University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2017 Unique Roles for ISE2 in Chloroplast RNA Metabolism and Regulation of Plasmodesmata-mediated Intercellular Trafficking Tyra Nadine McCray University of Tennessee, Knoxville, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Recommended Citation McCray, Tyra Nadine, "Unique Roles for ISE2 in Chloroplast RNA Metabolism and Regulation of Plasmodesmata-mediated Intercellular Trafficking. " PhD diss., University of Tennessee, 2017. https://trace.tennessee.edu/utk_graddiss/4415 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Tyra Nadine McCray entitled "Unique Roles for ISE2 in Chloroplast RNA Metabolism and Regulation of Plasmodesmata-mediated Intercellular Trafficking." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Life Sciences. Tessa Burch-Smith, Major Professor We have read this dissertation and recommend its acceptance: Albrecht vonArnim, Elena Shpak, Brad Binder, Barry Bruce Accepted for the Council: Dixie L. Thompson Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Unique Roles for ISE2 in Chloroplast RNA Metabolism and Regulation of Plasmodesmata- mediated Intercellular Trafficking A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Tyra Nadine McCray May 2017 Copyright © 2017 by University of Tennessee All rights reserved. ii ACKNOWLEDGEMENTS Thank you to my advisor, Tessa Burch-Smith for her mentorship and guidance. And thank you to the present and previous Burch-Smith lab members. Thank you also to my committee members: Albrecht von Arnim, Barry Bruce, Brad Binder and Elena Shpak and for their assistance and helpful suggestions. Thank you to the sequencing center employees Veronica and Joe for experimental assistance and helpful discussions. Thank you to the Program for Excellence and Equity in Research (P.E.E.R), Plant Research Center (PRC), American Society of Plant Biologists (ASPB) and International Conference of Arabidopsis Research (ICAR) for academic support and funding. I would also like to thank members of the BCMB/GST administration: Sekeenia, Anamika, Donna, Terry, and Roger for their assistance and to family and friends for support. iii ABSTRACT INCREASED SIZE EXCLUSION LIMIT 2 (ISE2) is a nuclear gene encoding a chloroplast-localized RNA helicase that is essential for Arabidopsis thaliana embryogenesis, chloroplast RNA metabolic events and the regulation of plasmodesmal permeability. Here I report that ISE2 is essential for the editing of several chloroplast transcripts. Emb175/PPR103 is a nuclear gene encoding a pentatricopeptide repeat (PPR) protein that was previously reported to be required for embryogenesis in Arabidopsis thaliana and for seedling survival in Zea mays. EMB175/PPR103 was previously identified in our lab in a yeast-two-hybrid interaction screen with ISE2 and subsequently named ISE2 PROTEIN INTERACTOR (IPI)1. Confocal fluorescence microscopy illustrates that IPI1-YFP, similar to ISE2-YFP, localizes to chloroplasts, consistent with its predicted chloroplast N-terminal targeting sequence. In Nicotiana benthamiana, silencing of emb175/PPR103/IPI1 in mature leaf tissue produces a chlorotic phenotype coupled to defective chloroplast structural integrity. Interestingly, virus induced gene silencing (VIGS) of N. benthamiana emb175/PPR103/IPI1 or N. benthamiana ISE2 revealed defects in the RNA editing of N. benthamiana chloroplast transcripts. However, ISE2-silenced plants displayed increased plasmodesmata-mediated intercellular trafficking, whereas no intercellular trafficking defect was observed in N. benthamiana plants silenced for emb175/PPR103/IPI1. These results indicate that ISE2 performs unique functions in the regulation of PD permeability. Collectively, our results identify IPI1 as an ISE2 interacting protein that localizes to the chloroplast and that participates in the proper RNA editing of select N. benthamiana chloroplast transcripts. These observations add to the rapidly growing knowledge base of RNA helicase and PPR protein function in plants. iv TABLE OF CONTENTS Chapter One Introduction…………………… ......................................... …………1 Intercellular Trafficking in Higher Plants ........................................................... 3 Regulation of Intercellular Trafficking ............................................................... 6 Molecular Factors that Regulate Intercellular Trafficking ..................................... 7 Chloroplast-derived Signals May Regulate Intercellular Trafficking ............ 14 Regulation of Organelle RNA Metabolism ..................................................... 19 Chloroplast Editing Factors ............................................................................ 24 Ph.D Dissertation Aims .................................................................................. 29 Chapter Two Results ......................................................................................... 30 Generation of Arabidopsis Plants That Were Used to Measure Editing ......... 30 Generation of Amplicons from Arabidopsis Chloroplast Transcripts That Were Used to Measure Editing…………………………..…………………………...…31 ISE2 is Necessary for the Full Editing of a Subset of Transcripts in Arabidopsis Mature Leaf Tissue .................................................................... 32 Editing Sites are Conserved in Nicotiana benthamiana Four Week Leaf Tissue ........................................................................................................................ 36 Editing Defects are Detected in Nicotiana benthamiana ISE2-silenced Plants ........................................................................................................................ 39 Characterization of ISE2 Interacting Protein (IPI1) ......................................... 41 IPI1 physically Interacts With ISE2 via the C-terminal Region ............................ 43 IPI1 is a Conserved PPR Protein in Plants ....................................................... 46 IPI1 is Localized to Chloroplasts ..................................................................... 49 Phenotypic Characterization of Plants with Reduced NbIPI1 Expression ............ 50 IPI1 is Necessary for the Accumulation of Chloroplast rRNA Species ................. 50 IPI1 is Necessary for Editing Chloroplast Transcripts ........................................ 54 No IPI1 Intercellular Trafficking Defects are Seen in IPI1-silenced Leaves .......... 57 Transcriptome Wide Analysis ......................................................................... 59 General Pipeline ........................................................................................... 61 Detection of RNA Editing Defects in IPI1-silenced Leaves ................................. 66 Identification of Novel Chloroplast RNA Editing Sites in Nicotiana benthamiana Leaves ......................................................................................................... 67 Conclusion ................................................................................................... 74 Chapter Three Discussion ................................................................................. 76 Biological Implications of ISE2’s Role in RNA Editing .................................... 76 Functions of Arabidopsis ISE2-affected Edited Transcripts ................................ 76 Differences in the Role of ISE2 in Editing Arabidopsis thaliana vs. Nicotiana benthamiana Transcripts ................................................................................ 78 The Role of ISE2 in Editing May be Exclusive to its Role in PD Regulation ........ 79 Biological Implications of RNA Editing Defects in IPI1-silenced leaves .......... 81 Function of Edited Transcripts Affected in IPI1-silenced Leaves ........................ 81 IPI1 Does Not Affect Plasmodesmata Function ................................................ 83 Biological Significance of ISE2 and IPI1 Protein Interaction ............................... 87 Transcriptome Wide Profiling of IPI1 .............................................................. 88 v Chapter Four Materials and Methods ................................................................. 90 Materials ......................................................................................................... 90 Plant Materials .............................................................................................. 90 Agrobacteria and Yeast Strains ...................................................................... 90 Vectors ......................................................................................................... 92 Oligonucleotides ........................................................................................... 94 Methods 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