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1 CHARACTERIZATION OF THE MAT LOCUS GENES IN THE FUNGAL PLANT PATHOGEN Sclerotinia sclerotiorum (Lib.) de Bary By BENJAMIN DOUGHAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013
2 2013 B enjamin Doughan
3 To my family
4 ACKNOWLEDG E MENTS I would like to thank Dr. Rollins for his patience, motivation, tolerance and guidance over the past five years and without which none of my work would have been possible. I would like to th ank my committee members Dr. Fo lta, Dr. Keyhani, and Dr. Chung for their critical analysis and guidance of my work and writing There would be no work done without Ulla Be nny without whom all is lost. I would like to thank my entire family for the love and support.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ..................... 4 LIST OF TABLES ................................ ................................ ................................ ................................ 7 LIST OF FIGURES ................................ ................................ ................................ .............................. 8 LIST OF ABBREVIATIONS ................................ ................................ ................................ ............... 10 ABSTRACT ................................ ................................ ................................ ................................ ...... 11 CHAPTER 1 LITERATURE REVIEW ................................ ................................ ................................ .............. 13 Introduction to the Ascomycotal life cycle ................................ ................................ ............ 1 3 Defining Characters ................................ ................................ ................................ ........ 13 Sex in the Ascomycota ................................ ................................ ................................ ... 1 5 Introduction to S. sclerotiorum (Lib) de Bary ................................ ................................ ........ 1 8 Taxonomy ................................ ................................ ................................ ....................... 18 Defining Characters ................................ ................................ ................................ ........ 1 9 Sclerotia ................................ ................................ ................................ .......................... 20 Sexual Fruiting Body Production ................................ ................................ .................... 2 1 Epidemiology, Host Range, and Control of S. sclerotiorum ................................ ........... 2 2 Disease Management ................................ ................................ ................................ ..... 2 5 Genome Sequence ................................ ................................ ................................ ......... 2 6 Ascomycota Mating Type Genes ( MAT ) ................................ ................................ ................ 2 7 Genetic Approach to Study MAT Gene Loci ................................ ................................ ... 30 2 CHARACTERIZATION OF THE MATING TYPE LOCUS GENES IN THE FUNGAL PLANT PATHOGEN S. sclerotiorum (LIB) de BARY ................................ ................................ ............. 3 3 Introduction ................................ ................................ ................................ ........................... 3 3 Materials and Methods ................................ ................................ ................................ ......... 3 6 Nucleic Acid Extraction and Tissue Manipulation ................................ .......................... 3 7 Gene Knockout Design and Execution ................................ ................................ ............ 3 8 Quantitative Reverse Transcription P olymerace C hain R eaction ................................ .. 3 9 Tissue Fixation, Embedding, and Sectioning for Microscopic Observations .................. 40 Macroscopic Observations ................................ ................................ ............................. 4 1 Results ................................ ................................ ................................ ................................ .... 4 2 Gene Knockouts ................................ ................................ ................................ .............. 4 2
6 Mycelial Growth ................................ ................................ ................................ ............. 4 3 Pathogenicity ................................ ................................ ................................ .................. 4 4 Sclerotia Development ................................ ................................ ................................ ... 4 4 Apothecia Development ................................ ................................ ................................ 4 5 Gene Expression ................................ ................................ ................................ ............. 4 6 MAT Gene Expression ................................ ................................ ................................ ..... 4 6 Putative Pheromone and Pheromone Receptor Gene Expression ................................ 4 8 Discussion ................................ ................................ ................................ .............................. 4 9 3 CONCLUSIONS ................................ ................................ ................................ ........................ 8 8 APPENDIX A THE 250 BASEPAIR INVERSION IN THE MAT LOCUS OF S. sclerotiorum ................................ 90 B S. trifoliorum MAT LOCUS INVESTIGATION ................................ ................................ ........... 9 6 LIST OF REFERENCES ................................ ................................ ................................ ..................... 9 8 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ............... 1 10
7 LIST OF TABLES Table page 2 1 Primers used for UTR amplification and cloning ................................ .............................. 5 8 2 2 Primers used for generation of the probes in Southern blot analysis of the MAT locus genes in S. sclerotiorum ................................ ................................ ........................... 5 9 2 3 Primers designed for QPCR analysis ................................ ................................ ................. 60 2 4 Compos ite of the gene expression comparisons in the MAT locus mutants ................... 61
8 LIST OF FIGURES Figure page 1 1 Life cycle of S. sclerotiorum ................................ ................................ .............................. 3 2 1 2 The MAT locus of S. sclerotiorum ................................ ................................ ..................... 3 3 2 1 The apothecial stages of S. sclerotiorum ................................ ................................ ........... 62 2 2 The split marker strategy for MAT 1 1 5 gene replacement ................................ ............ 64 2 3 The spl it marker strategy for MAT 1 1 1 gene replacement. ................................ ............ 6 6 2 4 The spl it marker strategy for MAT 1 2 4 gene replacement. ................................ ............ 6 8 2 5 The spl it marker strategy for MAT 1 2 1 gene replacement. ................................ ............ 70 2 6 The average daily mycelial growth of the MAT locus mutants ................................ ....... 71 2 7 Pathogenicity assays for MAT locus mutants ................................ ................................ .. 72 2 8 Average mass per sclerotium for MAT locus mutants ................................ ..................... 73 2 9 Relative spermatia production for MAT locus mutants ................................ .................. 74 2 10 Sclerotial cross sections for MAT locus mutants ................................ ............................. 75 2 11 The average apothecia pro duction time in wild type and mutant strains ...................... 7 6 2 12 Tubular MAT 1 2 4 apothecia and arrested stipes ................................ ........................... 7 7 2 13 Cross sections of tubular MAT 1 2 4 apothecia ................................ ............................... 7 8 2 14 Crennulate apothecia of MAT 1 2 4 ................................ ................................ ................. 7 9 2 15 Crenulate and tubular apothecia of the mat 1 2 4 mutant ................................ ............. 80 2 16 Ascus and ascospore formation in the mat 1 2 4 mutant ................................ ................ 81 2 17 MAT gene expression in mycelial tissue of the MAT locus mutants ............................... 82 2 18 MAT gene expression in stage three sclerotial tissue of the MAT locus mutants .......... 83 2 19 MAT gene expression in stage five scle rotial tissue of the MAT locus mutants .............. 84 2 2 0 Putative pheromone and pheromone receptors gene expression in mycelial tissue of the MAT locus mutants ................................ ................................ ................................ 8 5
9 2 2 1 Putative pheromone and pheromone receptors gene expression in stage three sclerotial tissue of the MAT locus mutants ................................ ................................ ...... 8 6 2 22 Putative pheromone and pheromone receptors gene expression in stage five sclerotial tissue o f the MAT locus mutants ................................ ................................ ...... 8 7 A 1 MAT locus in S. sclerotiorum tetrad number one. ................................ ............................ 91 A 2 arrangement of the MAT locus in S. sclerotiorum tetrad number one. ........................... 92 A 3 PCR analysis showing the MAT locus inversion in second tetrad of S. sclerotiorum ...... 93 A 4 Protein sequence alignment of inverted MAT 1 2 1 against consensus protein sequence of HMG domain ................................ ................................ ................................ 94 A 5 MAT 1 1 1 protein sequence alignment of S. sclerotiorum compared to ancest ral form of alpha box domain. ................................ ................................ ............................... 95 B 1 The partial MAT locus arrangement of S. trifoliorum ................................ ...................... 97
10 LIST OF ABBREVIATIONS bp Base p air C Celsius CWDE Cell wall degrading e nzymes DAPI diamino 2 phenylindole DNA Deoxyribonucleic a cid EST Expressed sequence t ag H3 Histone protein t hree HMG High mobility g roup Kb Kilobase MAT Mating t ype g ene mat Mating type gene k nockout Mb Megabase PCR Polymerase chain r eaction PDA Potato dextrose a gar PEG Polyethylene g lycol RNA Ribonucleic a cid g Microgram UTR Untranslated r egion UV Ultra v iolet WT Wild t ype
11 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZATION OF THE MAT LOCUS GENES IN THE FUNGAL PLANT PATHOGEN Sclerotinia sclerotiorum (Lib.) de Bary By Benjamin Doughan December 2013 Chair: Jeffrey Rollins Major: Plant Pathology Sclerotinia sclerotiorum (Lib.) de Bary is an omnivorous, polyphagus, phytopathogenic fungus that relies on the completion of t he sexual cycle to initiate new disease cycles. The sexual cycle is characterized by the development of apothecia that forcibly discharge ascospores for local and, under suitable conditions, long distance dissemination. A strategy for understanding the regulation of apothecial multicellular development was pursued through functional characterization of the mating type genes in S. sclerotiorum These gen es are hypothesized to encode master regulatory proteins required for aspects of sexual development ranging from fertilization through fertile fruiting body development. Experimentally, gene deletion strategies were performed to create loss of function mu tants in the two conserved specific genes found only in S. sclerotiorum and closely related fungi. The mat 1 1 1 mat1 1 5 and mat 1 2 1 mutants are able to form ascogonia but are blocked in all aspects of apothecia development. These mutants also exhibit defects in secondary sexual characters including lower number s of spermatia and altered rates of mycelial growth. The mat 1 2 4 mutants
12 exhibited delayed apothecia production with altered disc morphogenesis and ascospore production. They too produce lower numbers of spermatia and exhibit a faster hyphal growth rate. All four MAT gene mutants showed alter ations in the expression of putat ive pheromone precursor ( PPG 1 ) and pheromone receptor ( PreA, PreB ) gene s Our findings demonstrate that MAT genes are in volved in both sexual fertility, gene regulation and development in S. sclerotiorum.
13 CHAPTER 1 LITERATURE REVIEW Introduction to Ascomycotal Life Cycle Defining Characters The phylum Ascomycota or sac fungi, of which S. s clerotiorum is a member, contains over 32,000 species and 3 400 genera (Kirk et al ., 2001). The defining morphological characteristic of the group is the ascus, a sac like structure formed during the sexual life cycle in which t he non motile sexual spores, ascospore s, are formed. Ascomycetes may be saprotrophs, necrotrophic or biotrophic parasites of plants and animals, and mutualistic symbionts (Webster and Weber, 2007). Ascomycete environmental niches are equally as diverse ranging from the soil, above and below ground par ts of plants, freshwater and salt water environments (Webster and Weber, 2007). The vegetative growth habits of the Ascomycota separates the members into two distinct s ubphyla : (1) the unicellular Saccharomycotina which reproduce by budding or fission and (2) the mycelial Pezizomycotina with septate hyphae (Moore, 1998; Pggler et al ., 2006; Webster and Weber, 2007). Some s pecies in both groups have the ability to switch between the two states and are described as dimorphic (Moore, 1998; Webste r and Weber, 2007). A unifying feature of all members of the Ascomycota is the limitation of the dikaryotic state to specialized hyphae which form diploid cells that proceed directly into meiosis. The asexual spore or conidia l states of Ascomycetes a re highly variable ranging from complex multicellular spores and supporting tissues to simple fragmentation or int ercalary modification of hyphae to a complete lack of sporulation. Most conidial spores form on specialized hyphae called conidiophores and these asexual spores are more resilient than the hypha e state when in
14 a suspended state of growth (Adams, 1995; Ebbole, 1996) There are many different class ifications of conidiogenesis filling a spectrum from blastic to thallic methods but most share som e common themes (Cole, 1986). The conidia are formed from a conidiogenous cell and usually form a stalk called a conidiophore (Webster an d Weber, 2007). Conidia have different morphological feature s such as germ pores and slits, ornamentations, and origi nation of spore walls (Read and Beckett, 1996). The condiophores may bundle together to form different types of conidioma. Synnemata or coremia are parallel bundles of which there are simple, compound or parallel, conidiophores may also develop from a cus hion or stroma, and sporodochium is a cushion like conidiomata bearing a layer of short conidiophores (Seifert, 1985; Webster and Weber, 2007). A relationship between the asexual spore and sexual spore production has been described in A. nidulans wherein an oxylipin driven mechanism helps regulate the two developmental pathways ( Tsitsigiannis et al ., 2004 ). Individual hyphal cells of a scomycete fungi are generally haploid, multinucleate and homokaryotic containing genetically iden tical nuclei. Vegetative hyphal fusion, governed by vegetative compatibility loci ( Leslie, 1993 ; Saupe, 2000 ) or spontaneous mutations can give rise to heterokaryotic hyphae, containing genetically unique nuclei. Parasexual cycles are known to function in some ascomycete fungi w hich can generate genetic variation via mitotic recombination ( Papa, 1973; Leslie, 1993 ). Ascomycota h yphae are delimited by cross walls known as septa which contain a single pore that allows cytoplasm and membrane bound organelles to flow throughout the mycelia and use Woronin bodies, globose or hexagonal proteinaceous crystals, to block the pore (Moore, 1998; Webster and Weber, 2007). These Woronin bodies act as gate keepers of the septa and are unique to the Ascomyc ota
15 Sex in the Ascomycota Mycelial Ascomycetes typically form fruiting bodies called ascomata or ascocarps (Pggler et al 2006; Webster and Weber, 2007). These take on many different morphologies including the perithecium, a flask like structure; the cleistothecium, a completely enclosed structure; the apothecium, a cup like structure ; and the pseudothecium ,forming locules containing the asci within a n ascostroma (Webster and Weber, 2007). These structures develo p from haploid hyphal tissue. Meiosi s in Ascomycetes occurs in the sac like structure, the ascus, which is the namesake character only produced during the sexual life cycle and distinguishes the phylum. The sexual life cycle of Ascomycetes may be homothallic (self compatible) where individu al isolates are all of the same mating type or heterothallic (self incompatible) where individual isolates exists as one of two mating types. Heterothallic and some forms of homothallic reproduction are preceded by plasmogamy, the fusion of two different gametangia, and has three different forms: gametangio gametangiogamy specifying fusion between a n antheridium and an ascogonium gameto gametangiogamy specifying fusion between a spermatium and an ascogonium, and somatogamy specifying fusion between undiff erentiated hyphae (Webster and Weber, 2007). A third form of reproduction known as pseudohomothallism or secondary homothallism gives the appearance of self compatible but occurs when two nuclei of differing mating types are incorporated in ascospores and resulting hyphae ( Debuchy and Turgeon, 2006; Whittle and Johannesson, 2011). Regardless of mechanism, the first step of sexual reproduction in mycelial fungi occurs when compatible nuclei are combined in the same cell.
16 In gameto gametangiogamy and gametangio gametangiogamy fusion the gametangia are morphologically distinct and i n some species the female ascogonium is surrounded by sterile hyphae to form a pre fruiting body which then bears a specialized receptive hypha called the trichogyne (Pggle r et al ., 2006). The male gamete may be formed on the mycelium or in specialized structures called spermogonia and defined as either uninucleate spermati a microconidia, or multinucleate macroconidia The female and male gametangia then fuse and a fertil izing nucleus from the male gametangia migrates to the ascogonium and this female organ undergoes a series of mi totic divisions creating a multi nucleate aggregate of cell s (Bistis, 1981; Debuchy and Turgeon, 2006; Pggler et al ., 2006). Following fertil ization nuclei of opposite mating types migrate and form heterokaryons and ultimately a dikaryon within the ascogenous hyphae maintaining a 1:1 ratio of parental nuclei (Debuchy and Turgeon, 2006; Pggler et al ., 2006 ). The tip of the ascogenous hyphae differentiates into a crozier made up of two uninucleate and one dikaryo tic cell where the nuclei fuse and meiosis immediately follows (Saupe, 2000; Debuchy and Turgeon, 2006; Pggler et al ., 2006 ). The cell elongates forming the ascus and a post meiotic mitotic division occurs after which the ascospores are delineated within the ascus (Saupe, 2000; Debuchy and Turgeon, 2006; Pggler et al ., 2006 ). The post meiotic divisions determine the number of ascospores within th e ascus and varies in a species specif ic manner yet the most common number of ascospores is eight (Read and Beckett, 1996 ; Raju and Perkins, 2000 ; Pggler et al ., 2006; Webster and Weber, 2007). The timing of this process varies but the steps are mainly conserved throughout the Ascomycetes (R ead and Beckett, 1996). Ascospores between species and sometimes within a
17 single ascus may be morphologically distinct having different sizes and shapes (Read and Beckett, 1996). At the tip of the ascus there may be a cap like structure called an opercu lum, these are operculate fungi, co nversely inoperculate fungi have a pore and make up the majority of the Ascomycetes (Read and Beckett, 1996) including S. sclerotiorum The ascus is surrounded by a protective layer of filamentous tissue which includes p araphyses, that grows from different vectors along the ascus ; this sterile tissue makes up the ham a thecium (Read and Beckett, 1996). The asci are arranged scattered or in a defined pattern in the area of the fruiting structure called the hymenium The four major types of fruiting bodies described before act as a platform from which ascospores are forcibly discharged o r not (as in the cleitothecial/protunicate species) The release of the ascospores is either passive or active in nature with a polarized ascus shape correlating to the active mechanism (Read and Beckett, 1996). The polarized shape allows for hydrostatic pressure buildup by solute production and water uptake necessary to forcibly discharge spores through the apical pore (Read and Beckett, 1996; Fischer et al ., 2004). The release of spores may be simultaneous among asc i of a fruiting body or singular (Meredith, 1973). Passive release relies on other mechanisms such as desiccation, wind, rain, dew (Meredith, 19 73; Read and Beckett, 1996; Fi s her et al ., 2004). A scospores delivered to a suitable environment germinate forming hypahe and restart the life cycle of the fungus.
18 Introduction to Sclerotinia sclerotiorum (Lib.) de Bary Sclerotinia sclerotiorum (Lib.) de Bary is an omnivorous, necrotrophic fungal pathogen that affects a wide range of plants. More than 60 names have been used to refer to diseases caused by this fungal pathogen (Purdy, 1979), with many of the more common names including rot and/o r mold in the description reflecting the symptoms created. S. sclerotiorum has a wide host range of over 400 crops and a global distribution of which contain many important agricultural crops: soybeans, canola, sunflowers, lettuce, potatoes, tomatoes an d (Boland and Hall, 1994). Based on estimated yield losses from 1996 through 2009, it was estimated that Sclerotinia stem rot in USA soybeans caused yield losses of 10 million bushels (270 million kg) in seven of the 14 y ea r s (Wrather and Koenning 2009, Koenning and Wrather 2010, Peltier et al ., 2012). In Germany potato yield loss was as high as 30% in some areas (Quentin, 2004) and canola yield losses vary from 11.1 75% across the world (Morrall and Dueck, 1982; Shivpuri and Ghasolia, 2009; Peltier et al ., 2012). According to the reports of the National Sclerotinia Initiative in 2005, collective annual losses in the United States caused by S. sclerotiorum for canola, soybeans, dry beans, sunflowers and pulse crops have been as high as 250 280 million US dollars in a single year (Anonymous, 2005a; Anonymous, 2005b), making it one of the most devastating and important plant pathogens of agriculture crops. Ta xonomy The Sclerotiniaceae is a family of fungi within the order Helotiales in the phylum Ascomycota. T he Sclerotiniaceae family is defined by stromatal forming inoperculate discomycetes that produce stipitate apothecia containing ellipsoid ascospores wit h globose spermatia and produc e tuberoid sclerotia that do not incorporate host tissue within the
19 sclerotial medulla, develop an apothecial ectal excipulum composed of globose cells and lack a disseminative conidial state (Whetzel, 1945; Kohn, 1979; Bolton et al ., 2005). This family has been modified multiple times cu rrently including 33 genera T he Sclerotinia genus contains three economically important plant pathogens S. sclerotiorum S. minor Jagger and S. trifoliorum Eriks (Dumont and Korf, 1971; Korf 1973; Holst Jensen et al 1997; Wille t ts, 1997). The earliest description of S. sclerotiorum came from Libert (1837), who placed it within the Pizizomycetes as Peziza sclerotiorum Subsequently the taxonomic placement of this species has been revised several times (Wakefield, 1924). The current name, Sclerotinia sclerotiorum (Lib.) de Bary which was first used by Heinrich Anton de Bary in 1884 was accepted as the conserved name in 1979 (Kohn, 1 979) and approved by the International Botanical Congress in 1981. Defining Characters S. sclerotiorum produces simple, septate hyphae containing membrane bound vesicles, mitochondria, lipid bodies, rough and smooth endoplasmic reticulum and ribosomes (Max well et al ., 1972). Hyphae are hyaline, septate, branched, multinucleate, and look white to tan in culture and in planta (Bolton et al ., 2006). Vegetative hyphae are haploid and the number of nuclei vary per cell ( Maxwell et al ., 1972 ; Willets and Wong, 1979 ). Spermatia produced in chains are uninucleate from phialides on branched microconidiophores (Willets and Wong, 1979). Spermatia have been observed on aerial mycelia in culture, on the surface of sclerotia, and on the apothecial hymenium ( Le Tournea u, 1970 ; Kohn, 1979 ). The function of these spermatia in S. sclerotiorum has not been confirmed as the species is homothallic and fertilization by spermatia has not been resolved (Kohn, 1979).
20 Sclerotia S. sclerotiorum produces a resting phase stroma described as a scleroti um and may remain dormant and viable for years in soil which add s to the difficulty of controlling this plant pathogen (Adams and Ayers, 1979, Bourdot et al 2001). Sclerotial development has been defined to encompass three stages : 1) Initiation (aggregation of hyphae to form a white mass called a sclerotial initial) ; 2) Development (hyphal growth and further aggregation t o increase size) ; and 3) Maturation (surface delimi t ation, melanin deposition in peripheral rind cells and inte rnal consolidation). Sclerotia development involves a complex collaboration of environmental and nutritional factors but generally sclerotia are produced after a nutritionally competent mycelial growth encounters a physically limiting environment (Christi as and Lockwood, 1973; Le Tourneau, 1979; Willets and Wong, 1980; Hausner and Reid, 1999 ; Rollins and Dickman, 2001; Erental et al 2008; Liang et al ., 2010). Sclerotia are hyphal aggregates that are composed of two distinct layers, the medulla or inner mass, and the rind which is darkened by melanin and protects from microbial degradation in many fungi (Bell and Wheeler, 1986; Wille t ts, 1997; Henson et al ., 1999). The medulla is imbedded in a fibrillar matrix composed of carbohydrates and proteins (Le Tourneau, 1979). This is a character of the Sclerotiniaceae although the shape and size of the sclerotia varies and even within the species depending on the plant host nutritional conditions and physical environment (Bolton et al ., 2006). It is an over wintering structure used to survive harsh environmental conditions for up to 8 years in soil and to disperse the forcably discharge d ascospores from the apothecia produced from sclerotia (Adams and Ayers, 1979; Willetts and Wong, 1980). Hyphae may also em erge from the sclerotia depending on the conditions and
21 directly infect host tissue specifically lettuce, carrots and sunflower (Le Tourneau, 1979; Bardin and Huang, 2001). Sexual Fruiting Body Production Prior to carpogenic apothecia development, non propagative spermatia are formed from the hyphae associated with sclerotia and then hypothesized fertilization occurs (Kohn, 1979). Apothecial development is not time dependent but relies on an external signal or signal combinations of soil temperature, moisture and sclerotial stratification (Morrall, 1977; Willets and Wong, 1980; Mylchreest and Wheeler, 1987; Bardin and Huang, 2001). Apothecial development begins with an etiolated stipe which is photore sponsi ve growing towards or away from different spectrums of light when present, and then proceeds to form an expanding circular concave receptacle (3 6 mm diameter) containing the hymenial layer which is tan in color ( Thanning and Nilsson, 2000; Bolton et al. 2006). The stipe initials may form in complete dark the dev elopment of the apothecial cup is light dependent which directs the development of the apothecia above ground when the sclerotia are under soil or debris (Willets and Wong 1980 ; Thanning and N ilsson, 2000 ) Normal a pothecia production is triggered in wavelengths of light between 276 and 319 nm and the stipe phototropism was observed under light wavelengths not responsible for apothecial development indicating two photosystems (Thanning and Nil sson, 2000). Multiple apothecia may develop from a single scleroti um Histological studies by Kosasih and Willets (1975) separated the disc into four parts: 1) ectal exipulum 2) medullary excipulum 3) hymenium 4) subhumenium. The hymenial layer contains rows of asci which are cylindrical sac like zygote cells that contain eight hyaline ellipsoid binucleate ascospores (4 6 x 9 14 m) per ascus (Kohn 1979).
22 Ascospores are formed from selfing (Codron, 1974) or outcrossing by heterokaryon formation and rec ombination (Ford et al ., 1995) which are forcibly discharged under optimum conditions for more than 10 days in the field at a rate of 1600 spores/h (Clarkson et al ., 2003). Often S. sclerotiorum releases ascospores in mass by puffing when disturbed by cha nges in relative humidity or physical interactions (Hartill and Underhill, 1976). These ascospores may be distributed over long distances but the vast majority of them land in the field from where they were produced (Li et al ., 1994 ; Wegulo et al ., 2000). If landing on dead or dying tissue, the ascospores will germinate producing infectious hyphae which may colonize the rest of the healthy plant completing the life cycle. Epidemiology, Host Range and C ontrol of S. sclerotiorum The ability to survive for l ong periods of time in a dormant stage and then disperse ascospores over long distances makes the sexual cycle a key factor for the epidemiology of S. s clerotiorum (Fig 1 1). When soils are shaded, moist and cool (4 16C), sclerotia within the top five c entimeters of the soil profile can germinate to produce apothecia (Adams and Ayers 1979, Grau and Hartman 1999, Wu and Subbarao 2008). Infection is also favored by cool to moderately warm temperatures and a higher moisture content (Workneh and Yang, 2000) A single apotheci um may produce 2.3 x 10 8 ascospores and multiple apothecia may be produced from a single scleroti um (Schwartz and Steadman, 1978 ). Infection by S. sclerotiorum occurs typically after a saprophytic growth stage, the initial infection is commonly by the landing of ascospores, which have a sticky mucilage, on the above ground senescent or dead plant tissue of the host (Bolton et al ., 2006; Grau and Hartman 1999). A white mycelia and compound appressoria are produced from the initial colonization and are able to penetrate the
23 cuticle of a healthy host plant using cell wall degrading enzymes, the toxic metabolite oxalic acid and mechanical force via appressoria unless a natural opening or wound is present (Lums den and Dow, 1973; Lumsden, 1979). Host cells are killed and the middle lamella destroyed in advance of fungal colonization (de Bary, 1884). Anton de Bary (1884) was also the first to describe the oxalic acid production of S. sclerotiorum and determined that it solely was not responsible for symptom development but other hypothesized factors were also required Since then a multitude of enzymatic activities produced by S. sclerotiorum during infection have been characterized. Pectinolytic enzymes, pol ygalacturonases, endopolygalacturonases, exopolygalacturonases, proteases, cellulases, and glucoamylases are all produced by S. sclerotiorum during infection (Riou et al ., 1991; Alghisi and Favaron, 1995; Fra i ssinet and Fevre, 1996; Poussereau et al ., 2001 ; Bolton et al ., 2006). Oxalic acid production is increased during infection which creates a low pH environment (Bateman, 1964). Bateman also showed that treatment with oxalic acid alone caused injury and host tissue bleaching while treatment with oxalic acid and an extracted enzyme mixture produced tissue damage and hypocotyl collapse. This study determined that oxalic acid and polygalacturonase activity functioned synergistically to cause disease symptoms. Later a dynamic among oxalic acid productio n, environment pH acidification, multiple enzyme activities, enzyme gene regulation, carbon food source and ambient pH signal transduction was shown to play a significant role in Sclerotinia disease development and plant responses respectively ( Vega et al 1970; Marciano et al ., 1983; Godoy et al 1990; Dutton and Evans, 1996; Cessna et al ., 2000; Rollins, 2003; Guimar e aes and Stoltz, 2004). S. sclerotiorum is also able to secret several different molecular forms or isozymes of polygalacturonases that
24 ex hibit similar enzymatic activities which target the plant cell wall component pectin and has been hypothesized to allow for large host range (Bolton et al ., 2006) Similar enzymatic arsenals are found in the closely related plant pathoge n Botrytis cinerea where five endopolygalaturonases were recently found in this fungus that display different biochemical properties and necrotizing activity on different hosts (Kars et al ., 2005, Bolton et al ., 2006). Altered pH levels also provides a fa vorable environment for sclerotia development (Maxwell and Lumsden, 1970; Rollins and Dickman, 2001). N ecrotrophic colonization of host tissues is followed by a saprotrophic growth and the development of sclerotia to complet e the life cycle. Three mecha nisms by which oxalic acid aids in pathogenicity having been proposed (Bolton et al ., 2006): 1) Provide a favorable acidic pH environment for optimum CWDE activities (Favaron et al ., 2004) or pH regulated genes necessary for the pathogenesis ( Rollins, 2003 ; Kim et al ., 2007) 2) Suppress oxidative burst initiating the plant defense response (Cessna et al ., 2000) 3) Facilitate hyphal penetration by guard cell deregulation to induce stomatal opening (Guimares and Stotz, 2004). Oxalic acid appears to be a primary physiological determinant of pathogenicity and shows an important regulatory role as well. Full virulence of S. sclerotiorum relies on the correct regulation of genes and timing in response to the ambient pH and nutrient source. Current evidence for this is that Pac1 activating mutants can constitutively accumulate oxalic acid under both low and hi gh pH but exhibit an attenuated virulence phenotype (Kim et al ., 2007).
25 Disease Management Sclerotinia disease management has a variety of means: Chemical and biological control, transgenic and naturally derived resistance, and agricultural practices. Chemical control has been successful with Remedier Phenylthiourea, Difenoconazole, Actigard, Flint, Rovral, Switch, Topsin, Botran and Quadris ( Kuckarek, 2003; Gengotti et al ., 2011). Biological control strategies involve Coniothyrium minitans and Sporid esmium sclerotivorum two mycoparasites which degrade sclerotia (Ayers and Adams, 199 1) and antagonistic Pseudomonas spp (DF 41 and PA 23) has shown success in greenhouse and field plots (del Rio et al ., 2002; Li et al ., 2006; Partridge et al ., 2006). B iological control agents which have been approved for use on S. sclerotiorum and S. minor include: Intercept WG and Contans WG. Variations and alterations to plant canopy morphology has shown to decrease disease severity as well as agricultural practices such as: field flooding, crop rotation, control of alternate weedy hosts, removal of crop residues post harvest, deep plowing of fields, maintaining a well spaced plant density, avoiding excessive irrigation and selecting crop varieties with open canopies that may decrease disease incidence ( Moore, 1949; Steadman, 1979 ; Savchuk and Fernando, 2 004; Jurke and Ferna ndo, 2008; Pohronezny and Purdy, 1981 ). Partial inherited resistance to Sclerotinia diseases has been observed in a few agricultural crops but complete inherited resistance has not been observed in any crop to date ( Grau et al ., 1982; Boland and Hall, 1987; Nelson et al ., 1991; Kim and Diers, 2000; Rousseau et al ., 2004; Diers et al 2006; Yin et al ., 2010). Evaluation of this resistance has proven difficult and dependent heavily on environmental conditions ( Rousseau et al ., 2004; Die rs et al ., 2006 ; Bastien et al 2012; Ebrahi mi et al ., 2013). Transgenic resistance focusing on the degradation of
26 oxalic acid by oxalate oxidase and other enzymes has been successful in sunflower, soybean and peanut ( Donaldson, et al 2001; Hu et al ., 2 003; Ke r sarwani et al ., 2000; Livingstone et al ., 2005 ; Cunha et al ., 2010). The introduction of lipid transfer proteins and polygalacturonase inhibitor proteins has also shown to increase resistance to S. sclerotiorum infection (Bashi et al ., 2013; Fan et al. 2013). To date there is no completely successful method of control for S. sclerotiorum and most of the genetically derived methods of control center around the production of oxalate and interruption of the life cy cle at various points. There are a large number of targets both from chemicals produced by the pathogen and genes regulating the life cycle which may be exploited in the future for a more comprehensive and successful mechanism of control. Genome Sequence The genome of S. sclerotiorum wa s released in 2005. The project was carried out by the Broad Instit ute for Biomedical Research in p artnership with academic labs at the University of Florida (Dr. Jeffrey A. Rollins), the University of Nebraska (Dr. Martin B. Dickman) and the University o f Toronto (Dr. Linda M. Kohn) ( S. sclerotiorum Sequencing Project: Broad Institute of Harvard and MIT. http://www.broad.mit.edu ). Genome sequencing information has been used to promote identification of genes for genetic and functional studies as well as serve as a template for comparative genomics. Sequence information may also aid in the identification of potential anti fungal targets and facilitate the elucidation of signal transduction pathways. Whole genome shotgun sequencing provided an average of 8X coverage. It is estimated that the S. sclerotiorum genome is 38Mb in size with 14,522 genes where 41.3% of the genome contains coding sequence. Automated calling of gen ome sequence data was released in late
27 20 05 and was done by analyzing the genome sequence data via FGENESH and GeneID programs combined with the analysis of expressed sequence tags (ESTs). Analysis of genome data revealed that there is ~1 gene for every 2.6 kb of sequence with an average intron length of 140 bp. The shortest intron designated was 24 bp and the longest was 1494 bp. The average exon length was estimated at 38 9 bp with the longest being 17, 212 bp and the shortest as 1 bp ( S. sclerotiorum Sequencing Project. Broad Institute of Har vard and MIT. http://www.broad.mit.edu ). Ascomycota Mating Type Genes ( MAT ) In fungi mating type is used to define individuals that are compatible for mating and was discovered and defined by Blakeslee (1904) in Rhizopus There are two states defined by the mating type system: homothalism is the state of being self fertile and heterothallism is the state of being self infertile. The mating type genes at loci of heterothallic species appear not to be evolutionar ily related and the term idiomorph rather than allele has been put forth to describe the very dissimilar sequences at the opposite mating type loci (Metzenberg and Glass, 1990; Souza et al ., 2003 ; Debuchy and Turgeon, 2006 ). These mating type genes encode transcription factors in mycelial ascomycetes (Souza et al ., 2003). Ascomycetes have a bipolar system with two mating type determinants and one mating type locus. The core mating type genes in filamentous Ascomycetes share functional sequences that box proteins both of which are transcription factors (Turgeon et al ., 1993 ; Debuchy and Turgeon, 2006 ). The MAT 1 1 idiomorph is defined by the MAT 1 1 1 gene that encodes box protein, and MAT 1 2 idiomorph is de fined by the
28 MAT 1 2 1 gene which encodes the HMG protein These two genes make up the conserved genes of the two mating types in the filamentous Ascomycetes, subsequent novel genes found at each locus are labeled sequentially e.g., MAT1 1 2 encoding a HPG protein and MAT 1 1 3 encoding for another HMG protein at the MAT1 1 locus in Neurospora crassa (Ferreira et al ., 1996). In heterothallic species one isolate will carry only one of the MAT idiomorphs and a sexually compatible isolate will carry the other, specifically one MAT1 1 1 (alpha 1) and one MAT1 2 1 (HMG box) gene ( Debuchy and Turgeon, 2006; Dubuchy et al ., 2010). In homothallic species there is only one version of the MAT locus often referred to as a fused idiomorph in that it usually carries MAT 1 1 and Mat 1 2 genes The genus Cochliobolus contains both homothallic and heterothallic species and exchanging genes within these loci may convert one mating type system for another (Lu et al ., 2011) S. sclerotiorum is homothallic wh ere the MAT1 1 and MAT1 2 idiomorphs are fused together in one locus (Amselem et al ., 2011). There are four genes present at the MAT locus: MAT1 1 1 MAT1 1 5 MAT1 2 1 and MAT1 2 4 (Amselem et al ., 2011). Botrytis cinerea a related member of the Sclerotiniaceae has the same two novel genes MAT1 1 5 and MAT1 2 4 with the first being present in other L eotiomycetes and the latter being found exclusively in B. cinerea and S. sclerotiorum ( Amselem et al ., 2011 ). B cinerea has these genes arranged in a heterothallic MAT arrangement where MAT1 1 isolates have the MAT1 1 1 and MAT1 1 5 genes and MAT1 2 isolates have MAT1 2 1 and MAT1 2 4 (Amselem et al ., 2011). In preliminary reports of B. cinerea MAT gene functions, k nockouts of the MAT1 1 5 ORF are unable to develop an apothecial disc in the dikaryon ( van Kan et al ., 2010). In mutants of B. cinerea where the M AT 1 2 4 or MAT1 1 5 gene is deleted and crossed with the wild type
29 isolate of the opposite mating type stipe formation is successful but disc differentiation is interrupted ( Terhem et al ., 2011.) Mating type switching from heterothallism to homothallism and homothallism to heterothallism is a rare occurrence in the filamentous Ascomycetes but a few instances of switching have been describe d (Mathieson, 1952; Uhm and Juji, 1983; Leslie et al., 1986; Faretra and Pollastro, 1996; Samuels and Lodge, 1996; Harrington and McNew, 1997). Recently, S. sclerotiorum has been shown to have a 3.6kb region which is inverted between meiotic generations a nd correlates with changes in MAT gene expression (Chitrampalam et al 2013) T he authors of this study speculate that a similar inversion region may be involved in mating type switching in the filamentous ascomycetes Chromocrea spinulosa Sclerotinia trifoliorum and in certain Ceratocystis species The role of the MAT genes include s regulation of pheromone and pheromone receptor production that are involved in mating partner recognition, transcription factor production, heterokaryon incompatibility an d possibly nuclear pairing post fertilization (Bistis, 1981; Zickler et al ., 1995; Arnaise et al ., 1997; Debuchy, 1999; Shiu and Glass, 2000; Bobrowicz et al ., 2002; Glass and Kaneko, 2003 ; Debuchy and Turgeon, 2006; Bidard et al ., 2011; Whittle and Johann esson, 2011 ). Phenotypes associated with mating type switching involving ascospore size and appearance have been described in S. trifoliorum and Chromocrea spinulosa where large ascospores are self fertile homothallic and smaller ascospores were self sterile heterothallic but in this case the switching does not reverse (Harrington and McNew, 1997).
30 Genetic Approach to Studying MAT Gene Loci The MAT locus in Ascomycetes is often associated with conserved flanking r egion s specifically the APN 2 (DNA Lyase) and SLA2 (Cytoskeleton Assembly Control) genes. Targeting these genes and exploring the intervening regions has proven effective in identifying MAT loc i for individual species (Turgeon et al ., 1993; Pggler and Kuck, 2001; Casselton, 2002; Ryd h olm et al ., 2007). Gene deletion or gene interruption has also been proven successful in determining the roles of the genes at the MAT locus giving detectable phenotypes focusing around various parts of the fungal life cyc le as previously mentioned (Bistis, 1981; Zickler et al ., 1995; Arnaise et al ., 1997; Debuchy, 1999; Shiu and Glass, 2000; Bobrowicz et al ., 2002; Glass and Kaneko, 2003 ; Pggler et al ., 2010; Bidard et al ., 2011). A study of the interactions of the MAT g ene mutants and their regulation of pheromone and pheromone receptor genes was done in F. graminearum (Zheng et al ., 2013) and blast searches of the S. sclerotiorum genome for the genes characterized was performed. Three genes were identified: SS1G_04155.3 the putative pheromone precursor gene 1 ( PPG 1 ), SS1G_10310.3 the putative pheromone receptor protein B ( PreB ) and SS1G_07464.3 the putative pheromone receptor pr otein A ( PreA ) and included for genetic expression analysis. The functions of the genes found at the MAT locus of S. sclerotiorum have not been characterized to date T his study seeks to understand the function of these genes by creating loss of function mutants Four genes have been selected for complete gene knockout: MAT 1 1 5 (SS1G_04003.3), MAT 1 1 1 (SS1G_04004.3), MAT 1 2 4 (SS1G_04005.3) and MAT 1 2 1 (SS1G_04006.3) (Fig 1 2). These genes have been described by Amselem et al ., ( 2011 ) where they wer e shown to have high sequence similarity but different structural organization as in B.
31 cinerea My hypothesis is that through gene deletion and phenotypic characterization, functions involving sexual fruiting body development and other aspects of the sexual life cycle of S. sclerotiorum will be defined for the mating type genes
32 Figure 1 1. The life cycle of S. sclerotiorum
33 Figure 1 2. The MAT locus of S. sclerotiorum The orientation of all four MAT locus genes, location of the alpha and HMG domains and their Broad Institute S. sclerotiorum database gene calls.
34 CHAPTER 2 CHARACTERIZATION OF THE MAT LOCUS GENES IN THE FUNGAL PLANT PATHOGEN Sclerotinia sclerotiorum (Lib.) de Bary Introduction Sclerotini a sclerotiorum (Lib.) de Bary is an omnivorous, polyphagus, phytopathogenic fungus that has a wide host range of over 400 plant species (Boland and Hall, 1994) Included within the host range are many important agricultural crops: soybeans, canola, sunflowers, lettuce, potatoes, tomatoes and peanuts. The disease cycle relies on the completion of the sexual cycle characterized by the development of apothecia that forcibly discharge ascospores for local and, under suitable conditions, long distance dissemination. A single apothecia may produce in excess of 2 x 10 8 ascospores and multiple apothecia may be produced from a single scleroti um (Schwartz and Steadman, 19 78 ). Infection by S. sclerotiorum occurs typically after a saprophytic stage, the initial infection is commonly achieved by the landing of ascospores, which have a sticky mucilage, on above ground senescent or dead plant tissue of the host (Grau and Hartman 1999 ; Bolton et al ., 2006 ). A white myceli um is produced from the germinated ascospores, commonly on flower blossoms and hyphae are able to penetrate the cuticle of the host plant mediated by cell wall degrading enzymes, oxalic acid and mechanical force via compound appressoria (Lumsd en and Dow, 1973; Lumsden, 1979). Th e lack of asexual conidia spores and the dependence on ascospores for initiating disease makes the sexual cycle of S. sclerotiorum an enticing target for disease control. In Ascomycetes the role of MAT genes include regulation of pheromone and pheromone receptor s required for mating partner recognition, heterokaryon incompatibility sexual fruiting body development, and possibly nuclear pairing post fertilization (Bistis, 1981; Zickler et al ., 1995; Arnai se et al ., 19 97; Debuchy, 1999; Shiu and Glass, 2000; Bobrowicz et al ., 2002; Glass
35 and Kaneko, 2003 ; Bidard et al ., 2011; Whittle and Johannesson, 2011 ) These wide ranging roles make them major regulators of the sexual life cycle. The core mating type genes in fila mentous Ascomycetes share functional sequences that encode the high mobility group box protein transcription factors (Turgeon et al ., 1993). Ascomycetes mating systems may be homothallic which allows for self compatibility but also the potentia l for outcrossing or heterothallic requiring obligate outcros sing (Debuchy et al ., 2010). In addition to t he two core MAT genes subsequent genes found at MAT loci are lineage specific with conservation observed at the family and class levels ( Debuchy et al ., 2010 ). In heterothallic species one isolate will carr ies an idiomorph of the MAT locus and a sexually compatible isolate carr ies the opposite MAT locus specifically one MAT1 1 1 (alpha 1) and one MAT1 2 1 (HMG) gene (Dubuchy et al ., 2010). In homothallic species there is only one version of the locus that usually carries both core MAT genes. In the Helotiales only a few species have had their MAT locus characterized: Pyrenopeziza brassicae (Singh and Ashby, 1998), Tapesia yalludae (Singh et al ., 1999), Rhynchosporium secalis (Foster and Fitt, 2004), S. sclerotiorum and B. cinerea (Amselem et al ., 2011). The two core MAT genes ( MAT1 1 1 and MAT1 2 1) are present in all species. The l ineage specific gene MAT1 1 3 is present in R. secalis and MAT1 1 4 is present in P. brassicae S. sclerotiorum and B. cinerea share the Mat1 1 5 and the MAT1 2 4 genes. These lineage specific genes have not been described from any other species to date. Little is known about the function of the core or lineage specific MAT genes within the Helotiales as characterized gene mutants have not been published.
36 S. sclerotiorum is homothallic with fused MAT1 1 and MAT1 2 idiomorphs containing MAT1 1 1 MAT1 1 5 MAT1 2 1 and MAT1 2 4 (Amselem et al ., 2011). The heterothallic MAT locus of B. cinerea has an identical gene content but is organized as two idiomorphs. MAT1 1 isolates contain the MAT1 1 1 and MAT1 1 5 genes and MAT1 2 isolates have MAT1 2 1 and MAT1 2 4 genes (Amselem et al ., 2011). In preliminary reports, B. cinerea k nockout mutants of the MAT1 1 5 ORF are unable to expand the apothecial disc structure in the dikaryon ( van Kan et al ., 2010) and MAT1 2 4 gene deletion mutants crossed with a wild type MAT1 1 isolate, is able to form stipe s but disc differentiation is interrupted ( Terhem et al ., 2011.) In this study each of the four MAT locus genes of S. sclerotiorum were targeted for functional analysis to test the hypothesis that MAT genes effect multiple aspects of the sexual life cycle. Phenotypic consequences of these mutants in the context of the entire lifecycle were characterized. Each of the four MAT genes was found to function in both the sexual and the vegetative portions o f the life cycle. Evidence that MAT gene products function cooperatively is evidenced by altered expression of the MAT genes in independent mutants and the altered regulation of putative pheromone and pheromone receptors Materials and Methods S. sclerotiorum wild type isolate 1980 was used for the generation of complete gene knockouts by homologous recombination and was maintained on potato dextrose agar (PDA) (Difco, Mi., USA) at room temperature. Gene deletion mutants were maintained on PDA with 100g/ml hygromycin at room temperature. All strains were propagated by mass hyphal tip transfer. Liquid shake cultures in YPsucrose medium (4g/L yeast extract (Difco, Detroit, MI), 15 g/l sucrose, 1g/L K 2 HPO 4 and 0.5 g/L MgSO 4 pH 6.5) were grown for mycelial harvest and later
37 DNA extraction. Mutants and wild type strains were grown on potato plates (15g/L agar, two large mashed potatoes, 500ml H 2 0 20 cm diameter plates) until mature sclerotia were produced These sc lerotia were used for apothecia induction as described below and harvested for cytological analysis at developmental stage s 3 and 5 described in Li, 2008 Apothecia were induced from surface sterilized (0.5% bleach solution), dried, mature sclerotia which we re frozen at 20 degrees C for 24 h and thawed to room temperature for 24 hours three times in a sterile plastic tube. They were grown in water saturated vermiculite and maintained under constant light conditions in a Percival growth chamber (Boone, IA USA) at lighting (40 moles/m 2 /s) Apothecia were harvested at e very stage defined in Figure 2 1 for tissue dissection and RNA extraction. Nucleic Acid Extraction and Tissue Manipulations Mycelia from liquid shake cultures were flash frozen in liquid nitrogen, lyophilized a nd stored at 80 degrees C Lyophilized mycelia were used to isolate genomic DNA in a modified procedure from Yelton et al (1984), a phenol chloroform 24:1 extraction step was added to the procedure and Lithium Chloride precipitation was removed in order to increase the quality of the DNA extracted. RNA was extracted from lyophilized mycelia using Trizol reagent (Gibco s. RNA electrophoresis was conducted as previously described (Rollins and Dickman, 2001). F or quantitative PCR analysis RNA was extracted from lyophilized mycelia using the RNeasy Plant Mini Kit and treated with the RNase free DNase kit according to man (QIAGEN, Hilden, Germany).
38 Escherichia coli isolations, agarose gel electrophoresis, DNA restriction digests, ligation reactions, and transformations of E. coli were conducted using standard laboratory procedures (Sambrook and Russell, 2001). For Southern hybridization analyses, digested genomic DNA was transferred to MagnaGraph Nylon Membrane (Micron Separations Inc. Westborough, MA) by downward alkaline tr ansfer (Chomczynski, 1992) then UV cross linked. For Northern hybridization (Ausubel et al ., 1991). RNA and DNA hybridization analyses was carried out with 10 g of nucl eic acids at high stringency as defined by Ausubel et al (1991). Probes for all hybridizations were made using the DIG High Prime DNA Labeling and Detection Kit (Roche) according to any). Northern and Southern hybridization procedures were performed to the specifications for the DIG labeling kit. Gene Knockout Design and Execution Complete gene knockout constructs were designed for each MAT gene using a split marker technique (Fairhe ad et al 1996; Fu et al 2006). This strategy is described for each gene with restric tion enzymes used in Figures 2 2 through 2 5 A plasmid with ~1 kb o f an intervening hyg romycin resistance cassette as described by Jurick and Rollins (2007) (Table 6 reverse (R1) primer for each construct had an added Asc I restriction enzyme sequence at Asc I restriction enzyme sequence added to the to facilitate hygromycin resistance sequence ligat ion P rimer pair s for the amplification
39 of the partial hygromycin cassette (~2/3 total) and complete amplification of the respective UTR for split marker r ecombination were designed and are detailed in Table 2.1 The hygromycin cassette was a derivative of pCSN43 (Staben et al ., 1989) in the PGEM HPH plasmid (Hutchens, 2005) DNA Purificat ion System (Promega, WI, USA). All primers used were made by Integrated DNA Technology (Coralville, IA, USA). Transformations were performed in wild type protoplasts using PEG mediated induction by the method described in Rollins (2003). Successful repl acement of the coding sequences with the hygromycin resistance cassette (gene knockouts by homologous recombination ) were confirmed by Southern hybridization and PCR analysis utilizing probes of the UTR sequence or coding sequence of each gene (Table 6 2). Quantitative Reverse Transcription P olymerase Chain Reaction Quantitative reverse transcription PCR (qRT PCR) analysis was performed to measure the affect of gene mutation on select gene expression at various points of the life cycle. Actively growing mycelia, enlarging sclerotia (stage 3) and pigmented sclerotia ( stage 5) were chosen for analysis by measuring expression of the four MAT one putative pheromone, and two putative pheromone receptor genes for each tissue type of all four mat gene mutants. One microgram of total RNA was used to synthesize cDNA for ea ch sample using the ProtoScript II First Strand cDNA Sy nthesis Kit by oligo DT priming specifications (New England Biolabs, MA, USA). One microliter of the reaction was used for subsequent PCR analysis. Each sample was checked for DNA contamination by PCR with histone H3 coding sequence ( H3 ; GenBank accession XM_001589836 ) primers and a wild type DNA sample prior to quantitative reverse transcription PCR (qRT PCR ) analysis. Quantitative
40 RT PCR was performed using the iQ SYBR G reen Supermix #170 8880 kit from BIO RAD (C A qRT PCR reactions were performed on a BIO RAD CFX Connect RT System using the CFX managing program for design and later analysis (BIO RAD, CA, USA). qRT PCR 3 were used for PCR reactions. All pri 200 250 bp. The data was analyzed with the CFX managing software using the comparative C T method described in Schmitt e gen and Livak ( 2008 ) where the internal control ( H3 ) was calculated to have no difference in gene expression between control and test samples by a t test (0.95). Wild type gene expression data with three technical replications was compared to absolute zero with H3 gene expression as a reference sample and two negative controls. The Cq method of normalized expression was selected to measure the gene expression data of each sample using the H3 gene expression as a reference (BIO RAD CFX Manager, BIO RAD, CA, USA) A two way ANOVA of the fold changes was performed utilizing Prism 6 software (Graphpad Software, Inc.) to determine the significance of the changes, fold changes with a p value less than 0.05 or less than three fold difference in gene expression were considered statistically significant for this study. The p value was an adjusted p value calculated by the Prism 6 software as defined by Wright, 1992. Tissue Fixation, Embedding, and Sectioning for Microscopic Observations Sclerotia and apothecia were harvested at defined stages of development for mutant and wild type strains fixed and embedded for sectioning using the method described in Kladnik
41 et al (2004). Embedded samples were sectioned (3m) using a rotary microtome HM 325 (Richard Allan Scientific, MI, USA) and heat fixed onto ProbeOne Pl us microscope slides (Fisher Scientific, USA). Samples to be analyzed were de waxed using Histoclear (National Diagnostics, GA, USA) and rehydrated in an ethanol series where they were stained in methylene blue and mounted in cytoseal (Rochard Allan Scien tific, MI, USA). All slides and microscopic material was observed on a Leica DMR microscope (Leica Microsystems, Germany) using the SPOT Digital Camera and software (SPOT Microsystems, MI, USA). Thin s ections of sclerotia and apothecia from mutant strain s were compared to the wild type for phenotyp ic analysis Squash slides were also made of mutant apothecia and analysis of apothecia structures, ascus morphology and ascospore production was compared with wild type Macroscopic Observations Pathogenici ty comparisons were completed using a detached leaf assay with tomatoes in a sterile moist chamber (Rubbermaid Tupperware, moistened paper towels, constant cool fluorescent light, room temperature) where they were inoculated with a 3 mm plug of actively growing mycelia on PDA and then covered in Tupperware under constant light conditions at room temperature where they were documented with digital photography daily This experiment was repeated three times and there was no visible difference between wild type and the mutant abilities to infect and cause disease symptoms Apothecia and other macroscopic material were digitally photographed with a Canon EOS T2i camera at multiple focal planes and combined using the CombineZM software program and edited for publication using P hotoshop CS3 software (Adobe, CA, USA)
42 Growth rates of the mutants were analyzed with race tubes made from modified 25 ml sterile plastic pipettes with PDA media (Park and Lee, 2004 ). Three independent gene knockout strains of mat 1 1 1 mat 1 2 4 and mat 1 2 1 plus two independent knockouts of mat 1 1 5 were analyzed with three replications each and grown under constant temperature and moisture with 12 hours light (cool fluorescent light) and 12 hours dark in a growth chamber until they reached the end of the tube. Measurements of each day s growth were used to compare the mutants and wild type isolates using a two sample independent t test with a significance of greater than 0.95. Results Gene Knockouts Replacement of each of the four S. sclerotiorum MAT gene coding sequence s with a hygromycin resistance marker was carried out using a split marker strategy A probe in the coding region and the UTR of each gene in conjunction with PCR amplifications were used to confirm gene deleti on and replacement of the coding sequences With each UTR probe restriction fragment size s were detected by S outhern hybridization and compared with sizes predicted from the genome sequence for each gene (Fig, 2.2B, 2.3B. 2.4B. 2.5 B) H eterokaryotic transformant s produced both wild type and homologous recombinant band s. Tr ansformants positive for homologous integration were taken through a process of genetic purification involving hyphal tipping and when required, carpogenic germination to produce a pothecia for single ascospore purification. Following purification, PCR amplification of coding sequences from each MAT gene produced a positive amplicon of predict ed size in the wild type but failed to amplify the respective gene sequence in the correspo nding mutant strain
43 indicative of their homokaryotic gene deletion status (Figs. 2. 2 B, 2. 3 B. 2. 4 B. 2. 5 B) Multiple gene knockout strains for all four MAT locus genes were created: MAT 1 1 5 (two independent knockouts, Fig 2 2 ); MAT 1 1 1 (three independent knockouts, Fig 2 3 ); MAT 1 2 4 (three independent knockouts, Fig 2 4 ) and MAT 1 2 1 (three independent knockouts, Fig 2 5 ). Ectopic integration of transforming DNA was evidenced by multiple hybridizing bands of sizes inconsistent with homologous recombinati on and were observed in 80 to 90% of transformants across all transformations (data not shown) These ectopic and heterokaryotic transformants acted as wild type in every assayed phenotype (data not shown). Mycelial Growth Gene deletion mutants of each MAT gene were assayed across all major stages of growth and development to determine the phenotypic consequences of MAT gene loss of function throughout the lifecycle. M utants were grown on PDA media and microscopic examination revealed n o differences bet ween wild type and mutant hyphae size or branching patterns (data not shown) Growth rates were measured using race tube assays and a significant difference between the wild type and all mutants was measured (p<0.0 1). mat 1 1 5 (20.3 mm/day) and mat 1 2 4 (20.6 mm/day) grew at a faster rate than wild type and mat 1 1 1 (19.8 mm/day) and mat 1 2 1 (19.4 mm/day) grew at a significantly slower rate than wild type (20.2 mm/day) (Fig 2 6 ). Mycelial compatibility was tested for all MAT gene mutants and no discerni ble changes were observed between pairs of mutants or between mutant and wild type pairings (data not shown)
44 Pathogenicity Pathogenicity trials using detached tomato leaves were performed for each mutant and compared against wild type for general symptom development and for changes in the level of virulence There was no visible difference between the mutant and wild type strains in the timing or rate of disease symptom development (Fig 2 7 ). Sclerotia l Development Each mutant produced sclerotia with rind, cortex and medulla layers visibly indistinguishable from wild type. Average sclerotium mass (Fig 2 8 ), total number of sclerotia produced and spermatia production (Fig 2 9 ) were calculated for each mutant. The average individual s clerotium mass appeared to vary from wild type in the mat 1 2 4 mutant exhibit ing an increase in mass while the other MAT locus gene mutants exhibited a decrease when compared to wild type. Despite t he apparent qualitative differences variation in the s amples proved to be too large to be statistically significant ( p>0.20). Spermatia production was also affected in all four mutants with a significant decrease in production compared to wild type (p<0.015) and no significant difference (p<0.05) among the four MAT locus gene mutants Thin sections for microscopic observation were made of the sclerotia on a weekly basis from the stage of maturation through wild type apothecia development. At week two of sclerotial incubation for apothecia induction al l mutants formed multiple ascogoni a within their sclerotia with numbers, morphology and developmental timing indistinguishable from the wild type (Fig 2 10 ).
45 Apothecia Development Sclerotia from each mutant and the wild type were assayed for apotheciu m development under standard conditions of light, and controlled moisture and temperature After a period of 4 6 weeks (35.7 days on average) wild type apothecia began to develop. The mat 1 2 4 mutant began produc ing apothecia a n average of 1 51.3 days after the wild type controls. The mat 1 1 5 mat 1 1 1 and mat 1 2 1 mutants never exhibited signs of carpogenic germination or apothecia development f ollowing 40 weeks of incubation an d assaying greater than 500 to 1400 sclerotia per mutant over 4 6 experiments ( Fig 2 11 ). The majority of apothecia produced by mat 1 2 4 were arrested at the initial stage of stipe development Of 258 total apothecia observed only 42 (16%) proceed ed beyond stage one (undifferentiated stip es) of apothecia development Of the developmentally arrested apothecia stipes developed in bundles of one (54), two (72), three (68) and four (22) and did not develop beyond the point of undifferentiated, arrested stipe emergence ( Fig 2 1 2) Over the course of all experiments, the number of apothecia produced per scleroti um was calculated at 0.03 for the mat 1 2 4 mutant and 0.47 for the wild type. The apothecia of mat 1 2 4 mutants develop ing beyond stage 1 showed a wide variety of aberrant disc morpholog ies This phenotype ranged from the development of long tubular apothecia (Fig 2 1 2 ) with uniform undifferentiated hyphae throughout the length of the stipe and failing to form an apical depression indicative of apothecial disc initiation. Other apothecia had a long tubular morphology with a partial apical depression that originated early but never fully expanded (Fig 2 1 3, Fig 2 15) The final morphological class included stipes producing partially expanded crenulated discs (Fig 2 1 4 Fig 2 15 ). Apothecia in this class produced viable
46 ascospores and tissue layers including the ectal excipulum, the medullary excipulum, subhymenium and hymenium with microanatomies as in wild type. Several examples of aberrant apothecial morphologies were selected for thin section and ascospore analysis. Fresh a pothecia from the mat 1 2 4 mutant were imaged with differential interference contrast and brightfield microscopy for ascus and ascospore formation. Of the 100 observations generated from the various preparations, an ascus exhibiting the wild type number of eight ascospores was nev er observed. Variations of two, four and six ascospores were observed and commonly when six ascospores were found, two were distorted and shriveled (Fig 2 16 ). Ascospores exhibiting wild type morphology were examined following DAP I staining and containe d two nuclei as observed in the wild type (data not shown). Ascospores plated on PDA and later transferred to PDA with 100g/ml hygromycin were viable, stably expressed hygromycin resistance and recapitulated the phenotypes observed with the progenitor ma t 1 2 4 mutant. Gene Expression Quantitative reverse transcription PCR (qRT PCR) analysis was performed to measure the affect of gene mutation on select gene expression at various points of the life cycle. Actively growing mycelia, enlarging sclerotia (stag e 3) and pigmented sclerotia (stage 5) were chosen for analysis by measuring expression of the four MAT one putative pheromone and two putative pheromone receptor gene s for each tissue type of all four mat gene mutants MAT Gene Expression During vegetative mycelial growth overall MAT gene expression increased when compared to wild type with one exception. T he mat 1 1 5 mutant showed a significant increase
47 in MAT 1 2 4 (+3.39) and MAT 1 2 1 (+5.05 ) gene expression The mat1 1 1 mutant showed a decrease in MAT1 1 5 ( 4.38) gene expression and an increase in MAT1 2 1 (+3.07) gene expression. The mat 1 2 4 mutant displayed an increase in MAT 1 2 1 (+3.07 ) gene expression. The mat 1 2 1 mutant showed an increase in MAT 1 2 4 (+41.79 ) gene expression (Fig 2 17 ). During stage 3 of sclerotial development a trend of decrease d or no change in MAT 1 1 5 and MAT 1 1 5 gene expression and an increase or no change in MAT 1 2 4 and MAT 1 2 1 gene expression was observed T he mat 1 1 5 mutant exhibited no significant alterations in MAT gene expression (Fig 2 18 ). The mat 1 1 1 mutant showed very large decreases in MAT 1 1 5 ( 11.07) and MAT 1 2 4 ( 8.16) gene expression. The mat 1 2 4 mutant showed a large decrease in MAT 1 1 5 ( 8.14) and a large increase in MAT 1 2 1 (+5.38) gene expression. The mat 1 2 1 mutant showed an increase in MAT 1 1 1 (+ 3.32) and a very large increase in MAT 1 2 4 ( + 24.42) gene expression. There was one observed difference in the trends described fo r the mat 1 1 1 mutants where MAT 1 2 4 gene expression decreases, an effect also seen with the putative pheromone and pheromone receptor gene expression (see below) During s tage 5 sclerotia l development an overall trend of no change or a decrease in MAT gene expression was observed with only one exception. T he mat 1 1 5 mutant showed an increase in MAT 1 2 1 (+3.41) gene expression (Fig 2 19 ). The mat 1 1 1 mutant showed a very large decrease in MAT 1 2 4 ( 21.93) and an increase in MAT 1 2 1 (+3.55) gene expression. The mat 1 2 4 mutant showed a very large decrease in MAT 1 1 5 ( 31.67) gene expression. The mat 1 2 1 mutant showed a small decrease in MAT 1 1 1 ( 3.02) and a very large increase in MAT 1 2 4 (+22.14) gene expression. The one exceptio n in the trend was the mat 1 2 1 mutant and the significant increase in MAT 1 2 4 gene expression
48 Putative Pheromone and Phero mone Receptor Gene Expression In mycelial tissue putative pheromone and pheromone receptor gene expression was increased or showed no change with only one exception. T he mat 1 1 5 mutant showed an increase in P pg 1 (+5.91), PreA (+8.86) and PreB (+3.06) gene expression (Fig 2 20). The mat 1 1 1 mutant showed a large increase in P pg 1 (+5.64) and PreA (+20.28) and a large decr ease in PreB ( 6.16) gene expression. The mat 1 2 4 mutant likewise showed a large increase in P pg 1 (+5.1) and PreA (+11.07) gene expression. The mat 1 2 1 mutant showed a large increase in P pg 1 (+5.72) and PreB (+5.3) gene expression. The one exception to the trend was in the mat 1 1 1 mutant where PreB gene expression was decreased, this coincides with other early trends of down regulation within the mat 1 1 1 mutant for other MAT genes. In stage 3 sclerotial tissue there was an in crease or no change in putative pheromone and pheromone receptor gene expression in all mutants except for mat 1 1 1 where all three genes exhibit decreased gene expression. T he mat 1 1 5 showed no significant change in the putative pheromone and pheromone receptor gene expression (Fig 2 20). The mat 1 1 1 mutant showed a significant decrease in P pg 1 ( 22.09), PreA ( 20.65) and PreB ( 4.5) gene expression. The mat 1 2 4 mutant showed a significant increase in PreB (+6.39) gene expr ession. The mat 1 2 1 mutant showed no significant changes in the putative pheromone and pheromone receptor gene expression. The extreme decreases of gene expression in the MAT genes and the putative pheromone and pheromone receptor genes shown in the ma t 1 1 1 gene mutant supports an earl y role for regulation of other MAT locus and pheromone genes. In stage 5 sclerotial tissue all putative pheromone related genes in all mutants showed a drastic decrease of more than 80 fold when compared to wild type gene expression except for
49 the mat 1 2 4 mutant with the PreB ( 4.37) gene (Fig 2 21) F old decrease s below 20 00 fold of wild type levels were int erpreted as no gene expression (T able 2 4) The smallest decrease in PreB for the mat 1 2 4 mutant may support a role for PreB in the pre fertilization or fertilization events necessary for apothecia development as the mat 1 2 4 mutant was the only MAT gene mutant to show apothecia devel opment Discussion Many MAT loc i genes have been described and used for epidemiological identification of isolates; yet, the number of studies characterizing the function of MAT genes in respective species are few (Debuchy and Turgeon, 2006). This study has shown that the MAT locus of S. sclerotiorum has functions throughout the lifecycle. The mat1 1 5 gene mutant displayed a complete lack of apothecia development although succ essfully produc ed ascogonia with the timing and frequency of wild type This mutant, and all of the mat gene mutants, had a significant decrease in the production of spermatia compared to wild type. These two characters indicate that MAT1 1 5 is involved in pre fertilization or fertilization events which when mutated prevented differentiation leading to stipe development from the ascogonium. When performing homology searches with the protein sequence of the MAT1 1 5 gene, the only other fungal species co ntaining a significant match is B. cinerea which has been reported in a preliminary study to require this ortholog in matings with MAT1 2 idiomorph strains for apothecial disc development (van Kan et al ., 2010). The most significant alteration noted in gen e expression of mat 1 1 5 gene mutants are in the putative pheromone and pheromone receptors which supports the involvement in pre fertilization and fertilization events In vegetative mycelial tissue there is an elevation of gene expression for all three pheromone
50 related genes similar to what has been observed in the F. graminearum MAT genes (Zheng et al ., 2013). In stage 3 sclerotial and stage 5 sclerotia l tissue we see a dramatic down regulation of all three putative pheromone related genes analyzed with the most significant decrease of P pg 1 where the transcript is virtually undetectable. Functional characterization other MAT locus gene mutants located proximal to the MAT 1 1 1 gene has reveal ed a varied set of phenotypes in other fung al species Altered regulation of pheromone and pheromone receptors in Sordaria macrospora and arrested sexual development at the point of protoperithecia formation in SmtA 2 ( mat1 1 2) gene mutants adjacent to the core MAT1 1 1 gene (Klix et al ., 2010). It is interesting to note that the mat1 1 2 gene of S. macrospora does not have an HMG domain but rather a PPF domain which plays a role in N. crassa fertility and in P. anseri n a blocks protoperithecia production despite lack of evidence for D NA binding (Klix et al ., 2010). These examples show that mating type genes other than the core MAT genes may have significant morphological phenotypes and alter gene expression without demonstrated transcription factor activity. In S. sclerotiorum there is no evidence of co regulation between the proximal MAT 1 1 5 and MAT 1 1 1 genes. This is evident as only a slight decrease of MAT 1 1 1 ( 1.81 to 2.07 fold) gene expression in the mat 1 1 5 gene mutant This finding supports independent roles in apot hecial development for these two genes The findings presented here demonstrate that loss of function mutants of the core mating type genes, mat1 1 1 and mat1 2 1 completely blocked apothecia development despite producing ascogoni a in week two of sclerotia incubation. Both genes encode well documented transcriptional regulators. This phenotype is found in other Ascomycetes but is not universal.
51 Deletion or altered gene expression of either of the core MAT genes may result in infe rtility ( N. crassa, C. heterostrophus ) and/or inability to form sexual structures ( N. crassa, P. anserin a C. heterostrophus, G. zea, B. cinerea ) (Debuchy and Turgeon, 2006 ; van Kan et al ., 20122; Zheng et al ., 2013 ). I ndirectly regulated products of the core MAT genes include pheromone and pheromone receptors genes responsible for the attraction of the male and female gametangia in Ascomycetes which when mutated arrest sexual development prior to fruiting body development (May r hrofer et al ., 2005; Debuchy and Turgeon, 2006; Pggler et al ., 2006). The homothallic Ascomycete Giberella zeae differs slightly in that the ability to form sexual structures (perithecia) is not impeded with core MAT gene mutations but the perithecia show altered mo rphology and lack of ascospores (Desjardins et al ., 2004). These mutants can be made to reproduce heterothallically by the removal of the MAT1 1 1 or MAT1 2 1 gene and crossing with a strain containing the other gene (Lee et al. 2003). The SmtA 1 ( MAT1 1 1 ) and SmtA 3 ( MAT1 1 3 ) genes in S. macrospora show no role in fruiting body development (Klix et al ., 2010); however, the Smta 1 ( MAT1 2 1 ) gene in S. macrospora is essential to fruiting body development and is arrested after ascogonium and protoperith ecia development (Pggler et al ., 2006). This range of phenotypes makes the characterization of each individual MAT gene a necessary step to understanding each respective MAT locus and thus sexual regulation of that species. As seen here, MAT 1 1 5, MAT 1 1 1 and MAT 1 2 1 are essential for regulating early stages of sexual compatibility and MAT 1 2 4 late stages of sexual compatibility including apothecium morphogenesis and ascospores development The MAT locus of S. sclerotiorum undergoes a 250 bp inversion described by Chitrampalam et al ( 2013 ) resulting in truncation of the MAT 1 1 1 coding sequence. Strains
52 carrying the inverted form of the MAT locus remain fertile and develop apothecia indistinguishable from non inverted st rains In this study I simultaneously confirmed th e occurrence of the MAT locus inversion and observed both the inverted and non inverted strain apothecium production. Upon further analysis of the M AT 1 1 1 alpha protein domain encoding sequences it was e stablished that the three essential alpha helices which make up the ancestral form of the alpha box (Martin et al ., 2010) are retained in the inversion strains (Fig A 4). The retention of the ancestral alpha box, the development of apothecia in both inverted and non inverted strains, and the lack of development of apothecia in the mat 1 1 1 gene mutant suggests that the truncation of the alpha 1 protein at the inverted locus does not effect its function All MAT gene mutations altered gene expression of other MAT locus genes. The most significant change that occurr ed in the mat1 2 1 mutant was that MAT1 2 4 gene transcription levels increased across vegetative hypha and sclerotial development stages (+41.79, +24.42, +22.14) above wild type and far ab ove any of the other genes analyzed The mat1 1 1 mutant showed a significant decrease in MAT1 2 4 gene transcription beginning in stage 3 sclerotia ( 8.16) and continuing to stage 5 sclerotia ( 21.93) As the homologous replacement events were designed to not alter the promoter sequences of adjacent mating type genes the changes in gene expression are interpreted to result from the loss of regulatory interactions among the MAT proteins MAT1 2 4 is flanked by the core MAT locus genes so this still may be a form of structural regulation or direct gene product interaction similar to the cis regulation seen in N. crassa (Ferrei r a et al ., 1997).
53 There is a decrease ( 11.07) in MAT 1 1 5 gene expression in the mat 1 1 1 gene mutants that appears in stage 3 sclerotial tissue but subsides in stage five sclerotial tissue ( 1.53) The mat 1 1 1 gene mutants also showed the earliest change in the putative pheromone and pheromone receptor gene expression in stage 3 sclerotial tissue. In this tissue most of the other MAT gene mutants show elevated putative pheromone gene expression and the mat 1 1 1 gene mutants have begun to down regulate all three putative pheromone genes. By stage five mat 1 1 1 gene mutants show the most severe down regulation of all the MAT locus mutants in all three putative pheromone genes. The mat 1 2 1 gene mutant showed increases in all putative pheromone genes until stage 5 sclerotia where it showed a decrease in all putative pheromone genes. The MAT 1 1 1 gene appears to be involved in regulatory events of both the other MAT genes and the three putative pheromone and pheromone receptor genes before the other three MAT locus genes. By stage five all four MAT locus genes are highly active in regulation of either other MAT genes or the three putative pheromone and pheromone receptor genes analyzed. The mat1 2 4 gene mutant was the only mutant to retain apothecia production although they were unable to form fully expanded discs and the majority of stipes did not proceed beyond stage 1 of apothecial development. The production of apothecia was also extremely delayed, ranging from 49 to 196 days beyond the time of wild type apothecia initiation Following carpogenic germination, the mutation appears to aff ect the timing of apical disc depression and disc expansion consistent with a defect in interpreting signaling processes such as light recept ion or e nd ogenous spatial orientation signals required for proper patterning of tissue development. A subset of ap othecia from this mutant displayed tubular
54 forms in which disc expansion appears to initiate early but manifests as an expansion parallel to the stipe axis rather than horizontal. Tissue differentiation reminiscent of early disc development appears in mic roscopic sect io ns of these tissues A more readily observed phenotype in the mat1 2 4 mutants was a pothecia with malformed discs of crenulated morphology or completely folded in upon themselves These malformations occurred very early in the process of disc expansion and were never corrected as the apothecial disc matured. These observation s would be consistent with a hypothesis that that the signals to widen and invaginate the stipe apex to form a fully developed apothecia are present but not coordi nated well and perhaps the signals for such morphological development are being interpreted through secondary sub optimal pathways as a result of the primary pathway being interrupted. A distortion in the formation of ascospores of mat1 2 4 mutants was a lso observed such that a single ascus with a full set of eight ascospores was never found. Sets of two, four, and six were common. When six ascospores were present, two of the ascospores were shriveled and much smaller than the wild type ascospores. A similar observation has been made in the homothallic Ascomycete Coniochaeta tetraspora where a non random disintegration of sets of two ascospores, four in total, result in four spored asci and is hypothesized that similar to other fungi where MAT locus ge nes are involved in nuclear pairing and an epigenetic trigger may be signaling ascospore loss (Raju and Perkins, 2000). The disrupted arrangement of the MAT locus is N. crassa MATA 2 mutants followed with ectopic complement ation, restored sexual developme nt up to the point of ascospore formation but no ascospores are produced (Ferriera et al ., 1997). The arrangement of the MAT locus has also been shown to have a n effect on cytoskeleton stability and arrangement Yeast homokaryotes expressi ng MATa/MATa sh owed
55 decreased abilities to undergo c ytokenisis, chromosomal pairing and microtubule formation when compared to a heterokaryote expressing which exhibited increased abilities to do the same ( Steinberg Neifach and Eshel, 2002). Cytoskeletal disru ptions have also been found to disrupt fruiting body development in Aspergillus nidulans (Pggler et al ., 2006). This leads me to propose three hypotheses for the disrupted ascospore formation in mat1 2 4 gene mutants: 1) a regulatory pathway disruption involving the MAT locus itself has led to a deficiency in ascospore formation 2) improper nuclear pairing due to cytoskeletal instability and nuclear pair recognition error s lead to a deficiency in ascospore formation, or 3) programed death of ascospore s from meiotic progeny which do not recognize each other at the nuclear level. The hypothesis that MAT1 2 4 regulates fundamental aspects of cytoskeleton function could most parsimoniously account for the gross morphological phenotypes and well as the disto rted ascospores numbers and could be tested in the future with specific cytoskeletal mutants or chemical treatments known to disrupt cytoskeleton functions. The effect of the mat 1 2 4 gene deletion on MAT 1 1 1 and MAT 1 2 1 gene expression were not as se vere as the effects of the mat 1 1 1 and mat 1 2 1 gene deletions on MAT 1 2 4 gene expression. This supports a hypothesis that MAT 1 2 4 may be regulated by MAT 1 1 1 and MAT 1 2 1 In stage five sclerotia of mat 1 2 1 mutants MAT 1 2 4 gene transcripti on is up regulated (+23.14) this would indicate that it is down regulated in the wild type at the earliest stages of apothecia development but required later for proper apothecial disk development as seen in the mat 1 2 4 mutants apothecial phenotypes. Th e largest MAT gene expression effect of the mat1 2 4 mutant was on the MAT1 1 5 gene beginning in sclerotial stage 3 tissue ( 8.14) and continuing in stage 5 sclerotial growth ( 31.67 fold ) This may indicate that MAT1 2 4 is an
56 upstream regulator of MAT1 1 5 in sclerotial tissue but does not impede the function of Mat1 1 5 to the level that affects apothecium development Sexual and non sexual developmental phenotypes involving pheromone and pheromone receptor gene double knockouts and individual pheromon e gene knockouts have been well documented (P ggler and K ck, 2001; Seo et al ., 2004; Mayrhofer et al., 2005; Lee et al., 2008; Zheng et al., 2013) The alteration of putative pheromone production in the mat 1 2 4 gene mutant was seen as an increase in transcript levels in mycelial tissue and stage 3 sclerotial tissue and then a decrease in stage 5 sclerotial tissue. The PreB ( 4.37) gene expression was the least down regulated putative pheromone g ene observed and may support a role in the pre fertiliz ation and fertilization events that allowed the mat 1 2 4 gene mutant to dev elop apothecia. In the other three MAT gene mutants PreB is down regulated from 129.3 to 1075 fold relative to wild type which may prevent them from fertilizing successfully or may play a role in developing elongated stipes and expanded discs later The P pg 1 and PreA genes down regulation may also support a role in the phenotypes observed in the stages of development after a mature sclerotia are formed in S. sclerotiorum Gene knockouts of the P pg 1 P pg 2 Pre1 ( PreA ) and Pre2 ( PreB ) genes in G. zeae were analyzed for expression and function (Lee et al ., 2008). P pg 2 which was not found in S. sclerotiorum was not found to be expressed in any G. zeae tissue analyzed Th is short (25 AA) protein that is not well conserved among Ascomycete species (Lee et al ., 2008) Pre2 / P pg 1 were found to enhance but not be essential for selfing and outcrossing while P pg 2 / Pre1 had no discernible role (Lee et al ., 2008). This lack of function or possible redundant function for P pg 2 may have led to the eventual loss of the sequence completely in S. sclerotiorum
57 This study has shown the MAT locus is involved in a broad range of developmental processes includin g regulation of sexual development, sexual structures, mycelial growth ( MAT1 1 1 and MAT 1 2 1 ), and MAT locus and putative pheromone and pheromone receptor gene regulation. Further study into the roles of the putative pheromone and pheromone receptors by functional analysis and the characterization of other genes regulated by the MAT locus will provide a more complete understanding to the sexual regulation of S. sclerotiorum and the roles of endogenous and exogenous signaling in coordinating complex fru iting body development
58 Table 2 1 Primers used for UTR amplification and cloning. Primer Name Primer Sequence MAT 1 1 5 5' UTR F1 AGG TCT GCC GTC TAG ATC AT MAT1 1 5 5' UTR R1 AGG CGC GCC TGT TAC GAG GTT TGC CGT CT MAT 1 1 5 3' UTR F1 AGG CGC GCC ACC GTT TAA GGG AAA TCC AG MAT 1 1 5 3' UTR R1 ACG TCC AAA TCT GTC AAG GT MAT 1 1 1 5' UTR F1 CGC CAG GAA CGA AAT GCG AAA GAA MAT 1 1 1 5' UTR R1 AGG CGC GCC TAT GAA TTG ACA GAG CGC CGA GGA MAT 1 1 1 3' UTR F1 GGC GCG CCA GGC ATT GTC CTG TTC CCA CGA TA MAT 1 1 1 3' UTR R1 TCC AAG ACG ACA ACT CCA CAA CCA MAT 1 2 4 5' UTR F1 AGG AAA GCT GAT GGA AGA GGT GGT MAT 1 2 4 5' UTR R1 AGG CGC GCC TCT CAA GCG GTA TGA TCC CAC CAA MAT 1 2 4 3' UTR F1 GGC GCG CCC GGC AAG CTT GAT GCT TAA CAG GT MAT 1 2 4 3' UTR R1 TAC GA G ACA AAT CGG GTG GGT TGA MAT 1 2 1 5' UTR F1 AGG GAG GAG ATG GGA CAT AGT TCT MAT 1 2 1 5' UTR R1 AGG CGC GCC TTG GTG GGA TCA TAC CGC TTG AGA MAT 1 2 1 3' UTR F1 GGC GCG CCT TGG TGC GAA CAG CAA TTA CGA GC MAT 1 2 1 3' UTR R1 HY Split Marker YG Split Marker AAG AAC AAC CGA TGG ACT GAG GGT AAA TTG CCG TCA ACC AAG CTC TGA TAG TTT CAG CTT CGA TGT AGG AGG GCG
59 Table 2 2 Primers used for generation of the probes in Southern blot analysis of the MAT locus genes in S. sclerotiorum. Primer Name Primer Sequence `5 3` MAT 1 1 5 Coding Probe F1 ATA TTC GCC CCT GCG CCC TT MAT 1 1 5 Coding Probe R1 TGG ATT CTC CTG CCG TCT GA MAT 1 1 5 3' UTR Probe F1 GTT AGC CGA CTC CAG CCA CA MAT 1 1 5 3' UTR Probe R1 GAG CCT CAA CCC ACC CGA TT MAT 1 1 1 Coding Probe F2 TTG CTC CAC CTC CCA AGC CA MAT 1 1 1 Coding Probe R2 AGA GAT ATC GCC AGG AAC AT MAT 1 1 1 3' UTR Probe F1 ACA CTC CCC AGT ATG GAT MAT 1 1 1 3' UTR Probe R1 ACG AGG AAG CCT GAT GCG TA MAT 1 2 4 Coding Probe F2 AGC CGA TTT GGG GCT GGT GA MAT 1 2 4 Coding Probe R2 AAA GGG AGG AGA TGG GAC AT MAT 1 2 1 Coding Probe F1 TCA ACC CAT GGT GTG AAC TA MAT 1 2 1 Coding Probe R3 AAG CCT GCG ACG GCT AAC AT MAT 1 2 1 3' UTR Probe F1 GGC GCG CCT TGG TGC GAA CAG CAA TTA CGA GC MAT 1 2 1 3' UTR Probe R1 AAG AAC AAC CGA TGG ACT GAG GGT
60 Table 2 3 Primers designed for QPCR analysis. Primer Name Primer Sequence `5 3` MAT 1 1 5 QPCR Primer F1 CGG TGA AAT TGA GGG TGG TA MAT 1 1 5 QPCR Primer R1 GGC ATG AAA AGG ATC ACG TC MAT 1 1 1 QPCR Primer F1 TTG GAT CCA TCG ACT TTT CC MAT 1 1 1 QPCR Primer R1 CCG CAT ACT TCG TGG GTA AT MAT 1 2 4 QPCR Primer F1 AAA GGG AGG AGA TGG GAC AT MAT 1 2 4 QPCR Primer R1 AGC CGA TTT GGG GCT GGT GA MAT 1 2 1 QPCR Primer F1 TCA ACC CAT GGT GTG AAC TA MAT 1 2 1 QPCR Primer R1 AAG CCT GCG ACG GCT AAC AT P PG 1 QPCR Primer F1 CAA ACG CAA TTG CTC TCG CT P PG 1 QPCR Primer R1 CCG CAC CAT GCC TCA GCA TT PreA QPCR Primer F1 AAC CGC TGG TCC GGC CAT AT PreA QPCR Primer R1 AAG TGC TCA TCA GGT GTG GT PreB QPCR Primer F1 TCT CTT CGC GCT CAA TAC CC PreB QPCR Primer R1 CTC CGT ACT ATC TCG CGG AA
61 Table 2 4 Composite of the gene expression comparisons in the MAT locus mutants. Mycelia Stage 3 Sclerotia Stage 5 Sclerotia Transcript Mutant Fold Change Standard Deviation Fold Change Standard Deviation Fold Change Standard Deviation MAT 1 1 1 mat1 1 5 2.07 1.61 1.84 0.86 1.81 0.2 MAT 1 2 4 mat1 1 5 3.39 2.04 1.64 0.56 1.12 0.35 MAT 1 2 1 mat1 1 5 5.05 1.37 1.13 0.81 3.41 0.35 P pg 1 mat1 1 5 5.91 1.24 1.21 0.52 9630.09 4.4 PreA mat1 1 5 8.86 0.59 1.13 0.64 895.57 1.35 PreB mat1 1 5 3.06 0.97 2.1 2.47 1074.91 3.05 MAT 1 1 5 mat1 1 1 4.38 1.06 11.17 1 1.53 0.24 MAT 1 2 4 mat1 1 1 2.06 1.6 8.16 0.75 21.93 1.77 MAT 1 2 1 mat1 1 1 3.38 1.06 1.08 0.97 3.55 0.35 P pg 1 mat1 1 1 5.6 4 0.83 22.09 0.84 14197.34 3.22 PreA mat1 1 1 20.28 1.27 20.65 0.51 2836.7 1.69 PreB mat1 1 1 6.16 3.49 4.5 0.51 925.02 1.35 MAT 1 1 5 mat1 2 4 1.96 2.34 8.14 3.43 31.67 0.77 MAT 1 1 1 mat1 2 4 1.09 1.58 1.28 1.41 2.64 1.04 MAT 1 2 1 mat1 2 4 3.07 1.72 5.38 2.59 1.66 0.44 P pg 1 mat1 2 4 5.1 0.81 2.52 0.37 482.15 4.57 PreA mat1 2 4 11.07 1.11 2.35 0.71 80.08 3.6 PreB mat1 2 4 1.56 0.78 6.39 1.14 4.37 2.78 MAT 1 1 5 mat1 2 1 2.74 2 2.32 1 1.1 0.35 MAT 1 1 1 mat1 2 1 1.02 1.93 3.32 0.41 3.02 1.3 MAT 1 2 4 mat1 2 1 41.79 1.93 24.42 0.23 22.14 0.34 P pg 1 mat1 2 1 5.72 2.21 2.67 1.48 954.32 1.99 PreA mat1 2 1 2.57 0.86 1.43 0.44 94.68 1.46 PreB mat1 2 1 5.3 0.83 2.56 0.32 129.34 2.93
62 Figure 2 1 The apothecial stages of S. sclerotiorum Apothecia are numbered sequentially and defined by easily measurable morphological changes: Stage 1 ( undifferentiated stipe elongation ); Stage 2 ( concave stipe tip ), Stage 3 ( deepening of the stipe tip ); Stage 4 (widening of the stipe ), Stage 5 ( disc expansion) Stage 6 (outturning of the disc ) and Stage 7 (mature apothecia with no further expansion ).
64 Figure 2 2 The split marker strategy for MAT 1 1 5 gene replacement. A) The MAT locus of S. sclerotiorum B) Graphic representation of the split marker strategy. C) Genetic confirmation of the homologous recombinations. All homologous recombinations required along with the probes designed for identifi cation of successful gene knockout (B ). Genomic DNA was isolated from wild type (C, 1) and each transformant (C, 2 3) and digested with Pst I which would produce either a 7146 wild type bp band during Southern analysis or a 4999 bp homologous recombinatio n band wh ). The transformants were also analyzed by PCR utilizing the coding sequence probe primers (C ).
66 Figure 2 3 The split marker strategy for MAT 1 1 1 gene replacement. A) The MAT locus of S. sclerotiorum B) Graphic representation of the split marker strategy. C) Genetic confirmation of the homologous recombinations. All homologous recombinations required along with the probes designed for identification of successful gene knockout (B ). Genomic DNA wa s isolated from wild type (C, 1) and each transformant (C, 2 4) and digested with Hind III which would produce either a 1657 wild type bp band during Southern analysis or a 2858 bp homologous recombination band wh ). The tra nsformants were also analyzed by PCR utilizing the coding sequence probe primers (C ).
68 Figure 2 4 The split marker strategy for MAT 1 2 4 gene replacement. A) The MAT locus of S. sclerotiorum B) Graphic representation of the split marker strategy. C) Genetic confirmation of the homologous recombinations. All homologous recombinations required along with the probes designed for identification of successful gene knockout (B ). Genomic DNA wa s isolated from wild type (C, 1) and each transformant (C, 2 4) and digested with Hind III which would produce either a 1745 wild type bp band during Southern analysis or a 2494 bp homologous UTR probe (C ). The transformants were also analyzed by PCR utilizing the coding sequence probe primers (C ).
70 Figur e 2 5 The split marker strategy for MAT 1 2 1 gene replacement. A) The MAT locus of S. sclerotiorum B) Graphic representation of the split marker strategy. C) Genetic confirmation of the homologous recombinations. All homologous recombinations required along with the probes designed for identification of successful gene knockout (B ). Genomic DNA wa s isolated from wild type (C, 1) and each transformant (C, 2 4) and digested with Bam HI which would produce either a 7759 wild type bp band during Southern analysis or a 5840 bp homologous recombination band when uti ). The trans formants were also analyzed by PCR utilizing the coding sequence probe primers (C ).
71 Figure 2 6 The average daily mycelial growth of the MAT locus mutants. All transformants were grown under 12hr light and 12 hr dark cycles in race tubes. Daily mea surements of nine technical replications of each mutant were made and compared against wild type growth and showing one standard deviation Growth was analyzed with a 2 sample t test and all mutants exhibited statistically significant growth different fro m wild type and each other (p<0. 01). 18 18.5 19 19.5 20 20.5 21 mat1-1-5 mat1-1-1 mat1-2-4 mat1-2-1 WT mm/day Daily Average of Mycelial Growth of the MAT Locus Mutants
72 Figure 2 7 Pathogenicity assays for MAT locus mutants. A) mat 1 1 5 assay. B) mat 1 1 1 assay. C) mat 1 2 4 assay. D) mat 1 2 1 assay. E) WT assay. Detached tomato leaf assays were performed and inoculated with a 3mm plu g of actively growing mycelia from a PDA plate. There was no differences in ability to infect and cause disease symptoms.
73 Figure 2 8 Average mass per sclerotium for MAT locus mutants. The average mass for sclerotium was determined by sample statistic from ten sclerotia grown in 20 cm potato plate cultures and shown with one standard deviation mat 1 2 4 showed a higher average than wild type sclerotial mass with the remaining MAT locus mutants showing a decr ease in average sclerotial mass however this was not found to be statistically significance due to the high variance of all the sclerotia sampled (p>0.20). 0 0.01 0.02 0.03 0.04 0.05 0.06 WT mat1-1-5 mat1-1-1 mat1-2-4 mat1-2-1 mass (g) Average Mass per Sclerotium for MAT Locus Mutants
74 Figure 2 9 Relative spermatia production for MAT locus mutants. T he average number of sp ermatia produced was calculated for 10 sclerotia from each mutant and wild type in to 1 ml of water f or three replications and shown with one standard deviation All mutants showed significantly decreased spermatia production in rela tion to wild type (p<0.015) however little difference between each other (p<0.05). 0.00E+00 1.00E+01 2.00E+01 3.00E+01 4.00E+01 5.00E+01 6.00E+01 7.00E+01 8.00E+01 9.00E+01 1.00E+02 mat1-1-5 mat1-1-1 mat1-2-4 mat1-2-1 WT Spermatia/10 sclerotia/1ml H 2 O Relative Spermatia Production
75 Figure 2 1 0 Sclerotial cross sections for MAT locus mutants. A) mat 1 1 5 sclerotial cross section. B) mat 1 1 1 sclerotial cross section. C) mat 1 2 4 sclerotial cross section. D) mat 1 2 1 sclerotial cross section. Cross sections stained with methyle ne blue of the mutant sclerotia with ascogonium indicated by arrows N o differences in timing or morphology of ascogonium development were observ ed among m utants and the wild ty pe.
76 Figure 2 1 1 The average apothecia pro duction time in wild type and mutant strains Apothecia were grown in a growth chamber under constant cool white lights, moisture and temperature. The time each apothecium was fi rst detected was recorded and the average incubation time to first detection for mat 1 2 4 (187.1 days) was found to be significantly longer (p<0.025) than wild type (35.7 days) incubation and shown with one standard deviation T he other MAT locus mutants neve r exhibited any signs of apothecia production.
77 Figure 2 1 2 Tubular mat 1 2 4 apothecia and arrested stipes. A) mat 1 2 4 sclerotium and apothecia. B) Additional mat 1 2 4 sclerotium and apothecia. mat 1 2 4 apothecia showed a variety of phenotypes ranging from arrested stipe initials in bundles of two (A, arrowed) and three (B, arrowed) to long tubular stipes with no disc widening (A ,B ) and long tubular stipes with arrested disc widening.
78 Figure 2 1 3 Cross sections of tubular mat 1 2 4 apothecia. A) Cross section of point A in panel D. B) Cross section of point B in panel D. C) Cross section of point C in panel D. D) mat 1 2 4 apothecia from figure 2 16 labeled for cross sections. The mat 1 2 4 apothecia showed partial disc invagination sho wing the uniform tissue type (A ) throughout the tubular portion of the ttempt (B, C ) at an invagination and disc formation at the more basal end of the stipe shown by the differential tissue types in circular rings from the center of the stipe cross section and small opening at the tip Apothecia with no invagination showed no tissue differentiation in the stipe.
79 Figure 2 1 4 Crennulate apothecia of mat 1 2 4 A) Arrested stipes and apothecia of the mat1 2 4 mutant. B) Close up of the mat 1 2 4 mutant apothecia. mat 1 2 4 apothecia showing a large number of stipes that did not develop past stage one (A, arrowed) and the commonly found widening of the stipe in the apothecia that did proceed beyond stage one (B). The apothecia that did invaginate their disc and proceed to widening of the cup did so in a variety of crenulated folded patterns.
80 Figure 2 1 5 Crenulate and tubular apothecia of the mat 1 2 4 mu tant A) Multiple apothecia of the mat 1 2 4 mutant. B) Additional apothecia of the mat 1 2 4 mutant. The mat 1 2 4 apothecia showed a wide variety of phenotypes of the stipes that proceeded beyond stage one. Apothecia commonly had widened stipes and ult ra folded crenulated discs (A, B)
81 Figure 2 16 Ascus and ascospore formation in the mat 1 2 4 mutant A) mat1 2 4 ascus. B) mat1 2 4 ascus. C) WT ascus. D) mat1 2 4 ascus. E) mat1 2 4 ascus. The a scus from mat 1 2 4 (A,B, D,E) showed an abnormal number of ascospores formed compared to wild type (C ). When six ascospores were found two of the ascospores were comm only found to have a shriveled appearance (A,B,D, arrowed).
82 Figure 2 17 MAT g ene expression in mycelial tissue of the MAT locus mutants. Gene expression in mycelial tissue of the four MAT locus gene mutants were compared against wild T method. Three biological replications with three tec hnical replications were performed and statistically significant differences (p<0.05) are marked with a star Each of the MAT locus genes were compared against wild type expression levels and the fold change in gene expression is displayed showing standar d deviations for the mean
83 Figure 2 18 MAT g ene expression in stage three sclerotial tissue of the MAT locus mutants. Gene expression in stage three sclerotial tissue for the four MAT locus gene mutants was compared against wild type expression using the T method. Three biological replications with three technical replications were performed and statistically significant differences (p<0.05) are marked with a star. Each of the MAT locus genes were compared against wild type expression levels and the fold change in gene expression is displayed showing standard deviations for the mean
84 Figure 2 19 MAT g ene expression in stage five sclerotial tissu e of the MAT locus mutants. Ge ne expression in stage five sclerotial tissue for the four MAT locus gene mutants was compared T method. Three biological replications with three technical replications were performed and statistically significant differences (p<0.05) are marked with a star. Each of the MAT locus genes were compared against wild type expression levels and the fold change in gene expression is displayed showing standard deviations for the mean
85 Figure 2 20 Putative pheromone and pheromone receptors g ene expression in mycelial tissue of the MAT locus mutants. Ge ne expression in mycelial tissue for the four MAT locus gene T method. Three biological replications with three technical replications were performed and statistically significant differences (p<0.05) are marked with a star. Each of the putative pheromone and pheromone receptor genes were compared against wild type expression levels and the fold change in gene expression is displayed with standard deviations of the mean
86 Figure 2 21 Putative pheromone and pheromone receptors g ene expression in stage three sclerotial tissue of the MAT locus mutants. Ge ne expression in mycelial tissue for the four MAT T method Three biological replications with three technical replications were performed and statistically significant differences (p<0.05) are marked with a star. Each of the putative pheromone and pheromone receptor genes were compared against wild type expre ssion levels and the fold change in gene expression is displayed with standard deviations of the mean
87 Figure 2 22 Putative pheromone and pheromone receptor g ene expression in stage five sclerotial tissue of the MAT locus mutants. Ge ne expression in mycelial tissue for the four MAT T method. Three biological replications with three technical replications were performed and statistically sign ificant differences (p<0.05) are marked with a star. Each of the putative pheromone and pheromone receptor genes were compared against wild type expression levels and the fold change in gene expression is displayed with standard deviations of the mean
88 C HAPTER 3 CONCLUSIONS Mutation of the MAT genes of S. sclerotiorum manifest as distinct phenotypic alterations in the sexual life cycle and various aspects of the secondary sexual characteristics. I have determined that the MAT 1 1 1 MAT 1 1 5 and MAT 1 2 1 genes are necessary for the initiation of apothecia while the MAT 1 2 4 gene is necessary for the proper timing and development of apothecial discs. The two core mating type gene mutants mat 1 1 1 and mat 1 2 1 appear to slow mycelial growth and decrease s permatia production. The mat 1 1 5 and mat 1 2 4 both show an increase in mycelial growth rate and decrease in spermatia production. The mat 1 2 4 apothecial phenotype is consistent with phenotypic observations of the orthologous gene in B. ciner e a (van Kan, 2011) where a disruption in disc formation and apical stipe depression was observed. The seven to twenty eight week delay of apothecia initiation along with the various types of apothecia produced where disc elongation was attempted at prematur e to delayed states shows a function in the MAT 1 2 4 gene in timing of stipe development and initiation and timing of disc differentiation. The MAT 1 2 4 gene also plays a role in ascospore formation where a full eight ascospores were never observed and th e possible degeneration of ascospores was observed. This could possibly be due to a genetic or epigenetic determinant, cytoskeletal instability in meiotic and mitotic divisions or nucleic acid recognition as in other Ascomycetes. All MAT locus mutation s showed a change in gene expression and instances where the mutant altered or reversed wild type trends in gene expression of mat genes. There appears to be a link between MAT 1 2 1 gene regulation and MAT 1 2 4 regulation in tissues of all types and I hyp othesize that downstream regulation or their colinearity is responsible for
89 this P utative p heromone and pheromone receptor transcripts were altered as seen in other MAT gene mutants, when measuring mycelial tissue an increase in gene expression is observed This is reversed once the MAT gene mutants started stage 5 sclerotial development where very significant decreases to essential elimination of any transcripts. Futu re studies into the roles of the putative pheromone and pheromone receptors and the characterization of other genes regulated by the MAT locus hopefully will provide a more complete understanding to the sexual regulation of S. sclerotiorum
90 APPENDIX A T HE 250 BASEPAIR INVERSION IN THE MAT LOCUS OF SCLEROTINIA SCLEROTIORUM During investigation of the MAT locus of S. sclerotiorum probes were made for the coding sequence of the MAT 1 1 1 and MAT 1 2 1 genes. These coding sequences were located in the coding sequence replaced with a hygromycin gene cassette. A second probe was made using UTR sequences which would exhibit a size change in the banding pattern for homologously recombined knockouts that were homokaryotic. PCR was finally used as a third method of confirming the knockouts where the coding sequence probe primers were used to detect the presence or absence of the genes (Fig 2 3 C; Fig 2 5, C ). When a transformant showed signs of being a homologously recombined homokaryote for the UTR probe and PCR analysis the transformant would also show an absence of the band predicted in the coding sequence. However in some primer combinations, secondary band was observed in both the wild type and successful transformants. This band was a result of a stretch of sequence that was found in both the MAT 1 1 1 sequence and MAT 1 2 1 sequence which was later defined to be part of an inversion point ( Chitrampalam et al ., 2013). This was confirmed by me at the two isolates of S. sclerotiorum and sequencing of the inversion sites in both types to determine exactly what was being inverted (Fig A 1 through A 3). I determined that the inversion was happening 50% of the time in each ascus and that the inversion would replace the critical HMG enco ding domain of MAT 1 2 1 along with the rest of the lineage specific DNA sequence and split the alpha domain sequence in MAT 1 1 1 between helices three and four of the DNA binding domain (Fig A 4, Fig A 5). MAT 1 1 1 reads directly into MAT 1 2 1 retaining critical helices 1 3 which make up the ancestral form of the alpha box sequence composed of 368 base pairs or 122 amino acids from the start site (Martin et al ., 2010).
91 Figure A 1 MAT locus in S. sclerotiorum tetrad number one.
92 Figure A of the MAT locus in S. s clerotiorum tetrad number one.
93 Figure A 3. PCR showing the MAT locus inversion in a second tetrad of S. sclerotiorum
94 Figure A 4. Protein sequence alignment of inverted MAT 1 2 1 against consensus protein sequence of HMG domain. This ClustalW alignment shows that the inverted portion of the gene is replaced and maintained with the HMG domain intact.
95 Non Inverted Sequence: MILNVLGNIPQKYKSAILSVLWGRDPFHAKWSILARAYTLMRDTNVRRTVSE YLALVCPYIGILAVNDYLTDLN WIFETNEEGIVCLRQTSPSDIRSFPAHIARTTLTDLDVITFCGSQGYLPAATAA Sequence Translocated During Inversion: GIVQGWNRMHPNANMAIQGTLPVTQHIASTQNAYGAWEMPKGVIVAPPPKPAFDHPLTYTLSTPLPTGTL PPPPPWSTGMMAGYWSQDNGSIDLNGLENQFDQFNPTSLGDANSLYVPGDISPPSDLTDS* Figure A 5. MAT 1 1 1 p rotein sequence alignment of S. sclerotiorum against the ancestral form of the alpha box domain. This alignment shows that ~40% of it is left behind in the inversion which contains the critical three helices of the ancestral alpha box DNA binding domain. The portion of the inverted sequence not inverted would contain helices 1 3, the primary DNA binding domains, and the remaining MAT 1 1 1 se quence of the inverted sequence that is translocated would be helix four, just outside of the cruci al DNA binding domain and will contain any lineage specific sequences of the alpha box.
96 APPENDIX B SCLEROTINIA TRIFOLIORUM MAT LOCUS INVESTIGATION The mating type locus of Sclerotinia trifoliorum has been demonstrated to perform unidirectional mating type switching (Glass and Kudlau, 1992). A mechanism of altering the mating type locus of Sclerotinia sclerotiorum has recently been described and hypothesized to be present in S. trifoliorum (Chitra malam et al., 2013). Yet the arrangement of the MAT locus of S. trifoliorum has yet to be described Contig analysis of S. trifoliorum has confirmed the presence of t he four MAT genes found in S. sclerotiorum but in a different orientation. The MAT 1 2 4 reads into MAT 1 1 1 which ends with the SLA2 gene running in the opposite direction (Fig B 1). This alone indicates a novel mechanism of MAT locus rearrangement is occurring since the position and orientation of the MAT 1 2 4 and MAT 1 1 1 genes in the same direction next to SLA2 are not possible with the method described in Chitramalam et al ., 2013. MAT1 1 1 in S. sclerotiorum would not transfer the entire portion of the MAT1 1 1 gene and would n ot place it in the same direction as MAT1 2 4 The MAT 1 2 1 MAT 1 2 1 and APN2 genes were not able to locate proximal to the identified MAT locus genes but sequences identifying them are located on other contigs Amplification of the APN2 and MAT 1 1 5 coding sequence was achieved but sequencing reactions were unable to link the two genes together in sequencing reactions. This sequence may represent repetitive sequence found next to APN2 and MAT 1 1 5 predicting an absence of the APN2 gene o r a more distal relations hip than expected when compared to the S. sclerotiorum MAT locus MAT 1 2 1 and the contig it was located in never produced an amplification product with any of the MAT locus gene primers indicating a distal placement in the genome A short sequence (113 bp) that matched with 98% identity to the S. sclerotiorum MAT 1 2 1 sequence start s MAT 1 2 4 is the only indication to date that MAT 1 2 1 exists until new sequencing data is available
97 Figure B 1. The partia l MAT locus arrangement of S. trifoliorum PCR amplification of the coding sequences of contigs containing MAT locus gene sequences that matched S. MAT locus was performed and the reactions sequenced providing the orientation of two of the four members of the S. sclerotiorum MAT locus in S. trifoliorum
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110 BIOGRAPHICAL SKETCH Benjamin Doughan was born in Iowa, United States of America and grew up on a family farm with his two sisters. His parents are Dwight and Conni Doughan who farm there still. He is an avid hockey and physical fitness enthusiast. Benjamin attended the University of North ern Iowa where he majored in biotechnology and chemistry. His undergraduate research project subject was Fusarium graminearum wh ere he worked on restriction fragment length polymorphism analysis to discern different isolates of the fungus. After graduati on he took a job in the private sector for a company inspecting pharmaceutical companies and identifying microorganisms. After that position he worked at Integrated DNA Technology working on sequencing and oligonucleotide platforms. With a plant pathol ogy career in mind, Benjamin decided t o go back and get a Master of Science in b otany at the University of Northern Iowa on a full academic scholarship. His research project was on the leaf development of two species of grapes, Ampelopsis arborea and Ampe lopsis cordata This along with some vineyard management set him on his path to Sclerotinia sclerotiorum and was funded by the alumni fellowship for the first four years. While working in Dr. Rollins lab he worked on a variety of subjects such as light regulation, sclerotial mutants, potential polycomb proteins, Sclerotinia trifoliorum mating type locus (MAT) and finally resolving to work on the MAT locus and characterize the genes within S. sclerotiorum Benjamin Doughan was awarded his PhD in December of 2013 and hopes to pursue a career as a research scientist in the future where he can combine his experience in botany along with plant pathology.