UNIVERSITI PUTRA MALAYSIA
TAN YUNG CHIE
ITA 2013 3
EXPRESSION PROFILING AND FUNCTIONAL CHARACTERIZATION OF SELECTED OIL PALM GENES IN HOST-MICROBIAL INTERACTION
© COPYRIG
HT UPM
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
© COPYRIG
HT UPM
COPYRIGHT
All material contained within the thesis, including without limitation text, logos, icons,
photographs and all other artwork, is copyright material of Universiti Putra Malaysia
unless otherwise stated. Use may be made of any material contained within the thesis
for non-commercial purposes from the copyright holder. Commercial use of material
may only be made with the express, prior, written permission of Universiti Putra
Malaysia.
Copyright © Universiti Putra Malaysia
© COPYRIG
HT UPM
ii
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
© COPYRIG
HT UPM
iii
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
© COPYRIG
HT UPM
iv
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.
© COPYRIG
HT UPM
v
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.
© COPYRIG
HT UPM
vi
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
© COPYRIG
HT UPM
vii
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.
© COPYRIG
HT UPM
viii
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.
© COPYRIG
HT UPM
ix
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
Institute of Tropical Agriculture
Universiti Putra Malaysia
(Internal Examiner)
Parameswari Namasivayam, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(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
Universiti Putra Malaysia
Date: 19 September 2013
© COPYRIG
HT UPM
x
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
Institute of Tropical Agriculture
Universiti Putra Malaysia
(Chairman)
Wong Mui Yun, PhD
Associate Professor
Institute of Tropical Agriculture
Universiti Putra Malaysia
(Member)
____________________________
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
© COPYRIG
HT UPM
xi
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
© COPYRIG
HT UPM
xii
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
© COPYRIG
HT UPM
xiii
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 Materials and Methods 74
4.2.1 Plant materials and treatments 74
© COPYRIG
HT UPM
xiv
4.2.2 RNA extraction, DNase treatment, and cDNA
conversion 75
4.2.3 Quantitative Reverse Transcription (qRT)-PCR 75
4.3 Results and Discussion 76
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 Materials and Methods 90
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
© COPYRIG
HT UPM
xv
5.3 Results and Discussion 107
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