1 Transcriptional Profiling of Chromera velia Under Diverse Environmental Conditions Thesis by Annageldi Tayyrov In Partially Fulfillment of the Requirements For the Degree of Master of Science King Abdullah University of Science and Technology Thuwal, Kingdom of Saudi Arabia May, 2014 2 EXAMINATION COMMITTEE APPROVALS FORM The thesis of Annageldi Tayyrov is approved by the examination committee. Committee Chairperson: Dr. Arnab Pain Committee member: Dr. Liming Xiong Committee member: Dr. Christian Voolstra 3 © 2014 Annageldi Tayyrov All Rights Reserved 4 ABSTRACT Transcriptional Profiling of Chromera velia Under Diverse Environmental Condi- tions Annageldi Tayyrov Since its description in 2008, Chromera velia has drawn profound interest as the closest free-living photosynthetic relative of apicomplexan parasites that are significant pathogens, causing enormous health and economic problems. There- fore, this newly described species holds a great potential to understand evolu- tionary basis of how photosynthetic algae evolved into the fully pathogenic Apicomplexa and how their common ancestors may have lived before they evolved into obligate parasites. Hence, the aim of this work is to understand how C. velia function and respond to different environmental conditions. This study aims to reveal how C. velia is able to respond to environmental perturbations that are applied individually and simultaneously since, studying stress factors in separation fails to elucidate complex responses to multi stress factors and un- derstanding the systemic regulation of involved genes. To extract biologically significant information and to identify genes involved in various physiological processes under variety of environmental conditions (i.e. a combination of vary- ing temperatures, iron availability, and salinity in the growth medium) we pre- pared strand specific RNA-seq libraries for 83 samples in diverse environmental conditions. Here, we report the set of significantly differentially expressed genes as a re- sponse to the each condition and their combinations. Several interesting up- regulated and down-regulated genes were found and their functions and in- 5 volved pathways were studied. We showed that the profound regulation of HSP20 proteins is significant under stress conditions and hypothesized that the- se proteins might be involved in their movements. 6 ACKNOWLEDGEMENT First of all, I would like to thank my supervisor Dr. Arnab Pain for all that I have learned from him and his continued guidance and support in all stages of this project. Also, I am sincerely grateful to Dr. Yong H. Woo for his valuable advices, discus- sions, directions and tremendous help with the computational part of the work. I also thank the thesis committee members Dr. Liming Xiong and Dr. Christian R. Voolstra. I would like to thank Center for Desert Agriculture for letting us to use their in- cubators, especially to Dr. Ali Mahjoub for his assistance in this. Furthermore, I am sincerely grateful to all of my colleagues in the Pathogen Ge- nomics Group and all my friends in KAUST. Finally, my sincere gratitude goes to my family for always believing in me, for their continued love and support in my decisions, without which I could not have made it here. 7 TABLE OF CONTENTS EXAMINATION COMMITTEE APROVAL FORM………………………………………………2 COPYRIGHT…………………………………………………………………………………………………3 ABSTRACT…………………………………………………………………………………………………..4 ACKNOWLEDGEMENTS……………………………………………………………………………….5 LIST OF ABBREVIATIONS…………………………………………………………………………….8 LIST OF FIGURES…………………………………………………………………………………………9 LIST OF TABLES …………………………………………………………………………..……………10 Chapter 1 Introduction 11 1.1 Discovery of C. velia……………………………………………………………………………12 1.2 Life cycle and morphology of C. velia……………………………………………………..12 1.3 C. velia as a “missing link” in the evolution of apicomplexan parasites… 14 1.4 C. velia as a model organism……………………………………………………………….15 1.5 Environmental stresses and Chromera velia………………………………………..16 1.5.1 Heat shock response……………………………………………………………………..16 1.5.1.1 Small heat shock proteins in adhesion and movement of cells..17 1.5.2 Iron deficiency………………………………………………………………………………18 1.5.3 Effect of salinity…………………………………………………………………………….19 1.6 Transcriptomics with strand specific RNA-seq…………………………………….20 1.7 ANOVA………………………………………………………………………………………………22 1.7.1 Multi-way analysis of variance……………………………………………………….22 1.7.2 Factorial Experiment……………………………………………………………………..23 Chapter 2 Methods 2.1 Medium preparations and C. velia culturing……………………….……………….24 2.2 Heat stress application……………………………………………………………………....24 2.3 Iron deficiency application……………………………………………………………...….25 2.4 Combination of stress application……………………………………………………….26 2.5 Sampling and RNA extraction……………………………………………………………..27 2.6 Strand specific RNA-seq library preparation……………………………………….27 2.7 RNA-seq data processing……………………………………………………………………28 Chapter 3 Results and Discussion 3.1 Effect of Iron deficiency and induction………………………………………………..30 3.2 Heat shock treatment…………………………………………………………………………31 3.3 Multi-factorial experiment………………………………………………………………….33 3.4 Differentially expressed genes and cellular pathways…………………………..44 Chapter 4 Conclusions 50 Future studies 52 References 54 Appendices 59 8 ABBREVIATIONS ANOVA analysis of variance cDNA complementary DNA CV Chromera velia FDR false discovery rate FPKM Fragments Per Kilobase (of transcript) per Million (mapped reads) HSPs heat shock proteins KAAS KEGG Automatic Annotation Server KEGG Kyoto Encyclopedia of Genes and Genomes RH relative humidity RIN RNA integrity number RNA-seq RNA sequencing RPM revolutions per minute RT-qPCR reverse transcription-quantitative polymerase chain reaction 9 LIST OF FIGURES Figure 1. Life cycle of C. velia Figure 2. Transcriptome profiles of iron depressed and induced C. velia Figure 3. Transcriptome profiles of heat shock response in C. velia Figure 4. Factorial experiment design Figure 5. General outcome of the factorial experiment Figure 6. Correlation of gene expression patterns between individual experi- ment and factorial experiment after 2 h. of heat treatment Figure 7. Effect of heat shock treatment on genes with HSP domains Figure 8. Effect of salinity and iron on expression of HSP20 and dynein genes Figure 9. General representation of KEGG metabolic pathways Figure 10. Number of significantly up- and down- regulated genes for each condi- tion Figure 11. Precursory metabolic pathways for synthesis of proteins Figure 12. Some of the crucial pathways for synthesis and regulation of new RNA molecules Figure 13. Pathways that are directly involved in multiplication of the cells Appendix E - Global metabolic map for differentially expressed genes 10 LIST OF TABLES Appendix A – Pilot Experiment: Heat Shock treatment Hiseq-2000 sequencing output data summary Appendix B – Pilot Experiment: Fe deficiency and induction treatments Hiseq- 2000 sequencing output data summary Appendix C – Multi-Factorial Experiment Hiseq-2000 sequencing output data summary (Single end) Appendix D - Number of DE genes and their involved KEGG pathways 11 Chapter 1 Introduction Chromera velia is newly described marine species that turned out to be the clos- est known living photosynthetic organism to the apicomplexan parasites [1]. The Apicomplexa is a critical group of parasites that include malaria causative agent -Plasmodium spp. that kills more than million people annually; Toxoplasma gondii- causes fatal injuries in immune-deficient pregnant women and their fe- tus; Eimera and Neospora – responsible for large loss of poultries and beef each year [2-5]. In fact, it is virtually believed that almost every animal on the planet has at least one species of apicomplexan parasites that challenges it. Thus, this group of parasites has been extremely successful in term of host adaptation. What makes this newly described species of Alveolata very important is that it is known to be the “missing link” between photosynthetic algae and the parasitic Apicomplexa [6]. Furthermore, one of the major bottlenecks to finding cures for the diseases caused by Apicomplexa is that these obligate parasite organisms are difficult to study as they require specific host cells. On the other hand, C. velia can be cultured simply and cheaply in a laboratory. Since, C. velia is closely relat- ed to the apicomplexan parasites, it could be a suitable model organism to work on in development of cures against those parasites. Because, apicomplexan par- asites have leftover chloroplast remnants, indicating their evolutionary past as algae, this newly described species, C. velia, will also help us in understanding evolutionary basis of how photosynthetic algae evolved into the fully parasitic Apicomplexa and how their ancestors may have lived before they evolved into obligate parasites of land animals and human[1]. 12 1.1 Discovery of C. velia Chromera velia was first found in 2001 while trying to get pure culture of Symbi- odinium, one of the major symbionts of the stony corals. However, it is described and introduced to the literature in 2008 by Moore et al. [1] It was first isolated from the stony corals of the Sydney harbor, and can live either freely or as an as- sociated manner with the corals. Later on, another group isolated C. velia from another coral species, Montipora digitata, at Magnetic Island, Austria, while aim- ing to culture Symbiodinium from the corals [7]. Phylogenetic analysis suggests that C. velia belongs to the Alveolata superphylum, but due to its significant dif- ferences