Establishment of Biotrophy by the Maize Anthracnose

University of Kentucky UKnowledge Theses and Dissertations--Plant Pathology Plant Pathology 2015 ESTABLISHMENT OF BIOTROPHY BY THE MAIZE ANTHRACNOSE PATHOGEN COLLETOTRICHUM GRAMINICOLA: USE OF BIOINFORMATICS AND TRANSCRIPTOMICS TO ADDRESS THE POTENTIAL ROLES OF SECRETION, STRESS RESPONSE, AND SECRETED PROTEINS Ester Alvarenga Santos Buiate University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Alvarenga Santos Buiate, Ester, "ESTABLISHMENT OF BIOTROPHY BY THE MAIZE ANTHRACNOSE PATHOGEN COLLETOTRICHUM GRAMINICOLA: USE OF BIOINFORMATICS AND TRANSCRIPTOMICS TO ADDRESS THE POTENTIAL ROLES OF SECRETION, STRESS RESPONSE, AND SECRETED PROTEINS" (2015). Theses and Dissertations--Plant Pathology. 17. https://uknowledge.uky.edu/plantpath_etds/17 This Doctoral Dissertation is brought to you for free and open access by the Plant Pathology at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Plant Pathology by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Ester Alvarenga Santos Buiate, Student Dr. Lisa J. Vaillancourt, Major Professor Dr. Lisa J. Vaillancourt, Director of Graduate Studies ESTABLISHMENT OF BIOTROPHY BY THE MAIZE ANTHRACNOSE PATHOGEN COLLETOTRICHUM GRAMINICOLA: USE OF BIOINFORMATICS AND TRANSCRIPTOMICS TO ADDRESS THE POTENTIAL ROLES OF SECRETION, STRESS RESPONSE, AND SECRETED PROTEINS ______________________________________ DISSERTATION ______________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture, Food and Environment at the University of Kentucky By Ester Alvarenga Santos Buiate Lexington, Kentucky Director: Dr. Lisa J. Vaillancourt, Professor of Plant Pathology Lexington, Kentucky 2015 Copyright © Ester Alvarenga Santos Buiate 2015 ABSTRACT OF DISSERTATION ESTABLISHMENT OF BIOTROPHY BY THE MAIZE ANTHRACNOSE PATHOGEN COLLETOTRICHUM GRAMINICOLA: USE OF BIOINFORMATICS AND TRANSCRIPTOMICS TO ADDRESS THE POTENTIAL ROLES OF SECRETION, STRESS RESPONSE, AND SECRETED PROTEINS Colletotrichum graminicola is a hemibiotrophic pathogen of maize that causes anthracnose leaf and stalk rot diseases. The pathogen penetrates the host and initially establishes an intracellular biotrophic infection, in which the hyphae are separated from the living host cell by a membrane that is elaborated by the host, apparently in response to pathogen signals. A nonpathogenic mutant (MT) of C. graminicola was generated that germinates and penetrates the host normally, but is incapable of establishing a normal biotrophic infection. The mutated gene is Cpr1, conserved in eukaryotes and predicted to encode a component of the signal peptidase complex. How can we explain why the MT is normal in culture and during early stages of pathogenicity, but is deficient specifically in the ability to establish biotrophy? To address this, first I characterized the insertion in the 3’ UTR of the MT strain in detail, something that had not been done before. The wild-type (WT) transcript did not differ from predictions, but the MT produced several aberrant transcript species, including truncated and non-spliced transcripts, and the normal one. Aberrant splicing of MT cpr1 was observed both in RNAseq transcriptome data and reverse- transcription polymerase chain reaction (RT-PCR), under different growth conditions and in planta. I also conducted a bioinformatic analysis of other conserved components of the secretory pathway in the MT and WT in planta. One explanation for nonpathogenicity of the MT is that it cannot cope with an increase in secretory activity during infection, and fails to produce necessary pathogenicity factors. With the transcriptome data, I was able to identify effector proteins that were expressed in the WT but not in the MT. Another possible explanation for the MT phenotype is that the MT can’t adapt to stress imposed by the plant. I developed a growth assay to characterize the effect of chemical stressors in vitro. The MT was more sensitive to most stressors, when compared to the WT. The transcriptome data indicates that the genes involved in different stress pathways are expressed in planta in both WT and MT, although very few genes are differentially expressed across the different growth stages. KEYWORDS: corn anthracnose, secretion pathway, fungal stress pathway, fungal effectors, bioinformatics. Copyright © Ester Alvarenga Santos Buiate 2015 ESTABLISHMENT OF BIOTROPHY BY THE MAIZE ANTHRACNOSE PATHOGEN COLLETOTRICHUM GRAMINICOLA: USE OF BIOINFORMATICS AND TRANSCRIPTOMICS TO ADDRESS THE POTENTIAL ROLES OF SECRETION, STRESS RESPONSE, AND SECRETED PROTEINS by Ester Alvarenga Santos Buiate Lisa J. Vaillancourt Director of Dissertation Lisa J. Vaillancourt Director of Graduate Studies March 23, 2015 Date Aos meus pais, que foram meus primeiros professores com seu exemplo de força e caráter. O constante apoio e amor incondicional de vocês se refletem nas minhas conquistas. Obrigada! Für Hermann, der mir Stärke gab als ich selbst keine hatte. Acknowledgements I’ve known Dr. Lisa Vaillancourt for almost nine years, and she has not only been a great mentor and a colleague, but a great friend. Her patience, concern and dedication to her work and her students, and her faith in me are what motivated me during those years. I am forever grateful for all that you taught me. I would like to thank my dissertation committee, Dr. Mark Farman, Dr. Peter Nagy and Dr. Arthur Hunt, for their academic support and insightful advice over the years. In addition, my gratitude is extended to the faculty members of the department, from whom I learned much through classes and our conversations. I am extremely grateful to Etta Nuckles and Doug Brown not only for their great work, essential for this dissertation to be completed, but for their encouragement and friendship. To all the staff of the Department, especially (my maid of honor) Alicia Landon and Shirley Harris, many thanks for always been a tremendous help and for the laughs when needed. During these years at Vaillancourt Lab, I was extremely lucky to have had incredible PhD students as my colleagues. I would like to thank Dr. Sladana Bec, you taught me so many things in the lab, as well as to live life in a sweeter way. Your friendship has been invaluable inside and outside the lab. Dr. Maria Torres, we’ve shared benches, too many corn sheaths, a few worries and incredibly funny moments. I am grateful to have met such an amazing scientist and a life-long friend. Many thanks to Katia Xavier, for always providing the much-needed coffee, encouragement and for being a tremendous help whenever I needed. Your friendship made the completion of this dissertation easier. To all the other past and current members of the Vaillancourt Lab whom I’ve had the pleasure to work with, thank you for your help, conversations and support. Finally, I would like to thank the friends I made over the years at UK, most now scattered around the world. Your support, near or far, was essential. iii Table of contents Acknowledgements ......................................................................................... iii Table of contents ............................................................................................. iv List of Figures ................................................................................................ viii List of Files ...................................................................................................... xi Chapter 1 – Colletotrichum graminicola, the causal agent of maize anthracnose ........................................................................................................................ 1 1.1 Overview ................................................................................................ 1 1.2 The economic impact of maize anthracnose .........................................

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