Investigating the role of the Mediator complex in plant

defence

Thorya A Fallath

Master of Biotechnology Bachelor of biology

A thesis submitted for the degree of Doctor of Philosophy at

The University of Queensland in 2016

School of Agriculture & Food Sciences

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Abstract The Mediator complex is an evolutionarily conserved protein complex involved in transcriptional

regulation. In plants, the Mediator complex plays an important role in regulating development as

well as the response to abiotic and biotic stress. In this thesis, I investigated three subunits of the

Arabidopsis thaliana Mediator complex MEDIATOR25, MEDIATOR18 and MEDIATOR20

(MED25, MED18 and MED20) in plant defence.

MED25 has been shown to interact with several transcription factors (TFs) to control jasmonate

(JA)-associated transcription. Here, I investigated these interactions further and characterized the

minimum protein domains of the TFs needed for interaction and identified new proteins that

interact with MED25. The results showed that the protein sequence corresponding to amino acids

138-218 of ETHYLENE RESPONSE FACTOR1 (ERF1) appears to be the minimum protein length

for interaction with MED25 and contains the conserved motif (CMIX). Although the protein motif

has been found in different proteins in A. thaliana that belong to a variety of families, only

members from the IXc AP2/ERF family were found to interact with the MED25 ACID domain.

Apart from MED25, I found that the MED18 and MED20 subunits interact within the Mediator

complex and infection of the corresponding mutants with Fusarium oxysporum revealed that they

display a high level of resistance. RNAseq analysis of F. oxysporum infected med18 and med20

roots showed that salicylic acid (SA)-associated PATHOGENESIS RELATED genes and (ROS)

reactive oxygen producing genes were up-regulated while some JA-regulated genes were repressed.

We also noticed induction and repression of genes involved in glucosinolate production. This may

suggest that ROS and antifungal metabolites are induced in med18 and med20 mutants and could

potentially contribute to the reduced colonization of the mutants by F. oxysporum. Overall, the

results reveal a new role for MED18 and MED20 in susceptibility to F. oxysporum, most likely via

their involvement in JA signalling whose up-regulation would compromise SA and ROS signalling

pathways that can confer resistance against this hemibiotrophic pathogen.

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Declaration by author

This thesis is composed of my original work, and contains no material previously published or

written by another person except where due reference has been made in the text. I have clearly

stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical

assistance, survey design, data analysis, significant technical procedures, professional editorial

advice, and any other original research work used or reported in my thesis. The content of my thesis

is the result of work I have carried out since the commencement of my research higher degree

candidature and does not include a substantial part of work that has been submitted to qualify for

the award of any other degree or diploma in any university or other tertiary institution. I have

clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and,

subject to the policy and procedures of The University of Queensland, the thesis be made available

for research and study in accordance with the Copyright Act 1968 unless a period of embargo has

been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright

holder(s) of that material. Where appropriate I have obtained copyright permission from the

copyright holder to reproduce material in this thesis.

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Publications during candidature

1- Journal papers Fallath TA, Kidd BN, Stiller J, Manners J, Kazan K and Schenk PM (2016). Mediator head

domain subunits, MEDIATOR18 and MEDIATOR20 control susceptibility to Fusarium oxysporum

in Arabidopsis thaliana (submitted to PLoS ONE)

2-Book chapters Fallath TA, Bin Rosli A, Kidd BN, C. Carvalhais L and Schenk PM (2016). Towards plant defense

mechanisms against root pathogens. In Agriculturally important microbes for sustainable

agriculture. (submitted to Springer) 3- Conference papers

Fallath TA, Kidd BN, Stiller J, Kazan K, Schenk PM (2015). Investigating the role of Mediator

complex in plant defence. The 11 Annual SCMB Research Student Symposiums,

Brisbane, Australia, 26 November.

Fallath TA, Kidd BN, Stiller J, Kazan K, Schenk PM (2015). Investigating the role of Mediator

complex in plant defence. ComBio Conference, Melbourne, Australia, 27 September-1

October.

Schenk PM, Rincon-Florez V, Rosli A, Chen YC, Liu H, Pretorius LS, Fallath T, Mirzaee H,

Shuey L, Al-Amery A, Dennis P, Iram S, Kidd B, Carvalhais LC (2014) Metagenomics

and transcriptomics to elucidate new plant-microbe interactions at the rhizosphere. 15th

International Symposium on Microbial Ecology, Seoul, South Korea, August 24-29.

Publications included in this thesis None

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Contributions by others to the thesis

RNA sequencing was carried out by the Australian Genome Research Facility (AGRF). The initial

analysis of RNAseq data was assisted by Dr. Jiri Stiller (CSIRO). The conception and design of

this project was guided by Dr. Brendan Kidd and Prof. Peer Schenk.

Statement of parts of the thesis submitted to qualify for the award of another degree

None

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Acknowledgements

First and foremost, I would like to express my gratitude to Allah “God” (subhana wa taala) for

endowing me with health, patience, and knowledge to complete this thesis.

Secondly, I would like to thank my supervisor Prof Peer Schenk who gave me the opportunity to

work in a wonderfully vibrant and hardworking lab and for his guidance, advice and invaluable help

during my project. My deepest gratitude and thanks go to Dr Brendan Kidd; he was a wonderful

source of knowledge and whose assistance and advice was greatly appreciated throughout my

project. He also was never too busy to help with all my ridiculous inquiries and problems and who

provided a greatly deal of support and guidance. Without them this project would not have been

possible.

I would like to thanks Dr. Max Chen, Dr. Lilia Carvalhais, Dr. Ahmad Radhzlan Rosli, Dr. Lara

Pretorius and Dr. Anaheed Al-Ameir, Dr. Hongwei Liu, Asmaa Ali and Amreen Kwaja for helping

me and providing a great advice and knowledge since the beginning of my work in the lab. I would

like to thank all the members of the Schenk Lab for the very educational and entertaining lab

meetings, making Mondays easier to handle and more importantly for their friendship.

Also, my gratitude and thanks goes to the Saudi Arabian government in particular The Ministry Of

High Education for providing me with this great opportunity to study overseas at the University of

Queensland and all the financial support during my studies.

My greatest thanks go to my dearest darling mum for all her night prayers and moral support and

blessings. I would like to express a deep sense of gratitude to my wonderful husband Sami

Howsawi for his endless patience, support, encouragement and sacrifices to achieve my dreams and

I simply could not have done it without him. Special thanks are due to my loving brothers and

sisters for all their emotional and financial support. Sweet thank is also due to my lovely sons

Mohammed, Abdulrahman and Ahmad for their puerile support. Last but not the least I would like

to gift all of my work to my dad may God keep his soul in peace in heaven.

Finally, I am also thankful to my friends here in Brisbane who helped make my stay in Australia a

very pleasant one.

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Keywords

Arabidopsis thaliana, Mediator complex, med18, med20, Fusarium oxysporum, root defence,

disease resistance,gene expression, jasmonate, susceptibility

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 060702 PLANT CELL AND MOLECULAR BIOLOGY 50%,

060704 PLANT PATHOLOGY 50%

Fields of Research (FoR) Classification

FoR code: 0607, Plant Biology, 100%

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Table of Contents

LIST OF FIGURES..................................................................................................................................................IXLIST OF TABLES......................................................................................................................................................XLIST OF ABBREVIATIONS.................................................................................................................................XICHAPTER I..................................................................................................................................................................1

INTRODUCTION...................................................................................................................................................................21.1 REVIEW OF LITERATURE.................................................................................................................................................31.21.2.1 Plant Disease and Resistance..................................................................................................................................31.2.2 The role of Mediator complex in plant defence.................................................................................................9a) The Mediator Complex Subunit8 (MED8).............................................................................................................15a) The Mediator Complex Subunit18 (MED18)........................................................................................................16b) The Mediator Complex Subunit20 (MED20)........................................................................................................18c) The Mediator Complex Subunit21 (MED21)........................................................................................................19

SUBSEQUENT CHAPTERS.................................................................................................................................22PH.D. RESEARCH PLAN.....................................................................................................................................22

RESEARCHRATIONALE....................................................................................................................................................231.3 RESEARCH AIMS..............................................................................................................................................................231.4 THE RESEARCH OBJECTIVES........................................................................................................................................231.51.5.1 Aims of the project.....................................................................................................................................................241.5.2 Aim 1...............................................................................................................................................................................241.5.3 Aim 2...............................................................................................................................................................................241.5.4 Aim 3...............................................................................................................................................................................24 RESEARCH TIMELINE....................................................................................................................................................251.6

2 CHAPTER II:.....................................................................................................................................................26INVESTIGATING THE MINIMUM DOMAIN FOR MED25 INTERACTION IN TFS......................26

INTRODUCTION................................................................................................................................................................272.1 MATERIALS AND METHODS........................................................................................................................................282.22.2.1 Determine the minimum domain for MED25 interaction..........................................................................282.2.2 Search for novel proteins containing the ELLEL motif..............................................................................292.2.3 Cloningintoentryvector.......................................................................................................................................292.2.4 Yeast (Saccharomyces cerevisiae) two-hybrid (Y2H) screening.............................................................31 RESULTS............................................................................................................................................................................322.32.3.1 Identification of ELLEL motif containing proteins.......................................................................................322.3.2 Cloning of novel ELLEL motif containing genes...........................................................................................322.3.3 Screening of MED25 and its candidate interacting TFs............................................................................342.3.4 Y2H Screening for protein’s fragments that interact with the conserved ACID domain in Arabidopsis MED25.................................................................................................................................................................36 DISCUSSION......................................................................................................................................................................372.42.4.1 The role of ELLEL conserved motif for MED25 interaction....................................................................372.4.2 Screening for protein’s fragments that interact with the conserved ACID domain in Arabidopsis MED25..........................................................................................................................................................................................39 CONCLUSION....................................................................................................................................................................402.5

3 CHAPTER III:...................................................................................................................................................41IDENTIFYING THE ROLE OF MED18 AND MED20 IN CONFERRING F. OXYSPORUM RESISTANCE...........................................................................................................................................................41

INTRODUCTION................................................................................................................................................................423.1 MATERIALS AND METHODS........................................................................................................................................433.23.2.1 Plasmid preparation.................................................................................................................................................433.2.2 Y2H screening.............................................................................................................................................................43

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3.2.3 Filter lifting assay......................................................................................................................................................433.2.4 Bimolecular Fluorescence Complementation (BiFC) assay.....................................................................443.2.5 Plant growth and growth conditions..................................................................................................................453.2.6 F. oxysporum inoculation.......................................................................................................................................453.2.7 RNA sequencing experiment..................................................................................................................................453.2.8 Root colonisation analysis......................................................................................................................................453.2.9 Disease resistance assays with F. oxysporum................................................................................................46 RESULTS AND DISCUSSION..........................................................................................................................................463.33.3.1 MED18 and MED20 interact with each other in plant...............................................................................463.3.2 Bimolecular Fluorescence Complementation (BiFC) assay.....................................................................473.3.3 med18 and med20 show different growth patterns to WT..........................................................................473.3.4 med18andmed20rootsdisplayreducedcolonisationofaGFP-taggedF.oxysporum.............513.3.5 MED18 and MED20 regulate similar gene sets in Arabidopsis.............................................................523.3.6 med18 and med20 display different responses to F. oxysporum infection compared to WT Arabidopsis plants.....................................................................................................................................................................543.3.7 The role of glucosinolates in F. oxysporum resistance...............................................................................58 CONCLUSION....................................................................................................................................................................613.4

4 CHAPTER VI:...................................................................................................................................................62GENE EXPRESSION PROFILING OF JA, SA AND ROS MARKER GENES IN MED18 AND MED20 MUTANTS..................................................................................................................................................62

INTRODUCTION................................................................................................................................................................634.1 METHODS:.........................................................................................................................................................................644.24.2.1 Plant growth and growth conditions..................................................................................................................644.2.2 Methyl jasmonate, ethylene and salicylic acid treatment...........................................................................644.2.3 F. oxysporum inoculation.......................................................................................................................................644.2.4 Primer design for RT-Q-PCR................................................................................................................................644.2.5 RT-Q-PCR analysis...................................................................................................................................................664.2.6 DAB Staining...............................................................................................................................................................67 RESULTS.............................................................................................................................................................................684.34.3.1 JA signalling genes show altered expression in Arabidopsis med18 and med20 roots.................684.3.2 SA pathway associated genes show high expression in Arabidopsis med18 and med20 roots...684.3.3 MeJA, ET and SA treatment of med18 and med20 leaf tissue..................................................................694.3.4 med18 and med20 show higher expression of ROS related genes..........................................................704.3.5 ROS production in the roots..................................................................................................................................71...................................................................................................................................................................................................794.4 CONCLUSIONS..................................................................................................................................................................804.5

5 GENERALCONCLUSIONS...............................................................................................................................81REFERENCE:..........................................................................................................................................................87

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List of Figures

Figure 1: The structure of the Mediator complex..............................................................................................11Figure 2: Arabidopsis MED25 conserved domains..........................................................................................11Figure 3: The conserved motif (CMIXc) in MED25 interacting IXc AP2/ERF.....................................17Figure 4: Model showing the functions of MED18 and its partners...........................................................18Figure 5: Schematic diagrams of MED20 paralogs MED20a, MED20b, and MED20c......................19Figure 7: Typical disease phenotypes of wild-type (Columbia-0), med18 and med20a plants after

inoculations of the roots with F. oxysporum..............................................................................................20Figure 8: (A) Phylogenetic tree of the proteins containing the ELLEL motifs identified using a

PROSITE scan of Arabidopsis proteins......................................................................................................33Figure 9: (A) The conserved motif ELLEL amino acid residues that are found in the carboxy-

terminal region sequence of RNA55, AGL, ERF14 and ERF96........................................................34Figure 10: Y2H analyses of interaction between ACID domain of MED25 with the other ELLEL

proteins...................................................................................................................................................................35Figure 11: Schematic representation of the ERF1 protein..............................................................................36Figure 12: The Y2H analyses of interaction between ACID domain of MED25 with the different

fragments of ERF1 and DREB2A Arabidopsis TFs...............................................................................37Figure 13: Y2H analyses of interaction between different Mediator subunits in Arabidopsis...........48Figure 14: The in vivo interaction between MED18 and MED20 subunits in the leaves of N.

benthamiana.........................................................................................................................................................49Figure 15: Growth and development characteristics of WT, med18 and med20....................................50Figure 16: Confocal images for the colonisation of a GFP-tagged F. oxysporum in different plants

roots.........................................................................................................................................................................52Figure 17: Venn diagram of differentially regulated genes between each Arabidopsis mutant and the

WT...........................................................................................................................................................................53Figure 18: Genes induced or repressed in each Arabidopsis mutant and the WT at early time-points

after F. oxysporum infection...........................................................................................................................54Figure 19: Top ten most significantly enriched GO terms for genes induced in Arabidopsis WT vs.

med18 or med20..................................................................................................................................................56Figure 20: Top ten most significantly enriched GO terms for genes induced in med18 and med20 vs.

WT...........................................................................................................................................................................56Figure 21: Proportion of plant processes identified by GO term enrichment analysis in WT

compared to med18 or med20 in response to F. oxysporum infection in roots..............................57Figure 22: Disease symptoms and scores of tgg1, tgg2, pen2, pen1/pen2/pen3 plants after F.

oxysporum infection...........................................................................................................................................60Figure 23: RT-Q-PCR data for different JA associated genes in Arabidopsis WT, med18 and med20

roots with and without F. oxysporum infection........................................................................................73Figure 24: RT-Q-PCR data of SA related genes in WT, med18 and med20 roots with and without F.

oxysporum infection...........................................................................................................................................74Figure 25:RT-Q-PCR data for hormone associated genes in WT, med18 and med20 leaves after

MeJA, ET or SA treatment..............................................................................................................................76Figure 26: RT-Q-PCR data of ROS-related genes shown as the different relative abundance in WT,

med18 and med20 Plants..................................................................................................................................78Figure 27: DAB staining of WT, MED18 and MED20 roots........................................................................79

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List of Tables

Table 1: A comprehensive overview listing all of the known TFs interacting with MED25, MED8, MED18 and MED21 Mediator subunits, and the method by which they have been identified....................................................................................................................................................................................21

Table 2: Primers that were used in amplification of the different fragments of TFs. F= forward primer, R= reverse primer................................................................................................................................28

Table 3: Primers that were used in amplification of the different genes that contain the ELLEL protein motif. F= forward primer, R= reverse primer............................................................................30

Table 4: List of primers that were used in amplification of the different Mediator subunits. F= forward primer, R= reverse primer...............................................................................................................43

Table 5: Primer pairs used in real time RT-Q-PCR. F= forward primer, R= reverse primer.............65Table 6:LIST OF GENES Accession Numbers................................................................................................86

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List of Abbreviations

GTF General Transcription Factor

RNA Pol II RNA Polymerase II

TF Transcription Factor

MED25 Mediator Subunit 25

ERF1 ETHYLENE RESPONSE FACTOR1

ORA59 OCTADECANOID RESPONSIVE AP2/ERF59

DREB2A DROUGHT RESPONSE ELEMENT PROTEIN B

MYC2 BASIC HELIX- LOOP-HELIX PROTEIN

PCR Polymerase Chain Reaction

Y2H Yeast Two-Hybrid

cDNA Complementary Deoxyribonucleic Acid

RNA Ribonucleic Acid

ACID Activator-Interacting Domain

SC Synthetic Complete Medium

dpi days post infection

SC-Leu-Trp-His

(SC-LWH)

Synthetic Complete Medium that lacks Leu-Trp-His amino acid

SC-Leu-Trp

(SC-LW)

Synthetic Complete Medium that lacks Leu-Trp amino acid

LB Lysogeny Broth

AGRF Australian Genome Research Facility

JA Jasmonic Acid

SA Salicylic Acid

Real time RT-PCR Real-Time Reverse-Transcriptase-PCR

WT Wild-Type

T-DNA Transfer DNA

ROS Reactive Oxygen Species

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Chapter I

1 Introduction and Literature Review

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Introduction 1.1Plants are a very important source of food for many organisms such as bacteria, fungi, protists,

insects, and vertebrates. Therefore, to keep themselves save from invading by these organisms and

inhibit them before they are able to cause extensive harm, plants need to develop a remarkable

variety of structural, chemical, and protein-based defences. Plants are vital for people not only for

food, but also for several essential non-food products such as wood, dyes, textiles, medicines,

cosmetics, soaps, rubber, plastics, inks, and industrial chemicals. Thus it is necessary to understand

the defence process that plants use to protect themselves from pathogens in order to defend this key

supply and develop highly disease-resistant plant varieties.

The transcription process in eukaryotes is highly complex due to the involvement of a variety of

proteins that work together in synchrony. The core proteins involved during this transcriptional

machinery are the general transcription factors (GTFs), the RNA polymerase II (pol II) enzyme, a

range of transacting activators and repressors, and the Mediator complex. The Mediator and its

subunits play a crucial role as a bridge between the pol II enzyme and DNA-bound TFs. Until now

there is a limited understanding of how different TFs interact with different subunits of the

Mediator complex.

There are approximately 1,500 TFs in the genome of Arabidopsis thaliana (Ratcliffe and

Riechmann, 2002) that are allocated into several gene families based on the similarity of the DNA

binding domains (McGrath et al., 2005). It is generally agreed that TFs in plants play a critical role

in how plants respond to both abiotic and biotic stress. In response to a pathogen attack, TFs can

either activate or suppress the expression of particular defence pathways, depending on the

pathogen that is invading. In addition, they can fine-tune other plant signalling pathways to

suppress non-essential pathways to prioritise the defence response (Anderson et al., 2004).

Recently, an interaction between one of the Mediator complex subunits, MED25, and a number of

TFs has been reported (Elfving et al., 2011; Blomberg et al., 2012; Chen et al., 2012; Cevik et al.,

2012). Some of the TFs were known to play an important role in plant hormone signalling pathways

during plant defence against pathogens (Chen et al., 2012; Cevik et al., 2012).

In light of this, the study of MED25 and the Mediator complex subunits, not only offers a better

understanding of plant defence mechanisms, it also provides an understanding of how the Mediator

complex can interact with TFs to control the broader transcriptional process. This literature

background will provide a broad overview of findings and progression that has been made on the

research of the Mediator complex and also their subunits.

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Review of literature 1.2Plant health can be disrupted by a wide variety of living agents (biotic), like viruses, bacterial and

fungal pathogens, or by environmental (abiotic) factors including nutrient shortage, drought stress,

oxygen deficiency, extreme temperature, ultraviolet radiation, or pollution. In order to respond to

the vast array of potential biotic stresses, plants have evolved a multi-level defence system that

includes both constitutive and inducible defences. Constitutive (continuous) defence is the first

layer that plants use to defend themselves from infection by potentially harmful organisms. This

layer consists of various preformed barriers such as cell walls, waxy epidermal cuticles, and bark

(Hematy et al., 2009; Reina-Pinto and Yephremov, 2009). These physical barriers play an important

role in protecting plants from invasion as well as giving them strength and rigidity. The only way

for pathogens to pass these barriers is either by possessing specific mechanisms that allow them to

enter the plant tissue such as physical pressure (e.g. appressoria) or by producing enzymes that can

degrade the cell wall. The second layer that plant cells use to stop pathogen entry is inducible

defences that include chemical defence mechanisms. After a pathogen penetrates the cell wall

plants not only produce toxic chemicals and enzymes that can degrade pathogens, they may also

initiate hypersensitive response (HR)-mediated programmed cell death in order to isolate the

pathogen from the rest of the plant (VanEtten et al., 1994).

1.2.1 Plant Disease and Resistance

1.2.1.1 The Plant Immune System

A vast array of microorganisms surrounds plant roots in the rhizosphere. These microorganisms can

be either harmful or beneficial to plants (Whipps, 2001). Thus plants have developed basal

resistance, also known as innate immunity. There are two layers of sophisticated surveillance used

to detect potential pathogens: pathogen-associated molecular pattern (PAMP)-triggered immunity

(PTI) and effector-triggered immunity (ETI) (Jones and Dangl, 2006). PAMPs include microbes’

cell wall components or specific pathogen proteins (such as bacterial flagellin). PTI is activated

when PAMPs of potential pathogens have been recognised by PAMP receptors in plants (Nicaise et

al., 2009). PAMP receptor activation leads to reactive oxygen species (ROS) production (Lamb and

Dixon, 1997) as well as activation of the MAP Kinase signalling cascades which then leads to

increased ion flux, phytoalexins, hormone production and defence gene expression (Nicaise et al.,

2009).

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On the other hand, pathogens have evolved proteins known as effectors that can suppress PAMP-

triggered immunity and allow the pathogens to colonise inside the plant. In this situation, plants

might respond by recognising these effector proteins and activting the second line of defence

known as ETI that may lead to HR at the infection site. If pathogens overcome ETI as well, the

plants must respond by evolving new resistance (R) genes that are able to recognise these effectors

and again recruit a successful defence response to protect the plant (Bent and Mackey, 2007).

It seems that plants and pathogens are in constant competition to develop new methods to outsmart

one another.

1.2.1.2 Plant Pathogens

Plant pathogens can be classified according to their lifestyles into three categories, biotrophic,

necrotrophic or hemi-biotrophic. Biotrophs keep their host alive and extract nutrients from its

tissues (Voegele et al., 2003; Ellis et al., 2006), and include examples such as the powdery mildew

fungus Blumeria graminis and the bacterial rice pathogen Xanthomonas oryzae. Necrotrophic

pathogens produce toxins and/or enzymes that degrade the plant cell so they can overwhelm plant

defences and absorb nutrients, such as the gray mold fungus Botrytis cinerea and the bacterial soft-

rot pathogen Erwinia carotovora (Agrios, 2005). The third type of pathogen is the hemi-biotrophic

pathogen. This type is a combination of the two previous categories as it starts its infection as a

biotroph and after initial infection it alters its strategy to necrotrophy to complete its life cycle

(Brown and Ogle, 1997). An example of hemi-biotrophic pathogens is the wilt causing fungus

Fusarium oxysporum.

Pathogen effectors evolved to play a positive role in virulence by enabling growth and reproduction

in host plants (Dong et al.,. 2015 and Lo Presti et al.,. 2015). Nonetheless, effectors can meet their

match with host immune receptors that recognise their presence, a result that is detrimental for the

pathogen. Such immunity-triggering proteins are known as avirulence (Avr) effectors. When an Avr

effector triggers a host immune receptor, the pathogen’s survival depends upon generating variants

that escape, suppress, or alter this recognition event in ways that allow the pathogen to grow and

reproduce. This can be accomplished by numerous means. Transposon insertions or mutations to

the DNA sequence encoding the Avr gene, or its complete loss, are commonly encountered as gain

of virulence mechanisms (Na and Gijzen, 2016). The immunity can also be overcome without

modification of the Avr gene. One approach is by gaining or developing an additional, epistatic

effector that subdues the immune-triggering incident caused by the Avr effector. The best examples

are, in the potato late blight pathogen Phytophthora infestans (Chen et al.,. 2012), the wheat

5

powdery mildew pathogen Blumeria graminis (Bourras et al.,. 2015), the tomato wilt pathogen F.

oxysporum (Houterman et al.,. 2008), and the canola blackleg pathogen Leptosphaeria maculans

(Plissonneau et al.,. 2016). Another approach is by changing the expression of the Avr gene to

defeat immunity. This could simply result from a DNA mutation in the Avr gene’s regulatory region

or in an epistatic location in the genome that affects Avr gene transcription (Na and Gijzen, 2016).

1.2.1.3 Plant defence hormones

The plant hormones most commonly associated with plant defence include salicylic acid (SA),

ethylene (ET) and jasmonate (JA). The former is commonly induced in response to biotrophic

pathogens, while the latter two are often associated with a response to necrotrophic pathogens

(Glazebrook, 2005). SA plays a role in mediating the systemic acquired resistance (SAR) pathway

that results in the systemic activation of defence mechanisms such as PR (Pathogenesis-Related)

gene expression, like PR1 and PR5 (Uknes et al., 1993). The JA-dependent defence response is

mediated by jasmonoyl isoleucine (JA-ile) and leads to the induction of defence related genes such

as PLANT DEFENSIN1.2 (PDF1.2) and THIONIN2.1 (THI2.1) (Epple et al., 1995; Penninekx et

al., 1996). In addition to its involvement in plant defence, the ET pathway is involved in a variety of

plant growth and developmental processes, such as seed germination, root hair development, root

nodulation, flower senescence, abscission and fruit ripening (Johnson and Ecker, 1998). The JA, ET

and SA pathways interact with each other and this cross-talk can be quite complex. Generally, the

JA and SA pathways are said to be antagonistic to each other but at low concentrations they can

also be synergistic (Schenk et al., 2000; Mur et al., 2006). For example, SA has been stated to stop

not only the JA-biosynthesis, but also JA-responsive gene expression (Pennickx et al., 1996),

whereas, JA has been shown to suppress the pathway of SA signalling. Defence hormone cross-talk

is controlled by TFs that can activate target genes of one pathway, while inhibiting those of the

other pathway. The three closely related Arabidopsis basic leucine zipper (bZIP) TFs, TGA2,

TGA5 and TGA6 are examples of TFs that are required to activate SA regulated defence genes and

at the same time repress the JA pathway (Zander et al., 2010).

1.2.1.4 Defence against the root pathogen Fusarium oxysporum

More than 100 different plant species are infected by the root pathogen F. oxysporum, including

cotton, tomato, banana and Arabidopsis. Being a root infecting pathogen poses some unique

challenges in studying the plant-pathogen interaction. However, a number of scientific studies have

established some general methods to study the early infection process and response of the plant, in

tomato (Beckman et al., 1989; Gao et al., 1995; Olivain et al., 2006), cotton (Shi et al., 1991;

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Rodriguezgalvez and Mendgen, 1995), banana (Vandermolen et al., 1987; Visser et al., 2004) and

Arabidopsis pathosystems (Czymmek et al., 2007). These investigations have uncovered some

strategies that the host plant uses to resist the pathogen.

The infection of F. oxysporum starts from the root toward the vascular tissue, where it proliferates

and causes the plant to wilt. The disease symptom progression mainly relies on the extent to which

the xylem is blocked or not. Several studies that conducted electron microscopy have indicated that

a variety of physical alterations occur in response to infection. One of the prior studies in F.

oxysporum infection illustrated that within 1 h post inoculation of resistant tomato plants, the

contact parenchyma cells (parenchyma cells adjacent to infected xylem cells) display an expanded

cytoplasm opposite the infection (Beckman et al., 1991). In addition, Beckman et al., (1989) and

Mueller et al., (1994) have reported that these contact cells deposit apposition layers made of

callose to secure the cell wall. According to the results of these studies, the plants can sense the

presence of the pathogens in the xylem; therefore, callose-containing apposition layers are

synthesised in the nearby cells to inhibit the lateral spread of the pathogen.

Moreover, plants possess numerous armaments to block off longitudinal extent of the fungus. Shi et

al. (1992) have showed that after cotton plant infection, the contact cells play an important role to

secrete a combination of gels and gums to block off the vasculature. Tyloses can be produced as

well to impede the infection. Tyloses are parenchyma cells that balloon out into the lumen through

the xylem pits (Vandermolen et al., 1987). Also, wide ranges of phenolic compounds are released to

support both tyloses and gel-gum secretions (Beckman, 2000). The phenolic compounds oxidise in

the xylem and polymerise together to form stable lignified barriers (Beckman et al., 1974).

Nevertheless, it is not known if all plants can produce defence mechanisms such as tyloses or gum

secretions to block F. oxysporum infection. Furthermore, more research is needed to determine the

signalling pathway that is required to trigger these defence responses.

1.2.1.5 Defence against F. oxysporum in Arabidopsis

Investigating the plant defence signalling network is a worthy alternative approach to understanding

F. oxysporum resistance. These signalling processes can be modified to study which genes may be

required for resistance or susceptibility to pathogens. Arabidopsis is a good model system to study

these defence signalling pathways as numerous genes encoding mutants of known defence

regulators have been studied to assess their involvement in plant defence.

7

Mutants deficient in SA-mediated defences were shown to be more susceptible to F. oxysporum.

For example, the sid2 mutant that is impaired in SA biosynthesis is more susceptible to F.

oxysporum f.sp. conglutinans (Berrocal-Lobo and Molina, 2004; Diener and Ausubel, 2005). In

addition, treatment of SA to the leaves of Arabidopsis plants resulted in a partial increase in

resistance (Edgar et al., 2006).

The Arabidopsis R genes, RESISTANCE TO FUSARIUM OXYSPORUM (RFO1, RFO2, RFO3 etc),

have also been successfully cloned. Diener and Ausubel, (2005) discovered the RFO1 gene by

using a cross between the moderately resistant ecotype Columbia-0 (Col-0) and the susceptible

ecotype Taynuilt-0 (Ty-0). RFO1 encodes a wall-associated kinase-like kinase 22 (WAKL22).

RFO1 is an atypical R gene in that it provides broad-spectrum resistance to three dissimilar types of

F. oxysporum as opposed to a single race. Furthermore, the resistance provided by RFO1, RFO2,

RFO3 is quantitative and hence provides only partial resistance. However, RFO3 only confers

specific resistance to F. oxysporum f.sp. matthioli, and provides no resistance to two other species;

F. oxysporum f.sp. conglutinans or F. oxysporum f.sp. raphani. A recent study has shown that

RFO2 provides strong pathogen-specific resistance (Shen and Diener 2013). The extracellular

leucine-rich repeats (eLRRs) in RFO2 and the related receptor like protein (RLP) 2 protein are

interchangeable for resistance and remarkably similar to eLRRs in the receptor-like kinase PSY1R,

which perceives tyrosine-sulfated peptide PSY1 (Shen and Diener, 2013). The study also found

reduced infection in psy1r. Consequently, the results of this suggest that F. oxysporum produces an

effector that prevents the normal negative feedback regulation of PSY1R, which stabilises PSY1

signalling and induces susceptibility. However, RFO2, acting as a decoy receptor of PSY1R, is also

stabilised by the effector and instead induces host immunity (Shen and Diener 2013). The RFO R

genes are unique in that they provide protection to multiple formae speciales of F. oxysporum and

provide only partial resistance in some host-pathogen combinations. Nonetheless, the process by

which these genes provide F. oxysporum resistance is still unknown.

There is also evidence that plants insensitive to JA are more resistant to F. oxysporum.MYC2,

BASIC HELIX- LOOP-HELIX PROTEIN transcription factor, is a positive regulator of insect

defence, wound responses, flavonoid metabolism, and oxidative stress tolerance during JA

signalling (Dombrecht et al., 2007). In contrast, MYC2 is thought to act predominantly as a negative

regulator of JA/ET-associated pathogen defence (Kazan and Manners, 2008). However, the JA

signalling-compromised MYC2 mutant shows increased resistance to F. oxysporum (Anderson et

al., 2004), and has increased SA defence gene expression which could explain the enhanced

resistance to F. oxysporum.

8

Similarly, the coi1 mutant has also been shown to possess increased resistance to F. oxysporum

(Thatcher et al., 2009; Trusov et al., 2009). COI1 (CORONATINE INSENSITIVE1) is an integral

part of the SCF (Skp-Cullin-F-box) type E3 ubiquitin ligase complex that acts as co-receptor with

the JAZ repressor for the active form of jasmonate, jasmonoyl isoleucine, (JA-ile) (Thines et al.,

2007; Yan et al., 2009; Sheard et al., 2010). Mutation of COI1 abolishes JA function, including the

expression of JA-associated defence genes, such as PDF1.2 (Penninckx et al., 1996). Therefore the

increased resistance of the coi1 mutant suggests that functional JA signalling is required for

susceptibility to F. oxysporum and that activation of this susceptibility program may overwhelm

any benefit of increased JA defence gene expression. Grafting studies performed with the coi1

mutant showed that the root section is required for resistance/susceptibility to F. oxysporum. A coi1

rootstock grafted to a WT scion resulted in almost complete resistance, suggesting that roots play a

significant role in plant defence against F. oxysporum. In additional, Pantelides et al. (2013)’s study

showed that mutant on receptor 1 ethylene (etr1-1) plants exhibited statistically less Fusarium wilt

symptoms compared to the other mutant col-0 plants. Quantitative polymerase chain reaction

analysis associated the decrease in symptom severity shown in etr1-1 plants with reduced vascular

growth of the pathogen, suggesting the activation of defence mechanisms in etr1-1 plants against F.

oxysporum in the roots. This study suggested F. oxysporum also requires ETR1 mediated ethylene

signalling to promote disease development in plants.

A microarray analysis carried out by Chen et al., (2014) showed that in contrast to the leaf response,

root tissue did not show a strong induction of defence-associated gene expression and instead

showed a greater proportion of repressed genes. Screening insertion mutants from differentially

expressed genes in the microarray uncovered a role for the transcription factor ETHYLENE

RESPONSE FACTOR72 (ERF72) in susceptibility to F. oxysporum. Due to the role of ERF72 in

suppressing programmed cell death and detoxifying reactive oxygen species (ROS), they examined

the pub22/pub23/pub24 U-box type E3 ubiquitin ligase triple mutant which is known to possess

enhanced ROS production in response to pathogen challenge. they found that pub22/23/24 is more

resistant to F. oxysporum infection, suggesting that a heightened innate immune response provides

protection against F. oxysporum (Chen et al., (2014). This demonstrates again that root-mediated

defencess against soil-borne pathogens could be provided at multiple levels.

9

1.2.2 The role of Mediator complex in plant defence

1.2.2.1 The discovery of the Mediator complex

The first identification of the Mediator complex was from yeast, Saccharomyces cerevisiae by

Flanagan and colleagues during their studies of in vitro transcription (Flanagan et al., 1990;

Kelleher et al., 1990; Flanagan et al., 1991). In the following years, investigation of the Mediator

complex established its role as an essential requirement for activator-dependent stimulation of Pol

II-mediated transcription.

1.2.2.2 The Mediator complex in plants

Thirteen years after the discovery of the Mediator complex in yeast, Bäckström and colleagues

reported their purification of the Mediator complex and its subunits in Arabidopsis thaliana and

classified these proteins as new components of the transcriptional machinery in plants (Bäckström

et al., 2007). The Mediator complex is a multi-protein complex and is present in all eukaryotes

(Bäckström et al., 2007; Chadick et al., 2005; Malik and Roeder 2005), although the primary

sequence of Mediator subunits diverges significantly between eukaryotic kingdoms.

To date, there are 21 conserved and six plant-specific subunits that have been identified in

Arabidopsis thaliana (Bäckström et al., 2007). The investigation of these proteins as subunits of the

Mediator complex has demonstrated the important as well as diverse roles that single subunits can

possess. A single subunit is expected to differentiate and respond to diverse TFs within the

Arabidopsis genome (Kidd et al., 2011). Consequently, determining which TFs that individual

Mediator subunits can recognise would definitely speed up our understanding of how the Mediator

complex manages to integrate the complex transcriptional information in plants.

1.2.2.3 The structure of the Mediator complex

The subunits of the Mediator complex are organised in four sub-modules; the head, the middle, the

tail and the Cyclin Dependent Kinase module (CDK) (Dotson et al., 2000). The head sub-module

(most conserved) is thought to serve as an interface for RNA Pol II–binding and interacts through

the Pol II C-terminal domain. While the tail sub-module (least conserved) is responsible for linking

to sequence-specific transcription factors (Chadick and Asturias, 2005, Malik and Roeder, 2005).

The middle module of Mediator is responsible for the signal transfer from the tail to the head, and

the CDK module usually functions as a transcriptional repressor (Figure 1; Casamassimi and

Napoli, 2007). In addition to these direct interactions, contemporary publications illustrate that the

10

subunits of this complex have further multiple functions and can link to and facilitate the roles of

diverse co-activators and co-repressors, including those acting at the level of chromatin (Malik and

Roeder, 2005; Chen and Roeder, 2011; Hentges, 2011; Kidd et al., 2011). Although the subunit

composition and the structure of Mediator have been intensely studied, relatively little is known

about how Mediator operates to integrate and convey signals from DNA-bound transcriptional

regulators.

1.2.2.4 The function of the plant Mediator complex

Since its identification in plants, the multi-protein complex has been discovered to regulate

numerous biological activities, such as the response to biotic and abiotic stress, controlling growth

and development of plant organs as well as regulating flowering time and fertility (Kidd et al.,

2011). Furthermore, a number of subunits from this complex have been indicated to play an

important role in nuclear functions; for example, some subunits possess DNA helicase activity, or

are involved in RNA processing e.g. mRNA splicing or rRNA methylation (Kidd et al., 2011). In

addition, a recent study identified an important role for this large protein complex in noncoding

RNA production (Kim et al., 2011).

1.2.2.5 The structure of the Mediator subunit 25 (Med 25)

MED25 encodes a protein of 836 amino acids in length. It contains three different conserved

domains (Figure 2). The conserved Von Willebrand factor type A (vWF Pfam · PF00093) domain

in its amino terminus that is crucial for Mediator binding. An ACID domain that is required for the

interaction with transcription factors as well as a glutamine rich region in the carboxyl terminus

(Bäckström et al., 2007; Cerdán and Chory 2003; Mittler et al., 2003). Cerdan and Chory (2003)

proposed that MED25 is a transcriptional co-regulator due to its ability to activate transcription in

yeast when fused to the LexA DNA binding domain. The structure of the MED25 activator

interaction domain (ACID) was recently solved using NMR (Milbradt et al., 2011; Vojnic et al.,

2011; Landrieu et al., 2015). They proposed that the ACID domain consists of a closed β-barrel

with seven strands framed by three α-helices (H1-H3).

11

Figure 1: The structure of the Mediator complex. The head sub-module serves as interface of RNA

Pol II, interacting through the C-terminal domain, while the tail sub-module links to sequence-

specific transcription factors. The middle module of Mediator is responsible for the signal transfer

from the tail to the head. GTFs – general transcription factors, TF- transcription factor. (reproduced

from (Sohail and Robert, 2010))

Figure 2: Arabidopsis MED25 conserved domains. The MED25 protein contains a vWF-A domain

(blue), a middle domain (MD) (white), an ACID domain (red), and a GD domain (gray). Bar = 100

amino acids (aa) (Chen et al., 2012).

GTFs

Middle

Med24

Tail

Head

Mediator

TF

GTFs

Med21

theMediatorcomplex

12

1.2.2.6 The identification and role of MED25 in plant development

The MED25 subunit was first identified as PHYTOCHROME AND FLOWERING TIME1 (PFT1)

based on its role in stem elongation and floral transition when there is a change in light quality

(Cerdán and Chory, 2003). When red light is decreasing and far red light is increasing this acts as

an indicator of increasing shade and can act as a warning signal of potential competition for light

from neighboring plants. As a result the plant will decide whether to initiate stem elongation to

reach more light or induce flowering if the shade is prolonged and unavoidable (Franklin and

Whitelam, 2005). The studies of this mechanism showed that MED25 has an important role in

regulating this adaptive process called shade avoidance (Cerdan and Chory, 2003; Wollenberg et

al., 2008).

1.2.2.7 The role of MED25 in biotic stress responses

Besides controlling flowering time, recent publications reported that MED25 has an essential

function in pathogen defence (Kidd et al., 2009). The authors of this report showed MED25 mutant

plants to be more susceptible to the leaf infecting necrotrophic fungal pathogens Alternaria

brassicicola and Botrytis cinerea. On the other hand there was an increase in resistance to the root

infecting hemi-biotrophic fungal pathogen, Fusarium oxysporum (Kidd et al., 2009). Remarkably,

there was a decrease in the level of the transcription of numerous plant defence genes, including

those responsive to JA (Kidd et al., 2009). Importantly this mechanism justified the varied

phenotypes in response to these fungal pathogens, as defences regulated by JA signalling are

recognised to be needed for resistance to necrotrophic pathogens (On ate-Sanchez and Singh, 2002),

whereas F. oxysporum is believed to hijack JA signalling for stimulating senescence responses

hypothesised to increase symptoms in response to this pathogen (Thatcher et al., 2009). In addition,

the reduced JA responses also result in increased resistance to the hemibiotrophic pathogen

Pseudomonas syringae pv tomato (Pst) DC3000 which also requires JA signalling to induce disease

(Chen et al., 2012). Therefore these data possibly implicate MED25 as an interacting partner for

JA-associated TFs in the Mediator complex.

1.2.2.8 Interaction of MED25

To date researchers have identified 25 TFs that interact with MED25. These TFs belong to a variety

of transcription factor families including AP2/ERF, bHLH, MYB, WRKY, bZIP and zinc finger,

demonstrating that Arabidopsis MED25 plays regulatory roles in diverse physiological processes

including JA-dependent defence response (Chen et al., 2012, Elfving et al., 2011, C evik et al., 2012,

Ou et al., 2011). A recent study found that the MED25 subunit could interact with eight different

13

transcription factors by using a high-throughput Y2H analysis (Ou et al., 2011). Five of these TFs

identified can interact with the promoter region of the PDF1.2 gene, a commonly used marker for

the JA signalling pathway (Ou et al., 2011). Three of these TFs, all from the AP2-EREBP family,

interact directly both with MED25 and the GCC-box of PDF1.2, suggesting that MED25 regulates

PDF1.2 expression through these three TFs. A failed interaction with these transcription factors in

the MED25 mutant could potentially explain why JA-associated gene expression is reduced with

the associated effects on plant–microbe interactions.

Subsequently, an additional three transcription factors were found to interact with MED25 (Elfving

et al., 2011). Two of the transcription factors that were identified, ZFHD1 and DREB2A, have

published roles in abiotic stress tolerance (Sakuma et al., 2006, Maruyama et al., 2006, Tran et al.,

2007). The authors were able to show that MED25 mutant plants also displayed alterations in both

drought and salt stress; however the increased drought resistance phenotype of MED25 was not

consistent with the increased drought sensitivity of the two transcription factor mutants (Elfving et

al., 2011). The increased drought tolerance of the MED25 mutant may also be related to the positive

role that MED25 plays in jasmonate signalling, as this pathway is known to antagonise abscisic acid

(ABA) signalling that is associated with drought tolerance responses (Anderson et al 2004).

Moreover, Cevik and colleagues (2012) have pointed out that MED25 physically interacts with

several key transcriptional regulators of the JA signalling pathway, including the APETALA2

(AP2)/ETHYLENE RESPONSE FACTOR (ERF) transcription factors such as ERF1

and OCTADECANOID RESPONSIVE ARABIDOPSIS AP2/ERF59 (ORA59) as well as the

master regulator MYC2. Physical interaction detected between MED25 and four group IX AP2/ERF

transcription factors was shown to require the activator interaction domain of MED25 as well as the

recently discovered Conserved Motif IX-1/EDLL transcription activation motif of MED25-

interacting AP2/ERF (Figure 3). Using transcriptional activation experiments, they also indicated

that ORA59 and ERF1-dependent activation of PDF1.2 as well as MYC2-dependent activation of

VEGETATIVE STORAGE PROTEIN1 (VSP1) requires a functional MED25. In addition, MED25 is

required for MYC2-dependent repression of pathogen defence genes. Together these findings

demonstrate the versatility of the MED25 subunit in regulating multiple pathways, from hormone

signalling and flowering to biotic stress responses.

Recently, research by Yang et al. (2014) has shown that MED25 interacts with another subunit of

the Mediator complex, YID1/MED16, to carry out its role in iron homeostasis. Their study

demonstrated that disruption of MED25 led to the hypersensitivity of plants to iron deficiency.

14

They found that MED25 interacts with Ethylene - Insensitive 3 (EIN3) and Ethylene - Insensitive 3

- Like 1 (EIL1), two transcription factors in ethylene signalling involved in iron homeostasis

regulation in Arabidopsis. Moreover, the transcripts of Fe-deficiency Induced Transcription factor

(FIT), Ferric Reduction Oxidase 2 (FRO2) and Iron–Regulated Transporter 1 (IRT1) were reduced

during an iron deficiency in yid1/med16 and med25 mutants when compared with those in the wild

type. Their results demonstrate that MED25, together with YID1/MED16, regulates iron

homeostasis by interacting with EIN3/EIL1 to regulate the expression of downstream genes (Yang

et al., 2014).

Direct interactions between transcriptional regulators and different Mediator subunits are believed

to recruit and stabilise Mediator at the promoter, and there are indications that interactions with

transcriptional regulators induce conformational changes or structural shifts in Mediator (Wands et

al., 2012 and Meyer et al., 2010). It is possible that such conformational changes in Mediator are

propagated to induce further structural shifts in components of the general transcription machinery.

These changes, combined with other signal pathways that cause posttranslational modifications of

different transcription factors, might therefore determine the level of transcription from each gene

(Blomberg et al., 2012). Consistent with this idea, Blomberg and colleagues have found that

binding of DREB2A to its canonical DNA sequence leads to an increase in secondary structure of

the transcription factor. Similarly, interaction between the DREB2A and MED25 in the absence of

DNA results in conformational changes. However, the presence of the canonical DREB2A DNA-

binding site reduces the affinity between DREB2A and MED25. They stated that transcription

regulation is facilitated by small but distinct changes in energetic and structural parameters of the

involved proteins (Blomberg et al., 2012).

Surprisingly, Blomberg et al. (2012) demonstrated that DREB2A from Arabidopsis can interact

with the human MED25 ACID domain and displayed similar complex kinetics as the Arabidopsis

MED25 ACID domain with DREB2A interaction. This result not only suggests that there is

sequence similarity in the ACID domain between the two species, but also it proposes that whatever

is discovered in plant gene regulation through the Mediators complex may be applied to the human

Mediator complex. Indeed, physical interactions between MED25 and a diversity of transcription

factors suggest that MED25 acts as an integrative hub within Mediator to regulate a wide range of

physiological processes including the plant defence response (Chen et al., 2012; Cevik et al., 2012)

15

1.2.2.9 Human MED25

Similar to the MED25 protein in plants, the human MED25 protein also seems to have a function in

host defence. The human MED25 has been shown to be the cellular target of a number of viral

activators through its C-terminus such as the well-studied transcriptional activator belonging to

Herpes Simplex Virus, VP16, (Mittler et al., 2003; Yang et al., 2004) and Lana-1 the activator from

the Kaposi Sarcoma associated herpes virus (Roupelieva et al., 2010), as well as the activator IE62,

from the closely related Varicella Zoster virus, the virus responsible for chicken pox and shingles

(Yang et al., 2008), whereas the N-terminus of human MED25 forms contact with the Mediator

complex (Mittler et al., 2003). The Arabidopsis MED25 protein shows similarity to human MED25

in its N-terminus, however the sequence is less conserved in the C-terminus. In addition to the

defence functions, human MED25 has been shown to be involved in Retinoic acid signalling,

xenobiotic and lipid metabolism, cranofacial development as well as the motor and sensory

neuropathy Charcot Marie Tooth Disease (Lee et al., 2007; Nakamura et al., 2011; Rana et al.,

2011; Youn et al., 2011).

1.2.2.10 Other significant Mediator subunits

Intriguingly however, the MED25 subunit is not the only Mediator subunit to control multiple

pathways and a number of other Mediator subunits have been reported to have regulatory roles in

flowering time [MED8, MED17, MED18, MED20a; (Kidd et al., 2011; Kim et al., 2011)], biotic

stress [MED8, MED21; (Kidd et al., 2011; Dhawan et al., 2009)] or abiotic stress [MED16

(Wathugala et al., 2011 and Knight et al., 2009)].

a) The Mediator Complex Subunit8 (MED8) MEDIATOR8 (MED8) is part of the head module of the Mediator complex. Studies on MED8

show that the mutation of this Mediator subunit not only has a stronger delay in flowering time than

MED25 under both LD and SD conditions, but also plays a significant role in plant defence. The

med8 mutant is susceptible to A. brassicicola but resistant to F. oxysporum in Arabidopsis (Kidd et

al., 2009). Moreover, the med8 mutant shows enhanced susceptibility to Pseudomonas syringae

DC3000, but has no significant defects in biological induction of SAR (Zhang et al., 2012). The

study of Kidd and co-worker (2009) also state that even though there is only a minor reduction in

MeJA-induced PDF1.2 expression in the med8 mutant, the effect of med8 on PDF1.2 expression is

more visible in the med25 mutant background, as the double mutant shows a further decrease in

16

PDF1.2 expression. As expected, the med8 mutation has an additive effect with the med25 mutation

on F. oxysporum resistance (Kidd et al., 2009). Therefore, MED8 and MED25 may act

independently within Mediator to influence the plant defence response.

a) The Mediator Complex Subunit18 (MED18) MEDIATOR18 (MED18) is part of the head module of the Mediator complex. It is a

multifunctional protein that has a key role in regulating plant immunity, development, flowering

time and responses to hormones through interactions with distinct transcription factors as well as its

role in non-coding RNA production. Mutant med18 plants possess similar developmental

phenotypes to med20a and display reduced miRNA levels (Kim et al., 2011). MED18 represents a

unique immunity regulator that functions distinctly from other immune response factors. The

immune response function of MED18 is carried out through at least two mechanisms involving

activation (PTR3) and suppression (thioredoxins and glutaredoxin) of gene expression. PTR3 is a

di- and tri-peptide transporter implicated in plant responses to wounding and infection (Stacey et

al., 2002; Karim et al., 2007). It is possible that deficiency in the uptake of amino acids

compromises the synthesis of some defence compounds. A recent paper demonstrates that MED18

interacts with zinc finger TF YY1 (YIN YANG1) to suppress disease susceptibility genes

glutaredoxins GRX480, GRXS13 and thioredoxin TRX-h5. Consequently, yy1 and med18 mutants

exhibit deregulated expression of these genes and enhanced susceptibility to fungal infection.

Intriguingly, MED18 is also required for pathogen-induced expression of PTR3, a positive regulator

of plant defence (Lai et al., 2014). Moreover, MED18 is recruited to ABI5 and FLOWERING

LOCUS C (FLC) promoters through interaction with TFs ABI4 (ABA INSENSITIVE 4) and SUF4

(SUPPRESSOR OF FRIGIDA4) to regulate responses to ABA and flowering time, respectively.

Interestingly, in addition to transcription initiation, MED18 is involved in transcription elongation,

termination, as well as recruitment of RNA Pol II to target genes in the various pathways (Lai et al.,

2014). Notably, RNA polymerase II occupancy and histone H3 lysine tri-methylation (H3K36me3)

on chromatin of target genes are affected in the med18 mutant (Lai et al., 2014), reinforcing

MED18’s function in different mechanisms of transcriptional control. Overall, MED18 conveys

distinct cues to engender transcription underpinning plant responses (figure 4).

17

Figure 3: The conserved motif (CMIXc) in MED25 interacting IXc AP2/ERF. The group IX family

of AP2/ERF can be divided into three groups (Nakano et al., 2006). All Group C proteins have the

conserved domain CMIXc (showing in pink) in their carboxy- terminal region and six of these

proteins interact with MED25 (A). Also shown is the sequence of the conserved domain CMIXc

which is important for interaction with MED25 and the conserved ELLEL motif identified in

MED25 interacting AP2/ERF proteins (Cevik et al., 2012) (the conserved amino acids are shaded in

grey boxes) (B). The EDLL motif previously identified by Tiwari et al., (2012) is also shown in red.

A

B

18

Figure 4: Model showing the functions of MED18 and its partners. MED18 is required for

signalling from upstream regulators to downstream components through interaction with

transcription factors ABI4, YY1 and SUF4 to regulate plant responses to ABA, infection and

flowering time, respectively. These interactions directly regulate the expression of the

corresponding target genes ABI5, TRX-GRX, PTR3 and FLC genes. The transcription factor,

mediating PTR3 regulation by MED18, is unknown (Lai et al., 2014).

b) The Mediator Complex Subunit20 (MED20) MEDIATOR20 (MED20) is also belong to the head module of the Mediator complex. In

Arabidopsis there are three different paralogs of MED20. They were named MED20a, MED20b,

and MED20c (Figure 5). MED20a and MED20b have 85% identity at the nucleotide level.

MED20c is shorter than MED20a and MED20b. There is 93% identity at the nucleotide level in

exon 2 among the three genes (Kim et al., 2011). Kim et al., (2011) have provided evidence that

MED20a is crucial in regulating development and non-coding RNA production. med20a has

reduced fertility and altered leaf phyllotaxis and this gene is important for non-coding RNA

biogenesis (Kim et al., 2011)

The interactions between MED18, TFs and their target genesare summarized in the model for MED18 functions (Fig. 9,Supplementary Fig. 12). MED18 interacts with ABI4, YY1 andSUF4 TFs that are known to bind to different DNA sequences intheir respective target genes performing different physiologicalfunctions. First, MED18 interaction at the ABI4-binding site ofABI5 modulates ABI5 gene expression, consistent with thepositive role of MED18 on ABA responses. The impairedresponses to ABA, reduced expression of ABI5 in med18 mutantand physical interaction with ABI4 demonstrate the critical roleof MED18 in ABA responses. Interestingly, the disease resistanceand ABA response functions of MED18 appear independent.Second, the negative regulation of GRX480, GRXS13 and TRX-h5is clearly mediated by MED18 interaction with the transcriptionalrepressor YY1. YY1 maintains normal expression of GRX480,GRXS13 and TRX-h5 that are plant susceptibility factors to

infection. Third, SUF4 interaction with MED18 plays a criticalrole for normal expression of the floral repressor FLC. However,the regulation of FLC is generally more intricate with otherfactors and regulatory processes involved. Mutation in SUF4results in early flowering by reducing FLC gene expression39. Thegenetic data suggest that SUF4 and MED18 have contrastingfunctions. In the wild-type background MED18 may suppress thefunction of SUF4 in promoting FLC expression. Congruent withthis, the med18 mutant exhibits enhanced FLC expression andextremely delayed flowering, which suggests the function of thewild-type MED18 protein is to promote flowering. This isconsistent with our observation that MED18 suppresses SUF4function through physical interaction and association at the FLClocus. Fourth, MED18 regulates PTR3 through direct associationwith the PTR3 regulatory regions. The intermediary TF throughwhich this association occurs is not known. Together, the abovedata suggest that MED18 functional specificity and its role inactivation or repression of gene expression are conferred by thesequence-specific DNA-binding zinc finger (YY1, SUF4) or ERF/AP2 (ABI4) domain TF proteins in response to upstream extra-and intracellular cues. Overall, MED18 is involved in multipleand seemingly independent biological functions and cellularprocesses.

Plant responses to infection involve specific and broadreprogramming of gene expression. MED18 is required tointegrate and transmit defence-related signals from TFs to thetranscriptional machinery contributing to the up and down-regulation of diverse immune factors. Interestingly, markers ofcanonical immune response pathways such as effector-triggeredimmunity, particularly R-genes, PAMP-triggered immunity,systemic acquired resistance and JA/ET-mediated such asresponses show no altered gene expression responses in med18mutant. Thus, MED18 represents a novel immunity regulator thatfunctions distinctly from other immune response factors. Theimmune response function of MED18 is carried out through atleast two mechanisms involving activation (PTR3) and suppres-sion (thioredoxins and glutaredoxin) of gene expression. PTR3 isa di- and tri-peptide transporter implicated in plant responses towounding and infection30,40. It is possible that deficiency in the

PTR3 TATA box ABI5 TATA box FLC TATA box

PTR3 coding ABI5 coding FLC coding

2 0.3 2

1

0

0.2

0.1

0

1a

a

a

a

b

c

d

c

b bb

b

c

c

b

b

aa

b b b

c

c

b

0

4 1

0.5

0

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0

Wt med18-1

Wt med18-1 Wt med18-1 Wt med18-1

Wt med18-1 Wt med18-1

%C

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put

%C

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%C

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Figure 8 | MED18 modulates histone H3K36 tri-methylation at the coding region of target genes. (a) H3K36 tri-methylation at PTR3, (b) ABI5 and (c)FLC loci in wild-type and med18 mutant plants. ChIP was conducted with H3K36me3-specific antibodies (Abcam). ChIP for PTR3 was performed on4-week-old plants at 2 dai with B. cinerea. AB15 ChIP was on 2-week-old seedlings treated with 50mM ABA for 3 h. FLC ChIP was conducted on 2-week-olduntreated seedlings. Data are representative of two independent experiments. Error bars indicate s.e. (n¼ 3).

Bioticstress

TF

Targetgenes

Process: ABAresponse

TRX and GRXABI5

ABI4(ERF/AP2)

YY1(Zinc finger)

SUF4(Zinc finger)

Flowering

FLCPTR3

ResistanceFungal(B. cinerea)

?

Light Hormones Abioticstress

MED18

Figure 9 | Model showing the functions of MED18 and its partners.MED18 is required for signaling from upstream regulators to downstreamcomponents through interaction with transcription factors ABI4, YY1 andSUF4 to regulate plant responses to ABA, infection and flowering time,respectively. Those interactions directly regulate the expression of thecorresponding target genes ABI5, TRX-GRX, PTR3 and FLC genes. Thetranscription factor mediating PTR3 regulation by MED18 is unknown.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4064 ARTICLE

NATURE COMMUNICATIONS | 5:3064 | DOI: 10.1038/ncomms4064 |www.nature.com/naturecommunications 11

& 2014 Macmillan Publishers Limited. All rights reserved.

19

Figure 5: Schematic diagrams of MED20 paralogs MED20a, MED20b, and MED20c. The asterisk

indicates the mutation causing a premature stop codon in the med20a mutant. Black rectangles

represent exons and lines represent introns. MED20a and MED20b are composed of three exons

showing 85% identity at the nucleotide level. MED20c only encodes the conserved second exon

compared with MED20a and MED20b.

c) The Mediator Complex Subunit21 (MED21) MEDIATOR21 (MED21) is part of the middle module of the Mediator complex. MED21 is

believed to have a critical role in regulating development and plant defence. It regulates defence

against necrotrophic fungal pathogens by interacting with HUB1 (Histone Monoubiquitination1).

HUB1 is a RING E3 ligase functioning in histone H2B monoubiquitination and contributes to

resistance against A. brassicicola and B. cinerea in Arabidopsis (Dhawan et al., 2009). RNA

interference (RNAi) lines with reduced MED21 gene expression exhibit increased susceptibility to

A. brassicicola and B. cinerea, whereas MED21 over-expressing plants do not show any significant

difference compared to the wild type (Dhawan et al., 2009). In addition, T-DNA insertion mutants

are embryo lethal (Dhawan et al., 2009). An and Mou (2013) have pointed out that MED21 may

relay signals from upstream regulators and chromatin modifications to the RNA polymerase

1.2.2.11 The role of med18 and med20 mutants in F. oxysporum susceptibility

Fusarium oxysporum is considered to be one of the most significant fungal pathogens for a wide

range of plant species. It is a hemi-biotrophic pathogen that has a complicated, multi-stage infection

lifestyle. The infection process starts with a biotrophic root colonisation stage, followed by invasion

repeats and transposons (reviewed in Chen, 2009), raises thequestion of whether Mediator acts in transcriptional genesilencing (TGS) and whether Mediator promotes the activitiesof Pol IV or Pol V.

Here, we evaluate the effects of three Mediator mutationsin the expression of protein coding as well as noncoding RNAgenes in Arabidopsis. We show that Mediator is likely ageneral transcription factor that promotes Pol II transcriptionof a large number of protein-coding genes. We also show thatMediator promotes the transcription of miRNA genes (MIR)by recruiting Pol II to their promoters. In addition, we reveal apreviously unsuspected role of Mediator in noncoding RNAproduction at, and TGS of, loci regulated by endogenoussiRNAs. These findings broaden our knowledge of the roleof Mediator in gene expression and genome defense.

Results

Arabidopsis mutants in three Mediator subunit genesdisplay pleiotropic developmental defectsA mutant with pleiotropic developmental phenotypes wasisolated in a forward genetic screen in the Columbia (wildtype) background. The mutant was smaller than wild type instature and late flowering (Figure 1A). The leaves had shorterpetioles and curled downward (Supplementary Figure S1A),the phyllotaxy of flowers was abnormal (Supplementary

Figure S1B), and fertility was reduced (Figure 1B; Supple-mentary Figure S1D). At a frequency of B20%, the mutantdeveloped three cotyledons and three first true leaves insteadof a pair of each in wild type (Supplementary Figure S1A).

The mutation was mapped to a 120-kb region covered bythe BACs T3B23 and T1B3 on chromosome 2. Sequencingcandidate genes in this region revealed a C-to-T mutation inAt2g28230, which encodes a subunit of the head submodulein Mediator (Figure 1C). The mutation is at the 58th nucleo-tide of the coding region in the second exon and results in apremature stop codon (Figure 1C). A genomic constructcovering 2.5 kb of the promoter and the entire coding regionof At2g28230 was introduced into the mutant—the morpho-logical phenotypes of the mutant were rescued in all 39independent transgenic lines (Supplementary Figure S1Cand data not shown). Therefore, the med20a mutation wasresponsible for the morphological defects. A homology-basedsearch identified two more paralogs in Arabidopsis:At2g28020 and At4g09070. Therefore, At2g28230,At4g09070, and At2g28020 were named MED20a, MED20b,and MED20c, respectively (Figure 1C). MED20a and MED20bhave 85% identity at the nucleotide level. MED20c is shorterthan MED20a and MED20b; there is 93% identity at thenucleotide level in exon 2 among the three genes.Transcriptome studies using Affymetrix ATH1 microarraysfound MED20a and MED20c to be expressed throughout the

B

E

med20aCol

MED20a

Col med17

MED17

Col med18

MED18

UBQ5

RT(–)

A

Col

Col

med20a

med20a

med17

med17

med18

med18

nrpb2-3

C

MED20a

MED20b

MED20c

*med20a

D

MED17(At5g20170)

MED18(At2g22370)

med17

med18SALK_102813

SALK_027178

Figure 1 Isolation and characterization of mutants in Mediator genes. (A) Four-week-old plants of wild type (Col), med20a, med17, med18,and nrpb2-3. (B) Siliques from Col, med20a, med17, and med18 plants. (C) Schematic diagrams of MED20 paralogs MED20a, MED20b, andMED20c. The asterisk indicates the mutation causing a premature stop codon in themed20amutant. Black rectangles represent exons and linesrepresent introns. MED20a and MED20b are composed of three exons showing 85% identity at the nucleotide level. MED20c only encodes theconserved second exon compared with MED20a and MED20b. (D) Schematic diagrams of T-DNA insertion mutants of MED17 and MED18.Large triangles represent T-DNA insertions. Black rectangles represent exons. (E) RT–PCR analysis of MED20a, MED17, and MED18 expressionin med20a, med17, and med18 mutants, respectively. The images in the top row represent the indicated Mediator genes. UBIQUITIN5 (UBQ5)was used as an internal loading control. The ‘!RT’ reactions were performed with the UBQ5 primers. The PCR primers used are indicated bysmall arrowheads in (C, D).

Mediator in noncoding RNA productionYJ Kim et al

&2011 European Molecular Biology Organization The EMBO Journal VOL 30 | NO 5 | 2011 815

20

of the vascular system through the xylem. The pathogen then switches to a necrotrophic lifestyle

involving host wilting, stem browning, senescence and finally necrosis.

Recently, new research in the Schenk laboratory, has found that mutations in the MED18 and

MED20 subunits of the Mediator complex lead to surprisingly strong resistance to F. oxysporum

(figure 7). Initial qRT-PCR experiments suggest that the resistance of med18 plants seems to be

independent of JA signalling as the med18 mutant shows WT levels of PDF1.2 expression (data not

shown). We therefore would like to investigate the cause of resistance in the med18 and med20

mutants further using RNAseq expression analyses.

Figure 6: Typical disease phenotypes of wild-type (Columbia-0), med18 and med20a plants after

inoculations of the roots with F. oxysporum. The med18 and med20 mutants show resistance to F.

oxysporum and also delayed flowering.

21

Table 1: A comprehensive overview listing all of the known TFs interacting with MED25, MED8, MED18 and MED21 Mediator subunits, and the method by which they have been identified.

22

SUBSEQUENT CHAPTERS

Ph.D. Research Plan.

23

Research Rationale 1.3

Consistent with what has been discussed in the above literature review, there are fundamental

questions that arise in our understanding.

• For example, the mechanism of how Mediator subunits regulate gene expression in plants

through interaction with TFs is currently unknown.

• Transcriptional activation domains in TFs have long been identified through functional

testing, but how these domains interact on the Mediator surface is unknown.

• What is the role of additional subunits such as MED18 and MED20 in controlling plant

defence and particularly in defence against F. oxysporum?

Research aims 1.4

To date, the potential roles of Mediator subunits have not been systematically analysed in plants.

Research in the Schenk laboratory at UQ and the Kazan laboratory at CSIRO Agriculture has

shown that manipulating genes encoding individual Mediator subunits offers yet unexplored control

points to regulate innate immunity and other important physiological processes in plants. Therefore,

the first aim of this project is to identify and characterise the potential functions of the MED25

subunit by identifying new interacting proteins and characterising further its interaction with the

known MED25 interacting TFs. The interacting proteins are likely to belong to a set of transcription

factors which provide additional control points to regulate physiological pathways in plants. In

addition, other Mediator subunits, in particular MED18 and MED20 will be characterised for their

role in plant defence. The outcome of this project will not only be a new set of molecular tools

enabling us to better understand and bioengineer important traits in plants, but also will provide an

increased understanding of how Mediator-regulated gene expression functions in plants and in

humans.

The research objectives 1.5The following three objectives chosen to be investigated in the present PhD study are:

1. To investigate the domain required for MED25 subunit interaction in TFs.

2. To identify the role of MED18 and MED20 in regulating gene expression in med18 and

med20 mutants in response to F. oxysporum infection.

3. To explore stress response signalling of med18 and med20, particularly ROS, SA and JA

responses, and their function in F. oxysporum resistance.

24

1.5.1 Aims of the project

1.5.2 Aim 1 Investigating the domain for MED25 interaction in TFs.

• Perform Y2H assays with MED25 and its interacting TFs: ERF1, ORA59, DREB2A and

MYC2 to test whether we can identify the minimum domain for MED25 interaction

amongst different TFs.

• Using sequence comparisons to identify novel TFs that may interact with MED25

through the identified domain.

1.5.3 Aim 2 Identifying the role of MED18 and MED20 in conferring F. oxysporum resistance

• Characterise the resistance phenotype of med18 and med20 mutants by identifying the

extent of colonisation of F. oxysporum using microscopy at early time-points.

• Perform an RNAseq experiment to identify candidate genes that may be providing

resistance in these mutants. The RNAseq experiment will be performed with AGRF and

analysed together with bioinformatics support from CSIRO Agriculture.

• Investigate this resistance phenomenon further by screening mutants of the candidate

genes to confirm the source of resistance in the med18 and med20 mutants.

1.5.4 Aim 3

Gene expression profiling of JA, SA and ROS marker genes in med18 and med20 mutants infected with F. oxysporum.

• Time course experiment for jasmonate acid and salicylic acid treatment.

• Perform qRT-PCR to measure the gene expression of some JA, SA, ET and ROS related

genes.

• Study the ROS activity of both mutants after F. oxysporum infection.

25

Research Timeline 1.6Research activity 20

13 2014 2015 2016

Q2

Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1

• Chapter1: Literature review.

• Chapter2: Investigating the minimum domain for MED25 interaction in TFs

• Determine the minimum domain for MED25 interaction

• Identify novel TFs contain CMIXc conserved motif

• Test binding capability using Y2H

Mileston 1:PhD Confirmation document Chapter3: Identifying the role of med18 and med20 in conferring F. oxysporum resistance

• Investigate other MED subunits that have roles similar to MED25 in flowering time and plant defence by Y2H

• Bimolecular Fluorescence Complementation (BiFC) assay

• Gus measurement (MUG assay) •

• Look at colonization of F.oxysporum in med18 and med20 roots.

• Perform RNAseq of the WT and med18 roots

• Disease resistance assays with F.oxysporum

Mileston 2:PhD Mid-candidature review Chapter4: Gene expression profiling of JA, SA and ROS marker genes in med18 and med20 mutants.

• Real time qRT- PCR following RNA-

seq • Real time qRT-PCR to study ROS

related genes. • GUS production in med18 and med20

• Methyl jasmonate and salicylic acid treatment

Mileston 3:PhD thesis reviewand and final write-up

26

2 Chapter II:

Investigating the minimum domain for MED25 interaction in TFs.

27

Introduction 2.1

The MEDIATOR25 (MED25) subunit is one of a few genetically characterised Arabidopsis

Mediator subunits and it illustrates the various functions that one Mediator subunit can have in

diverse plant activities. MED25 has not only been shown to have role in organism development and

propagation (Xu and Li, 2011), but also in numerous abiotic and biotic stress responses (Kidd et al.,

2009; Elfving et al., 2011; Iñigo et al., 2012; Sundaravelpandian et al., 2013; Raya-González et al.,

2014; Carvalhais et al., 2015; Seguela-Arnaud et al., 2015).

MED25 has been shown to interact with numerous TFs that belong to several families including

AP2/ERF, bHLH, MYB, WRKY, bZIP and zinc finger. Thus, MED25 in Arabidopsis takes an

important part in regulating various physiological activities including the JA-dependent defence

response (Chen et al., 2012, Elfving et al., 2011, C evik et al., 2012, Ou et al., 2011).

Moreover, a study done by Cevik and colleagues (2012) illustrated that MED25 physically interacts

with several key transcriptional regulators of the JA signalling pathway, including ERF1 and

ORA59 as well as the master regulator MYC2. Physical interaction detected between MED25 and

four group IX AP2/ERF transcription factors was shown to require the activator interaction domain

of MED25 as well as the recently discovered Conserved Motif IX-1/EDLL transcription activation

motif of MED25-interacting AP2/ERF.

There are eight members of the AP2/ERF IXc subfamily. Cevik et al., (2012) identified six of the

eight in a large screen of Arabidopsis TFs, but ERF14 and ERF96 were not identified and have not

been studied before. Therefore in this thesis I have examined three members of the AP2/ERF IXc

subfamily, ERF1 (as a positive control), as well as ERF14 and ERF96. Figure 3 (Page 17) and

Figure 9 (Page 34) show the members of the IXc subfamily with and without the ELLEL motif

This chapter aimed to identify new interacting proteins that have the Conserved Motif IX-1/EDLL.

Also investigated was the minimum domain required for the interaction between MED25 and

ERF1. To achieve this, the interaction results were compared with the known MED25 interacting

TFs. Determining the minimal protein domain that is necessary for activation of MED25 may

enable the creation of engineered activation domains and provide additional control points to

regulate physiological pathways in plants.

28

Materials and Methods 2.2

2.2.1 Determine the minimum domain for MED25 interaction

A cDNA sequence encoding amino acids 551–680 of A. thaliana MED25 (ACID) was amplified by

PCR using the appropriate forward and reverse primer pairs (Table 1). Also, the nucleotide

sequence corresponding to amino acids ERF1 1-138 and ERF1 138-218, DREB2A 1-168 and DREB2A 168-

335, and MYC2 1-188, MYC2 149-188, MYC2 189-624 and MYC2 100-200, were amplified from Arabidopsis

cDNA using the appropriate forward and reverse primer pairs (Table 2). All PCR products were

cloned into the pCR8 vector using the pCR 8/GW/TOPO TA Cloning Kit (Invitrogen), and the Pure

Yield Plasmid Miniprep System kit (Invitrogen) was used to purify plasmids according to the

manufacturer’s instructions. All plasmid constructs were confirmed by restriction analysis and

sequencing. The PCR conditions, plasmid cloning and purification steps were as stated previously.

Table 2: Primers that were used in amplification of the different fragments of TFs. F= forward

primer, R= reverse primer.

Gene name Primer

type

Primer

Med 25 F 5′-ACTTCACAATCCAAATATGTG

(ACID) R 5′-ATTTGGAATTTGTGGTTTAA

ERF11-138 F 5’-ATGGATCCATTTTTAATTCAGTCCCC

R 5’-TCGGCGGAGAGAGTTCAAGA

ERF1138-218 F 5’-TCGGCGGAGAGAGTTCAAGA

R 5′-TCACCAAGTCCCACTATTTTCAGA

DREB2A1-168 F 5′-ATGGCAGTTT ATGATCAGAGTGGA

R 5′-ATCTGGATCCTCTGTTTTCACATG

DREB2A168-335 F 5′-GATTGTGAATCTAAACCCTTCTCC

R 5′-TTAGTTCTCCAGATCCAAGTAACTC

MYC21-188 F 5′-ATGACTGATTACCGGCTACAACCA

R 5′-TGCAAACGCTTTACCAGCTAATCC

MYC2149-188 F 5′-GGTGTTGCTCCGTCGGATGACG

R 5′-TGCAAACGCTTTACCAGCTAATCC

5’- -3’

29

MYC2189-624 F 5′-ACGGGTAACGCGGTTTGGGTT

R 5′-TTAACCGATTTTTGAAATCAAACTTG

MYC2100-200 F 5′- CTCGGATGGGGAGATGGTTATTA

R 5′-TCATTGATCTGACCCGGAAACCC

2.2.2 Search for novel proteins containing the ELLEL motif A PROSITE scan (http://prosite.expasy.org/scanprosite/) using the ELLEL (ExLxxxxLExL) motif

was performed to identify Arabidopsis proteins that contain the ELLEL conserved motif and may

potentially interact with MED25.

2.2.3 Cloningintoentryvector

2.2.3.1 PCR amplification

Four candidate genes (RNA55, AGL, ERF14 and ERF96) that contain the ELLEL protein motif

were amplified by PCR. The PCR reaction was made up by adding 11.7 μL of dH2O, 4 μL of 5x

phire II buffer, 0.4 μL of 10mM dNTPs, 0.4 μL of phire II and 2 μL of (forward and reverse) gene-

specific primer pair (see Table 1) with 1 μL of cDNA template in each PCR tube. After that, the

tubes were incubated at 98 ºC for 5 min, 60 ºC for 20 s, then 72 ºC for 1.5 min. These incubation

periods were repeated for 40 cycles before the reaction was incubated at 72 ºC for 5 min.

2.2.3.2 Transformation

The PCR products were separated on a 1% agarose gel and the bands were cut and measured before

they were cleaned up using a Wizard ® SV Gel and PCR Clean Up System (Promega). After the

clean up, a mixture of 2 μL of 10x Taq Polymerase buffer, 1 μL of 10 mM dNTPs and 1.5 μL of

Taq Polymerase was added to all PCR tubes. Then these tubes were incubated at 72 ºC for 15 min.

Thereafter, each PCR product was ligated into the pCR8 vector using the pCR8/GW/TOPO® TA

Cloning ® Kit (Invitrogen) according to the manufacturer’s instructions and then transformed into

E. coli Top10 cells and plated on Lysogeny broth (LB) agar medium containing the antibiotics

spectinomycin with a final concentration of 50 µg/mL.

The reference vectors; pDEST32 and pDEST22 with MED25 or other TFs previously identified to

interact with MED25 were a kind gift from Volkan Cevik. A total of 2 μL of these plasmids were

30

transformed into competent DH5α E. coli cells for plasmid bulking. Cloned TF genes were

transferred from pCR8/GW/TOPO using LR Clonase II (Invitrogen) into either pDEST32 or

pDEST22 according to the manufacturer’s protocol. Finally, 150 μL of bacterial culture was spread

on LB agar plates containing gentamycin with a final concentration of 10 µg/mL for pDest32

plasmids that contain MED25 fragments or Ampicillin with a final concentration of 100 µg/mL for

pDest22 plasmids that contain the different TF genes and plates were incubated overnight at 37 ºC.

Table 3: Primers that were used in amplification of the different genes that contain the ELLEL protein motif. F= forward primer, R= reverse primer.

Gene

name

Primer

F

type R

RNA55

ATGGATTCATCGCCACCGAACAC

CTATAATCTCAAATTCATTTCTTCTTC

AGL

ATGGCGAGAGAGAAGATAAGGA

TCATTCCCAAGATGGAAGC

ERF14

ATGGATCAAGGAGGTCGTAGC

TTATTGCCTCTTGCCCATGT

ERF96

ATGGATCAAGGAGGTCGAGGT

TCATTTCTTCTTGCCCTTGTT

2.2.3.3 Plasmid purification

10 colonies of each plate were streaked out into a new plate and incubated overnight at 37 ºC. The

next day, four colonies from the new plates were inoculated into 5 mL of LB liquid medium in a 50

mL Falcon tube and incubated on a shaker overnight at 37 ºC to grow. The tubes contained either

the antibiotics spectinomycin, gentamicin or ampicillin. Next, the Pure Yield Plasmid Miniprep

System kit (Invitrogen) was used to purify plasmids according to the manufacturer’s instructions

with the exception of using nuclease-free water in eluting the plasmid DNA instead of TE buffer.

For the restriction digestion EcoRI restriction enzyme (New England Biolabs) was used to cut the

PCR8/ GW/ TOPO vector and BsrgI or BamHI restriction enzymes (New England Biolabs) were

used to cut pDest32 and pDest22 to confirm that the genes of interest were cloned into the plasmid.

5’- -3’

31

The plasmid DNA yields were measured by a spectrophotometer (NanoDrop® ND-1000). The

plasmid DNA was sent for sequencing to make sure that the reading frame of the entry clones is

correct for insertion into pDEST22.

2.2.4 Yeast (Saccharomyces cerevisiae) two-hybrid (Y2H) screening

2.2.4.1 Preparation of media

Three types of media were prepared to carry out Y2H screening. YPAD agar and liquid contained

20 g of Bacto Peptone, 20 g of agar (except for the liquid YPAD), 10 g of Yeast Extract, 50 mL of

40% glucose solution and 100 mg of adenine sulfate. The pH of these media was adjusted to pH 6

before they were autoclaved and poured into plates. Two forms of Synthetic Complete Medium

(SC) were made; SC-L-W-H which contains an amino acid mix that does not contain leucine,

tryptophan and histidine amino acids and SC-L-W which does not contain leucine and tryptophan

amino acids (Sunrise Science Products, San Diego, CA).

2.2.4.2 Yeast Two-Hybrid (Y2H) Screen.

To carry out Y2H screening of the TFs in the prey constructs with the MED25 (ACID) 551–680 in the

bait constructs, I co-transformed 10 μL of both bait and prey plasmids into the yeast strain

Saccharomyces cerevisiae cells (MaV203) according to the S. c. EasyComp Transformation Kit

system manual (Invitrogen). The transformed reaction was then plated into two different plates, SC-

Leu-Trp (SC_LW) plates (50 μL and 25 μL) and YPAD plates (10 μL and 1 μL). Selection of the

positive clones was carried out on SC-Leu-Trp-His plates, SC- Leu-Trp-His with 12.5 mM

analogue 3-amino-1,2,4-triazole (3-AT) and SC- Leu-Trp-His with 25 mM 3-AT plates.

2.2.4.3 Filter lifting assay The X-Gal filter assay was carried out on YPAD plates according to the protocol of Gietz et al.

(1997) with the exception of using 10.7 g of Na2HPO4x2H2O and 6.2 g of Na2PO4x2H2O instead of

16.1 g of Na2HPO4x7H2O and 5.5 g of Na2PO4xH2O respectively in the Z-buffer solution. The filter

papers were wrapped with a plastic wrap and incubated at 37 ºC until colonies turn blue.

32

Results 2.3

2.3.1 Identification of ELLEL motif containing proteins

Previous research identified six AP2/ERF TFs that interact with MED25 (Cevik et al., 2012). These

six TFs belong to the same sub class of AP2/ERF proteins (group IX) and share a common domain

in the C-terminus with a conserved sequence of ELLEL (see Figure 9B below). This motif had

previously been described to be a potent transcriptional activation domain and was termed EDLL

(Tiwari et al., 2012). In addition mutation of the last two L amino acids in the sequence inhibited

MED25 interaction. As the ELLEL amino acids were conserved amongst the six TF proteins, we

performed a PROSITE scan using the ELLEL motif and identified 48 Arabidopsis proteins that

contain the ELLEL motif and may potentially interact with MED25 (Figure 8). Included in this list

were additional ERF TFs, ERF96, and AGL24, as well as RNA helicase proteins and defence

associated proteins. ERF96 along with ERF14 are two AP2/ERF TFs from subgroup IX that had not

been identified in our original screens (Cevik et al., 2012). While ERF96 was identified by the

PROSITE scan, ERF14 TF was not identified as it contains a modification of the conserved

sequence replacing the last E with a D (Figure 9A). It is interesting to determine whether this motif

can still interact with MED25.

2.3.2 Cloning of novel ELLEL motif containing genes

To test whether the identified proteins interact with MED25, PCR amplification was carried out for

three different candidate genes that have the conserved ELLEL motif (RNA55, AGL, ERF96). These

proteins were chosen as they represented TFs or RNA associated proteins that may interact with

MED25. We also included ERF14 which was not identified by the PROSITE scan. The cloned PCR

products were of the expected size; RNA55 1498 bp, AGL 663 bp, ERF14 402 bp and ERF96 496

bp and were subsequently sequenced in their cloning vectors before transferring to the Gateway

compatible Y2H vectors pDEST22 and pDEST32.

33

Figure 7: (A) Phylogenetic tree of the proteins containing the ELLEL motifs identified using a

PROSITE scan of Arabidopsis proteins.

34

Figure 8: (A) The conserved motif ELLEL amino acid residues that are found in the carboxy-

terminal region sequence of RNA55, AGL, ERF14 and ERF96. The ELLEL motif is highlighted in

blue, and the non conserved D amino acid in ERF14 is highlighted in red. (B) The conserved

ELLEL motif identified in MED25 interacting proteins (Cevik et al., 2012); the EDLL motif

previously identified by Tiwari et al., (2012) is also shown in red, the conserved amino acids are

shaded in grey boxes.

2.3.3 Screening of MED25 and its candidate interacting TFs

To identify the interaction between MED25 and the proteins that contains the ELLEL conserved

motif, I carried out Y2H screening using MED25DvWF-A/Q-rich as bait construct and the ERF14,

ERF96, RNA55 or AGL24 as prey constructs. I also co-transformed ERF1 as a control prey

construct which has previously been shown to interact with MED25 (Cevik et al., 2012).

MED25DvWF-A/Q-rich consists of the MED25 activator interaction domain (ACID; amino acids

551–680), minus the vWF-A domain for Mediator interaction and the C-terminal Q-rich domain.

This domain was previously used to test interaction with the DREB2A TF and therefore the same

domain was chosen for the candidate interactions in this study (Blomberg et al., 2012). The bait and

the prey construct were co-transformed into MaV203 yeast and streaked out onto plasmid selective

media SC_LW plates and non selective YPAD plates. In the two previous media (SC- LW and

YPAD), we observed growth with ERF14, ERF96, RNA55 and AGL24 as well as the control TF

ERF1 as expected. In parallel I performed a serial dilution of the yeast colonies onto selective plates

A

! 22!

AGL as well as RNA helicase and defense associated proteins. The ERF14 TF contained a

modification of the conserved sequence replacing the last E with a D (Figure 3). It will be

interesting to determine whether this motif can still interact with MED25.

ERF1 VVVF E D L GEQY L E E L L

ERF15 LVVF E D L GAEY L E Q L L

ORA59 LVVL E D L GAEY L E E L M

TDR1 VFEF E Y L DDKV L E E L L

ERF091 LFEF E D L GSDY L E T L L

ERF95 VIEF E Y L DDSL L E E L L

: . : * * . * * * :

Conserved E L L E L

Figure 3. The conserved ELLEL motif identified in MED25 interacting proteins (Cevik et al.,

2012). The EDLL motif previously identified by Tiwari et al., (2012) is also shown in red. The

conserved amino acids are shaded in grey boxes

Cloning of novel ELLEL motif containing genes

To test whether the identified proteins interact with MED25, PCR amplification was carried out

for the 10 different candidate genes that have the conserved ELLEL motif (see figure 4 below).

DCL4, RNA49, DRL31 and RNA55 were amplified by using the erf6 cDNA samples as

templates. While AGL, RPS2, RNA18, FH6, ERF14 and ERF96 were amplified by using the WT

cDNA samples as templates. The sizes of PCR products were determined by Agarose gel

electrophoresis. Eight out of 10 genes were detected in the gel. No bands were detected for DCL4

and RNA49 genes although we performed six PCR reactions using a sensitive polymerase.

Compare to the ladder our candidate genes fragments were in the expected size; DRL31 2625

base pair (bp), RNA55 1498 bp, AGL 663 bp, RPS2 2730 bp, RNA18 1782 bp, FH6 2700 bp,

B

! 23!

ERF14 402 bp and ERF96 496 bp (see figure 5a, and 5b). We managed to get the others genes

that did not produce any bands under different annealing temperatures (Data not shown).

After amplification, we gel extracted the PCR products. As RNA55 produce two very close bands

we cut out both bands and added A-tails to the PCR products using Taq polymerase to allow

them ligate into PCR8 vector that has T in its open site. The transformation procedure was carried

out according to PCR TM 8/GW/TOPO® TA Cloning ® Kit (Invitrogen). After the incubation time,

we checked the plate and we noticed that there were more colonies in our candidate genes plates

compare to the control plates.

DCL4 E R L DEES L E H L

RNA49 E F L DFRN L E I L

DRL31 E S L HCPK LE T L

RNA55 E N L DFRN L E R L

AGL E D L DGLN L E E L

RPS2 E F L SREN L E R L

RNA18 E I L DFRN L E I L

FH6 E S L GAEL LE T L

ERF14 E Y L DDSV LD E L

ERF96 E Y L DDSV LE E L

* * * * *

Conserved E L L E L

Figure 4. Shows the conserved motif ELLEL amino acid residues that found in the carboxy- terminal

region sequence of DCL4, RNA49, DRL31, RNA55, AGL, RPS2, RNA18, FH6, ERF14 and ERF96. The

ELLEL motif is highlighted in blue, and the non conserved D amino acid in ERF14 is highlighted in red.

! 23!

ERF14 402 bp and ERF96 496 bp (see figure 5a, and 5b). We managed to get the others genes

that did not produce any bands under different annealing temperatures (Data not shown).

After amplification, we gel extracted the PCR products. As RNA55 produce two very close bands

we cut out both bands and added A-tails to the PCR products using Taq polymerase to allow

them ligate into PCR8 vector that has T in its open site. The transformation procedure was carried

out according to PCR TM 8/GW/TOPO® TA Cloning ® Kit (Invitrogen). After the incubation time,

we checked the plate and we noticed that there were more colonies in our candidate genes plates

compare to the control plates.

DCL4 E R L DEES L E H L

RNA49 E F L DFRN L E I L

DRL31 E S L HCPK LE T L

RNA55 E N L DFRN L E R L

AGL E D L DGLN L E E L

RPS2 E F L SREN L E R L

RNA18 E I L DFRN L E I L

FH6 E S L GAEL LE T L

ERF14 E Y L DDSV LD E L

ERF96 E Y L DDSV LE E L

* * * * *

Conserved E L L E L

Figure 4. Shows the conserved motif ELLEL amino acid residues that found in the carboxy- terminal

region sequence of DCL4, RNA49, DRL31, RNA55, AGL, RPS2, RNA18, FH6, ERF14 and ERF96. The

ELLEL motif is highlighted in blue, and the non conserved D amino acid in ERF14 is highlighted in red.

! 23!

ERF14 402 bp and ERF96 496 bp (see figure 5a, and 5b). We managed to get the others genes

that did not produce any bands under different annealing temperatures (Data not shown).

After amplification, we gel extracted the PCR products. As RNA55 produce two very close bands

we cut out both bands and added A-tails to the PCR products using Taq polymerase to allow

them ligate into PCR8 vector that has T in its open site. The transformation procedure was carried

out according to PCR TM 8/GW/TOPO® TA Cloning ® Kit (Invitrogen). After the incubation time,

we checked the plate and we noticed that there were more colonies in our candidate genes plates

compare to the control plates.

DCL4 E R L DEES L E H L

RNA49 E F L DFRN L E I L

DRL31 E S L HCPK LE T L

RNA55 E N L DFRN L E R L

AGL E D L DGLN L E E L

RPS2 E F L SREN L E R L

RNA18 E I L DFRN L E I L

FH6 E S L GAEL LE T L

ERF14 E Y L DDSV LD E L

ERF96 E Y L DDSV LE E L

* * * * *

Conserved E L L E L

Figure 4. Shows the conserved motif ELLEL amino acid residues that found in the carboxy- terminal

region sequence of DCL4, RNA49, DRL31, RNA55, AGL, RPS2, RNA18, FH6, ERF14 and ERF96. The

ELLEL motif is highlighted in blue, and the non conserved D amino acid in ERF14 is highlighted in red.

A

35

that lacked histidine (SC-LWH) with or without 12.5 mM 3-AT to assess the interaction with

MED25.

Our results from preliminary Y2H screening indicate that only AP2/ERF proteins belonging to the

group IX sub-class (ERF14 and ERF96) and not the other ELLEL motif containing proteins RNA55

or AGL can interact with MED25 (see Figure 10 below). Interestingly, ERF14 which has a non

conserved aspartate in place of a conserved leucine in the ELLEL motif, had reduced colony growth

relative to ERF96 and ERF1 constructs.

Figure 9: Y2H analyses of interaction between ACID domain of MED25 with the other ELLEL

proteins. Only ERF1, ERF96 and ERF14 showed positive results (growth) on SC-LWH 12.5 mM 3-

AT medium. Similar results were found on SC-LWH without the addition of 3-AT (data not

shown). ERF1 was used as a positive control for the reliability of our Y2H system. All transformed

colonies grew equally well on YPAD media (data not shown)

(+ve)

(-ve)

AD-ERF14

AD-RNA55

AD-ERF96

AD-AGL

AD-AGL

AD-ERF1

AD-GFP

-LW -LWH 12.5mM 3-AT

1 10-1 10-2 10-3

MED25-BD MED25-BD

1 10-1 10-2 10-3

36

2.3.4 Y2H Screening for protein’s fragments that interact with the conserved ACID domain in

Arabidopsis MED25.

To identify plant transcriptional regulators that operate by interaction with MED25, we used a bait

composed of amino acids 551–680 of Arabidopsis MED25, in a yeast-two-hybrid screen with prey

composed of different fragments of ERF1 (see Figure 11 below), DREB2A and MYC2. Cells were

plated on high-stringency media (SD-Trp/-Leu/-His, SD-Trp/-Leu/-His+12,5 AT, and SD-Trp/-

Leu/-His+ 25AT) and incubated at 30°C for 3 days. We localised the minimal domain of ERF1

required for interaction with MED25 551-680 to amino acids 138-218 (see Figure 12 below). We also

confirmed that the minimal domain of DREB2A required for interaction with MED25 551-680 is to

amino acids 169–254 (see Figure 12 below), however I was unable to determine the minimum

domain for MYC2 interaction as none of the three fragments that I tested were sufficient for

MED25 interaction (data not shown).

Figure 10: Schematic representation of the ERF1 protein. (A) full protein 218 aa. (B) ERF1 1-138

and (C) ERF1 138-218, the two fragments used in the Y2H analysis. The green box represents the

AP2/ERF binding domain while the red box represents the transcriptional activation domain (TAD-

The EDLL motif).

37

Figure 11: The Y2H analyses of interaction between ACID domain of MED25 with the different

fragments of ERF1 and DREB2A Arabidopsis TFs. Only ERF1 138-218 and DREB2A 168-335

fragments show positive results. Y2Hs were performed with SD-Leu-Trp as well as SD-Leu-Trp-

His supplemented with 12.5mM 3-AT. Positive interactions were confirmed with an X-Gal filter lift

assay.

.

Discussion 2.4

2.4.1 The role of ELLEL conserved motif for MED25 interaction

In this chapter, we examined the ability of Arabidopsis ERF transcription factors and ELLEL

containing proteins to interact with the Mediator complex through MED25. In 2007, the Mediator

complex was purified from Arabidopsis and identified as a new component of the transcriptional

machinery (Bäckström et al., 2007). The Mediator complex plays a key role as a universal adaptor

between the RNA polymerase II complex and gene-specific transcription factors to start

transcription (Malik and Roeder 2005). In addition, there are various biological activities that the

Arabidopsis Mediator complex has been discovered to control; including the abiotic and biotic

stress response, regulating of the organisms growth and development as well as controlling

flowering time and fertility (Kidd et al., 2011; Borggrefe and Yue, 2011; Mathur et al., 2011).

MED25(ACID)+BD-MED25(ACID)+BD- MED25(ACID)+BD-

(+ve)-(+ve)-(+ve)- (+ve)-(+ve)-(+ve)-GFP+BD-GFP+BD-GFP+BD-

SC+LW-SC+LWH-12.5mM-3+AT-

AD#ERF1(1+138-(

AD#ERF1-138+218-(

AD#DREB2A-1+168-(

AD#DREB2A-168+335-(

X+Gal-filter-assay--

X-Galfilterassay

(+ve)MED25(ACID)-BD

38

In agreement with this, the MED25 subunit has been shown to have a role in controlling plants

development (Xu and Li, 2011) and flowering time (Cerdán and Chory, 2003; Wollenberg et al.,

2008), but also in numerous abiotic and biotic stress responses (Kidd et al., 2009; Elfving et al.,

2011). In recent studies the MED25 subunit has also been shown to physically interact with several

key transcriptional regulators of the JA and ET defence signalling pathways by the ELLEL

conserved motif (Cevik et al., 2012). Therefore, in the chapter of my study, we investigated whether

a small subset of Arabidopsis proteins that contain the ELLEL conserved motif is able to interact

with MED25 in yeast 2 hybrid (Y2H) experiments.

We initially chose four proteins to study, based on their reported roles in transcription. Two of the

four candidate proteins (ERF14 and ERF96) belong to the subgroup IX family of AP2/ERF TFs that

are well known for their role in plant pathogen defence (Camehl and Oelmüller, 2010). Both ERF96

and ERF14 have published roles in plant defence signalling (Catinot et al., 2015; Oñate-Sánchez et

al., 2007), with ERF14 demonstrating a role in resistance against pathogens including Fusarium

oxysporum (Oñate-Sánchez et al., 2007) as well as being involved in the beneficial interaction

between Arabidopsis and Piriformospora indica (Nakano et al., 2006; Oñate-Sánchez et al., 2007).

Previous research showed six members of the AP2/ERF subgroup IX interact with MED25 through

the ELLEL conserved motif (Cevik et al., 2012). Therefore we hypothesised that these two proteins

would also interact. However, the ELLEL motif of ERF14 has one amino acid different from the

other members of group IX from the AP2/ERF TF family. Interestingly, it did not grow as strongly

as the other related AP2/ERF ERF1 and ERF96 (Figure 10). This could potentially suggest that the

binding of ERF14 to MED25 is either weak or transitory and may suggest that the ELLDL motif of

ERF14 is not optimal for MED25 binding. Further experimentation on the proteins using in vitro

binding studies or additional in planta transcriptional activation assays is needed to make firm

conclusions about the interaction strength of the ERF14 motif. Additionally, further experiments,

such as protein pull-downs or bimolecular fluorescence complementation experiments are required

to verify that the proteins physically interact in vivo with each other through the ELLEL conserved

motif. Interestingly, the MED25 ACID domain did not interact with AGL or RNA55. AGL

(At4g24540) is a MADS-box TF that has a role in flowering time (Torti and Fornara, 2012), and

RNA55 (At1g71280) is an uncharacterised DEA(D/H) box RNA helicase. We hypothesised that

these might be good candidates to test for an interaction with MED25, given MED25’s role in

transcription and flowering time. While a negative interaction in Y2H is not conclusive evidence of

non-interaction, the results suggest that the ELLEL motifs in these proteins are not sufficient for

interaction. Therefore either amino acids adjacent or within the ELLEL motif might be required or a

39

general topology of the surrounding protein may also play a role in interaction. These results are

intriguing, given that just 24 aa of the ELLEL domain of ERF98 were shown to activate

transcription when fused to a DNA binding domain (Tiwari et al., 2012). Further investigation of

the key amino acids of this domain, such as the amino acid changes in ERF14 will potentially

provide opportunities to modify a strong activation domain to a potentially weaker domain that

could be attached to proteins of interest for fine-tuned expression in plants through the MED25

subunit of the Mediator complex.

2.4.2 Screening for protein’s fragments that interact with the conserved ACID domain in

Arabidopsis MED25.

To further explore the transcriptional activation domain that interacts with MED25 we aimed to

clone the minimum protein domains of different TFs (ERF1, DREB2A, and MYC2) to test their

binding strength in vitro with MED25 purified from E. coli. We wished to determine the minimum

protein domain necessary to assist future studies such as NMR heteronuclear single quantum

coherence spectroscopy. To start this work we cloned fragments of the TFs for Y2H interaction. It

was previously reported that the transcription activation domain of DREB2a is located to amino

acids 254–335 (Sakuma et al., 2006) but the minimal domain required for interaction with MED25-

ACID in Y2H assays is located to amino acids 168–254 ( Elfving et al., 2011). In a study done by

Blomberg et al. (2012) they found that a fragment of DREB2a (residues 168–335; DREB2a 168–

335), which includes both the TAD and the MED25 interaction domain was required to bind

MED25-ACID with a high affinity in vitro (Blomberg et al., 2012). Also Chen et al. (2012) have

shown that the transcription activation domain of MYC2 is located to the residues 149–188 and

required the full length of MED25 (Chen et al., 2012).

In the current study, we localised the minimal domain of ERF1 required for interaction with

MED25-ACID to residues 138-218 (see Figure 12). We also confirmed that the minimal domain of

DREB2A required for interaction with MED25-ACID is amino acids 169–254. These data support

the finding by Blomberg et al. (2012). On the other hand, our result shows that the ACID domain of

MED25 is not enough for MYC2 interaction as this TF required the full MED25 protein for its

interaction, as stated by Chen et al. (2012). Our data also show that the AP2/ERF DNA binding

domain of ERF1 is not required for its interaction with MED25, whereas the transcriptional

activation domain (TAD-The EDLL motif) is important for interaction with MED25. More

investigations using smaller fragments of ERF1, such as ERF1178-218 and ERF1195-218 are to be done

40

to confirm this result. Although the DREB2A1-168 fragment showed a positive result in the X-gal

assay, there is no interaction between this sequence and MED25 (ACID) in the selective SC-LWH

plates. The positive result may stem from ACID overexpression, and the SC-LWH+3AT result

confirmed that (see Figure 12).

Conclusion 2.5In conclusion, the Y2H results suggest that the ELLEL conserved motif previously involved in

transcriptional activation is important for interaction of ERF14 and ERF96 to MED25. Although

the ELLEL conserved motif has been found in different proteins that belong to a variety of families,

only members from the subgroup IXc AP2/ERF family can interact with the MED25 domain.

Screening the other members of this family in the Y2H system will provide a framework to

determine which variations of the ELLEL motif bind and which do not. Further investigation of this

domain will potentially provide a strong activation domain that can attach to proteins of interest for

high expression in plants through the MED25 subunit. Further studies in other key motifs in

MED25 interaction could facilitate our knowledge of which motifs are required to control gene

expression through the MED25 subunit.

In addition, our results showed that the nucleotide sequence corresponding to amino ERF1 138-218

appear to be the key domain for its interaction with Med 25. This sequence represents the conserved

motif (CMIXc) from the IXc AP2/ERF family that previously has been reported for its importance

in transcriptional activation. With the identification of the minimum region of the ERF1 TF, it is

now possible to proceed to in vitro binding experiments. The interaction of TFs with Mediator

subunits has generated significant interest with several papers analysing both human and

Arabidopsis MED25 with TFs and viral activators (Vojnic et al., 2011; Milbradt et al., 2011;

Blomberg et al., 2012; Aguilar et al., 2014; Sela et al., 2013). Recent studies have analysed MED25

interaction with DREB2A and MYC3 in vitro (Blomberg et al., 2012; Zhang et al., 2015), and

interactions with ERF1 may assist in expanding our knowledge on how TFs from diverse families

containing different secondary structures are able to interact with the same ACID domain of

MED25. More investigation also needs to be performed in other members of the AP2/ERF super

family to determine what the key amino acids are, required for their interaction with MED25 and

other Mediator subunits.

41

3 Chapter IlI:

Identifying the role of MED18 and MED20 in conferring F.

oxysporum resistance

42

Introduction 3.1The Mediator complex plays an important role in plant defence and several plant Mediator subunits

have been identified with altered hormone and pathogen signalling (Samanta and Thakur, 2015).

For example, the MED25 subunit of the plant Mediator complex has been shown to be required for

jasmonate-dependent defence gene expression and resistance to leaf-infecting necrotrophic fungal

pathogens. A MED25 over-expressing line was found to be highly susceptible to F. oxysporum.

Therefore, MED25 has a key role in modulating plant susceptibility to F. oxysporum (Kidd et al.,

2009). As med25 mutants have reduced expression of defence genes, yet an increased resistance to

F. oxysporum, it indicates that F. oxysporum hijacks non-defensive aspects of the JA-signalling

pathway to cause wilt-disease symptoms that lead to plant death in Arabidopsis (Thatcher et al.,

2009). Kidd et al. (2009) also pointed out that although the med8 mutant displayed no change in the

expression of JA-associated marker genes, the double mutant med25 med8 exhibited additive

resistance over the single mutations. To investigate whether other head domain subunits play a role

in susceptibility to F. oxysporum, we investigated mutants in the head domain subunits MED18 and

MED20.

MED8, MED18 and MED20 from the yeast Mediator head module have been shown to depend on

each other for folding and complex formation (Shaikhibrahim et al., 2009). These subunitsbind to

the yeast TATA binding protein and other general transcription factors (Plaschka et al., 2015)

In this chapter I investigated if there is interaction between these Mediator subunits in plants by

using the methods of Y2H as well as Bimolecular Fluorescence Complementation (BiFC). I also

studied the role of med18 and med20 mutants in F. oxysporum susceptibility. Our results show that

there is an interaction between MED18 and MED20 suggesting that they interact within the plant

Mediator complex, but no interaction was found with MED8. In addition, infection with F.

oxysporum revealed that these two mutants have a high level of resistance compared to the WT and

other F. oxysporum resistant Mediator subunits. I also conducted an RNA sequencing analysis of

WT, med-18 and med-20 roots one-day post infection to identify root genes involved in defence

against F. oxysporum. Overall, our results suggest a potentially novel mechanism for susceptibility

to F. oxysporum that is conferred by the MED18 and MED20 genes.

43

Materials and Methods 3.2

3.2.1 Plasmid preparation To test the interaction between different plant Mediator subunits, the full length coding sequence of

MED8, MED8N (MED8 N-terminal sequence), MED18, MED20a, MED20b, MED20c, MED21

and MED25 were amplified with primers listed in Table 3 from cDNA. The PCR products were

cloned into the vector pCR8GW-TOPO according to the manufacturer’s instructions (Invitrogen).

All plasmid constructs were confirmed by restriction analysis and sequencing.

The bait constructs (BD-MEDs) were generated by LR recombination reaction with pCR8GW–

MEDs and pDEST32. The prey constructs (AD-MEDs) were generated by LR recombination

reaction with pDEST22 and each of pCR8GW–MEDs. The LR recombination reaction was done

using Invitrogen Gateway® LR Clonase II Enzyme kit according to the manufacturer’s instructions.

Pure Yield TM Plasmid Miniprep System kit (Invitrogen) was used to purify the plasmids according

to the manufacturer’s instructions. All plasmid constructs were confirmed by restriction analysis

and sequencing.

3.2.2 Y2H screening

To carry out Y2H screening of the MEDs in the prey constructs with the MED25 in bait constructs,

I co-transformed both of the bait and prey plasmids into the yeast strain S. cerevisiae (MaV203)

(Invitrogen) according to the S. c. EasyComp Transformation Kit system manual. The transformed

reaction was then plated into two different plates, SC-Leu-Trp (SC_LW) plates (50 μL and 25 μL)

and YPDA plates (10 μL and 1 μL). Selection of the positive clones was carried out on SC-Leu-

Trp-His plates, SC- Leu-Trp-His with 12.5 mM 3-AT and SC- Leu-Trp-His with 25 mM 3-AT

plates.

3.2.3 Filter lifting assay

The X-Gal filter assay was carried out on YPDA plats according to the protocol of Gietz et al.

(1997) with the exception of using 10.7 g of Na2HPO4x2H2O and 6.2 g of NaH2PO4x2H2O instead

of 16.1 g of Na2HPO4x7H2O and 5.5 g of Na2PO4xH2O, respectively, in the Z-buffer solution. The

filter papers were wrapped with a plastic wrap and incubated at 37ºC until colonies turned blue.

Table 4: List of primers that were used in amplification of the different Mediator subunits. F=

forward primer, R= reverse primer.

44

Gene name Primer

type

Primer

MED8 F ATGGAGACAC AGCCGCAG

R ACAAAGGCAC CAAAATCCTC AATAA

MED8N F ATGGAGACAC AGCCGCAG

R TTACTGCTGATTCGTCTGCATAT

MED20a F ATGCCCGTCAAGTGGGTT

R GTTCAAGCTGTGAGAGGCTAA

MED20b F ATGCCCGTCAAATGGCTTT

R ATTCAAGCTGGGAGAGGCTAA

MED20c F ATGGAAATAGCAAGAAAAGTGATGG

R GTTCAAGCTGTGAGTGGCTAA

MED 21 F ATGGATATAATCTCACAGTTGCA

R GAACATG AAGAAACCTGAATGA

MED 18 F ATGTCAATGGAGTGTGTGGT

R TTACAATGTAGTACCTCCTCCATC

MED25 F ATGTCGTCGG AGGTGAAACA

R GAGCTGGCTTCATGGGATA A

3.2.4 Bimolecular Fluorescence Complementation (BiFC) assay

The full-length MED8, MED18, MED20 and MED21 genes were cloned into either pSAT5A

DEST-cYFP-C1B (CD3-1097) or pSAT4A DEST-nYFP-C1 (CD3-1089) using LR recombination

reaction (Invitrogen) with pCR8GW–MEDs. To carry out Yellow Fluorescent Protein (YFP)

screening through infiltration, as the pSAT5-MEDs and pSAT4A-MEDs are not binary vectors, I

cut the MED-nYFP and MED-cYFP constructs using NotI/AgeI sites and then ligated into pGreenII

0229. The resulting vectors were transformed into Agrobacterium tumefaciens AGL1. The

infiltration of the constructs of interest was performed in Nicotiana benthamiana plants according

to the established protocol (Voinnet et al., 2003). The infiltrated plants were kept in long day (16:8

h day:night regime) growth cabinets for 2 days before observation with the confocal microscope

(Zeiss LSM700).

5’- -3’

45

3.2.5 Plant growth and growth conditions

The seeds of all Arabidopsis plant types used in this chapter were sown onto sterilised moist soil

(UC mix) in a 30-cell seedling tray and kept at 4ºC in the dark for 48 h. After the stratification

period, the trays transferred to the short day growth cabinets at 24ºC, with an 8 h photoperiod (160

µE m-2s-1) and humidity of around 60%, and 16 hours dark with temperature dropped to 21ºC and

humidity of 70%. After 2 weeks, seedlings were separated, and grown until the six to eight leaf

stage.

3.2.6 F. oxysporum inoculation

Arabidopsis plants were inoculated with F. oxysporum as described previously (Edgar et al., 2006).

Briefly, at 1 h after the start of the photoperiod (t = 0 h) the plants were gently uprooted and dipped

for 15 s in fungal spore suspension with a concentration of 106 spores/mL in water and then

replanted. Mock plants were dipped in water and replanted.

3.2.7 RNA sequencing experiment WT (Arabidopsis thaliana Col-0), med18 and med20 plants were used in this experiment. Root

tissues were harvested at 24 h after F. oxysporum inoculation or a mock treatment (three

independent biological replicates of twenty plants per genotype). Once the root samples were

harvested, the root tissue was snap-frozen in liquid nitrogen. The total RNA was isolated and

DNAse treated using an RNeasy Plant Mini kit (Qiagen) according to the manufacturer’s

instructions. RNA integrity was confirmed using the Agilent 2100 bioanalyser system (Agilent

Biotechnologies).

The RNA from F. oxysporum-infected samples was sent to the Australian Genome Research

Facility (AGRF) for library preparation and RNA- sequencing. Messenger RNA was selected using

Poly-A tail selection prior to preparation of 100 bp paired end read libraries. Sequencing was

performed on an Illumina HiSeq 2000 system generating an average of 23 million paired end RNA-

seq reads per sample. The initial analysis of RNAseq data was assisted by Jiri Stiller

(bioinformatics support from CSIRO Plant Industry) and Brendan Kidd (UQ).

3.2.8 Root colonisation analysis

med18, med20 and WT Arabidopsis plants were used in this experiment. At the age of three to four

weeks, plants were inoculated with F. oxysporum and replanted (three independent biological

replicates). Two days and three days post infection the plants were removed and washed carefully

46

with water to remove adhering soil particles. The root slides were observed under the confocal

microscope (Zeiss LSM700) using a 488nm laser line.

3.2.9 Disease resistance assays with F. oxysporum

To test whether the genes identified from the RNA-seq study play a role in defence against F.

oxysporum, I obtained T-DNA insertion mutants for, PEN2, TGG1 and TGG2 from the ABRC

stock center while the pen1pen2pen3 triple mutant was a kind gift from Mats Andersson. The seeds

were previously characterised mutants and were confirmed for homozygosity by PCR. The pen

mutants, for instance, are loss of function mutants due to a point mutation so I did not check for a

change in expression by RT-Q-PCR.”

Results and Discussion 3.3

3.3.1 MED18 and MED20 interact with each other in plant

To identify whether different subunits from the plant Mediator complex interact with each other

within the plant Mediator complex, I used a bait composed of either MED8, MED8 N-terminus,

MED18, MED20a, MED20b, MED20c, MED21 and Green Fluorescent Protein (GFP) in a two-

hybrid screen with prey composed of either MED8, MED8 N-terminus, MED18, MED20a,

MED20c, MED21 and GFP. Cells were plated on media (YPDA, SD-Trp/-Leu, SD-Trp/-Leu/-

His+12,5 AT, and SD-Trp/-Leu/-His+ 25AT) and incubated at 30°C for three days. MED20a,

MED20b and MED20c refer to paralogs according to the naming convention of Kim et al., (2011).

Our results show that only colonies corresponding to MED18 and MED20a displayed positive

results in the selected plates and also turned blue using the X-Gal assay on the YPDA colonies.

However, no interaction was found between MED8 with either MED18 or MED20 (Figure 13).

Previous studies in the yeast Mediator complex reported that these three subunits interact with each

other (Gugliemi et al., 2004; Lariviere et al., 2006; Shaikhibrahim et al., 2009) and they have been

shown to display similar pathogen and developmental phenotypes in Arabidopsis. Our result

potentially suggests that MED18 and MED20 may form a subdomain within the Arabidopsis

Mediator complex with roles distinct from that of MED8.MED20a, MED20b and MED20c refer to

paralogs corresponding to the naming convention of Kim et al., (2011).

47

3.3.2 Bimolecular Fluorescence Complementation (BiFC) assay To confirm the interactions between MED18 and MED20, I carried out a BiFC analysis. I co-

transformed the nYFP-MED18 with cCFP-MED20 or cCFP without MED genes into the leaves of

N. benthamiana. The obvious fluorescence in the nuclei was observed in both combinations of

nYFP-MED18 / cCFP-MED20 and nYFP-MED20 / cCFP-MED18 (Figure 14A and 14B),

suggesting that MED18 interacts with MED20 in vivo. No interaction was observed in the negative

control nYFP-MED18/ cCFP or nYFP-MED20/ cCFP (Figure 14C). As the Y-2-H and the X-GAL

assays show negative result of MED8 interaction with either MED18 or MED20, we did not pursue

it further.

3.3.3 med18 and med20 show different growth patterns to WT

To compare the growth and development of our mutants with WT, I measured the rosette diameter

of med18, med20 and WT plants before the F. oxysporum infection. There was a big difference in

growth between the WT and our mutants (Figure 15). However, med18 and med20 show similar

growth pattern. In addition, both mutants have thin and quite yellowish leaves compared to WT

plants (Figure 15C). Furthermore, these mutants displayed a delay in flowering time, especially

med20 with an increase in the number of leaves before flowering, as previously reported (Kim et

al., 2011). These results therefore confirm the role of MED18 and MED20 in controlling flowering

time. We initially expected that the pale green/yellowish leaves would lead to increased

susceptibility in these mutants so we were surprised that they were almost immune. However the

strong vein chlorosis phenotype induced by F. oxysporum is easily distinguished from the med18

and med20 leaves so it would not interfere with the detection of symptoms. We initially expected

that the pale green/yellowish leaves would lead to increased susceptibility in these mutants so we

were surprised that they were almost immune. However the strong vein chlorosis phenotype

induced by F. oxysporum is easily distinguished from the med18 and med20 leaves so it would not

interfere with the detection of symptoms. Nevertheless, more studies need to be carried out to test

wether these phenotypes play a role in the resistance/susceptibility of MED18 and MED20 to F.

oxysporum infection.

48

Figure 12: Y2H analyses of interaction between different Mediator subunits in Arabidopsis. (A)

The order of colonies in B-E. (B & F) show the growth of yeast colonies in normal YPAD media.

Only colonies corresponding to MED18 and MED20a showed positive results in the selected plates

(D, E, G & H) and also turned blue in the X-Gal assay (I). The order of colonies in F, G, H & I are

as following: 1&2: p22MED8+p32MED20a, 3&4: p22MED8N+p32MED20a, 5&6:

p22MED8+p32MED20c, 7&8: p22MED8N+p32MED20c, 9&10: p22MED8+p32MED18, 11&12:

p22MED8N+p32MED18, 13&14: p22MED8+p32MED21, 15&16: p22MED18+p32MED20c,

17&18: p22MED18+p32MED20a, 19&20: p22MED18+p32MED21, 21&22:

p22MED18+p32GFP, 23&24: p22MED8+p32 GFP, 25&26: p22MED20a+p32 GFP, 27&28:

p22MED21+p32 GFP. Y2Hs were performed with YPDA medium as well as SD-Leu-Trp-His

medium supplemented with 12.5 3-AT or 25 3-AT

SC-LW SC-LWH25mM3-ATSC-LWH12.5mM3-ATYPAD

A

B C D E

AD-MED21 AD-MED18 AD-MED20

AD-MED21 AD-MED8 AD-MED20

AD-MED21 AD-MED18 AD-MED8

AD-MED8 AD-MED18 AD-MED20

DBD-MED8

DBD-MED18

DBD-MED20

DBD-GFP

G H I J

G H I JIHGF

YPAD SC-LWH12.5mM3-AT

SC-LWH25mM3-AT

XGALassay

49

Figure 13: The in vivo interaction between MED18 and MED20 subunits in the leaves of N.

benthamiana. (A) nYFP-MED18 with cCFP-MED20. (B) nYFP-MED20 with cCFP-MED18. (C)

nYFP-MED18/ cCFP (-ve). The image on the left hand side is the fluorescence channel and the

right hand side is a transmitted light image of the leaf.

A

B

C

50

Figure 14: Growth and development characteristics of WT, med18 and med20. (A) The first two

columns are WT plants. The 3rd and 4th columns show med18 data. The last two columns show

med20 data. (B) Rosette diameter of WT, med18, med20. The y-axis indicates the plant size. The

result shown is from three independent biological replicates, each containing a pool of 20 plants.

(C) Both mutants have thin and quite yellowish leaves compared to WT plants.

A

WT med18 med20

C

51

3.3.4 med18andmed20rootsdisplayreducedcolonisationofaGFP-taggedF.oxysporum

The soil-borne fungus F. oxysporum causes vascular wilts of a wide variety of plant species by

directly penetrating roots and colonising the vascular tissue. To observe the invasive growth and

colonisation of F. oxysporum in wild-type and mutants, I inoculated 4 week-old plants with the

GFP-expressing F. oxysporum. 2-3 days after inoculation, roots were uprooted and gently washed

before microscopy observation. The confocal microscope scan readily detected hyphae of the GFP-

expressing F. oxysporum in the vascular tissue of WT roots. In contrast, less mycelial structures of

the GFP-expressing F. oxysporum were observed within the med18 and med20 tissues (Figure 16).

This observation suggests that the reduced colonisation of a GFP-tagged F. oxysporum in med18

and med20 might be the reason behind their resistance to F. oxysporum. Additional experiments

using a GUS-transformed F. oxysporum showed similar reductions in root colonisation in med18

and med20 relative to the WT (B. Kidd unpublished). Inoue and co-workers (2002) have shown that

the colonisation of F. oxysporum in tomato plants changes according to the days post infection.

They found that from 1 to 3 dpi, F. oxysporum hyphae started to colonise the root surface, growing

along the junctions of the epidermal cells and outlining the cell borders. By day 4, most of the root

surface was covered with dense mycelium, and only the root tips remained free of infection. By 5 to

6 dpi, mycelial growth expanded to larger areas along the root, and hyphae had colonised the

central cylinder and grew in the xylem vessels. During this time, hyphae also colonised the grooves

of cortex cells on the tertiary and secondary lateral roots (Inoue et al., 2002). Future studies of

different time points need to be performed to know when and where F. oxysporum pathogen spread

is arrested in med18 and med20 roots.

52

Figure 15: Confocal images for the colonisation of a GFP-tagged F. oxysporum in different plants

roots. (A) GFP-tagged F. oxysporum in the WT was clearly distinguishable from that of the med18

(B) and med20 (C), as it was found in more than 80% of WT roots, while med18( B) and med20(C)

showed reduced colonisation of the GFP-tagged F. oxysporum.

3.3.5 MED18 and MED20 regulate similar gene sets in Arabidopsis

To identify root genes that might be altered in med18 and med20 roots, I conducted an RNA

sequencing experiment using WT, med18 and med20 roots harvested 1 day after infection with F.

oxysporum. We chose this time point because we wanted to study the early defence response to F.

oxysporum infection, given we had observed penetration into the xylem by 2 days post infection.

Overall, according to the differential reads analysis by Cuffdiff, there were 1269 genes and 1818

genes in med18 and med20, respectively, that were differentially regulated by >2 fold and

significantly (FDR p<0.05) compared to the WT. Comparing the med18 and med20 RNAseq reads

A

B

C

53

to each other identified only 48 genes to be significantly different between the two mutants (21

expressed higher in med20 and 27 expressed higher in med18) (Figure 17). Comparing the

differentially expressed genes between WT and med18 or WT and med20 indicated a high ratio of

co-regulated genes (Figure 18). Thus the RNAseq analysis suggests MED18 and MED20 regulate

similar gene groups in Arabidopsis in response to F. oxsporum.

Figure 16: Venn diagram of differentially regulated genes between each Arabidopsis mutant and the

WT. The blue circle displays differentially expressed genes between WT and med18 (709 expressed

higher in WT and 560 expressed higher in med18), while the yellow circle represents genes that

were differentially regulated between WT and med20 (1007 expressed higher in WT and 811

expressed higher in med20). Most differentially expressed genes are shared between med18 and

med20. Therefore med18 and med20 have similar gene expression profiles.

54

A B

Figure 17: Genes induced or repressed in each Arabidopsis mutant and the WT at early time-points

after F. oxysporum infection. (A) Genes that were significantly up-regulated greater than 2-fold by

F. oxysporum infection in WT vs. either med18 or med20. (B) Genes that were significantly

induced greater than 2-fold by F. oxysporum infection in mutant(s) vs. WT.

3.3.6 med18 and med20 display different responses to F. oxysporum infection compared to

WT Arabidopsis plants

To investigate the genes that were expressed differentially in each mutant compared to the WT, I

performed Gene ontology (GO) term singular enrichment analysis (Zhou et al., 2010). Comparing

genes that were significantly up-regulated greater than 2-fold by F. oxysporum infection in

Arabidopsis roots of our samples, we found that around 622 genes were up-regulated in WT vs.

either med18 or med20. Whereas only 483 genes were up-regulated in mutant(s) vs. WT (Figure

18). The genes induced in WT versus each mutant belong to numerous defences and stress related

GO terms that were significantly enriched including: response to chitin, response to fungus,

response to hormones (jasmonate and abscisic acid) and plant cell wall modification. Looking at the

gene lists themselves, we found pattern triggered immunity (PTI) associated genes such as

FLAGELLIN SENSITIVE 2 (FLS2), PLANT U-BOX 23 (PUB23) and MITOGEN-ACTIVATED

PROTEIN KINASE 3 (MPK3) as being induced higher in the WT, in addition to other genes that

associated with defence induction, such as the cell wall synthase-encoding genes (CESA4 and

CESA8), the plant defensin genes PDF1.4 and PDF2.2, as well as PROPEP1, a gene which

activates PDF1.2 and reactive oxygen species (ROS). Interestingly, amongst the genes induced in

the WT compared with our two mutants we identified a number of JA-associated genes. The

55

induced JA-associated genes list include the JA-associated transcription factor-encoding gene

MYC2, the JASMONATE ZIM DOMAIN (JAZ) repressor proteins; JAZ1, JAZ5, JAZ7, JAZ8 and

JAZ10, JA biosynthesis genes ALLENE OXIDE SYNTHATSE (AOS), ALLENE OXIDE CYCLASE

(AOC3), LIPOXYGENASE4 (LOX4), the galactolipase DONGLE, a JA-ile-hydroxylase (CYP94B3),

SULFOTRANSFERASE 2A (ST2A) which acts specifically on 11- and 12-hydroxyjasmonic acid and

VEGETATIVE STORAGE PROTEINS; VSP1 and VSP2 (Figure 19-21). While we cannot rule out

that many of the JA genes seen in the RNAseq data may be induced by the wounding process that

occured during the F. oxysporum inoculation process, we have previously shown that JA gene

expression is higher in inoculated vs. mock infected roots (Kidd et al., 2009). Therefore the

differential gene expression of JA-associated genes between either med18 or med20 and WT roots

in the RNAseq experiment suggests that the Mediator subunits are required for maintaining normal

JA responsive gene expression in processes such as wounding and F. oxysporum infection.

Although, there were a small number of significantly enriched GO terms in the list of genes induced

in med18 compared to WT, the med20 compared to WT list includes high numbers of differentially

expressed genes including those from the med18 enriched GO terms together with other GO terms

relating to oxidative stress and biosynthetic processes (Figure 19-21). Expectedly, the GO terms

associated with transcription and RNA regulation were enriched in the two mutants’ induced genes

which suggests that mutation of MED18 and MED20 changes transcription. Thus several TFs are

included amongst the differentially expressed genes. Other enriched GO terms included ion

transport and multi–drug transport. One of the transporters significantly induced was PDR12,

encoding a pathogen and defence hormone inducible ABC transporter (Campbell et al., 2003).

However, we were unable to find induction of defence responsive gene expression in the med18 and

med20 mutants that could explain the significant F. oxysporum resistance phenotype. Overall, the

WT responds to F. oxysporum infection with induction of PTI and JA-associated gene expression,

whereas the med18 and med20 mutants seem to be encumbered in the expression of these gene sets.

56

Figure 18: Top ten most significantly enriched GO terms for genes induced in Arabidopsis WT vs.

med18 or med20.

Figure 19: Top ten most significantly enriched GO terms for genes induced in med18 and med20 vs.

WT.

Top ten most significantly enriched GO terms For genes induced in the WT vs med18 or med20

response%to%sFmulus%response%to%chemical%sFmulus%lipid%localizaFon%response%to%stress%response%to%organic%substance%response%to%wounding%response%to%endogenous%sFmulus%response%to%jasmonic%acid%sFmulus%response%to%external%sFmulus%response%to%abioFc%sFmulus%

response%to%sFmulus%response%to%chemical%sFmulus%response%to%organic%substance%response%to%endogenous%sFmulus%response%to%jasmonic%acid%sFmulus%response%to%stress%response%to%hormone%sFmulus%response%to%chiFn%lipid%localizaFon%response%to%abioFc%sFmulus%

Enriched%in%WT%vs%med18% Enriched%in%WT%vs%med20%

Fairly%similar%lists%–%see%Excel%for%the%full%set%

57

Figure 20: Proportion of plant processes identified by GO term enrichment analysis in WT

compared to med18 or med20 in response to F. oxysporum infection in roots. (A) Biological

functions terms that were upregulated in WT vs. med20 (B) Biological functions terms that were

upregulated in WT vs. med18.

58

3.3.7 The role of glucosinolates in F. oxysporum resistance

One of the highest genes expressed in the WT compared to both med18 and med20 was the

thioglucoside glucohydrolase known as TGG2. . Similarly, TGG1 was also highly induced in the

WT compared to both mutants. TGG1 and TGG2 encode myrosinases that convert glucosinolates

into toxic metabolites to combat herbivores. In addition, glucosinolate breakdown products play a

role in defence against both leaf and root pathogens (Barth and Jander, 2006). To test whether

glucosinolates play a role in defence against F. oxysporum, we obtained T-DNA insertion mutants

for tgg1, tgg2, as well as pen2 and the triple mutant pen1/pen2/pen3 The three proteins PEN1, PEN2 and PEN3 (penetration-resistance) have been found to act as central components in cell wall-

based defence against the non-adapted powdery mildew Blumeria graminis fsp. hordei (Bgh). A

study found that loss of function mutations in any of the three PEN genes cause decreased

hypersensitive cell death triggered by recognition of effectors from oomycete and bacterial

pathogens in Arabidopsis (Johansson et al., 2014). PEN1 encodes a syntaxin that plays an important

role in vesicle fusion in plant immunity (Collins et al., 2003), while PEN3 is a ATP binding cassette

transporter (Stein et al., 2006). I then performed disease resistance assays with F. oxysporum.

Disease was measured by visually assessing symptom development on the leaves at different time

points post inoculation using a scale of 0–5 with 0 being asymptomatic and 5 being dead as

described previously (Thatcher et al., 2012). At least 40 plants were assessed per line. Inoculation

of the mutant tgg2 with F. oxysporum revealed a relatively small but additive increase in disease

symptoms relative to the mutant tgg1 or WT (Figures 22A). By contrast, the triple mutant

pen1/pen2/pen3 showed no significant difference in F. oxysporum survival however interestingly

the single mutant pen2 was more susceptible (Figure 22B). At 17 days after inoculation, the

increased susceptibility of pen2 single mutant was more evident, with a noticeable difference in the

survival rate and overall vigor of the triple mutant pen1/pen2/pen3 compared with pen2 lines

(Figure 22B). It is possible that PEN1 and PEN3 might have opposite roles to PEN2 in F.

oxysporum resistance, however we consider this unlikely as its previously hypothesized thatPEN2

and PEN3 act in a similar pathway separate to PEN1. However it was shown that the triple pen

mutant has significant additive effects when it comes to plant defence and the hypersensitive

response (Johansson et al., 2014), so this may explain the difference in susceptibility between the

pen2 and pen triple mutant. More studies on the role of these two mutants in plant susceptibility to

F. oxysporum need to be undertaken. In both assays, tgg2 and pen2 showed increased disease

symptoms relative to WT, which suggests that these two genes may play an important role in

protecting the plant during F. oxysporum infection. However, given that the expression of TGG1

59

was reduced in the med18 and med20 mutants and this leads to increased susceptibility, these genes

are therefore not responsible for the increased resistance observed in the med18 and med20 mutants.

In addition to TGG1 and TGG2, a number of other glucosinolate modulating genes were

differentially regulated in the med18 and med20 gene sets. INDOLE GLUCOSINOLATE O-

METHYLTRANSFERASE 5 and FLAVIN-MONOOXYGENASE that encodes a glucosinolate S-

oxygenase were up-regulated along with MYB34/ATR1 encoding a transcription factor involved in

activation of the tryptophan metabolic pathway upstream of glucosinolate production.

One of the most highly expressed genes in med18 and med20 relative to the WT was the

cytochrome CYP79F1. CYP79F1 metabolises mono- to hexahomomethionine, resulting in both

short- and long-chain aliphatic glucosinolates. Differential regulation of these genes suggests that

glucosinolate profiles in med18 and med20 might be affected and may contribute to altered

resistance to F. oxysporum. It has previously been shown that the tryptophan pathway is induced in

response to root pathogens F. oxysporum and Verticillium longisporum (Kidd et al., 2009; Iven et

al., 2012).

Despite up-regulation of the TRP pathway, Iven et al. (2012) did not find an increase in potentially

antifungal glucosinolate breakdown products in the roots of V. longisporum infected Col-0 plants.

However, indole-3-carboxylic acid and the phytoalexin, camalexin, which are derived from TRP

metabolism were induced in response to infection, and camalexin was found to be induced to levels

that could potentially inhibit V. longisporum growth. Therefore, metabolite profiling of both

aliphatic and indole glucosinolates is required to assess their contribution to resistance in med18

and med20.

60

C

Figure 21: Disease symptoms and scores of tgg1, tgg2, pen2, pen1/pen2/pen3 plants after F.

oxysporum infection. (A) The disease score of WT, tgg1 and tgg2 plants at 8, 12 or 15 days after F.

oxysporum inoculation. (B) The disease score of WT, pen2 and pen1/pen2/pen3 plants at 7, 10, 13,

17 or 20 days after F. oxysporum inoculation. (C) Representative disease symptoms characteristic

of the 0-5 scoring system.

0

0.5

1

1.5

2

2.5

3

3.5

4

day8 day12 day15

disease

score

wt

tgg1

tgg2

0

1

2

3

4

5

6

day7 day10 day13 day17 day20

disease

score

wt

pen2

pen1+2+3

A

B

61

Conclusion 3.4To sum up our results in this chapter, I present evidence that MED18 and MED20 interact with one

another in the Arabidopsis Mediator complex and mutation of these genes present similar

developmental and defence associated phenotypes. MED8, MED18 and MED20 interact in the

yeast Mediator complex to form a sub domain in the head module and are thought to be dependent

on each other for correct folding (Shaikhibrahim et al., 2009). We did not find an interaction with

MED8 and either MED18 or MED20, however it is possible that the Arabidopsis MED8 protein is

not correctly folding in yeast. Further work using co-immunoprecipiation might determine whether

MED8 does interact with the Arabidopsis MED18-MED20 dimer. While the increased rosette and

delayed flowering phenotypes are similar to the previously characterised med8 mutant, the F.

oxysporum resistance of med18 and med20 is much stronger, suggesting that MED18 and MED20

are the primary subunits involved in conferring F. oxysporum susceptibility, and F. oxysporum

susceptibility in med8 may be through preventing MED18 and MED20 from properly interacting

with the Mediator complex. These results might suggest that MED18 and MED20 can partially

interact with the Mediator complex in the absence of MED8. While further work is needed to

properly assess the impact that removal of Mediator Head domain subunits such as MED18 and

MED20 has on plant transcription, the results presented here confirm the role of MED18 and

MED20 in controlling growth, development and flowering time and reveal a new role for MED18

and MED20 in susceptibility to F. oxysporum and controlling the expression of glucosinolate and

JA-associated genes.

62

4 Chapter VI:

Gene expression profiling of JA, SA and ROS marker genes in

med18 and med20 mutants.

63

Introduction 4.1A vast array of microorganisms surround plant roots in the rhizosphere. These microorganisms can

be either harmful or beneficial to plants (Whipps, 2001). Thus plants have developed basal

resistance, also known as innate immunity. There are two layers of sophisticated surveillance basal

resistance: pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-

triggered immunity (ETI) (Jones and Dangl, 2006). PAMPs include microbes’ cell wall components

or specific pathogen proteins (such as bacterial flagellin). PTI is activated when PAMPs of potential

dangerous pathogens have been recognised by PAMP receptors in plants (Nicaise et al., 2009).

PAMP receptor activation leads to reactive oxygen species (ROS) production (Lamb and Dixon,

1997), activation of the MAPK signalling cascades which then leads to hormone production and

defence gene expression (Nicaise et al., 2009), phytoalexins and increased ion flux.

In the previous chapter, the RNA-seq experiment identified numerous genes that play important

roles in the expression of JA-associated genes and glucosinolate biosynthesis. In this chapter I

studied the gene expression of JA, SA and ROS marker genes in med18 and med20 mutants in

response against F. oxysporum as well as after hormone treatment using RT-Q-PCR. We

hypothesised that increased SA signalling provides a better and earlier response to infection by this

hemi-biotrophic pathogen through hypersensitive response (HR) and programmed cell death.

64

Methods: 4.2

4.2.1 Plant growth and growth conditions

The growth condition remained the same as detailed in the previous chapter.

4.2.2 Methyl jasmonate, ethylene and salicylic acid treatment

Methyl jasmonate (MeJA) treatment was performed by spraying a piece of cotton wool with MeJA

in ethanol with a final concentration of 4 µM/L of air. The cotton was placed inside a sealed

phytocon vessel containing the plants. Leaf tissues were collected after 24 hours for gene

expression studies.

Salicylic acid (SA) treatment was performed by lightly and evenly spraying a solution of SA with a

final concentration of 100 µM over plants. Leaf tissues were collected at 24 hours after spraying for

gene expression studies.

Ethylene (ET) treatment was performed by injecting 20 mL of ethylene gas into each sealed 10 L

phytocon vessel containing the plants.

4.2.3 F. oxysporum inoculation

F. oxysporum inoculations were performed in the same manner as detailed in the previous chapter.

4.2.4 Primer design for RT-Q-PCR

Primers were designed using Primer3. The software finds PCR primers for RT-Q-PCR by searching

potential primer pairs against fixed parameters (Min Tm = 57, Max Tm = 63, optimum Tm = 60, Max

primer length = 24, amplicon length =100-200, the remaining parameters are as per default). The

selected primer pairs were checked by using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for

specificity. The primer pairs used in real time RT-Q-PCR are listed in Table 4.

65

Table 5: Primer pairs used in real time RT-Q-PCR. F= forward primer, R= reverse primer.

A Gene name Gene ID Primer

type Primer

MYC2 At1g32640 F TCATACGACGGTTGCCAGAA R AGCAACGTTTACAAGCTTTGATTG JAZ1 At1g19180 F CGGTAGCTTTGGAGATCTGAG R TCACAAGGGAATAAACTCATGG JAZ2 At1g74950 F CCTCTGGGACCAAAGAAGAT R CCATAACTCGACCACCGTAG JAZ3 At3g17860 F TTTATGATTCAACCCGCAAA R CGACATGGAAACTGCGTTAG JAZ4 At1g48500 F CAATCTTTTATGCCGGTTCA R CGGTTTAGCATGAGGTCCAT JAZ5 At1g17380 F CACGTAGAGCTTCCCTCCAT R TGGGAGGATAACGATGATGA JAZ6 At1g72450 F GGAACGCATTGCAAGAAGA R GGAAGATGACTACCGTGTTGG JAZ7 At2g34600 F GACTTGGAACTTCGCCTTCTT R TCTGAGATTCTTGCTTTGGTTG JAZ8 At1g30135 F CAGCAAAATTGTGACTTGGAA R TGGATTTCCAGAACTGGTTG JAZ9 At1g70700 F CACAATCTTTTATGGCGGAAC R TCCAGTTTCACCTTTCAAACC JAZ10 At5g13220 F ATCTCGTTCGGTTCCGTCTA R ACAGTTCCCGAAACGAGTTC JAZ11 At3g43440 F TTTGGTGGGAGTTTTAGCGTA R TTGTCTCAGTGGCTTTAGCTG JAZ12 At5g20900 F GCTGCACAGCCATTTCCTA R CTCGAGGAATCGTTGAAGC PDF1.4 AT1G19610 F TTCCACTGAGATGATGGCGG R GTGAAGCACGTTCCCATCTC PDF2.2 AT2G02100 F AGGGTACATGCGTGAGTGC R CTGGTGCAGAAGCAACGAC BID Primer F Primer R

PR2 TACCTTGCCAAGTCCATCGG AACTGGGAACGTCGAGGATG

PR5 AATGTCAAGCTGGGGA AGGTGCTCGTTTCGTC

VSP2 GAAAACCATCTTTGGGAACG CGGTTTTGGAGTCGTATTGG

GST6 GGGTACTGACTCCAAGGTGC TAACCTTAGCCCAAGCAGGC

5’- -3’

66

PR4 CGGACTACTGTTCTCCGACC ACGGCTTATCAGCATCCCAC

MPK3 TGCGCTTATTGACAGAGTTGC TTAGCTAAGGGCTGACGTGG

MPK6 GCGACTCTTAGCCATGGTGG CATAGCCGAACAAACGATGCC

RBOHD TTCGAGTGGTTCAAGGGAATAATG CGTACACACTCGTGCAATAATTGTG

RBOHB

AGGAAATGTACTTTCACTTTACATGTC

G

ATTGTAATGGTGAGACGTCAGAACAG

EX1 TCTGGTTTCCAGAGTTTCCTGC GATGAAATCCTTATCCACCCTTCC

OXI1 CCAAGAGATTTTTGCTGCAAGAC

CCTTAACCCATTCCCCACTAGTATTAT

C

ZAT12 CCTAACAACGACGCTTTGTCG GTCCCATCGGAAACTCCACTC

HSFA4A CCAGGGCTTGCTTTGAACC GGTTCATCGGGAAAGAACTCG

HSF1 TCCCAGATACCACAATTGACACG TGAATGCCTCTGGAACATTCTTC

MDAR1 TTGGGTTCAAGGTGGTAAAGTGG TCGAGCTTTGGCGACTTTAGC

MDAR2 GGAAAGTGGTTGGAGCATTTTTAG CACTTCAAGGCTCTCAACAGAAGG

MDAR3 CTGAAGCCTGGTGAACTCGC GGTCGGATTGACTTCGAGGTC

DHAR1 CTCTGACAAACCCCAGTGGTTC CAACGATGACGTCGGAATCA

APX4 GCAACAGAGGCTGATCCAGAAG CCAATCCAACAGCAATGAACTTATC

CATALASE

1 CGTGAAGCGTTTTGTTGAAGC

CGAGTTGCTAGTTTCTGTCCCAG

CATALASE

2 CTATCCGACCCACGCATCAC

TTCAGACGGCTTGCCAGC

VTC2 GATGGCAGCAAATTCAACTTCAC GGCATGCAAGGGAAGAACTG

HSP17 TCATGAGGAGGTTTCGGTTGC CTCTCCTGAACTTTCGGCACC

4.2.5 RT-Q-PCR analysis

Total RNA from roots for RT-Q-PCR analyses were isolated using the SV Total RNA Isolation Kit

(Promega). The concentration and quality of the RNA were measured with a spectrophotometer

(NanoDrop® ND-1000) and a 1% agarose gel, respectively. cDNA synthesis was performed with

67

0.2 µg root RNA in 8.7 µL, using the SuperScript™ III RT kit (Invitrogen) as follows. A total of

0.2 µL of 100 µM oligo-dT, 0.1 µL of 3 µg/µL random hexamers (Invitrogen) and 1 µL of 10 mM

dNTPs were added to a final volume of 10 µL. The mixture was denatured at 65°C for 5 min

followed by 2 min of chilling on ice. A total of 2 µL of 10x RT buffer, 4 µL of 25 mM MgCl2, 2 µL

of 0.1 mM DTT (Invitrogen) and 1 µL (200 U/µL) SuperScriptTM III Reverse Transcriptase were

added, followed by incubation at room temperature for 10 min. Tubes were then transferred to a

PCR machine and incubated at 50°C for 50 min and 85°C for 5 min. The resulting cDNA was

subsequently diluted to a final concentration of 20 ng/µL of input RNA for real time RT-Q-PCR.

Gene expression analysis by RT-Q-PCR was carried out in 384-well plates using an ePMotion

automatic pipetting (Eppendorf epMotion 5075) robot and an ABI ViiA7 Sequence Detection

System (Applied Biosystems). Each reaction contained 5 µL of SYBR green and 1 µL of 3 nM of

each gene-specific primer pair and 4 µL of cDNA template to a final volume of 10 µL. TheViiA7

cycling conditions were asfollows: 50°C for 2 min, 95°C for 10 min, then 40 cycles of: 95°C for 15

s, 60°C for 1 min.

The PCR primer efficiency (E) of each primer pair in each individual reaction was calculated from

the changes in fluorescence values (∆Rn) of each amplification plot, using LinReg PCR software

(Ramakers et al., 2003). E values for each gene were averaged across all samples, except in cases

where linear regression of amplification plots yielded a R2 value of less than 0.99, in which case the

derived E value for that sample was omitted from the calculation of mean E value. Amplification

plots were analysed using a threshold of 0.20 to give a cycle threshold (Ct) value for each gene and

cDNA combination. Gene expression levels relative to the Arabidopsis housekeeping genes β-

ACTIN 2 (At3g18780), β-ACTIN 3 (At3g53750) and β-ACTIN 7 (At1g49240) were calculated for

each cDNA sample using the following equation: Gene transcript levels relative to actin = (E

gene^(-Ct gene)) / (E Actin ^(-Ct Actin)). Three biological replicates and three technical replicates

were assessed per sample.

4.2.6 DAB Staining Detection of hydrogen peroxide was conducted using 3,3’-Diaminobenzidine (DAB) from Sigma-

Aldrich. Briefly, plants were inoculated with F. oxysproum spore suspension and the root tissues

were collected and mixed with 1 mL of the DAB liquid buffer solution with 30 µL of DAB liquid

chromogen. After staining, the plant tissues were rinsed with distilled water 5 times and then

observed under a microscope (Olympus BX60F5).

68

Results 4.3

4.3.1 JA signalling genes show altered expression in Arabidopsis med18 and med20 roots To independently confirm the results of RNAseq analyses, I performed real-time reverse

transcriptase quantitative PCR (RT-Q-PCR) on JA-associated genes that were differentially

expressed in the RNAseq experiment. Since the JAZ genes were found to be down-regulated in

both mutants (med18 and med20), we chose to investigate the expression of these genes as well as

the other members of the JAZ family.

The result illustrates that generally the expression of JAZ genes was higher in WT roots than med18

and med20 mutants (Figure 23). Some JAZ genes that were not identified in the RNAseq

experiment, such as JAZ6 and JAZ9, were found to have reduced transcript abundance in the

mutants. Furthermore, JAZ3, JAZ11 and JAZ12 had potentially higher expression compared with

the WT in the med20 mutant (Figure 23). MYC2 and two defensins PDF1.4 and PDF2.2 which

were identified in the RNAseq data to have reduced transcript abundance also had lower expression

in the mutants compared to WT using RT-Q-PCR. Therefore these results confirm the differential

expression for these genes noted from the RNAseq results. Lai et al. (2014) found increased

expression of the JA/ET marker gene PDF1.2 in med18, however we could find no significant

difference in PDF1.2 expression under our conditions (Davoine et al., 2015, submitted). However

we did observe decreased MYC2 expression in med18 and med20 and decreased expression of

MYC2 target genes, such as VSP1, VSP2, ATR1 and the JAZ genes (Dombrecht et al., 2006; Thines

et al., 2007). Overall, these results suggest that some aspects of JA signalling are reduced in the

med18 and med20 mutants. Given the role of JA in promoting F. oxysporum susceptibility, reduced

JA signalling may be the cause of F. oxysporum resistance in these mutants. The RNA-seq and GO

analysis were only performed on the infected samples. The mock samples have not been sequenced.

Therefore there is a significant difference in JA gene expression between the mediator mutants and

the WT, however no change between mock and infected samples was seen in by RT-Q-PCR.

4.3.2 SA pathway associated genes show high expression in Arabidopsis med18 and med20

roots

Given the reduced expression of JA-associated genes, I looked at the expression of SA pathway-

associated defence genes in F. oxysporum-infected med18 and med20 roots. Interestingly, med20 F.

oxysporum infected roots showed a significant induction of SA-associated marker genes compared

69

to WT (Figure 24). RT-Q-PCR data showed that SA marker genes PR1 and PR2 were expressed

significantly higher in the med20 mutant compared to WT and med18, whereas, PR5 appeared to be

induced in both med18 and med20 (Figure 24). Therefore, the heightened expression of SA-

associated marker gene PR5 could potentially contribute to the increased resistance of the mutants

to F. oxysporum.

4.3.3 MeJA, ET and SA treatment of med18 and med20 leaf tissue

To examine how med18 and med20 mutants may be generally affected in hormone signalling, we

also looked at how defence genes may be altered in the med18 and med20 mutants compared to WT

plants with and without exogenous hormone treatment. The expression of a number of JA, ET and

SA-associated marker genes was examined after treatment with either MeJA, ET or SA,

respectively. Interestingly, RT-Q-PCR experiments in the leaves showed an increase in the

transcript abundance of the SA-associated gene PR1 in both med18 and med20. Although med20 F.

oxysporum infected roots showed a greater induction of PR5 (Figure 24), the expression of this

gene in SA treated leaves was equivalent to that of the WT-SA treated samples (Figure 25A). By

contrast, SA-associated marker genes PR1 and PR2 showed higher expression levels in med18

mutants in response to SA treatment (Figure 25A). Therefore, the effect of med18 and med20 on

SA-associated PR gene expression is different in leaf tissue when treated with exogenous

hormones. Separate experiments on the roots with exogenous hormone treatment should be studied

in the future to determine the role of MED18 and MED20 in SA signalling.

We also investigated whether JA and ET-responsive defence genes showed any differential

expression in the med18 mutant in the leaf tissue after hormone treatment. Interestingly, while JAZ

gene expression was attenuated in leaf tissue, MYC2 expression was not reduced significantly in

med18 leaves, relative to the WT (Figure 25B). Overall, it is likely that a reduction in JA-responses

in med18 may be providing an increased tolerance to F. oxysporum, as is seen with the coi1 mutant

(Thatcher et al., 2009), however these results also suggest that MED18 and MED20 regulate

different gene sets in the roots and shoots.

We also looked at gene expression after ET treatment, however we could not find significant

changes in the expression level of JAZ1, JAZ2, JAZ5, JAZ7, JAZ8, JAZ9, JAZ10, JAZ11, JAZ12 as

well as PDF1.4 and PDF2.2 (data not shown). Given that we did not find any significant change in

70

expression in PDF1.2, other ET-associated genes will have to be investigated in the med18 and

med20 mutant.

4.3.4 med18 and med20 show higher expression of ROS related genes

Reactive oxygen species (ROS) are produced in plant cells in response to diverse biotic and abiotic

stresses as well as during normal growth and development. To investigate more the reason behind

resistance to F. oxysporum in med18 and med20 mutants, I conducted RT-Q-PCR expression

analyses of selected ROS-responsive genes. As shown in Figure 26 several ROS and stress

associated genes were upregulated, particularly in med20. Genes induced higher in med20 after F.

oxysporum infection included those encoding the ROS producing RESPIRATORY BURST

OXIDASE HOHMOLOGS, RBOHB and RBOHD, the ROS scavenging enzymes

MONODEHYDROASCORBATE REDUCTASE1 and MONODEHYDROASCORBATE

REDUCTASE3, and VITAMIN C DEFECTIVE2. Also induced were the genes encoding the ROS

responsive marker OXIDATIVE SIGNAL INDUCIBLE1 (Petersen et al., 2009) and MAP KINASE6.

OXI1 is a positively regulator for ROS production, and activation of OXI1 is required for dual

activation of MAPK3 and MAPK6 (Rentel et al., 2004). MAPK3 however was not differentially

regulated in either of the mutants. Also, the gene encoding HEAT SHOCK FACTORA4A,

(HSFA4A) was upregulated in med20, however HSF1 was not (Figure 26). HSFA4A is a oxidative

stress inducible HSF and has been suggested to be a target of MAPK3 and MAPK6 phosphorylation

(Pérez-Salamó et al., 2014).

Genes induced in both mutants included ROS scavenging genes such as CATALASE genes (CAT1

and CAT2) MONODEHYDROASCORBATE REDUCTASE2, and DEHYDROASCORBATE

REDUCTASE1. Also induced in both mutants was EXECUTER1 (EX1) which is a plastid gene that

is required for singlet oxygen mediated cell death (Lee et al., 2007). While stronger gene expression

patterns were seen in med20 root tissues, overall, the up-regulation of CATALASE, DHAR1 and

MDAR2 genes in both mutants support the increased ROS production observed in med18 and

med20 roots. This may also explain the leaf bleaching phenotype that is observed in the leaves after

F. oxysporum infection. It is interesting to find that these genes were up regulated in med18 and

med20 F. oxysporum treated roots, suggesting that ROS responses to F. oxysporum are up-regulated

in med18 and med20 mutants and that this may have contributed to the increased resistance

observed for these mutants. The hypothesis is that med18 and med20 mutants have higher ROS

production under control conditions and the accumulation of ROS through normal development

71

leads to less chlorophyll, and pale yellowish leaves. ROS levels may increase further in response to

infection and have a positive effect on F. oxysporum resistance, however future work will need to

confirm this experimentally.

4.3.5 ROS production in the roots

Activation of SA defence genes can often be associated with increased ROS production and stress

responses in plants. To investigate and compare the involvement of ROS in F. oxysporum infection

in the WT, med18 and med20, I undertook DAB staining experiments to detect local ROS

production in the root. After 2 days inoculation with F. oxysporum, Arabidopsis seedlings showed

strong accumulation of fungal growth around the root tip regions as previously identified

(Czymmek et al., 2007; Kidd et al., 2011). Our results show that med18 and med20 roots produced

more ROS in response to F. oxysporum infection. Despite large amounts of F. oxysporum hyphae

surrounding the WT root, ROS production was barely detected in WT roots at this time point

(Figure 27). This suggests that in med18 and med20 ROS is activated more strongly in the root

during F. oxysporum infection compared to WT.

72

a

b b

c

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wt med18 med20 wt med18 med20

MOCK INFECTED

rela

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in

JAZ7

73

Figure 22: RT-Q-PCR data for different JA associated genes in Arabidopsis WT, med18 and med20

roots with and without F. oxysporum infection. Results were obtained from three independent

biological replicates and three technical replicates; oneway ANOVA, LSD significant difference

test (a,b,c&d) indicates p-value < 0.05; error bars represent standard deviation of the biological

replicates.

a"

b" b"

a"

b"

b"

0.000"

0.050"

0.100"

0.150"

0.200"

wt" med18"med20" wt" med18"med20"

MOCK" INFECTED"

rela;

ve"ab

undance"t

o"ac;n

"" JAZ8

a"

b" b"

c"

b" b"

0.000"

0.005"

0.010"

0.015"

0.020"

wt" med18"med20" wt" med18"med20"

MOCK" INFECTED"

rela;

ve"ab

undance"t

o"ac;n

"" JAZ9

a"

b" b"

c"

b" b"

0.000"

0.050"

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wt" med18"med20" wt" med18"med20"

MOCK" INFECTED"

rela;

ve"ab

undance"t

o"ac;n

"" JAZ10

a"b" bc"

ab"

cd" d"

0.000"0.010"0.020"0.030"0.040"0.050"

wt" med18"med20" wt" med18"med20"

MOCK" INFECTED"rela;

ve"ab

undance"t

o"ac;n

"" JAZ11

a"ab" ab"

a"

ab""""

b"

0.000"0.020"0.040"0.060"0.080"0.100"0.120"

wt" med18"med20" wt" med18"med20"

MOCK" INFECTED"

rela;

ve"ab

undance"t

o"ac;n

"" JAZ12a"

b" b"

a"

b"b"

0.000"0.500"1.000"1.500"2.000"2.500"

wt" med18"med20" wt" med18"med20"

MOCK" INFECTED"

rela;

ve"ab

undance"t

o"ac;n

"" PDF2.2

a"

b"b"

a"

b"

b"

0.000"

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wt" med18"med20" wt" med18"med20"

MOCK" INFECTED"rela;

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undance"t

o"ac;n

""

PDF1.4

0"

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wt" med18" med20"

ATGRXS13

a"a"

a"

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a"

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wt" med18"med20" wt" med18"med20"

MOCK" INFECTED"

rela;

ve"ab

undance"t

o"ac;n

"" JAZ8

a"

b" b"

c"

b" b"

0.000"

0.005"

0.010"

0.015"

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wt" med18"med20" wt" med18"med20"

MOCK" INFECTED"

rela;

ve"ab

undance"t

o"ac;n

"" JAZ9

a"

b" b"

c"

b" b"

0.000"

0.050"

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wt" med18"med20" wt" med18"med20"

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rela;

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undance"t

o"ac;n

"" JAZ10

a"b" bc"

ab"

cd" d"

0.000"0.010"0.020"0.030"0.040"0.050"

wt" med18"med20" wt" med18"med20"

MOCK" INFECTED"

rela;

ve"ab

undance"t

o"ac;n

"" JAZ11

a"ab" ab"

a"

ab""""

b"

0.000"0.020"0.040"0.060"0.080"0.100"0.120"

wt" med18"med20" wt" med18"med20"

MOCK" INFECTED"

rela;

ve"ab

undance"t

o"ac;n

"" JAZ12a"

b" b"

a"

b"b"

0.000"0.500"1.000"1.500"2.000"2.500"

wt" med18"med20" wt" med18"med20"

MOCK" INFECTED"rela;

ve"ab

undance"t

o"ac;n

"" PDF2.2

a"

b"b"

a"

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b"

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wt" med18"med20" wt" med18"med20"

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undance"t

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""

PDF1.4

0"

0.5"

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1.5"

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wt" med18" med20"

ATGRXS13

a"a"

a"

74

Figure 23: RT-Q-PCR data of SA related genes in WT, med18 and med20 roots with and without F.

oxysporum infection. Results were obtained from three independent biological replicates and three

technical replicates; oneway ANOVA, LSD significant difference test (a&b) indicates p-value <

0.05; error bars represent standard deviation of the biological replicates.

a a a aa

b

0.0000.2000.4000.6000.8001.0001.2001.4001.600

wt med18 med20 wt med18 med20

MOCK INFECTED

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in PR5

RT-Q-PCR data of SA related genes in WT, med18 and med20 roots with and without F. oxysporum infection. Results were obtained from three independent biological replicates and three technical replicates; oneway ANOVA, LSD significant difference test (a&b) indicates p-value < 0.05; error bars represent standard deviation of the biological replicates.

75

A

RT-Q-PCR data for hormone associated genes in WT, med18 and med20 leaves after SA treatment.; oneway ANOVA, LSD significant difference test (a&b) indicates p-value < 0.05

a a a

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0123456789

101112

wt med18 med20 wt med18 med20

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76

B

Figure 24:RT-Q-PCR data for hormone associated genes in WT, med18 and med20 leaves after MeJA, ET or SA treatment. (A) SA associated genes in WT med18 and med20 leaves after SA treatment; oneway ANOVA, LSD significant difference test (a&b) indicates p-value < 0.05 (B) JA and ET associated genes in WT med18 and med20 leaves after MeJA or ET treatment; asterisk (*) indicates p-value < 0.05 according to Student’s T test. RT-Q-PCR was performed using three independent biological replicates and three technical replicates; error bars represent standard deviations of the biological replicates.

JAandET

0.000

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77

a a aab

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78

Figure 25: RT-Q-PCR data of ROS-related genes shown as the different relative abundance in WT, med18 and med20 Plants. Results were obtained using three independent biological replicates and three technical replicates; error bars represent SD of three independent biological replicates.

a a a a

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79

4.4

Figure 26: DAB staining of WT, MED18 and MED20 roots. The roots were inoculated by F.

oxysporum, and the increase production of ROS in med18 and med20 roots can be seen compared to

WT roots by darker DAB staining.

DABstaining

WTroots

med18

med20

80

Conclusions 4.5The aim of this chapter was to unravel the cause of F. oxysporum resistance in med18 and med20

mutants. The colonisation of F. oxysporum in the roots of these two mutants was found to be less

extensive than for the WT. JA pathway genes were reduced in the RNAseq experiment. Here we

confirmed the role of med18 and med20 in JA signalling, as MeJA treated leaves of med18 and

med20 showed decreased expression of JA related genes compared to WT. In contrast, med18 and

med20 leaves exhibited increased expression of SA marker genes after SA treatment. Moreover,

RT-Q-PCR analysis on ROS-related genes as well as DAB staining in these two mutants’ roots

indicated increased production of ROS in F. oxysporum infected roots. We propose that med18 and

med20 mutants provide a strong ROS response to infection by this hemibiotrophic pathogen that

might restrict the pathogen from colonising med18 and med20 roots.

81

5 General conclusions

82

Since the purification of the Mediator complex from A. thaliana in 2007, investigation into

individual subunits of the Mediator complex has revealed diverse roles spanning plant development

to abiotic and biotic stress responses. In this thesis I have studied three subunits MED25, MED18

and MED20 that have been identified to have a role in conferring susceptibility against F.

oxysporum. While the role of MED18 in resistance against Botrytis cinerea has recently been

published (Lai et al., 2014), the mechanism of conferring susceptibility to F. oxysporum by MED18

and MED20 was completely unknown. MED25 has been shown to interact with several JA-

associated TFs to control JA-associated transcription. I investigated these interactions further and

characterised the minimum domains of the TFs needed for interaction. I also looked to identify new

proteins that might interact with MED25 based on the TF interaction motifs. Our results showed

that the nucleotide sequence corresponding to amino acids ERF1 138-218 appear to be the key

domain for its interaction with MED25. This sequence represents the conserved motif (CMIX) from

the group IX AP2/ERF family. In addition, the Y2H results suggest that the ELLEL conserved

motif previously involved in transcriptional activation could also be important for interaction of

ERF14 and ERF96 with MED25. Although the ELLEL conserved motif has been found in different

proteins in A. thaliana that belong to a variety of families, only members from the IXc AP2/ERF

family can interact with the MED25 ACID domain. Mutational screening of the members of this

family in the Y2H system will provide a framework of which variations of the ELLEL motif bind

and which do not. Further investigation of this domain will potentially provide a strong activation

domain that can attach to proteins of interest for high gene expression in plants through the MED25

subunit. Further study in other key motifs in MED25 interacting TFs could facilitate our knowledge

of which motifs are required to control gene expression through the MED25 subunit and how they

interact with the ACID domain. Of particular interest is how diverse proteins can bind to the same

interaction site, and whether there are common amino acids required for this interaction.

In addition to studying the MED25 subunit interaction with TFs, this project also investigated the

interaction of other subunits from the Mediator complex, as well as their response to F. oxysporum

infection. By conducting Y2H, followed by BiCF assays, our results show an interaction between

the two Mediator subunits, MED18 and MED20a that share similar phenotypes and function, but

not with the MED8 subunit within the Mediator complex. This result suggests that MED18 and

MED20 may form a subdomain within the Arabidopsis Mediator complex with roles distinct from

that of MED8.

The med18 and med20 mutants were found to display a very strong resistance to F. oxysporum

infection, stronger than either med25 or med8 and this was similar to the previously identified coi1

83

mutant (Thatcher et al., 2009). Using the data from the RNAseq analysis experiment, the project

identified the types of processes that are induced or repressed in the med18 and med20 mutants

compared to WT during F. oxysporum infection and investigated their role in providing resistance

or susceptibility. From this analysis, it was revealed that JA responsive genes as well as numerous

defence and stress responses were higher in the WT compared to the med18 and med20 mutants. On

the other side, the two mutants showed increased expression of SA-associated marker genes.

Consequently, the increased SA and increased ROS production could potentially contribute to the

increased resistance of the mutants to F. oxysporum. The RNAseq analysis also suggests that

MED18 and MED20 regulate similar gene groups in Arabidopsis. A similar finding was found in

the yeast Mediator complex, with med18 and med20 mutants also having similar gene expression

patterns (Larivière et al., 2008).

Our investigation aimed to identify the cause of resistance in med18 and med20 mutants. The

observation of root colonisation after F. oxysporum treatment demonstrated reduced colonisation of

analysis on ROS related genes as well as DAB staining in the mutant roots indicated high

production of ROS that may lead to a fast activation of the defence response in med18 and med20

plants toward F. oxysporum infection. However, more investigation into the PAMP and ROS

response of med18 and med20 need to be performed to determine the key processes behind med18

and med20 resistance to F. oxysporum infection. Also as we only investigated in this project the

early response of these mutants to F. oxysporum infection, future experiments at several infection

stages will provide a better picture of the resistance process.

Nevertheless, med18 and med20 infected roots showed a remarkable reduction in the expression of

JA-associated genes. These genes not only affect the JA-accumulation such as AOS, AOC3, LOX4,

DONGLE, and a JA-ile-hydroxylase as but also, the JA-signalling genes such as MYC2 and the JAZ

genes. Our results propose that MED18 and MED20 play a role in the regulation of JA signalling in

Arabidopsis. However, more investigation is needed to determine how disruption of MED18 and

MED20 affects JA signalling and whether MED18 and MED20 can interact with MYC2 similarly

to MED25 (Cevik et al., 2012).

The RT-Q-PCR analysis revealed that PR1 and PR5 were significantly expressed in med18 and

med20 after treatment with SA and F. oxysporum respectively. It is generally agreed from previous

researches that there is antagonistic interactions between JA- and SA-associated defence pathways,

therefore these result are in line with these findings. Moreover, our result showed that the

expression of EX1, and OXI1 (singlet oxygen stress responsive genes) together with the expression

84

of ROS scavenging genes such as CAT1 and the ascorbate reductases in med18 and med20 were up-

regulated compare to the WT. Recent studied have shown that OXI1 and EX1 to regulate singlet

oxygen mediated cell death through independent pathways (Lee et al., 2007; Shumbe et al., 2016).

Jasmonate has also shown to have a major role in singlet oxygen mediated cell death through

research into the flu and ch1 mutants (reviewed by Laloi and Havaux et al 2015). Thus, It may be

suggest that both JA signaling and singlet oxygen stress signaling is controlled through the

Mediator complex via MED18 and MED20 or alternatively, mis-regulation of the JA pathway in

med18 and med20 leads to defects in ROS production and tolerance. Further study need to be done

to understand how the interactions of MED18 or MED20 subunits with other Mediator subunits or

with other transcriptional regulators work to produce these phenotypes.

Apart from being more resistant to F. oxysporum, previous finding revealed that MED8, MED18

and MED20 are also susceptible to necrotrophic leaf pathogens such as Alternaria brassicicola and

Botrytis cinerea (Kidd et al., 2009; Lai et al., 2014). Therefore, it is possible that that MED8,

MED18 and MED20 form a sub-domain within the Arabidopsis Mediator complex that regulates

flowering time and pathogen defence. As I have mention before med18 has been found to interact

with TFs YY1, ABI4 and SUF4 to suppress disease susceptibility genes, regulate responses to ABA

and flowering time, respectively (Lai et al., 2014) and it would be interesting to test whether these

TFs also affect F. oxysporum resistance. Notably, the stabilization of RNA Pol II and TFIIB

interactions have been found to be affect by med18 and med20 mutations (Plascha et al., 2015).

Thus any distraction to this sub-domain may possibly leads to an alteration in the binding surface

that could consequently change these phenotypes throughout indirect mechanisms such as reduced

Pol II occupancy and altered methylation (Lai et al 2014). ). Current research have identified

numerous mutants in an RNA binding KH domain protein termed SHINY1/ENHANCED STRESS

RESPONSES1 (SHINY/ESR1) using a forward genetic screen (Thatcher et al., 2015). . The esr1

mutants were found to have increased resistance to F. oxysporum as well as differential regulation

in some but not all aspects of the JA pathway (Thatcher et al., 2015). Previously SHINY1/ESR1

was shown to interact with FIERY2/RNA POLYMERASE II CARBOXYL TERMINAL DOMAIN

PHOSPHATASE-LIKE 1 (FRY2/CPL1), a protein that is able to de-phosphorylate the C terminal

domain (CTD) of RNA Pol II (Koiwa et al., 2004). CPL1 has been shown to be essential for

accurate miRNA processing and controls mRNA splicing and mRNA decay (Manavella et al.,

2012; Chen et al., 2015; Cui et al., 2016). It has previously been shown that mutations in MED18

and MED20 also affect miRNA levels and a mutation in RNA Pol II leads to similar developmental

phenotypes as med18 and med20 suggesting a connection between mutations in RNA binding

proteins (Thatcher et al., 2015), the Mediator head domain and mutations in RNA Pol II itself (Kim

85

et al., 2011; this report). Additional researches have to be done to study the impact of these

mutations on the function of RNA Pol II and might be speeded with recent progress in the structure

of the transcriptional holoenzyme.

Overall, we found that med18 and med20 plants are highly resistant to F. oxysporum, suggesting a

potentially novel mechanism for susceptibility to F. oxysporum that is conferred by the MED18 and

MED20 genes. However it remains to be seen whether the strong resistance in coi1 also involves an

upregulation of ROS. The investigations of these genes as subunits of the Mediator complex

demonstrate the diverse role that a single subunit can possess. Accordingly, identification of

interacting proteins of Mediator subunits will definitely speed up our understanding of their specific

regulatory mechanisms in plant immunity. Future research should reveal new insights into the

specific roles of the remaining Mediator subunits and help to advance our understanding of the

transcriptional regulation of gene expression in plants that might provide help for agricultural

improvement.

86

Table 6:LIST OF GENES Accession Numbers

Sequence data for genes that were used in this project can be found in the Arabidopsis Genome

Initiative or GenBank/EMBL databases under the following accession numbers:

Gene name Locus ID

ETHYLENE RESPONSE F ACTOR1 (ERF1) AT3G23240

A basic helix- loop-helix transcription factor (MYC2) AT1G32640

DROUGHT RESPONSE ELEMENT PROTEIN B (DREB2A) AT5G05410

AP2/ERF 59 ORA59 AT1G06160

MEDIATOR SUBUNIT 8 (MED8) AT2G03070

MEDIATOR SUBUNIT 20a ( MED20a) AT2G28230

MEDIATOR SUBUNIT20b (MED20b ) AT4G09070-2

MEDIATOR SUBUNITS2c (MED20c) AT2G28020-1

MEDIATOR SUBUNIT 18 (MED18) AT2G22370

MEDIATOR SUBUNIT 21 (MED21) AT4G04780

MEDIATOR SUBUNIT 25 (MED25) AT1G25540

(TGG1) AT5G26000

(TGG2) AT5G25980

(PEN2) AT2G44490

87

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