estradiol diluted from a 10mM stock suspended in DMSO or an equivalent

volume of DMSO without ß-Estradiol (mock).

Phototropism assay: Seedlings were sown on vertical oriented plates with 1/2x MS

media in darkness of 3 days and then exposed to unilateral low blue light

(0.1 mmol m−2 s−1) for 4 hours. Images were acquired using an Epson Perfection V37

Scanner and angles were determined using FIJI software.

Chloroplast movement assay: Chloroplast movements of live cells was assessed using

a 96-well plate reader to measure change is transmittance and a blue LED light source

to stimulate chloroplast movements. 96-well plates were loaded with 100 µL of 1/2x MS

with 0.6% phytagel and a leaf punches with a diameter of ~ 6mm were placed adaxial

side up with the addition of 20 µL of liquid 1/2x MS to avoid dehydration. The plate

reader followed a program taking readings at 10 min intervals while ejecting the plate for

outside exposure (darkness or blue light) between intervals.

23

2.4 Results

HopA1 mediated resistance does not show dependency on PHOT1, PHOT2 or

PIXL under normal or monochromatic light

Bacterial growth assays analyzing photoreceptor dependencies have shown

varying results. Because of this, we wanted to test for possible phototropin involvement

in resistance using our own growth conditions and experimental procedures. Due to

homology with RPS6, we chose to use Pst DC3000 (hopA1) to both test PIXL for a

possible role as a helper NB-LRR, and to assess TNL mediated ETI in phototropin

mutant backgrounds. Additionally, the gl1 mutation, while most famous for its role in

trichome development, has been implicated in cuticle formation and establishment of

SAR (Xia et al., 2010). Because the phototropin double mutant is in the gl1

background, we must compare phot1-5/2-1 specifically to gl1 to ensure any effect is due

to the phototropins and not the gl1 mutation.

Wild-type Pst DC3000 is normally a virulent pathogen on Col plants, but

recognition of a single effector molecule can result in substantially less apoplastic

growth, as can be seen by the 100-fold difference in CFU (Colony Forming Unit)

between wild-type Col-0, which has a functional RPS6 and EDS1, and rps6-3, which

cannot recognize HopA1 and initiate defenses (Figure 2.1). These experiments were

repeated several times, and while occasionally the phototropin mutants exhibited

increased susceptibility relative to gl1, the results were too inconsistent for us to infer

any significance, leading us to conclude that phototropins are not essential for HopA1

mediated resistance under our experimental conditions, nor was PIXL found to be

24

necessary as can be seen by comparison to Col (Figure 2.1). A more reproducible and

sensitive pathogen growth assay may reveal a subtle role for phototropins in

DC3000(hopA1) mediated resistance.

Similar to previous studies, we also conducted bacterial growth assays under

various light treatments in order to test active vs. inactive states of phototropins under

pathogen challenge (Wu and Yang, 2010). Resistance in mutants grown under blue or

red light was indistinguishable from wild-type against DC3000 (hopA1) (Figure 2.2).

25

Figure 2.1 HopA1 mediated resistance does not show a dependency on PHOT1,

PHOT2 or PIXL

Bacterial growth was measured in Col, gl1, rps6-3, phot1-5, phot2-1, phot1-5/2-1 (phot1-5/phot2-1) and

pixl-1 on day 0 (white columns) and day 3 (black columns) after inoculation with DC3000(hopA1). Plants

were inoculated with a bacterial suspension at a density of either 1 x 106 cfu/mL (A) or 5 x 104 cfu/mL (B).

Values represent log averages of cfu/cm2 leaf tissue from triplicate samples with error bars denoting

standard deviation. While individual replicates of this experiment varied, the most representative are

depicted above. Statistical significance was determined using a Student’s t-test comparing mutants to

their respective background, Col (rps6-3, phot2-1, pixl-1) or gl1 (phot1-5, phot1-5/2-1) (p, ** ≤ 0.01, *** ≤

0.001).

26

Figure 2.2 HopA1 mediated resistance does not show a phototropin dependency

under BL or RL conditions.

Col, gl1, eds1-2 and phot1-5/2-1 (phot1-5/phot2-1) that were light adapted under BL (A) or RL (B) 3 days

pre- and up to 4 days post-inoculation. Bacterial growth was measured on day 0 (lighter columns) and

day 4 (darker columns) after inoculation with DC3000(hopA1). Plants were inoculated with a bacterial

suspension at a density of 1 x 105 cfu/mL. Values represent averages of cfu/mg leaf tissue from triplicate

samples with error bars denoting standard deviation. Statistical significance was determined using a

Student’s t-test comparing mutants to their respective background, Col (eds1-2) or gl1 (phot1-5/2-1) (p, *

≤ 0.05, ** ≤ 0.01).

27

Phototropin double mutants show reduced PR2 accumulation

We chose to test the phot1-5/2-1 mutant for PR2 protein levels in order to assess

if phototropins are involved in defense protein abundance rather than transcript level as

seen before with phot2 and HRT (Jeong et al., 2010). Testing for PR2 abundance is a

more sensitive assay and would allow us to detect smaller changes in defense than

other methods such as pathogen growth assays. Previously, PR1 expression in

photoreceptor mutants under pathogenic stress has been analyzed with only

phytochrome mutants showing altered expression (Griebel and Zeier, 2008). To test

ETI via CNL or TNL mediated resistance, we challenged plants with either DC3000

(avrRpt2), or DC3000 (hopA1). AvrRpt2 detection is mediated by RPS2 and defense

initiation requires NDR1 (Kunkel et al., 1993; Century et al., 1995). EDS1 is required for

defense initiation following RPS6 mediated recognition of HopA1 (Gassmann, 2005;

Kim et al., 2009). As expected, eds1-2 and ndr1-1 have a delayed accumulation of

PR2. A srfr1-4 mutation results in constitutively active defense responses and thus PR2

can be seen at uniform abundance across all time points, even prior to pathogen

challenge. PR2 levels were increased in the gl1 background relative to Col wild type.

We were surprised to see decreased PR2 levels in the phot1-5/2-1 double mutant

(Figure 2.3). This result suggests a role in establishing or maintaining PR2 protein

levels.

28

Figure 2.3 Phototropin double mutant shows decreased PR2 accumulation.

2-3 week old seedlings were inoculated with DC300 secreting HopA1 (A) or AvrRpt2 (B). PR2 was

detected via western blot at time-points throughout the infection. Ponceau stain is included as a loading

control. hpi = hours post inoculation.

29

Prolonged induction of PIXL-HA results in seedling death

Overexpression of R-genes can result in seedling death (Zhang et al., 2003; Yi

and Richards, 2007; Kim et al., 2010; Kim et al., 2012). To prevent rapid silencing of a

PIXL transgene in the event its presence resulted in overstimulation of the immune

system, we chose to use an inducible expression system, specifically an estrogen

receptor based system originally developed by Zuo and colleagues but then modified by

Nicaise and colleagues to include a C-terminal HA epitope tag (Zuo et al., 2000; Nicaise

et al., 2013). Transgenic PIXL-HA lines were produced in the pixl-1 mutant background.

Control lines in the pixl-1 background were also generated with an empty binary vector

(EV). In a mock treatment, an equivalent volume of DMSO needed to create 2 µM ß-

estradiol plate media was not found to have any ill effects. Additionally, 2 µM ß-

estradiol did not appear to induce any stress in the wild-type Col, mutant pixl-1 or any of

the EV backgrounds. PIXL transgenic lines, however, were dead within one week of

growth, presumably due to prolonged induction of the PIXL transgene.

30

Figure 2.4 Prolonged induction of PIXL expression results in seedling death.

Transgenic PIXL-HA or EV-HA (empty vector) driven by a ß-Estradiol inducible promoter were sown on

plates with either 2 µM ß-Estradiol (bottom) or mock treated (equivalent volume DMSO) and allowed to

grow for one week. Each letter corresponds to an independent insertional event for the designated

construct.

31

TNL mutants tested have altered phototropic phenotypes

While many experiments have been done to assess possible phototropin

involvement in defense responses, there have been no studies investigating phototropin

dependent physiology in response to immune activity. We began to address this

question by performing a standard phototropism assay on various defense mutants.

This experiment was physiologically relevant as the hypocotyl was the organ where the

phot1-PIXL interaction was originally identified. These experiments were done under

low blue light conditions (0.1 mmol m−2 s−1) where phot1 is the primary photoreceptor as

can be seen by the lack of bending in phot1-5 mutants which still possess a functional

phot2 (Figure 2.5). It is important to note that exact bending response can vary from

experiment to experiment due to temperature and other environmental conditions.

Because of this, comparisons may only be made between plants from the same

replicate experiment. pixl-1, along with rps4-2 and rps6 were chosen to probe the TNL

side of ETI for possible effects along with eds1-2. rps2-201 was tested to investigate

the CNL side of ETI, and fls2 was assayed to investigate possible involvement of PRRs

in the PTI branch of immune function. pixl-1, rps4-2 and rps6-3 all displayed an

increased phototropic phenotype relative to wild-type suggesting roles as negative

regulators of phototropism (Figure 2.5). It is interesting to note that of all the lines

tested only the TNL mutants had altered phototropic phenotypes, while the CNL mutant

rps2-201 did not.

32

Figure 2.5 Mutants lacking PIXL, RPS6 or RPS4 exhibit increased phototropic

curvature.

Phototropic curvatures of Col, phot1-5, pixl-1, rps6-3, rps4-2, eds1-2, rps2-201 and fls2 seedlings

exposed to 4 h of unidirectional BL (0.1 mmol m−2 s−1). Columns represent mean response of

approximately 300 seedlings from at least three replicate experiments. Error bars represent standard

error. ANOVA revealed a significant effect of genotype on curvature (p ≤ 0.001). Significant differences

compared to Col were determined using Holm-Bonferroni post-hoc analysis (p, ** ≤ 0.01, *** ≤ 0.001).

33

No detectible difference in chloroplast movements in select group of defense

mutants

Chloroplasts have been implicated in a variety of physiological responses from

HR and ROS accumulation, cell death, SA biosynthesis and production of other defense

related compounds (Genoud et al., 2002; Dong and Chen, 2013; Zbierzak et al., 2013;

Serrano et al., 2016; Wang et al., 2016). For these reasons we wanted to test defense

mutants for alterations in chloroplast movements, which are phototropin mediated

responses. Under low light conditions, chloroplasts will accumulate upon the top and

bottom of palisade cells in order to maximize photocapture (Trojan and Gabrys, 1996;

Kong and Wada, 2014). However, under high light conditions chloroplasts will stack

underneath one another in order to prevent excess excitation energy and damage to

photosynthetic machinery (Park et al., 1996; Cazzaniga et al., 2013). As the

predominant low light photoreceptor, phot1 is primarily responsible for the accumulation

response (Luesse et al., 2010) as can be seen in the phot1-5 mutant which has a

diminished accumulation response but a typical avoidance response (Figure 2.6).

Under highlight, however, phot2 is the main photoreceptor (Jarillo et al., 2001; Kagawa

et al., 2001) as can been seen in the phot2-1 mutant which shows normal accumulation,

but chloroplasts fail to stack and display an avoidance phenotype under high light

(Figure 2.6). pixl-1, rps6-3, rps4-2 and rps2-201 were all tested due to their involvement

in several points of ETI signaling with none of these mutants showing any difference

from wild-type under low or high light conditions (Figure 2.6).

34

Figure 2.6 No change in chloroplast movement was observed in mutants lacking

PIXL, RPS6, EDS1-2 or RPS2

Leaf discs from 3-4 week old plants were suspended in 1/2x MS in a 96-well plate. The plate was

wrapped in foil for 3 hours to return chloroplasts to dark-state. Absorbance at 660nm and 800nm was

measured in the dark to establish a baseline (black line). The plate was exposed to 1 μmol·m-2·sec-1 (light

blue line) and 75 μmol·m-2·sec-1 (dark blue line) blue-light. Absorbance was measured at 10 minute

intervals. Change in ∆%Transmittance was calculated by conversion of absorbance to transmittance by

referencing against the final dark-state measurement (t = 0). Three-way repeated measures ANOVA

revealed a significant effect of genotype (p ≤ 0.001), time (p ≤ 0.001) and light treatment (p ≤ 0.001) on

transmittance. Significant differences compared to Col were determined using Holm-Bonferroni post-hoc

analysis (p, * ≤ 0.05).

35

Figure 2.7 Model summarizing the association between phot1 and defense

proteins

The schematic depicts the associations between phot1, phototropism, PIXL, resistance proteins, and PR2

accumulation. Arrows represent stimulation. Perpendicular lines represent inhibition. Circles represent

interaction. Broken lines represent hypothesized activity.

36

2.5 Discussion

While it can certainly be concluded that phototropins are not essential for

resistance against Pst, involvement can still not be ruled out. Specific environmental

conditions can have profound effects on resistance phenotypes, as well as

morphological phenotypes. For example, fluctuations in temperature disrupt R-protein

mediated resistance with ranges of change as low as 6ºC (Kim et al., 2010).

Additionally, phototropin involvement in defense may be more pronounced when

challenged with other pathogens such as the necrotrophic pathogen Botrytis cinerea.

Investigating this association under various lighting conditions is worth considering as

well, as populations of phototropin interaction partners change under quality and

quantity of incoming light (Goyal et al., 2013). A thorough investigation under multiple,

precisely controlled growing conditions and challenged with a broad array of pathogens

must be completed before any concrete conclusions regarding phototropin involvement

in pathogen defense can be made. Lastly, an effect of phototropin mutations on PR2

accumulation was observed (Figure 2.6). PR2 accumulation following R protein

activation has been demonstrated (Figure 2.3), but implicating photoreceptors is a novel

finding (Figure 2.7). This result is reminiscent of the phot2 requirement for HRT stability

(Jeong et al., 2010), although regulation of PR protein stability has not previously been

shown. Further analysis at earlier and more frequent time points will help decipher if the

phototropins have a more pronounced function in establishment or stability of PR2.

In potato, NRL1 (NPH3/RPT2-Like1) was identified as a susceptibility factor

targeted by a Phytophthora infestans effector. In Arabidopsis, NPH3 is a critical phot1

37

interactor and acts as a substrate adaptor in a CULLIN3-based E3 ubiquitin ligase

system, which ubiquitinates phot1 differentially under various lighting conditions,

targeting it for degradation or altering localization patterns. Incorporating this finding

with the role phot2, and to a lesser extent phot1, play in stabilizing levels of R-protein

HRT through inhibition of COP1 activity, generates the notion of phototropin

involvement in defense being mediated through NPH3 and its homologs (Jeong et al.,

2010). The hypothesis that phototropins are involved in stabilization of defense related

proteins could explain the decrease of PR2 accumulation seen in phot1-5/2-1.

In the ß-estradiol inducible PIXL-HA lines, seedling death due to prolonged

exposure to ß-estradiol is consistent with PIXL being able to initiate defense responses

(Figure 2.4). Induction over shorter periods of time and analysis of defense markers will

provide a better understandings of the actions that lead to this response and a

mechanistic look at PIXL as a R-protein. For example, it is still not known if stimulation

of PIXL can induce PR2 accumulation (Figure 2.7). Additionally, testing if these

responses remain the same while in the eds1-2 mutant background will allow for

designation of PIXL as a canonical TNL R protein if the death phenotype observed is

abolished in the absence of EDS1. Conversely, if the PIXL induced death phenotypes

observed remain the same in the eds1-2 mutant background it would suggest PIXL is a

novel member of the TNL sub-family in terms of defense initiation.

Removal of certain TNL proteins resulted in increased phototropic output (Figure

2.5) while no changes in behavior were seen in chloroplast movements (Figure 2.6).

Further analysis is necessary to determine the exact mechanism by which phototropic

38

inhibition occurs. Inhibition by PIXL could be mediated through the interaction with

phot1, however RPS2 is also a phot1 interactor and rps2-201 mutants show no change

in phototropic curvature (Figure 2.7). Additionally, rps6-3 and rps4-2 mutants also

display increased phototropic curvature, but there is no evidence for interaction with

phot1 (Figure 2.7). One possible explanation for differences in phenotypes seen

between phototropic curvature and chloroplast movements could be due to the function

of TNL R proteins in the tissues tested as mutants showed significant changes in

hypocotyls but not leaves (Figure 2.6). An alternative hypothesis is that modulation of

phototropin-mediated responses by TNL R proteins are dependent on specific signaling

partners as the mechanisms behind chloroplast movements and phototropism are very

different. It is interesting to again address the possibility of phototropins and defense

machinery being linked through NPH3. This hypothesis is supported by the fact nph3

mutants are defective in phototropism while chloroplast movements show no detectable

difference (Inada et al., 2004), and that TNL mutants have increased phototropic

curvature but show no changes in chloroplast movement. In depth analysis of

resistance responses with more attention drawn to NPH3 could reveal a better

understanding of how plants govern crosstalk between resistance responses and other

cellular processes.

39

CHAPTER 3

WHEN IN ROME: CROSS KINGDOM SUBSTITUTION OF A KEY IMMUNE

REGULATOR IN PLANTS

Authors: Daniel L. Leuchtman, Anthony Shumate, Stephen Shannon, Saikat

Bhattacharjee, Michael L. Garcia, Walter Gassmann, Emmanuel Liscum

3.1 Abstract

In order to prevent unnecessary energetic costs and detrimental side effects, the

plant immune system must be kept tightly in check to ensure utilization only when a

threat is present. The mechanisms behind immune system regulation are still an active

area of research. In Arabidopsis, SRFR1 has been identified as a negative regulator of

immune responses. Sequence analysis reveals several key features that suggest

involvement in transcriptional regulation, protein-protein interaction and complex

formation. Cross kingdom homology is seen across the length of the protein with the

most notable region being an amino-terminal tetratricopeptide repeat (TPR) domain.

Some TPR domain containing proteins in animal systems have also been implicated in

immune system regulation, although they are involved in many other cellular functions

as well. In certain genetic backgrounds, plants lacking SRFR1 have markedly different

morphology, reduced viability, appear stunted and constitutively express several

defense related genes. When a srfr1 mutant plant is transformed with orthologous

MmSRFR1 from Mus musculus, the drastic srfr1 phenotype is reduced. This

40

observation suggests a level of conserved function for MmSRFR1 in Arabidopsis.

Further investigation will enable a deeper understanding of immune system regulation

and TPR protein function across kingdoms.

3.2 Introduction

Initiation and maintenance of pathogen defense is an energetically costly process

that must be kept under tight regulation in order to prevent unnecessary expenditures at

the expense of growth and development (Huot et al., 2014). Stimulation of the immune

system over prolonged periods of time can result in detrimental morphological

phenotypes or even tissue collapse (Howles et al., 2005; Kim et al., 2010). For

example, constitutive activation of R genes can result in elevated defense responses

and cell death (Howles et al., 2005; Tameling et al., 2006). As ETI is typically

associated with HR and an oxidative burst, it is especially important to keep it under

strict control as to not unnecessarily destroy otherwise healthy tissues.

SRFR1 is a protein originally discovered in a genetic screen for mutations that

would re-establish resistance against DC3000 avrRp4 in the normally susceptible RLD

background (Kwon et al., 2004). Sequence analysis of SRFR1 reveals conserved TPR

domains, typically involved in protein-protein interaction, as well as a domain of

unknown function at the C-terminus of the protein that is also conserved among

SRFR1-like TPR proteins in other eukaryotes. TPRs are found across all kingdoms and

have been implicated in various cellular processes. Most notably, Ssn6 of

Saccharomyces cerevisiae and OGT1 of Caenorhabditis elegans are part of

41

transcriptional repressor complexes (Lubas and Hanover, 2000; Smith and Johnson,

2000; Yang et al., 2002).

In Arabidopsis, srfr1 mutants constitutively exhibit several markers for defense

and it is believed that mitigation of SRFR1 dependent defense repression leads to

increased resistance to DC3000 avrRps4 in a susceptible background. Additionally,

mutant srfr1 elevated resistance is dependent on EDS1, a central regulator of TNL R

protein mediated defense activation (Kwon et al., 2009). SRFR1 and EDS1 form a

protein complex in addition with at least two other R proteins, and this complex is

targeted by AvrRps4 and HopA1 (Bhattacharjee et al., 2011). In the Col and RLD

backgrounds, srfr mutants have increased defense gene expression including RPS4,

PR1 and PR2 (Kim et al., 2009; Kwon et al., 2009; Kim et al., 2010). At temperatures

around 22ºC, srfr1-4 null mutants have a severely stunted phenotype that is abated at

higher temperatures around 28ºC. This morphological phenotype is due to the TNL R

gene SNC1.

Orthologous SRFR1 can not only be found in the plant kingdom but outside as

well, including mammalian MmSRFR1 (TTC13). Although MmSRFR1 is much smaller,

approximately half the amino acid length, homology with SRFR1 can be seen across

the length of the protein. Homologs of MmSRFR1 in mammals, known as TTCs

(Tetratricopeptide repeat domain) are involved in processes ranging from cell cycle

control, cancer metastasis and immune regulation (Helms et al., 2005; Cao et al., 2008;

Dmitriev et al., 2009; Suizu et al., 2009). In mice, MmSRFR1 expression is pervasive

with transcript particularly high in the thymus, liver, testis and kidney and protein

42

detected in kidney, liver, testis, lung, heart and B-cells, consistent with the hypothesis of

MmSRFR1 being involved in immune regulation (Shannon, 2012). Flow cytometry

experiments suggest a role in cell cycle regulation as overexpression of pFLAG-

MmSRFR1 resulted in increased DNA synthesis (Shannon, 2012). Cell proliferation

assays show that overexpression of pFLAG-MmSRFR1 results in increased cell

proliferation and supports the hypothesis of MmSRFR1 functioning in cell cycle

regulation (Shannon, 2012). Further studies are necessary in order to elucidate precise

mechanistic roles of MmSRFR1 in mammals.

To gain a better understanding of SRFR1 function, we transformed mutant srfr1-

4 plants with GFP tagged MmSRFR1. These constructs would allow us to test for

conserved functions of SRFR1 across kingdoms and help reveal any additional roles

the protein might play. Tagging the protein with GFP would also allow us to examine

localization and interacting partners in addition to any physiological effects the

transgene may have. Through these studies we can gain a better understanding of

TPR protein activity across evolutionary time and further characterize both SRFR1 and

MmSRFR1.

3.3 Experimental Procedures

Root growth assay: Seeds were sown on vertical petri plates containing 1/2x MS, 2%

sucrose, 1x Gamborg Vitamins, and 90µg/mL Timentin for normal growth experiments.

Additions were added where indicated: Hygromycin B at 15 µg/mL (1x HYG), 30 µg/mL

(2x HYG) or 7.5 µg/mL (1/2x HYG), glufosinate ammonium at 45µg/mL (1x BAR),

43

Paraquat at 0.1µM or 1µM (PAR). Plates were then placed in a growth chamber under

16 h light/ 8 h dark cycle at an intensity of 50 to 70 µmol photons m-2 s-1 at 22ºC and

allowed to germinate and grow for four to five days. Seedlings were then selected

based on similar size and transplanted six per plate onto a fresh plate, identical to the

original in composition. The bottom of each root was then marked and images were

taken each subsequent day for two weeks. Length post-transplant was measured as

the distance between the bottom of the root and the initial marking created on the day

seedlings were transplanted using FIJI software.

Shoot growth assay: Seeds were sown on horizontal petri plates containing 1/2x MS,

2% sucrose, 1x Gamborg Vitamins, and 90µg/mL Timentin. Where indicated, 15 µg/mL

Hygromycin B was added. Plates were then placed on a light rack with constant light at

room temperature and allowed to germinate and grow for 10 days. Seedlings were then

selected based on similar size and transplanted onto soil with different genotypes

randomized within the flat to avoid positional effects. Images were taken on the

transplantation day and every seven days for the next three weeks. Shoot area was

measured using FIJI software and the SIOX segmentation plugin. Shoot weight was

measured using an analytical scale.

44

3.4 Results

srfr1-4 root growth is severely stunted in the presence of hygromcin, and this

phenotype is diminished in mutants transformed with MmSRFR1

In the presence of hygromycin, srfr1-4 transformed with empty binary vector

containing the hygromycin resistance gene, HPT (Hygromycin Phospho Transferase)

(srfrEV-1) displays a stunted root phenotype (Figure 3.1). This phenotype was first

noted during transgenic screens of MmSRFR1 transformed plants. We then wished to

quantify this response in the same conditions as those used for the transgenic screen.

Plants instead transformed with MmSRFR1 relieve this phenotype (Figure 3.1). Two

inferences can be made. First, the normally reduced root growth phenotype seen in

srfr1-4 plants is severely exaggerated in the presence of hygomycin. Hygromycin

toxicity is due to inhibition of protein synthesis in prokaryotic ribosomes and targets

mitochondria and chloroplasts in plants (Borovinskaya et al., 2008; Oung et al., 2015).

This finding suggests a novel role of SRFR1 in chloroplast/mitochondria activity and/or

management of oxidative stress, as increasing ROS accumulation was seen as an

influential part of hygromycin toxicity in plants (Oung et al., 2015). Second, this finding

suggests that MmSRFR1 can function in Arabidopsis in such a way as to relieve the

hygromycin-induced srfr1-4 stunted root phenotype.

45

Figure 3.1 In the presence of hygromycin, resistant srfr1-4 shows a stunted root

growth phenotype and MmSRFR1 transgenic lines show increased growth.

Root length was measured on plants grown in normal media (A) and those grown in the presence of

hygromycin (B). Seeds were sown on vertical plates and allowed to grow for 4 days. On day 0 seedlings

were then selected based on similar size and transplanted onto a fresh plate. Root length post-transplant

was measured each day thereafter and the mean of approximately 20 seedlings was calculated. Error

bars represent standard deviation. Two-way repeated measures ANOVA revealed a significant effect of

genotype (p ≤ 0.001), and day (p ≤ 0.001) on root length. Significant differences compared to srfr1-4 (A)

or srfrEV-1 (B) were determined using Holm-Bonferroni post-hoc analysis (p, * ≤ 0.05, ** ≤ 0.01, *** ≤

0.001). B) Significant differences between srfrEV-1 and each transgenic line tested were observed on

each day of the experiment.

46

Select MmSRFR1 transgenic lines also exhibit an increase in shoot growth

compared to srfr1-4 srfrEV-1 control

MmSRFR1 transgenic lines were also subjected to a shoot growth assay in order

to test if attenuation of the srfr1-4 rosette stunting phenotype could also be seen.

Relative to srfrEV-1, certain MmSRFR1 transgenic lines showed increased shoot size

while others exhibited a decrease in size (Figure 3.2). Transgenic seedlings at

generation T3 were first selected on hygromycin plates before being transplanted onto

soil for shoot growth analysis. Silencing of the transgenes would increase hygromycin

sensitivity and could explain the reduction in growth seen in N-1, N-9 and N-13. It is

worth noting that these transgenic lines did show increased root growth in the T2

generation (Figure 3.1). Increased rosette area was seen in lines N-4 and N-8, with N-8

falling out of statistical significance on the last week of the experiment (Figure 3.2).

These two lines also showed increased shoot weight (Figure 3.2). This result presented

another instance where presence of MmSRFR1 relieved the reduction in growth seen in

srfr1-4 plants. Further analysis of SRFR1 and MmSRFR1 activity in the root and shoot

is necessary to explore the extent that shoot size is influenced by root size and the role

SRFR1 plays in this relationship.

47

Figure 3.2. Shoot growth of MmSRFR1 transgenic lines.

Seeds were sown on horizontal plates containing normal media (Col and srfr1-4) or media containing

hygromycin (transgenic lines) and allowed to grow for 10 days. On day 0 seedlings were then selected

based on similar size and transplanted into soil in a randomized pattern. Shoot area (A) was measured

every week starting on day 0 and the mean of approximately 20 seedlings was calculated for each time

point. Average shoot weight (B) was calculated from the same seedlings at the end of the experiment.

Error bars represent standard deviation. Two-way repeated measures ANOVA revealed a significant

effect of genotype (p ≤ 0.001), and day (p ≤ 0.001) on rosette area (A). One-way between groups

ANOVA revealed a significant effect of genotype (p ≤ 0.001) on shoot weight (B). Significant differences

between srfr1-4 and Col, or srfrEV-1 and transgenic lines were determined using Holm-Bonferroni post-

hoc analysis (larger than control p, * ≤ 0.05 ** ≤ 0.01, *** ≤ 0.001; smaller than control p, † ≤ 0.05 †† ≤

0.01, ††† ≤ 0.001).

48

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

To test if the MmSRFR1 transgene increased root growth or relieved the srfr1-4

dependent hygromycin phenotype, the same transgenic lines in the T3 generation were

subjected to a root growth assay without hygromycin in the media. MmSRFR1

transgenic lines, srfrEV-1 and srfr1-4 were all indistinguishable from one another

(Figure 3.3). We conclude that MmSRFR1 does not cause increased root growth, but

instead relieves growth inhibition caused by presence of hygromycin and lack of

SRFR1.

To confirm hygromycin dependency, srfrEV-1 and Col lines were subjected to a

root growth assay on plates with either no hygromycin or various concentrations. As

hygromycin concentration increases, the stunting of the roots and decrease in growth

becomes more severe (Figure 3.4). Furthermore, Col and srfrEV-1 root growth was

analyzed in the presence of glufosinate (BAR). Glufosinate is a broad-spectrum

herbicide that is commonly used in transgenic screens with the combined use of the

bialaphos resistance gene. The srfr1-4 mutant is a member of the Syngenta

Arabidopsis Insertion Library (Sessions et al., 2002). This library was constructed to

generate mutant populations and used glufosinate resistance as part of the screen.

Thus, srfrEV-1 is resistant to glufosinate in addition to hygromycin. As expected, Col

growth was severely inhibited on glufosinate media and were eventually killed (Figure

3.4). It is interesting to note how, despite susceptibility, Col roots are much larger on

49

glufosinate media compared to those grown in the presence of hygromycin (Figure 3.4).

Conversely, srfrEV-1 plants showed no difference in root growth between the

glufosinate and control treatments (Figure 3.4).

Glufosinate induces toxicity by inhibition of glutamine synthesis, causing

accumulation of ammonia and eventual inhibition of photosystem I and photosystem II

reactions (Lea et al., 1984; Sauer et al., 1987). ROS accumulation is a consequence of

both hygromycin and glufosinate sensitivity, but is highlighted as being a key part of

hygromycin toxicity (Sauer et al., 1987; Oung et al., 2015). Differences in srfrEV-1 root

length seen between control, hygromycin or glufosinate treatments reveals a

hygromycin specific effect on root growth. We hypothesize that the precise

mechanism behind srfrEV-1 hygromycin sensitivity is due to novel SRFR1 activity in the

mitochondria, chloroplast, and/or involvement in managing ROS homeostasis.

50

Figure 3.3. In the absence of hygromycin, MmSRFR1 transgenic root growth is

indistinguishable from srfr1-4.

Root length was measured on plants grown in normal media. Seeds were sown on vertical plates and

allowed to grow for 4 days. On day 0 seedlings were then selected based on similar size and

transplanted onto a fresh plate. Root length post-transplant was measured each day thereafter and the

mean of approximately 20 seedlings was calculated. Error bars represent standard deviation. Two-way

repeated measures ANOVA revealed a significant effect of genotype (p ≤ 0.001), and day (p ≤ 0.001) on

root length. Significant differences compared to srfr1-4 were determined using Holm-Bonferroni post-hoc

analysis (p, * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001).

51

Figure 3.4. srfrEV-1 stunted root phenotype is hygromycin dependent

Root length was assessed on plants grown in normal media containing various concentrations of

hygromycin (A) or glufosinate (B). Seeds were sown on vertical plates with their respective treatment and

allowed to grow for 4 days. Seedlings were then selected based on similar size and transplanted onto a

fresh plate with the same composition as the original. Images are of plants 12 days after transplantation.

52

Non-lethal doses of paraquat induce root stunting

To further examine root growth in the presence of oxidative stress, we grew Col,

srfr1-4 and srfrEV-1 in the presence of paraquat. Paraquat is a common herbicide

known to cause oxidative stress through inhibition of electron transport during

photosynthesis (Moreland, 1982). All three lines were also grown on control plates with

no herbicide as well as those with hygromycin for comparison. Paraquat was lethal to

all lines tested at a concentration of 1µM and caused a large reduction in root growth at

a concentration of 0.1µM (Figure 3.5). As seen before (Figure 3.1, Figure 3.3), srfr1-4

and srfrEV-1 roots were slightly shorter when compared to Col on control plates (Figure

3.5). There was no noticeable difference between Col, srfr1-4 and srfrEV-1 in the

0.1µM paraquat treatment plates. This result suggests that the increased sensitivity to

hygromycin seen in srfrEV-1 is due to a mechanism independent of those that cause

paraquat sensitivity.

53

Figure 3.5 Root growth of Col, srfr1-4 and srfrEV-1 in the presence of paraquat.

Root length was assessed on plants grown in media with control, hygromycin or paraquat treatments.

Seeds were sown on vertical plates with their respective treatment and allowed to grow for 4 days.

Seedlings were then selected based on similar size and transplanted onto a fresh plate with the same

composition as the original. Images are of plants 12 days after transplantation.

54

Hygromycin resistant Col plants show increased root growth compared to

hygromycin resistant srfr1-4 plants.

To confirm the increased sensitivity of srfr1-4 plants to hygromycin, Col plants

were transformed and srfr1-4 plants were re-transformed with the same empty binary

vector used previously. This procedure yielded six independent insertion sites in Col

(ColEV) and one additional insertion site in srfr1-4 (srfrEV-2). The same srfrEV-1 line

used from previous experiments was included for comparison. ColEV lines showed no

apparent reduction in root growth (Figure 3.6), although no concrete conclusions can be

made until the same lines are grown alongside plates without hygromycin. srfrEV-2

displayed a great reduction in root growth when compared to the ColEV lines, but

slightly increased root growth when compared to srfrEV-1 (Figure 3.6). This disparity

could be due to positional effects from their respective insertional events or multiple

insertion events in the srfrEV-2 line. Multiple insertion events could increase levels of

the HPT protein, thus increasing tolerance. It can be concluded, however, that mutants

lacking SRFR1 have increased sensitivity to hygromycin as can be seen by a reduction

in root growth.

55

Figure 3.6 Root growth analysis of hygromycin resistant Col and srfr1-4

Root length was assessed on plants grown in media containing hygromycin. Seeds were sown on

vertical plates with hygromycin and allowed to grow for 4 days. Seedlings were then selected based on

similar size and transplanted onto a fresh plate with the same composition as the original. Images are of

plants 12 days after transplantation.

56

Figure 3.7 Model summarizing the association between hygromycin, SRFR1 and

the HPT hygromycin resistance protein.

The schematic depicts the associations between hygromycin induced toxicity, the HPT hygromycin

resistance protein and SRFR1 in A) susceptible wildtype Col B) resistant wildtype Col and C) resistant

srfr1-4 (srfrEV-1 and srfrEV-2). Arrows represent stimulation. Perpendicular lines represent inhibition.

Broken lines represent hypothesized activity.

57

3.5 Discussion

Additional roles for SRFR1, outside of immune regulation, have not been

thoroughly investigated. There are enticing pieces of information that suggest

involvement in other areas of physiology. For example, SRFR1 has been shown to

interact with several TCP transcription factors and can form protein complexes in both

the nucleus and the cytoplasm (Kim et al., 2014). While potential roles for at least a

subset of TCPs in immune function is still an active area of research, many have

already been implicated in growth and development (Lopez et al., 2015). Thus, SRFR1

may represent a potential node in molecular cross-talk between signals promoting

normal developmental progression and those involved in responding to potential

threats.

There is a clear hygromycin dependent root stunting phenotype in srfrEV-1 and

srfrEV-2 plants, which again is simply srfr1-4 with hygromycin resistance HPT and GFP

genes (Figure 3.1, Figure 3.3, Figure 3.4, Figure 3.6). Sensitivity to hygromycin is due

not only to inhibition of protein synthesis, but to ROS accumulation as well. In studies

conducted by Oung and colleagues (2015), it was discovered that introducing the

antioxidant ascorbate into hydroponics systems gave an increase in hygromycin

tolerance to otherwise susceptible rice plants. Additionally, overexpression of genes

involved in ROS scavenging reduced sensitivity while overexpression of ROS inducing

genes increased sensitivity (Oung et al., 2015). Because a burst of ROS is a canonical

part of several ETI responses, it is feasible that a negative regulator of ETI could be

58

involved in maintaining ROS homeostasis. This would represent a novel function of

SRFR1.

Further analysis is necessary, however, as disruption of protein synthesis in the

chloroplast and mitochondria is also believed to be a key part of hygromycin toxicity. A

more prevalent role for SRFR1 in chloroplast and mitochondria regulation is supported

by the lack of difference seen between Col, srfr1-4 and srfrEV-1 plants in the presence

of paraquat (Figure 3.5). If SRFR1 played a more prevalent role in regulating redox

homeostasis, one would expect to see shortened roots in srfr1-4 and srfrEV-1 lines

compared to Col in the presence of paraquat. However, implications of ROS on srfrEV-

1 and srfrEV-2 hygromycin sensitivity cannot be ruled out as mechanisms by which

paraquat induces oxidative stress may be independent of SRFR1 influence. In addition

to ROS accumulation and inhibition of prokaryotic protein synthesis, there could also be

other toxic influences of hygromycin that SRFR1 is also associated with (Figure 3.7).

Chloroplasts have also been implicated in immune function by processes such

as biosynthesis of several defense related compounds and SA, and serving as an

additional source of ROS during pathogen defense (Serrano et al., 2016). More

experiments are needed to test if ROS accumulation is 1) part of the srfrEV-1 and

srfrEV-2-hygromycin root phenotype 2) due to absence of SRFR1 involvement

suppressing oxidative bursts typically associated with HR and 3) due to absence of

SRFR1 association with chloroplasts and mitochondria, making ROS accumulation an

indirect effect of chloroplast or mitochondrial perturbation.

59

Kanamycin also works through disruption of protein synthesis, but has not been

implicated in increasing oxidative stress like hygromycin (Oung et al., 2015). By using a

similar system as above and transforming kanamycin resistance with and without

MmSFR1 into srfr1-4 plants we can test for similar results, which would suggest

disruption of normal chloroplast and/or mitochondrial function is a key part of the

mechanism giving rise to our original result. Or, root growth in this system could be

more similar to results found on media with no selection, which would suggest that ROS

accumulation is a causal agent of the srfrEV-1 shortened root phenotype.

Mitigation of the srfrEV-1-hygromcin phenotype by MmSRFR1 transgenic lines

was a striking result (Figure 3.1). Study of MmSRFR1 in the Col background, with native

SRFR1 present as well, would allow us to decipher if MmSRFR1 was originally rescuing

normal SRFR1 function. The alternative hypothesis is that MmSRFR1 was stimulating

mechanisms not associated with SRFR1. Determining the interacting partners of

MmSRFR1, as well as cross-testing with SRFR1, is an obvious next step. Investigating

interaction partners in planta would allow us to advance our understanding of SRFR1

activity in immune regulation and possibly other areas as well. The question then

becomes, are these interactions conserved throughout evolutionary time? Are we

studying ancient associations that have been present since at least the divergence of

plants and animals? Further investigation with SRFR1 orthologues from a wide array of

species and movement into other model systems will help unravel these questions.

60

CHAPTER 4

CONCLUSIONS

Immune regulation is of critical importance to plant health. If underutilized, plants

risk becoming consumed or exploited by the vast microbial community in which they

exist. Overutilization creates an energetic deficit and results in costs of growth and

development (Huot et al., 2014). It should be no surprise that plants have evolved strict

control of immune regulation, allowing for fine adjustment of defense responses under

an immense array of environmental stressors. Immune regulation thus becomes an

excellent subject in the quest for a better understanding of crosstalk between molecular

signaling networks. Light, as a source of energy and key spatial-temporal information, is

also a critical system for enabling plants to adapt to an ever-changing environment. As

plants are constantly surveying and responding to their microbial environment, they are

also doing so with their light environment. Investigating these two systems concurrently

creates an excellent model for studying signaling crosstalk and signaling integration

between biotic and abiotic stress.

While other photoreceptors have already been implicated in defense signaling, a

definitive role for phototropins has not yet been found (Ballare, 2014). On the pathogen

side, it has been demonstrated that light quality can determine pathogen behavior, such

as attachment to leaf surfaces or motility, which is crucial for entry into the leaf and

onset of pathogenesis. Under blue and white light, Pst DC3000 will attach to leaf

surfaces and reduce motility, whereas in red light or dark conditions will instead become

61

more mobile. Numerous Pseudomonads possess putative LOV domain containing

proteins and in Pst DC3000 mutation in the lone gene significantly reduced leaf entry

and proliferation (Río‐Álvarez et al., 2014). The causal agent of citrus canker disease,

Xanthomonas citri, also has a LOV domain containing protein that drastically changes

symptom development when mutagenized (Kraiselburd et al., 2012). Precise roles for

bacterial LOV proteins are still under investigation, but the fact that both plant and

pathogen are closely monitoring blue light conditions highlights a key point in

pathogenesis and suggests that exciting developments are still to come.

The phot1-PIXL interaction is an excellent opportunity for mechanistic exploration

of the interactions between photobiology and pathogen defense. To date, RPS2 has

been the only clear member of the immune signaling network found to interact with

phot1 (Qi and Katagiri, 2009; Jeong et al., 2010). In my dissertation, I have provided an

initial characterization of PIXL and influences on phototropin dependent physiology. In

addition to results from already published literature, I have also investigated phototropin

involvement in defense signaling. Lastly, while investigating the interaction between

light and immune signaling I have also furthered examination of immune regulation with

regard to normal growth and development during the investigation of MmSRFR1 in the

morphologically distinct srfr1-4 mutant background. These studies have provided novel

insights to the question of how molecular stress signals overlap and coalesce within the

same organism.

62

4.1 Resistance to Pst DC3000 hopA1 does not show a strong phototropin or PIXL

dependency

In an effort to further characterize phototropin/PIXL involvement in ETI, we chose

to challenge Arabidopsis mutants with Pst hopA1 due to sequence similarity between

PIXL and RPS6. Phototropin double and single mutants did not show a difference in

bacterial proliferation when compared to the gl control (Figure 2.1). Additionally, to test

if pathogenesis is differentially affected by light quality, like Pst DC3000, we challenged

phototropin mutants again with Pst hopA1, but under blue and red monochromatic light.

Similar to experiments done under normal lighting conditions, bacterial proliferation was

not altered under these lighting conditions, suggesting that the phototropins are not

essential for HopA1 mediated initiation of ETI (Figure 2.1, Figure 2.2).

4.2 Phototropins influence PR2 accumulation in response to Pst DC3000 hopA1

and avrRpt2

We also examined the phot1-5/2-1 double mutants for the presence of defense

associated protein PR2. To our surprise, we discovered that the phot1-5/2-1 double

mutant showed a marked reduction in PR2 accumulation (Figure 2.3). This result

suggests that any fine influence phototropins may have in defense signaling could be

involved in regulating stability of certain defense related proteins, in agreement with

results found by Jeong and colleagues (2010). Additionally, there was an increase in

PR2 accumulation in the gl1 mutant (Figure 2.3). Because GL1 has been implicated in

establishment of SAR (Xia et al., 2010), our results suggest that phototropin influence in

63

defense may be more pronounced when taken out of the gl1 background. These

results highlight the need to reassess previous results investigating phototropin

involvement in pathogen defense. To date, no study has tested the double mutant

specifically, which is essential as the phototropins act redundantly especially under

moderate lighting intensities, in the GL1 background to avoid any confounding

influences from absence of GL1. These studies must first be done in order for any

definitive conclusions to be made.

4.3 Extended induction of PIXL-HA results in seedling death

To test if PIXL could induce any defense responses, we transgenically introduced

PIXL-HA into the pixl-1 mutant background driven by a ß-Estradiol inducible promoter.

Within one week after plating, transgenic PIXL-HA plants did not survive on plates

containing ß-Estradiol while those on mock plates remained alive (Figure 2.4).

Overexpression of R proteins can result in stunting and damaging side effects, thus the

seedling death phenotypes we observed suggest PIXL involvement in defense

elicitation (Zhang et al., 2003; Yi and Richards, 2007; Kim et al., 2010; Kim et al., 2012).

Further analysis of defense markers such as PR2 would confirm this hypothesis. This

finding also adds noteworthiness to the phot1 interaction with a R protein which would

signify a closer association with immunity than if PIXL were, for example, a helper NB-

LRR.

64

4.4 Certain defense mutants exhibit increased phototropic curvature, but do not

show changes in chloroplast accumulation or avoidance

To assess the reverse hypothesis, that defense regulatory components influence

phototropin mediated physiological changes, we subjected several defense mutants to

phototropism and chloroplast movement assays. Despite the notable importance of

chloroplasts in pathogen defense, we observed no noticeable changes in chloroplast

avoidance or accumulation (Figure 2.6) (Serrano et al., 2016). We did, however,

observe an increase in phototropic curvature in select TNL mutants, but not in the eds1-

2 mutant (Figure 2.5). Several possible explanations could help clarify these results

such as the presence of an additional signaling hub common to TNLs and associated

with phot1 but independent of EDS1. An attractive candidate is NPH3, a substrate

adaptor in the CULLIN3-based E3 ubiquitin ligase complex essential for phototropism

(Liscum and Briggs, 1996; Roberts et al., 2011). This hypothesis is especially attractive

because in addition to blue light photoreceptors influencing defense through modulation

of an E3 ubiquitin ligase in Arabidopsis, COP1, a NPH3/RPT2-like protein was identified

as a susceptibility factor and target of a Phytophthora infestans effector in potato (Jeong

et al., 2010; Yang et al., 2016). Perhaps the phot1 association with PIXL and defense

machinery is through association with NPH3.

65

4.5 Hygromycin resistant srfr1-4 exhibits a hygromycin dependent shortened root

phenotype

While screening MmSRFR1 transgenic lines, we observed a severe stunting of

root growth in the srfrEV-1 control on hygromycin plates, but to a much lesser extent on

normal plates (Figure 3.1, Figure 3.3). To further assess hygromycin dependency, we

tested srfrEV-1 plants using a concentration gradient and saw a negative relationship

between root/shoot size and hygromycin presence, but did not see a noticeable root

growth phenotype with glufosinate treatment (Figure 3.4). This result confirms

hygromycin dependent sensitivity of srfrEV-1. The induction of ROS caused by

hygromycin combined with the pivotal role ROS plays in defense responses argues for

a potential role of SRFR1 in mediating ROS homeostasis (Pastor et al., 2013; Oung et

al., 2015). However, since hygromycin also has a toxic effect on plastids and

mitochondria, the alternative hypothesis of SRFR1 involvement in maintenance and

regulation of these organelles cannot be ruled out based on our findings (Borovinskaya

et al., 2008). The latter hypothesis becomes more attractive due to the fact that no

reduction in root growth was observed during glufosinate treatment (Figure 3.4), and no

noticeable difference was observed between Col, srfr1-4 and srfrEV-1 when challenged

with non-lethal doses of paraquat (Figure 3.5).

66

4.6 Transgenic MmSRFR1 in the srfr1-4 background shows an increase in root

and shoot growth

MmSRFR1 transgenic lines in the srfr1-4 background were created to investigate

potential cross kingdom conservation. When compared to the srfrEV-1 control and

exposed to hygromycin, all MmSRFR1 lines tested displayed increased root growth and

two lines tested showed a statistically significant increase in shoot growth (Figure 3.1;

Figure 3.2). Taken together these results suggest a level of conserved function for

MmSRFR1. Since no differences were seen between srfr1-4 and MmSRFR1

transgenic lines in the absence of hygromycin, it appears that any conserved function

that exists in the processes we tested relates to relieving the increased sensitivity of

srfrEV-1 to hygromycin (Figure 3.3). More experiments are needed in order to find a

definitive role of SRFR1 in relation to hygromycin toxicity and how MmSRFR1 can act

as a substitute.

67

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84

VITA

I was born in St. Louis, Missouri on April 15, 1988 to father Richard R.

Leuchtman and mother Elizabeth L. Leuchtman. I have one sister with which I am very

close. Science was my best subject throughout my education. In high school I took two

science courses at a time instead of taking electives. I completed all but two science

classes offered by my school. My love of plant biology probably stems from spending

summers working in my grandparent’s garden, where I developed my green thumb. In

high school, I fell in love with genetics and wanted to work on the human genome

project. Upon arriving to the University of Missouri for a bachelor’s degree, I quickly

learned that I could combine my love of genetics and plant biology. I then developed an

interest in research and started working in the Galen lab as a greenhouse assistant.

Through this work I was introduced to Dr. Liscum and began exploring molecular

genetics. After I graduated with a Bachelor of Science in Biological Sciencs in 2010

took a summer off before enrolling in my PhD program at the University of Missouri. I

will earn my Doctor of Philosophy in Biological Sciences at the University of Missouri in

2016.