Comparative analysis of genetic incompatibility in Aspergillus niger and Podospora anserina Károly Pál Promotor: Prof. Dr. R. F. Hoekstra Hoogleraar in de Genetica Wageningen Universiteit Co-promotors: Dr. Ir. A. J.M. Debets Universitair Hoofddocent, Laboratorium voor Erfelijkheidsleer Wageningen Universiteit Dr. J. Varga Associate professor, University of Szeged, Hungary CBS Fungal Biodiversity Centre Promotie Commissie: Prof. Dr. J. A. van den Berg, Wageningen Universiteit Prof. Dr. Th. W. Kuyper, Wageningen Universiteit Prof. Dr. H.A.B. Wösten, Universiteit Utrecht Dr. R. A. Samson, Centraalbureau voor Schimmelcultures, Utrecht Dit onderzoek is uitgevoerd binnen de C.T. de Wit Graduate School. Károly Pál Comparative Analysis of Genetic Incompatibility in Aspergillus niger and Podospora anserina Proefschrift Ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. Dr. M. J. Kropff, in het openbaar te verdedigen op woensdag 23 mei 2007 des namiddags te half twee in de Aula Comparative Analysis of Genetic Incompatibility in Aspergillus niger and Podospora anserina PhD-thesis Wageningen University, with references – with summaries in English and Dutch Károly Pál, 2007 ISBN: 978-90-8504-655-4 Családomnak Table of contents: CHAPTER 1. GENERAL INTRODUCTION................................................................ 9 1.1 Sexual reproduction.............................................................................................. 11 1.2 The parasexual cycle and vegetative (in)compatibility ........................................ 13 1.3 The Aspergilli and the black mould Aspergillus niger ......................................... 15 1.4 Sordariales and Podospora anserina................................................................... 18 1.5 Outline of this thesis ............................................................................................. 20 CHAPTER 2. HETEROKARYON INCOMPATIBILITY GENES IN ASPERGILLUS NIGER .................................................................................................. 27 1. INTRODUCTION .......................................................................................................... 29 2. MATERIALS AND METHODS ....................................................................................... 30 2.1 Heterokaryon compatibility tests.......................................................................... 30 2.2 Het- and apoptosis genes...................................................................................... 30 2.3 Identification of HET homolog sequences in the A. niger databases................... 31 2.4 Identification of HET homolog sequences in other Aspergilli ............................. 31 2.5 Genome databases used for BLAST search.......................................................... 31 2.6 Comparison of homologs of different origin ........................................................31 3. RESULTS..................................................................................................................... 33 3.1 Heterokaryon (in)compatibility ............................................................................ 33 3.2 In silico comparison of yeast and filamentous fungi ............................................ 33 3.3 In silico comparison of the Sordariales and the Eurotiales ................................. 34 3.4 In silico comparison of the different Aspergilli.................................................... 34 3.5 In silico comparison of the two A. niger genomes 35 4. DISCUSSION................................................................................................................ 43 CHAPTER 3. THE HET-C GENE IN ASPERGILLUS NIGER ................................. 53 1. INTRODUCTION .......................................................................................................... 55 2. MATERIALS AND METHODS ....................................................................................... 56 2.1 A. niger genome database searches for het-c, vib-1, and pin-c ...........................56 2.2 Strains, characterization and culture conditions ................................................. 56 2.3 DNA isolation ....................................................................................................... 58 2.4 PCR and sequencing for het-c polymorphism ...................................................... 58 2.5 Introducing N. crassa het-cPA, het-cOR, and het-cGR in A. niger........................... 59 2.6 Comparison of homologs of different origin ........................................................59 2.7 Genome databases used for BLAST search.......................................................... 60 3. RESULTS..................................................................................................................... 60 3.1 Comparing the A. niger and N. crassa genomes for het-c genes and related sequences.................................................................................................................... 60 3.2 Screening a population of wild-type black Aspergilli for het-c polymorphisms .. 65 3.3 Testing the functionality of the A. niger het-c gene.............................................. 67 4. DISCUSSION................................................................................................................ 67 CHAPTER 4. ANALYSIS OF THE HCH AND HET-C GENES IN NATURAL ISOLATES OF PODOSPORA ANSERINA .................................................................. 73 1. INTRODUCTION .......................................................................................................... 75 2. MATERIALS AND METHODS ....................................................................................... 76 2.1 Strains and culture collections .............................................................................76 2.2 DNA isolation ....................................................................................................... 76 2.3 CAPS (Cleaved Amplified Polymorphic Sequence) analysis of the hch fragment77 2.4 Primer design and PCR amplification of hch and het-c genes............................. 77 2.5 Sequencing............................................................................................................ 78 3. RESULTS..................................................................................................................... 80 3.1 Absence of polymorphism in the hch gene............................................................ 80 3.2 New polymorphisms in the het-c gene .................................................................. 80 4. DISCUSSION................................................................................................................ 83 CHAPTER 5. SEXUAL GENES IN THE ASEXUAL FILAMENTOUS FUNGUS ASPERGILLUS NIGER AND RELATED BLACK ASPERGILLI ........................... 89 1. INTRODUCTION .......................................................................................................... 91 2. MATERIALS AND METHODS ....................................................................................... 92 2.1 Genes involved in mating ..................................................................................... 92 2.2 Identification of MAT homolog sequences in the A. niger databases .................. 92 2.3 Identification of mating gene homologs in other filamentous fungi..................... 93 2.4 Comparison of homologs of different origin ........................................................93 2.5 Strains and culture conditions.............................................................................. 93 2.6 DNA isolation ....................................................................................................... 94 2.7 PCR amplification ................................................................................................ 94 2.8 Sequencing............................................................................................................ 95 2.9 Statistics................................................................................................................ 95 3. RESULTS..................................................................................................................... 97 3.1 Comparison of yeast and filamentous fungi .........................................................97 3.2 Comparing the different filamentous fungi...........................................................97 3.3 Comparison of two A. niger genomes................................................................. 106 3.4 Screening for MAT homologs among black Aspergilli....................................... 108 4. DISCUSSION.............................................................................................................. 109 GENERAL DISCUSSION............................................................................................ 115 SUMMARY.................................................................................................................... 123 SAMENVATTING........................................................................................................ 127 LIST OF PUBLICATIONS.......................................................................................... 131 ACKNOWLEDGEMENTS.......................................................................................... 133 CURRICULUM VITAE..............................................................................................