The Pennsylvania State University The Graduate School Department of Crop and Soil Sciences PARASITIC CASTRATION BY A STINKING SMUT REGULATES SEX DETERMINATION AND INFLORESCENCE ARCHITECTURE IN DIOECIOUS BUFFALOGRASS A Thesis in Agronomy by Ambika Chandra © 2007 Ambika Chandra Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2007 The thesis of Ambika Chandra has been reviewed and approved* by the following: David R. Huff Associate Professor of Turfgrass Breeding and Genetics Thesis Adviser Chair of Committee Dawn S. Luthe Professor of Plant Stress Biology Barbara J.Christ Professor and Head of Plant Pathology Paula McSteen Assistant Professor of Biology David M. Sylvia Professor of Soil Microbiology Head of the Department of Department of Crop and Soil Sciences *Signatures are on file in the Graduate School. ii ABSTRACT PARASITIC CASTRATION BY A STINKING SMUT REGULATES SEX DETERMINATION AND INFLORESCENCE ARCHITECTURE IN DIOECIOUS BUFFALOGRASS Buffalograss is a dioecious grass with male and female inflorescences separated onto two different individuals. Buffalograss presumably evolved its present day dioecious breeding system from hermaphroditic ancestors. Pistil smut infection phenotypically alters unisexual florets of dimorphic male and female buffalograss making them hermaphrodites which are morphologically indistinguishable from one another. In this way, pistil smut infection allows the phenotypic expression of a hermaphroditic ancestor, which existed back in time, in the form of a retrospective phenotype or simply a retrophenotype. Underdeveloped stamens (male sex organs) and fungal sporulation within ovaries of male and female plants renders hermaphroditic florets of infected buffalograss reproductively sterile, i.e. parasitically castrated. Parasitic castration is a disease affecting animals, mollusks, insects, and plants where host gonads are sterilized preventing evolution of host resistance and redirecting resource allocation. How and why fungi induce hermaphroditism and cause parasitic castration in their hosts is unknown. Here I show that pistil smut induces hermaphroditism in male buffalograss by down- regulating a female suppressor gene homologous to Tasselseed2 (Ts2). This molecular mechanism is supported by temporal and spatial expression analyses performed using quantitative real time PCR and in situ hybridization, respectively, on a cloned full length buffalograss Ts2 homolog. In addition to inducing hermaphroditism, pistil smut infection iii enhances overall sexual reproductive allocation (SRA) in both male and female sex forms of buffalograss. In female plants, the fungus induces a 12.6-fold increase in ovary production while in male plants the fungus induces a 2.3-fold increase in floret number. Furthermore, phylogenetic analyses of morphology and nuclear 28s large subunit of ribosomal DNA (nLSU-rDNA) sequences show that pistil smut is clearly distinct from any other species of Tilletia suggesting that it may not even be a species of Tilletia. The unusually large genetic distance of pistil smut from Tilletia clade indicates an accelerated rate of evolution possibly due to pistil smut’s long term coevolutionary struggle with its host culminating in induced hermaphroditism. Therefore, a new genus Salmacisia is proposed and described to accommodate pistil smut. iv TABLE OF CONTENTS LIST OF FIGURES .....................................................................................................viiii LIST OF TABLES.......................................................................................................x Chapter 1 INTRODUCTION............................................................................................1 BIBLIOGRAPHY .........................................................................................13 Chapter 2 LIFE CYCLE AND PHYLOGENY OF TILLETIA BUCHLOËANA; A FUNGAL PARASITE CAUSING INDUCED HERMAPHRODITISM IN DIOECIOUS BUFFALOGRASS (BUCHLOË DACTYLOIDES).......................................................................................19 ABSTRACT ..................................................................................................19 INTRODUCTION.........................................................................................20 MATERIALS AND METHODS ..................................................................23 Isolation and maintenance of pistil smut................................................23 Host infection by pistil smut ..................................................................23 Scoring and analysis of pistil smut morphology ....................................24 Nucleic acid extraction and PCR amplification .....................................24 Taxa examined for phylogenetic analyses .............................................25 Taxa sequence alignments......................................................................26 Phylogenetic analyses ............................................................................27 RESULTS......................................................................................................29 Induced hermaphroditism and parasitic castration of buffalograss by pistil smut........................................................................................29 Morphological characteristics of pistil smut fungus in vitro..................31 Life cycle of pistil smut in vivo .............................................................32 Phylogenetic analysis of pistil smut based on morphology and life cycle characteristics ........................................................................33 Phylogenetic placement of pistil smut based on nLSU and ITS sequence analyses ...........................................................................35 ITS...................................................................................................39 Gamma distribution.........................................................................39 DISCUSSION................................................................................................40 BIBLIOGRAPHY .........................................................................................45 Chapter 3 SALMACISIA, A NEW GENUS OF TILLETIALES: RECLASSIFICATION OF TILLETIA BUCHLOËANA CAUSING INDUCED HERMAPHRODITISM IN BUFFALOGRASS......................................................................................................62 TAXONOMY................................................................................................63 Salmacisia D. R. Huff & A. Chandra, gen. nov. ....................................63 v Sori..................................................................................................63 Type species ....................................................................................63 Etymology........................................................................................64 Salmacisia buchloëana (Kellerman & Swingle) D. R. Huff & A. Chandra comb. nov. ........................................................................64 Sori..................................................................................................65 Characteristic DNA sequences .......................................................65 Characteristic fixed DNA polymorphisms ......................................66 Specimen examined .........................................................................67 Known distribution..........................................................................67 Chapter 4 INDUCED HERMAPHRODITISM CAUSED BY PARASITIC CASTRATION DISPLAYS AN ANCESTRAL PHENOTYPE BY REMOVING A SEX DETERMINING ONTOGENETIC LAYER FROM DIOECIOUS BUFFALOGRASS........................................69 ABSTRACT ..................................................................................................69 INTRODUCTION.........................................................................................69 MATERIALS AND METHODS ..................................................................74 Plant Material .........................................................................................74 Morphological stages of buffalograss inflorescence development ........74 Scanning Electron Microscopy ..............................................................74 Isolation of full length Ts2 homolog (BdTs2) from buffalograss..........75 Quantitative Real-Time PCR .................................................................76 RNA in situ hybridization ......................................................................77 RESULTS......................................................................................................78 DISCUSSION................................................................................................82 BIBLIOGRAPHY .........................................................................................85 Chapter 5 TEN-FOLD INCREASE IN POTENTIAL SEED YIELD DUE TO FUNGAL INFECTION OF BUFFALOGRASS ...............................................................................99 ABSTRACT: .................................................................................................99 INTRODUCTION.........................................................................................99 MATERIALS AND METHODS ..................................................................102 Isolation of pistil smut............................................................................102 Plant