MIAMI UNIVERSITY the Graduate School Yunluan Cui Doctor Of
Total Page：16
File Type：pdf, Size：1020Kb
MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Yunluan Cui Candidate for the Degree: Doctor of Philosophy _________________________ Director Dr. Nicholas P. Money _________________________ Reader Dr. Qingshun Q. Li __________________________ Reader Dr. Richard C. Moore __________________________ Reader Dr. M.H. Henry H. Stevens ___________________________ Graduate School Representative Dr. David J. Berg ABSTRACT DEVELOPMENT AND FUNCTION OF CONSTRICTING RING-FORMING NEMATOPHAGOUS FUNGI by Yunluan Cui Nematode-trapping fungi form specialized hyphal traps to capture and consume nematodes for energy and nutrients. Among the diverse trapping devices, constricting ring-forming fungi produce the most sophisticated three-celled ring traps and actively capture nematodes. When a nematode wedges its way through the aperture of the ring trap, the friction between its body and inner side of the ring triggers the trap cells to inflate three times their untriggered volume within 100 milliseconds. The snared nematode becomes colonized and digested by the predacious hyphae for nutrients. In this project, constricting ring-forming fungi Arthrobotrys brochopaga and Arthrobotrys dactyloides are studied with the aid of microscopic and molecular techniques, and new facets behind this fascinating predatory behavior of nematode-trapping fungi are revealed. Rapid response to external stimuli and fast configuration change demonstrated by constricting ring traps has evolved to capture prey. With the aid of a high speed camera and the application of mathematical modeling, we verified that the tripled volume change in normal-sized ring trap was accompanied by a 1.7-fold increase in the surface area independent of trap sizes. The giant ring trap is an inefficient trap form unable to reach full expansion to capture nematodes, which is explained by the unachievable ratio of surface area to volume. During the rapid shape change, the plasma membrane and cytoskeletons play critical roles in reconstruction of the cell shape. By using fluorescent-labeling techniques, we labeled the targeting cell structures and investigated their distribution and dynamics that are associated with the ring cell changes. The video indicated that the preexisting plasma membrane redistributed into the expanded area. This process is coordinated by actin filaments that asymmetrically deposit the cell wall building composition to the new cell boundary. Proteomic studies also facilitate the exploration of the cytological and molecular mechanism behind the trap development and function. A set of proteins associated with the production of predatory organs are differentially expressed between vegetative cells and ring trap cells. The finding of trap-development related proteins provides evidence that the life cycle transition from the saprophytic to predatory stage requires the morphological adaptation and energy input. DEVELOPMENT AND FUNCTION OF CONSTRICTING RING-FORMING NEMATOPHAGOUS FUNGI A DISSERTATION Submitted to the faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biology Botany Program by Yunluan Cui Miami University Oxford, OH 2014 Dissertation Director：Dr. Nicholas P. Money TABLE OF CONTENTS Page CERTIFICATE FOR APPROVING THE DISSERTATION ABSTRACT TITLE PAGE i TABLE OF CONTENTS ii LIST OF TABLES iv LIST OF FIGURES v DEDICATION vii ACKNOWLEDGEMENTS viii CHAPTER 1. Background and literature review 1 Diversity and biology of nematophagous fungi 1 Classification and phylogeny of nematophagous fungi 2 Ecology of nematophagous fungi 5 Genomic and proteomic study of nematophagous fungi 7 Previous research on constricting ring-forming fungi 9 Research aims 10 References 12 CHAPTER 2. Rapid cell movement and associated membrane configuration change 26 Abstract 26 Introduction 26 Materials and Methods 28 Results 33 Discussion 36 References 39 CHAPTER 3. Actin filament distribution during the constricting ring trap development 52 Abstract 52 Introduction 52 Materials and Methods 54 Results 57 ii Discussion 59 References 61 CHAPTER 4. Analysis of ring trap development related proteins 77 Abstract 77 Introduction 77 Materials and Methods 79 Results 83 Discussion 84 References 88 CHAPTER 5. Significance and conclusions 96 Significance of research on constricting ring trap 96 Future research 98 References 100 iii LIST OF TABLES Page Table 1.1. Taxonomic groups of nematophagous fungi 17 Table 2.1. Parameter measurements of ring traps 41 Table 2.2. Comparison between increase in surface area and volume based on three models 42 Table 3.1. Plasmid and primers used in recombinant PCR for construction 66 of hybrid expression vector. Table 4.1. List of proteins and peptides identified by MASCOT 92 iv LIST OF FIGURES Page Figure 1.1. Diverse trapping organs formed by carnivorous Orbiliales 18 Figure 1.2. Constricting ring traps of Arthrobotrys dactyloides and a firmly captured nematode 19 Figure 1.3. Illustration of conidial traps of Arthrobotrys dactyloides and A. oligospora 20 Figure 1.4. Phylogenetic relationships among the nematode-trapping fungi based on 18S rDNA 21 Figure 1.5. A hypothesized pathway showing evolution of trapping devices formed by species in Orbiliaceae 22 Figure 1.6. A phylogenetic tree of nematode-trapping structures of Orbiliaceae 23 Figure 1.7. Development of ring traps through hyphal branch differentiation and accurate curvature 24 Figure 1.8. Inflation process of constricting ring traps of Arthrobotrys brochopaga 25 Figure 2.1. Conidia and constricting rings of Arthrobotrys dactyloides 43 Figure 2.2. Models used by Muller for volume calculation of constricted and non-constricted ring cells 44 Figure 2.3. Measurement of trap size using Image-Pro® Plus 6.2 45 Figure 2.4. Schematic diagrams of constricted and nonconstricted normal ring trap 46 Figure 2.5. Fungal material setup for scanning confocal microscopic imaging 47 Figure 2.6. Cryofixation equipment used in TEM sample preparation 48 Figure 2.7. Constriction rate of ring traps of different sizes 49 Figure 2.8. Configuration change of plasma membranes during trap constriction 50 Figure 2.9. Transmission electron micrographs showing the intracellular structure of the motor cells before and after trap constriction 51 Figure 3.1. Vector map of pBC74 67 Figure 3.2. Vector map of pAL3-Lifeact 68 Figure 3.3. A GFP expression vector harboring ToxA promoter, lifeact, and TagRFP 69 Figure 3.4. Recombinant PCR to generate hybridized pBC-hygro-Lifeact-TagRFP 70 Figure 3.5. Colony of Arthrobotrys brochopaga with massive conidia 71 Figure 3.6. Conidia with germination tubes and spherical shaped healthy protoplast 72 Figure 3.7. PCR products of ToxA promoter, Lifeact-TagRFP, and hybridized fragment 73 Figure 3.8. Transformed protoplasts containing pBChygro-Lifeact-TagRFP 74 Figure 3.9. Constricted and nonconstricted ring traps with RFP label 75 Figure 3.10. Effects of actin inhibitor cytochalasin D on the morphology traps 76 v Figure 4.1. Liquid culture of Arthrobotrys brochopaga with randomly distributed ring traps 93 Figure 4.2. Proteins extraction from lyoprized Arthrobotrys brochopaga and separation on 2D gels 94 Figure 4.3. MALDI-TOF mass spectrometry characterized peptide peaks of vacuolar ATP synthase B and RAX1 protein 95 Figure 5.1. Isolation of gene YOR301W from Arthrobotrys brochopaga 102 Figure 5.2. Multiple loops formed in liquid culture of Arthrobotrys brochopaga 103 vi Dedication I would like to dedicate this dissertation to my daughter Sequoia Jiaqi Pan, who brought me new meaning of life. Having her in mind always, I have more passion to try things I have been dreaming to do in the future. vii Acknowledgements There are several faculty and staff of Miami University to whom I owe gratitude for facilitating the successful completion of this dissertation. I would like to thank my committee members Dr. Qingshun Q. Li, Dr. M. Henry H. Stevens, Dr. Richard C. Moore and Dr. David J. Berg, all who have offered constructive insight in their reviewing over the years. I would like to recognize Dr. Richard E. Edelmann and Matthew L. Duley of the Electron Microscopy Facility at Miami University for their advice with microscopic sample preparation, imaging and graphics. I would like to thank Dr. Mark Fisher (College of Mount St. Joseph, Cincinnati, OH) for his help in the mathematical modeling used in this dissertation. Special thanks are given to Dr. John Howes and Xiaoyun Deng in the Center for Bioinformatics & Functional Genomics at Miami University for their patient instruction on molecular methods and techniques that are critical for the completion of this dissertation. Appreciation is given to Dr. Lynda J. Ciuffetti (Oregon State University) for providing pCT74 transformation vector and its genetic map. I also give my sincere thanks to Mrs. Barbara Wilson of the Department of Botany, who creates a relaxed and friendly ambience for us to conduct research. Sincere gratitude is given to my academic advisor Dr. Nicholas Money for his support of my research all the time. Many thanks to my fellow graduate students, especially Jie Wang, Jingyi Cao, Erin Stempinskim, and Aswati Subramanian. Your useful ideas and patient help with experimental problems allowed my research to make significant progress. I would like to thank my lab mate Mirabeth Oak. It has been fun sharing research progress with you. Finally, I would like to thank my family and friends for their support throughout this