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UNIVERSITI PUTRA MALAYSIA

TAN YUNG CHIE

ITA 2013 3

EXPRESSION PROFILING AND FUNCTIONAL CHARACTERIZATION OF SELECTED OIL PALM GENES IN HOST-MICROBIAL INTERACTION

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EXPRESSION PROFILING AND FUNCTIONAL CHARACTERIZATION OF

SELECTED OIL PALM GENES IN HOST-MICROBIAL INTERACTION

By

TAN YUNG CHIE

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirements for the Degree of Master of Science

July 2013

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the degree of Master of Science

EXPRESSION PROFILING AND FUNCTIONAL CHARACTERIZATION

OF SELECTED OIL PALM GENES IN HOST-MICROBIAL INTERACTION

By

TAN YUNG CHIE

July 2013

Chairman : Ho Chai Ling, PhD

Institute : Institute of Tropical Agriculture

ABSTRACT

Basal stem rot is a major disease of oil palm caused by a pathogenic fungus,

Ganoderma boninense. It reduces the oil palm yield and causes severe economic loss

to the oil palm industry. To better understand oil palm defence system during the

host-pathogenic interactions, the gene expression profiles of eleven defence-related

cDNAs in oil palm treated with G. boninense, Trichoderma harzianum, or

mycorrhizas were studied. These cDNAs encode putative bowman-birk serine

protease inhibitors (EgBBI1 and 2), defensin (EgDFS), dehydrin (EgDHN), early

methionine labelled polypeptides (EgEMLP1 and 2), glycine rich RNA binding

protein (EgGRRBP), isoflavone reductase (EgIFR), metallothionein-like protein

(EgMT), pathogenesis-related protein-1 (EgPRP), and type 2 ribosome inactivating

protein (EgT2RIP). These cDNAs were chosen because they are related to plant

defence and were differentially expressed in oil palm upon inoculation by

mycorrhizas or G. boninensen in a previous study. In this study, the transcript

abundance of EgIFR and EgBBI2 increased in G. boninense-treated roots at 3 and/or

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6 weeks post inoculation (wpi). While the gene expression of EgT2RIP, EgBBI1, and

EgDFS increased in G. boninense-treated roots at 6 and/or 12 wpi. Meanwhile,

EgDHN was up-regulated at all three time points. These reveal that these genes could

have different roles at different stages during the infection. Transcript profiles in

leaves showed two candidate genes encoding EgEMLP1 and EgMT with different

profiles in G. boninense-treated leaves compared to that of T. harzianum. They may

have the potential to be developed as biomarkers for early detection of G. boninense

infection. Comparison of the transcripts expression profiles in the roots inoculated by

G. boninense, T. harzianum, and mycorrhizas showed that some of the transcripts

were increased by specific fungi (EgBBI1 and EgMT were up-regulated by G.

boninense while EgPRP was up-regulated by mycorrhizas). However, EgDFS and

EgT2RIP were up-regulated by all three fungi probably as a result of plant general

defence mechanism. The putative functions of these cDNAs were identified by

sequence analyses with other homologous proteins. SignalP predicted that EgBBI1

and 2 , EgDFS, EgPRP, and EgT2RIP are secretory proteins. The complete open

reading frames (ORFs) of EgBBI1, EgDFS, EgIFR, EgPRP, and EgT2RIP were

cloned for recombinant protein production. EgDFSm, EgT2RIPm-CA, and

EgT2RIPm-CB were sub-cloned after removal of signal peptide and linker peptide

sequences. Soluble recombinant proteins were obtained for EgDFSm, EgIFR,

EgT2RIPm-CA while partially soluble protein was obtained for EgT2RIPm-CB. The

recombinant EgDFSm managed to inhibit the growth of G. boninense mycelium by

inhibiting the assimilation of starch, possibly by acting on α-amylase or calcium

channel of the fungus. The butanol fraction of oil palm root extract treated with

EgIFR showed some differences in its chemical profiles when analysed using a

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reverse-phase-high performance liquid chromatography (RP-HPLC). Lastly, the

recombinant EgT2RIPm-CA and CB that formed EgT2RIPm-AB was found to be

toxic to both mammalian cell lines, MCF-7 and MCF-10. In conclusion, the findings

of this study have provided insights on the molecular events that happened during the

plant-microbe interaction as well as functional roles of some proteins encoded by

these cDNAs.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

PROFIL EKSPRESI DAN PENCIRIAN FUNGSI GEN-GEN OIL PALM

TERPILIH DALAM INTERAKSI HOST DENGAN MIKROB

Oleh

TAN YUNG CHIE

Julai 2013

Pengerusi : Ho Chai Ling, PhD

Institut : Institut Pertanian Tropika

ABSTRAK

Reput pangkal batang adalah penyakit utama kelapa sawit yang disebabkan oleh

kulat patogenik, Ganoderma boninense. Ia mengurangkan hasil minyak sawit dan

menyebabkan kerugian yang besar kepada industri minyak sawit. Untuk memahami

sistem pertahanan kelapa sawit semasa interaksi host-patogen, profil ekspresi sebelas

gen yang berkaitan dengan sistem pertahanan kelapa sawit telah dikaji ke atas kelapa

sawit yang telah dirawat dengan G. boninense, Trichoderma harzianum, atau

mikoriza. DNA komplemen (cDNA) yang dikaji mengekod putatif perencat protease

Bowman-Birk (EgBBI1 dan 2), defensin (EgDFS), dehydrin (EgDHN), polipeptida

berlabel methionine (EgEMLP1 dan 2), protein pengikat RNA yang kaya dengan

glisin (EgGRRBP), isoflavon reduktase (EgIFR), metallothionein (EgMT), protein

berkaitan dengan patogenesis-1 (EgPRP), dan protein perencat ribosom jenis ke-2

(EgT2RIP). cDNA tersebut dipilih kerana mereka adalah berkaitan dengan sistem

pertahanan kelapa sawit dan menunjukkan perubahan tahap ekspresi di kelapa sawit

yang diinolukasi dengan mikoriza atau G. boninense dalam satu kajian yang lepas.

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Dalam kajian ini, transkrip EgIFR dan EgBBI2 didapati meningkat dalam akar pada

3 dan/atau 6 minggu selepas inokulasi (wpi) G. boninense. Selain itu, transkrip

EgT2RIP, EgBBI1, dan EgDFS didapati meningkat dalam akar pada 6 dan/atau 12

wpi selepas inokulasi G. boninense. Sementara itu, transkrip EgDHN meningkat

pada ketiga-tiga masa dikaji. Ini menunjukkan bahawa gen-gen tersebut mempunyai

peranan yang berbeza di peringkat jangkitan yang berbeza. Profil transkrip dalam

daun menunjukkan dua calon gen yang mengekod EgEMLP1 dan EgMT mempunyai

profil yang berbeza selepas inokulasi G. boninense berbanding dengan T. harzianum.

Transkrip-transkrip tersebut mempunyai potensi untuk diperkembangkan sebagai

petanda biologi untuk mengesan jangkitan awal G. boninense. Perbandingan profil

transkrip-transkrip akar yang diinokulasi G. boninense, T. harzianum, dan mikoriza

menunjukkan bahawa sesetengah ekspresi transkrip ditingkatkan oleh fungi yang

spesifik (ekspresi transkrip EgBBI1 dan EgMT dipertingkatkan oleh G. boninense

sahaja manakala ekspresi transkrip EgPRP dipertingkatkan oleh mikoriza sahaja).

Walaupun begitu, ekspresi transkrip EgDFS dan EgT2RIP didapati dipertingkatkan

oleh ketiga-tiga fungi mungkin hasil daripada mekanisme pertahanan yang umum.

Putatif fungsi cDNA yang dikaji ditentukan dengan membanding bebenang asid

amino dengan protein-protein homologi yang lain. SignalP meramalkan bahawa

EgBBI1 dan 2, EgDFS, EgPRP, dan EgT2RIP adalah protein-protein sekretori.

Bingkai bacaan terbuka (ORFs) daripada EgBBI1, EgDFS, EgIFR, EgPRP, dan

EgT2RIP telah diklonkan untuk menghasil protein-protein rekombinan. EgDFSm,

EgT2RIPm-CA, dan EgT2RIPm-CB telah disubklon selepas penyingkiran peptida

isyarat dan peptida pencantum. Protein-protein rekombinan yang larut telah

diperolehi bagi EgDFSm, EgIFR, EgT2RIPm-CA, manakala protein rekombinan

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separa larut telah diperolehi bagi EgT2RIPm-CB. Rekombinan EgDFSm berjaya

merencat pertumbuhan G. boninense miselium melalui penghalangan asimilasi kanji

yang mungkin bertindak pada α-amilase atau saluran kalsium fungi. Pecahan butanol

ekstrak daripada akar kelapa sawit yang telah dirawat EgIFR menunjukkan beberapa

perubahan profil kimia setelah dianalisasi dengan menggunakan fasa terbalik

kromatografi cecair berprestasi tinggi (RP-HPLC). EgT2RIPm-AB yang terbentuk

daripada gabungan EgT2RIPm-CA dan CB adalah toksik kepada kedua-dua titisan

sel mamalia, MCF-7 dan MCF-10. Sebagai kesimpulannya, hasil kajian ini telah

memberi gambaran di tahap molekul semasa interaksi tumbuhan dengan mikrob serta

fungi-fungsi beberapa protein yang dikodkan oleh cDNA tersebut.

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ACKNOWLEDGEMENTS

This dissertation arose to fulfil part of the requirement for Master of Science in

Molecular Biology. By the time, I have worked with a great number of people who

contributed in assorted ways to the research. It is a pleasure to express my gratitude

to them all in my humble acknowledgement.

First of all I would like to express my sincere gratitude to my supervisor, Assoc. Prof.

Dr. Ho Chai Ling who has provided a great help in constructive criticism on my

study, valuable suggestions, continuous encouragement in supervising throughout the

completion of the study. Sincere appreciation is also extended to co-supervisor, Dr.

Wong Mui Yun, and all other lecturers who have provided support to me, especially

to Assoc. Prof. Dr. Noorjahan who inspired me in research.

Other than that, I would like express my sincere gratitude to Dr. Ho Wan-Yong, Ms.

Yeoh Keat-Ai, Siow Rouh-San, Hariyanti Baharum, Nur Anisza Hanoum Naseron,

Tee Syin-Ying for their valuable advices, teaching, and guidance during the study. I

would also like to thank my fellow labmates, including Kalai Vani Maniam, Khew

Choy-Yuen, Lim Ee-Leen, Kanagamalar Silvarajoo, and Vasagi Ramachandran for

their help, support, and friendship.

Last but not least, this dissertation is dedicated to my father Tan Chin Hin, my

mother Teo Bin Choo, and my siblings Yung Lym and Yung Jing for their love,

financial support and help.

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APPROVAL

I certify that a Thesis Examination Committee has met on 23rd

July 2013 to conduct

the final examination of Tan Yung Chie on his thesis entitled “Expression Profiling

and Functional Characterization of Selected Oil Palm Genes in Host-Microbial

Interaction” in accordance with the Universities and University Colleges Act 1971

and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998.

The Committee recommends that the student be awarded the Master of Science.

Members of the Thesis Examination Committee were as follows:

Sariah Meon, PhD

Professor

Institute of Tropical Agriculture

Universiti Putra Malaysia

(Chairman)

Siti Nor Akmar Abdullah, PhD

Associate Professor Datin



(Internal Examiner)

Parameswari Namasivayam, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences


(Internal Examiner)

Wong Hann Ling, PhD

Associate Professor

Faculty of Science

Universiti Tunku Abdul Rahman

Malaysia

(External Examiner)

____________________________

NORITAH OMAR, PhD

Associate Professor and Deputy Dean

School of Graduate Studies


Date: 19 September 2013

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Master Science. The

members of the Supervisory Committee were as follows:

Ho Chai Ling, PhD

Associate Professor



(Chairman)

Wong Mui Yun, PhD

Associate Professor



(Member)

____________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which

have been duly acknowledged. I also declare that it has not been previously, and is

not concurrently, submitted for any other degree at Universiti Putra Malaysia or at

any other institution.

____________________________

TAN YUNG CHIE

Date: 23 July 2013

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK v

ACKNOWLEDGEMENTS viii

APPROVAL ix

DECLARATION xi

LIST OF TABLES xvi

LIST OF FIGURES xvii

LIST OF ABBREVIATIONS xx

CHAPTER

1 INTRODUCTION 1

1.1 Introduction 1

1.2 Objectives of the Study 3

2 LITERATURE REVIEW 4

2.1 Symbiotic Interactions 4

2.1.1 Oil Palm 5

2.1.2 Ganoderma 7

2.1.3 Mycorrhizas 11

2.1.4 Trichoderma 16

2.2 Plant-Pathogenic Interactions 18

2.2.1 Biotrophic and Necrotrophic Pathogens 22

2.2.2 Compatible and Incompatible Interactions 24

2.2.3 Classification of Pathogenesis-Related Proteins 25

2.3 Candidate PR Gene Products from Oil Palm 26

2.3.1 Bowman-Birk Protease Inhibitor 27

2.3.2 Defensin 28

2.3.3 Dehydrin 29

2.3.4 Early Methionine Labelled Polypeptide 30

2.3.5 Glycine-rich Proteins 31

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2.3.6 Isoflavone Reductase 33

2.3.7 Metallothioneins 34

2.3.8 Pathogenesis-related Protein-1 35

2.3.9 Type 2 Ribosome Inactivating Protein 36

3 GENE EXPRESSION PROFILES OF OIL PALM DEFENCE

RELATED cDNAs DURING HOST-PATHOGENIC

INTERACTION 39

3.1 Introduction 39

3.2 Materials and Methods 41

3.2.1 Sequence Analyses 41

3.2.2 Plant materials and treatments 42

3.2.3 RNA extraction 42

3.2.4 DNase treatment 43

3.2.5 cDNA Synthesis 44

3.2.6 Real-time Quantitative Reverse Transcription

(qRT)-PCR 44

3.3 Results and Discussion 47

3.3.1 Sequence Analyses 47

3.3.2 RNA quality and integrity 61

3.3.3 Stability of reference genes and validation of the

experiment 62

3.3.4 Gene Expression Profiles 63

3.4 Conclusion 71

4 GENE EXPRESSION PROFILES OF OIL PALM DEFENCE

RELATED cDNAs UPON INOCULATION OF

MYCORRHIZAS AND TRICHODERMA 73

4.1 Introduction 73


4.2.1 Plant materials and treatments 74

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4.2.2 RNA extraction, DNase treatment, and cDNA

conversion 75

4.2.3 Quantitative Reverse Transcription (qRT)-PCR 75


4.3.1 Effects of Mycorrhizal Treatment on Plant

Growth 76

4.3.2 RNA quality and integrity 79

4.3.3 Gene Expression Profiles in Mycorrhizas-treated

Roots 80

4.3.4 Gene Expression Profiles in T. harzianum-treated

Roots 84

4.3.5 Summary of Expression Profiles of Eleven

Defence-related Genes in Oil Palm Roots

Inoculated with G. boninense, T. harzianum and

Mycorrhizas 86

4.4 Conclusion 88

5 EXPRESSION, PURIFICATION AND

CHARACTERIZATION OF RECOMBINANT PROTEINS

RELATED TO THE DEFENCE OF OIL PALM 89

5.1 Introduction 89


5.2.1 Cloning of cDNAs into pET32a 90

5.2.2 Transformation of Expression Hosts using

Expression Vectors 95

5.2.3 Production and Extraction of Crude Recombinant

Proteins 96

5.2.4 Quantification of Proteins 96

5.2.5 SDS-PAGE Analysis 97

5.2.6 Characterization of EgDFSm 98

5.2.7 Characterization of EgIFR 100

5.2.8 Characterization of EgT2RIP 103

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5.3.1 Cloning and Expression of cDNAs 107

5.3.2 Characterization of EgDFSm 114

5.3.3 Characterization of EgT2RIPm-CA and CB 121

5.3.4 Characterization of EgIFR 125

5.4 Conclusion 127

6 SUMMARY, GENERAL CONCLUSION AND

RECOMMENDATION FOR FUTURE RESEARCH 128

REFERENCES 131

APPENDICES 157

BIODATA OF STUDENT 184

LIST OF PUBLICATIONS 185

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