This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Hevein‑like peptides in plants : discovery, characterization and molecular diversity Kini Shruthi Gopalkrishna 2016 Kini Shruthi Gopalkrishna. (2016). Hevein‑like peptides in plants : discovery, characterization and molecular diversity. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/69393 https://doi.org/10.32657/10356/69393 Downloaded on 07 Oct 2021 03:03:39 SGT HEVEIN-LIKE PEPTIDES IN PLANTS: DISCOVERY, CHARACTERIZATION AND MOLECULAR DIVERSITY KINI SHRUTHI GOPALKRISHNA SCHOOL OF BIOLOGICAL SCIENCES 2016 Hevein-like Peptides in Plants: Discovery, Characterization and Molecular Diversity KINI SHRUTHI GOPALKRISHNA A thesis submitted to the Nanyang Technological University In partial fulfillment of the requirement for the degree of Doctor of Philosophy 2016 Dedicated to Amma and Annu Acknowledgements The 4-year-long journey that culminates in this thesis has been a truly riveting one! Its completion is thanks to all the people that motivated, supported and challenged me along the way. First and foremost, I express my heartfelt gratitude to my supervisor, Prof. James P. Tam for giving me the opportunity to pursue my research in his laboratory. His constant guidance and faith in me kept me motivated even through the tough times. He taught me that “there is no substitute for hard work” and that nothing is impossible without dedication and perseverance. I would also like to thank my co-supervisor, Assoc. Prof. Kathy Luo Qian and my thesis advisory committee for their invaluable guidance and advice throughout my PhD. I am deeply grateful to Prof. Dr. Thomas Peters from the University of Lübeck for giving me the opportunity to work in his laboratory and learn about NMR-titrations. I would also like to thank Assoc. Prof. Peter Dröge for his positive critique and Prof. P.V Subba Rao for being a source of inspiration and motivation throughout this journey. This thesis would not have been possible without the motivation and support from my lab members. In particular, I would like to thank Dr. Nguyen Quoc Thuc Phuong for patiently teaching me the basics of extraction, isolation and cloning. I thank Dr. Wong Ka Ho whole-heartedly for guiding me with anti-fungal assays, EST-based data mining and for being a great friend. I am blessed to have found wonderful friends in my lab-mates, Tan Wei Liang, Dr. Nguyen Kien Truc Giang, Clarene Chan, Yunjiao and Geeta Kumari. Our daily lunch-breaks, dinners and outings will be cherished forever. I also wish to thank Dr. Alvaro Mallagaray and Dr. Sophie Weissbach from the University of Lübeck for making me feel at home in a new environment and helping me with the NMR-titration experiments. Special thanks to Areetha D’Souza and Dr. Shin Joon for helping me with NMR i experiments at NTU. I am thankful to all the URECA and FYP students who worked with me and helped me learn along with them. A strong support system is essential to survive the rigorous journey towards obtaining a PhD. I am lucky to have met some wonderful people that I can call friends-for-life during this journey. My time in Singapore has truly been a pleasant one thanks to Akshita and Lakshya. With friends like them constantly by my side I knew I could face my toughest days with ease. This journey would have been a dull one without Annanya, my partner-in-crime and closest friend. I deeply appreciate Garima and Dilraj for their guidance, both professional and personal. My PhD would not have been possible without the encouragement from all my friends back home in Bangalore. I specially thank Paru and Prithvi for sticking by me despite the distance and constantly motivating me. My fiancé, Akhil, has been my pillar of support, my confidant and my biggest motivator throughout this journey. I thank him for his unwavering faith in me and for putting a smile on my face even on my worst days. I appreciate his patience in dealing with me during the most stressful phase of my PhD. Whether it was treating me to ice-cream in the middle of the night or staying awake with me during an overnight experiment, he has been there for me all the way and I will be forever grateful to him for his unconditional love for me. There are no words to express how thankful I am to my parents for trusting me with the decision to pursue my studies abroad and for allowing me to follow my dreams. They have celebrated every tiny success and consoled me through the failures during this journey for which I am truly grateful. It is their prayers and blessings that have helped me come this far. I hope this thesis makes them proud and I have lived up to their expectations. ii Table of Contents Acknowledgements ............................................................................................. i Table of Contents .............................................................................................. iii List of Figures ................................................................................................... vii List of Tables ...................................................................................................... x Abbreviations ..................................................................................................... xi Abstract ............................................................................................................ xiii Chapter 1 ............................................................................................................. 1 Introduction ......................................................................................................... 1 1. Cysteine-rich peptides in plant defense ................................................... 1 2. Hevein-like peptides ................................................................................ 5 2.1. Discovery of hevein-like peptides ................................................................ 5 2.2. Sequence diversity of hevein-like peptides ................................................ 10 2.3. Structure of hevein-like peptides ............................................................... 16 2.4. The Hevein-Chito-Oligosaccharide complex ............................................. 20 2.4.1. Active site involved in the binding interaction ..................................... 20 2.4.2. Effect of mutations on carbohydrate binding ...................................... 23 2.4.3. Multivalent nature of the binding interaction ....................................... 27 2.5. Biosynthesis of hevein-like peptides .......................................................... 29 2.6. Anti-microbial activity of hevein-like peptides ............................................ 32 3. Alternanthera sessilis and Moringa oleifera ........................................... 37 4. Aims and Significance of this study ....................................................... 40 Chapter 2 ........................................................................................................... 42 Materials and Methods ..................................................................................... 42 1. Materials ................................................................................................ 42 1.1. Chemical reagents ..................................................................................... 42 1.2. Kits ............................................................................................................. 43 1.3. Plant materials ........................................................................................... 43 iii 1.4. Fungal strains ............................................................................................ 43 2. Genomics .............................................................................................. 43 2.1. RNA extraction .......................................................................................... 43 2.2. DNA extraction .......................................................................................... 44 2.3. Rapid amplification of cDNA ends (RACE) and PCR analysis .................. 45 2.4. Sequence analysis .................................................................................... 46 3. Proteomics ............................................................................................ 46 3.1. HPLC and UPLC analysis ......................................................................... 46 3.2. MALDI-TOF MS and MS/MS ..................................................................... 47 3.3. Protein extraction and purification ............................................................. 47 3.4. De novo sequencing with MALDI-TOF MS/MS ......................................... 48 3.5 LC-ESI-MS/MS analysis ............................................................................ 48 3.6. Spectrophotometric determination of protein concentration ...................... 49 4. Structural analysis ................................................................................. 50 4.1. NMR spectroscopy .................................................................................... 50 4.2. Structure calculations ................................................................................ 51 4.3. NMR titration experiments ......................................................................... 52 5. Stability Assays ....................................................................................