Cross-Species Translocation of Mrna from Host Plants Into the Parasitic Plant Dodder
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Cross-Species Translocation of mRNA from Host Plants into the Parasitic Plant Dodder Jeannine K. Flagg Thesis submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Master of Science in Plant Pathology, Physiology, and Weed Science James H. Westwood, Chair Glenda Gillaspy Jake Tu April 11, 2006 Blacksburg, Virginia Keywords: Cuscuta pentagona, phloem-mobile mRNA, tomato, pumpkin Copyright 2006, Jeannine K. Flagg Cross-Species Translocation of mRNA from Host Plants into the Parasitic Plant Dodder Jeannine K. Flagg (ABSTRACT) Dodders (Cuscuta spp.) are parasitic plants that live by tapping into the vascular tissue of a host plant. Contents of the host phloem translocate readily into the parasite, and shared plasmodesmata have been documented between host cortical cells and dodder searching hyphae. Dodder is known to transmit viruses from one host to another, which is consistent with viral ability to traverse plasmodesmata (PD) with the aid of movement proteins (MPs). Plant endogenous mRNAs may also associate with specific proteins to pass through PD and traffic long distances in the phloem, a process that appears to play a role in coordination of development. We have evaluated the hypothesis that dodder is able to accumulate host phloem-mobile mRNAs by assaying lespedeza dodder (C. pentagona) for the presence of host transcripts. Reverse transcriptase PCR (RT-PCR) and tomato microarrays were used to probe RNA from dodder parasitizing tomato. Transcripts from four tomato genes were detected in dodder grown on tomato, but were not detected in control dodder grown on other hosts. Notable among these was LeGAI, a transcript previously shown to be phloem translocated. In addition, RT-PCR of RNA from dodder grown on pumpkin detected three mobile pumpkin mRNAs (CmNACP, CmSUTP1, and CmWRKYP). These results imply the existence of an extraordinary situation in which mobile mRNAs move from one plant into another, and raise questions about the role of this phenomenon in plant development and parasite pathogenicity. ii TABLE OF CONTENTS Title Page ………………………………………………………………………........ i Abstract …………………………………………………………………………….. ii Table of Contents …………………………………………………………………... iii List of Figures ……………………………………………………………………… iv List of Tables ……………………………………………………………………….. v Abbreviations …...………………………………………………………………….. vi Dedication ………………………………………………………………………….. vii Acknowledgements ………………………………………………………………… viii Chapter I: Review of Literature …………………………………………………. 1 I.1 Parasitic Plants: Cuscuta ………………………………………………………... 2 I.1.1 Introduction ………………………………………………………………... 2 I.1.2 Distribution and Host Range ………………………………………………. 2 I.1.3 Host Connections ………………………………………………………….. 3 I.1.4 Macromolecular Movement between Host and Parasite ………………….. 4 I.2 Parasitic Plants: Orobanche aegyptiaca ……………………………………….. 5 I.3 Cell-to-Cell and Systemic Movement of Macromolecules ……………………... 6 I.3.1 Introduction ……...………………………………………………………… 6 I.3.2 Plasmodesmata and Intercellular Trafficking …...………………………… 7 I.3.3 Movement Proteins and RNP Complexes ……...………………………….. 8 I.3.4 Systemic Transport: The Phloem …..……………………………………… 9 I.4 Phloem-Mobile RNA in Plants …………………………………………………. 10 I.4.1 Introduction ………………………………………………………………... 10 I.4.2 Phloem-Mobile RNA in Pumpkin ………………………………………… 10 I.4.3 More Evidence for Phloem-Mobile RNA …………………………………. 11 I.4.4 Regulation and Characteristics of Phloem-Mobile RNA ………………….. 12 I.5 RNA Silencing ………………………………………………………………….. 13 I.6 Study of mRNA Movement using Dodder ……………………………………... 15 References ………………………………………………………………………….. 16 Chapter II: Detection of Host mRNA in Dodder ……………………………….. 24 Abstract …………………………………………………………………………….. 25 Introduction ………………………………………………………………………… 26 Results ……………………………………………………………………………… 28 Discussion ………………………………………………………………………….. 34 Materials and Methods …………………………………………………………….. 37 References ………………………………………………………………………….. 40 Tables ………………………………………………………………………………. 42 Figures ……………………………………………………………………………… 44 Appendix A: Summary of Relevant Experiments not Described in Chapter II ... 47 Appendix B: Summary of Tomato Putatively Mobile Transcripts …………….. 65 Vita …………………………………………………………………………………. 80 iii LIST OF FIGURES Fig. 1: Lespedeza dodder growing on tomato ………………………………………. 44 Fig. 2: Detection of pumpkin mobile transcripts in dodder using RT-PCR ………... 45 Fig. 3: Detection of tomato transcripts in dodder by RT-PCR ……………………... 46 Fig. A1: RT-PCR assay for pumpkin transcripts in broomrape grown on pumpkin .. 59 Fig. A2: PCR limit of detection …………………………………………………….. 60 Fig. A3: RT-PCR confirmation of A. thaliana transcripts in dodder grown on A. thaliana ……………………………………………………………………………... 61 Fig. A4: L16C and wild type N. benthamiana plants photographed under UV light .. 62 Fig. A5: Movement of TMV from N. tabacum to N. benthamiana ………………… 63 Fig. A6: A cut dodder stem exudes liquid that can be collected using microcapillary tubes………………………………………………………………………………… 64 iv LIST OF TABLES Table 1: Microarray detection of transcripts from tomato, dodder grown on tomato, and dodder grown on other hosts ……...…………………………………………………………... 42 Table 2: Tomato genes and specific primers characterized by RT-PCR ……………………... 43 Table A1: Detection of A. thaliana transcripts in dodder grown on A. thaliana using microarray analysis …………………………………………………………………………… 57 Table A2: Genes chosen from A. thaliana microarray data analysis for RT-PCR confirmation. 58 v ABBREVIATIONS CALM1LE tomato calmodulin 1 gene CF carboxyfluorescein CmCYCLINP pumpkin CYCLIN gene CmGAIP pumpkin GIBBERILLIC ACID-INSENSITIVE gene CmIMPORTIN α pumpkin IMPORTIN α gene CmNACP pumpkin NAC domain gene CmRABP pumpkin RABP gene CmRBCS pumpkin RUBISCO gene CmRINGP pumpkin RING gene CmSTMP pumpkin STMP gene CmSUTP1 pumpkin SUTP1 gene CmWRKYP pumpkin WRKY gene DNA deoxyribonucleic acid dsRNA double-stranded RNA ER endoplasmic reticulum GFP green fluorescent protein LeBEC1 tomato beclin 1 gene LeGAI tomato GIBBERILLIC ACID-INSENSITIVE gene LeIAA7 tomato IAA7 gene LeMOB1 tomato unknown function gene, mobile 1 Me Mouse ears miRNA microRNA NCAP non-cell-autonomous protein PCR polymerase chain reaction PD plasmodesmata PTGS post-transcriptional gene silencing RNA ribonucleic acid RNP ribonucleoprotein complex RT-PCR reverse transcriptase polymerase chain reaction SEL size exclusion limit siRNA small interfering RNA TMV tobacco mosaic virus VIGS virus-induced gene silencing vi DEDICATION I would like to dedicate this work to my parents, Michael and Brenda Flagg, and my grandparents, Dean and Joyce Seiber. They have consistently supported me with their love and encouragement, and are exemplary models of perseverance and diligence. Their example of a Godly life has spurred me towards leading a life pleasing to the Lord. vii ACKNOWLEDGEMENTS All honor and glory belong to the Lord Jesus Christ, and I thank Him for his guidance and provision in my life, and especially for His gift of salvation. I would also like to thank my advisor, Dr. James Westwood, for his patience and support during my undergraduate and graduate studies. I have developed many skills under his guidance and he has helped me to understand the qualities of a strong research scientist. I am grateful for the interactions with Dr. Jake Tu and Dr. Glenda Gillaspy, members of my advisory committee. Their advice and comments on my research has been invaluable. I am thankful for past and present members of Dr. Westwood’s lab. Verlyn Stromberg has been supportive and encouraging, and always brings a smile to my face. Without her, the lab would not be as pleasant and work would be dull. Colin Felts has also provided invaluable help by making solutions, cleaning the glassware, and the occasional joke. Earlier interactions with Noureddine Hamamouch, Peter Hurtado, and Christy Fagg helped introduce me to research. I am also grateful for the love and support of my fiancé, Matthew Roney, who has helped me to persevere and has faithfully encouraged me in my disappointments. I would also like to thank my brothers, Garret Flagg and Ian Flagg, and my sisters, Heather Eklund and Melanie Flagg, for their encouragement, conversations, phone calls, and pictures. Again, I would like to thank my parents for helping me to reach my goals and remember the higher calling. viii CHAPTER I: Review of Literature I.1 PARASITIC PLANTS: Cuscuta I.1.1 Introduction Cuscuta spp. (dodders) are parasitic plants that are marked by rapid, spreading growth that establishes a stranglehold on their host plants. Dodder species are found on nearly every continent, and display diversity in size and color, yet all dodders share close connections with the vascular system of the host. They rely on their hosts for all water and nutrients, and crops parasitized by dodders have reduced yields. Although dodders are an agricultural weed, they are also interesting organisms and have been used for the study of plant virus host specificity. The connections formed between dodder and its host are capable of transmitting viruses and dodder can be used to transfer virus from one host to another. I.1.2 Distribution and Host Range Dodders are obligate stem parasites with limited photosynthetic ability that become dependent upon a host for water and nutrients a few days after germination (Press and Graves, 1995). Once established on a host, the dodder root senesces and the plant consists of a thin, yellow-orange stem that twines around host leaves and stems (Fig. 1). A single