REGULATION OF USTILAGO MAYDIS SPORULATION IN MAIZE by BRIJESH B. KARAKKAT (Under the Direction of Sarah F. Covert) The plant pathogenic fungus Ustilago maydis causes corn smut disease on maize plants. The lifecycle of this semi-obligate fungus from phylum basidiomycota is completed when it releases teliospores from the galls it produces on its host. U. maydis is a model system for other obligate pathogens, such as the rusts and smuts, which are responsible for huge losses in agriculture worldwide. To better understand the development of these important fungal diseases of plants, we have studied the maize-dependent sporulation of U. maydis by undertaking two projects. In the first project, we examined the function of the fungal-specific velvet family in U. maydis. This gene family is known to regulate many aspects of growth and development in from ascomycota phylum. We found three members of the velvet family in the U. maydis genome. These genes, umv1, umv2 and umv3 were deleted individually and the resulting mutants were analyzed. In the umv1 deletion mutants, disease was blocked in maize seedlings before the teliospore formation stage. When umv2 was deleted, there was a delay in the formation of teliospores in planta and a reduction in virulence. The umv3 deletion mutants did not show any effect on disease. When the umv1 and umv2 deletion mutants were complemented with native copies of each gene, virulence rose back to normal levels. Thus, we show that two members of the velvet gene family function in teliospore development and disease progression in the basidiomycota fungus U. maydis. In the second project, we cloned and attempted to alter expression of a maize gene predicted to influence sexual development in U. maydis. This gene, called ZmfluG, is homologous to fluG in the saprobic fungus Aspergillus nidulans and nodGS in Arabidopsis thaliana. In A. nidulans, FluG secretes an extracellular factor required for asexual sporulation, while in A. thaliana NodGS expression increases during biotic stress. The absence of a FluG homolog in U. maydis led us to hypothesize that maize ZmfluG might produce an extracellular signal essential for U. maydis sporulation in planta. To find evidence of this gene’s role, first we introduced ZmfluG into U. maydis, but we did not see any effect on U. maydis’ growth or pathogenicity. Second, maize plants were transformed with a ZmfluG RNAi construct. When we analyzed the resulting maize lines for herbicide resistance, we found that leaf sensitivity to the herbicide spray did not match with the results from a PCR screen for the herbicide resistance gene. Out of the 12 ZmFluG RNAi lines that were grown, three lines did not germinate at all. We also made constructs that overexpress ZmfluG, which can be transformed into maize in the future. Future analysis to confirm silencing of ZmfluG in the RNAi lines would allow us to examine U. maydis’ sporulation in them. INDEX WORDS: Ascomycota, Basidiomycota, Ustilago maydis, Aspergillus nidulans, umv, ZmfluG, RNAi, velvet, FluG REGULATION OF USTILAGO MAYDIS SPORULATION IN MAIZE by BRIJESH B. KARAKKAT MS, University of Mumbai, INDIA, 2000 MS, Missouri State University, 2005 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2011 © 2011 BRIJESH B. KARAKKAT All Rights Reserved REGULATION OF USTILAGO MAYDIS SPORULATION IN MAIZE by BRIJESH B. KARAKKAT Major Professor: Sarah F. Covert Committee: Anthony Glenn Scott Gold Michelle Momany Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2011 iv ACKNOWLEDGEMENTS I thank my advisor Dr. Sarah F. Covert for providing an opportunity to work in her laboratory to obtain my Ph.D degree and helping me realize my education goal. I thank Dr. Covert and my committee members Dr. Glenn, Dr. Gold and Dr. Momany on their valuable suggestions and advice to help improve my research and communication skills. I thank past and present Covert lab members in bearing with me in moments of high levels of research difficulties. I thank Dave and Sydney for revisions. I thank Dave, Su, Nadia and past lab members of the Gold lab. I thank the Professors that were associated with my research needs: the Gold, Momany, Dawe, Merkle, Nairn, Teskey and Walcott laboratories for allowing me to use their facilities and equipment. I thank both Botany and Plant Pathology Greenhouse personnel for helping me set up my plants. I thank the students of Plant Pathology for fun social events. I thank my few friends Sanchali, Amit, Jinu, Atanu, Rakesh, Satish, Chakravarthy, and few others from Mumbai University and Athens, Ohio. I thank my Master’s advisor Dr. Qiu of Missouri State Univ. for giving me a second shot at graduate school that gave me the drive to get my Ph.D degree from Univ. of Georgia. I thank Dr. Covert and the Dept. of Plant Pathology for supporting me with assistantships for the majority of my period here and also the assistantships from Biotechnology and Microbiology where I thoroughly enjoyed teaching. I thank my parents- Dad who set me free to approach life from my vision and learn from it, my Mom -her love and food I always missed in this country, and my cool brother who is a great shock absorber. Lastly, in case I miss out, along the words of Danny Boyle (Film Director) - I would like to thank all of you who helped me make this and all those of you who didn’t- Thank you. v TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS .....................................................................................................iv CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW .......................................................6 REFERENCES..............................................................................................................12 2. TWO MEMBERS OF THE USTILAGO MAYDIS VELVET FAMILY INFLUENCE TELIOSPORE DEVELOPMENT AND VIRULENCE ON MAIZE SEEDLINGS....16 ABSTRACT..................................................................................................................17 INTRODUCTION.........................................................................................................18 METHODS ...................................................................................................................20 RESULTS.....................................................................................................................24 DISCUSSION ...............................................................................................................30 REFERENCES..............................................................................................................33 3. PROGRESS TOWARD SILENCING AN ASPERGILLUS FLUG HOMOLOG IN MAIZE............................................................................................................................50 INTRODUCTION.........................................................................................................50 METHODS ...................................................................................................................53 RESULTS.....................................................................................................................58 DISCUSSION ...............................................................................................................61 REFERENCES..............................................................................................................63 4. CONCLUSIONS ............................................................................................................79 6 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Obligate plant pathogens require a living host for nutrition and reproduction, among which, the rust and smut fungi affect many cultivated and ornamental crops. These fungi are very difficult to control and many management techniques rely on incorporation of disease resistance genes into plants. This has led to some success, however, obligate pathogens more readily overcome resistance due to greater selective pressure. Therefore, this project aimed to develop a new method for controlling plant disease by reducing the ability of the pathogen to produce spores. To accomplish this, I selected Ustilago maydis, the corn smut pathogen, because it is an economically important pathogen of corn causing hundreds of millions of dollars per year in losses and also because it is a model organism. U. maydis belongs to the fungal phylum (3) that encompasses mushrooms and plant pathogenic rust and smut fungi (2). Rusts and smuts are the same phylum, which are all pathogens that infect a variety of economically important plants and both groups are characterized as having dark-colored resistant spores that, in mass, give the appearance of soot or smut (2), hence the common names. U. maydis is a hemi-biotroph that infects only two hosts, maize (Zea mays) and teosinte (Zea mexicana), the progenitor of maize (2). The disease in maize is called corn smut and is characterized by several symptoms, such as chlorosis, anthocyanin pigmentation and tumor development (2). Corn smut reduces seed yield and the infected plants are sometimes stunted. 7 The lifecycle of the smut fungus U. maydis is tightly linked with its host, where primary inoculum, teliospores, are produced from affected corn kernels and serve to infect new plants. Teliospores germinate to form basidiospores that divide to generate unicellular yeast-like cells called sporidia