Doctoral Program in Integrative Life Science (ILS) Department of Agricultural Sciences Faculty of Agriculture and Forestry University of Helsinki Pathogens of green roof mosses and the use of a Physcomitrella mutant collection as a source for elucidating genes involved in the chitosan- induced signaling pathway Eeva Marttinen ACADEMIC DISSERTATION To be presented with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public examination in Metsätalo hall 2, Unioninkatu 40 on September 24th 2020, at 12 noon Helsinki 2020 Supervisor Professor Jari Valkonen University of Helsinki Department of Agricultural Sciences FIN-00014 Helsinki Follow-up group Professor Jaakko Kangasjärvi University of Helsinki Faculty of Biological and Environmental Sciences FIN-00014 Helsinki Docent Sari Tähtiharju Finnish Medicines Agency Fimea FIN- 00034 FIMEA Reviewers Professor David Collinge University of Copenhagen Department of Plant and Environmental Sciences DK-1871 Copenhagen, Denmark Docent Kirk Overmyer University of Helsinki Department of Biosciences FIN-0014 Helsinki, Finland Opponent Associate professor Mari-Anne Newman University of Copenhagen Department of Plant and Environmental Sciences DK-1871 Copenhagen, Denmark Custos Professor Jari Valkonen University of Helsinki Department of Agricultural Sciences FIN-00014 Helsinki Language check Timothy C. Taylor Chief Science Officer BiomEditor, International Bioscience Consultants ISBN 978-951-51-6467-4 (paperback) ISBN 978-951-51-6468-1 (PDF) ISSN 2342-3161 (print) ISSN 2342-317X (online) Unigrafia Oy 2020 ABSTRACT Knowledge of the defense responses of mosses against pathogens has gained less attention than has our knowledge of the pathogens of vascular plants. Recently, the use of mosses has gained attention because mosses are used for greenhouse production as well as landscaping. In particular, the landscaping and greening of buildings have become popular because these initiatives offer one solution for mitigating urban problems such as heat islands and flooding. Mosses are an easy and lightweight solution for greening purposes as they can survive the harsh rooftop environment and have great stormwater retention. However, the health of plants is fundamental to achieving the benefits of greening. Like vascular plants, mosses also are susceptible to plant diseases. Many fungi damage mosses by causing brown patches of greenish moss. Brown patches on mosses are a characteristic sign of fungal infection. However, plants have various defense mechanisms, the first of which consists of preexisting structural and chemical defenses. Second, the plant immune system uses specific receptors with which to recognize the molecular structures of microbes that are not present on the surface of the plant's own cells. Receptor-mediated sensing of these structures can trigger early defense responses of plant, which can make the plant resistant to the attacking microbe. The model moss Physcomitrella patens, like the vascular plants, senses the molecular structures of microbes. For example, exposure of P. patens to chitosan—a component of the fungal cell wall—significantly increases peroxidase activity and oxygen radical formation. Oxygen radicals in turn affect many biological events; they can directly damage the pathogen or stimulate the plant's defenses. Currently, little is known about the peroxidase-based defense as well as chitosan-induced signaling pathways of P. patens. The aim of the research presented in this thesis was to study the pathogens of green roof mosses and establishes the host range of the isolated pathogens; the study also utilized a Physcomitrella mutant collection to identify genes involved in chitosan-induced signaling pathway. Fungal species that naturally inhabit mosses at a moss farm in Japan and on green roof environments in southern Finland were isolated and the ability of these fungi to infect and cause disease symptoms on the model moss P. patens was tested. In addition, the pathogenicity of fungus species towards vascular plants was also assessed. To elucidate the genes involved in chitosan-induced peroxidase activity, part of the Physcomitrella mutant collection was screened using the oxidation of 2,2´-azino-bis(3- ethylbenzothiazoine-6-sulfonic acid) as an indicator of peroxidase activity. Genome walking analyses were used to identify which genes were mutated within each moss line of interest. 3 The work described in this thesis demonstrates that mosses used for greening are colonized by many different fungal isolates. These studies reveal that several fungal genera such as Fusarium, Trichoderma, Phoma and Alternaria cause severe symptoms in P. patens. Moreover, our results demonstrate that mosses and vascular plants have common pathogens. The fungal isolates Fusarium avenaceum and Cladosporium oxysporum obtained from moss panels caused disease symptoms on barley and carrots, respectively, and also on two different moss species. The results also demonstrate that the Physcomitrella mutant collection is a valuable source for identifying genes involved in the chitosan-induced signaling pathway. Screening of part of the mutant collection and further analyses revealed that Rossmann fold protein is a significant part of the signaling chain leading to upregulated peroxidase activity induced by chitosan. In addition, this Rossmann fold protein is an important factor for normal lipoxygenase (LOX) expression and might contribute to defense against fungal pathogens. The results from this doctoral thesis provide new insights into the pathogens of green roofs, the host range of the pathogens and the molecular mechanisms involved in disease control and defense responses in moss. The knowledge gained concerning the pathogenicity of Trichoderma isolates and the host range of pathogenic fungi should be considered when planting moss farms and cultivating crops in close proximity to each other or when applying biological control agents containing Trichoderma species to green roofs. Furthermore, these results may encourage the use of the Physcomitrella mutant collection to identify candidate genes for signaling pathways to elucidate the molecular mechanisms underlying the defense responses of mosses. 4 CONTENTS ABSTRACT ............................................................................................................................................... 3 ABBREVIATIONS .................................................................................................................................... 6 LIST OF ORIGINAL PUBLICATIONS .................................................................................................. 7 1 INTRODUCTION .................................................................................................................................. 8 1.1 Green roofs are important solutions for mitigating urban problems ............................................10 1.2 Bryophyte-fungi interactions ..........................................................................................................11 1.3 Plant defense against pathogens .....................................................................................................12 1.3.1 Preexisting structural and chemical defenses ..........................................................................13 1.3.2 Plant immune system: recognition and activation of defense responses .................................14 1.3.3 Induced defense responses: early and late responses in chitin-induced signaling pathways ..15 2 AIMS OF THE STUDY .........................................................................................................................18 3 MATERIALS AND METHODS ...........................................................................................................19 4 RESULTS AND DISCUSSION .............................................................................................................20 4.1 Fungi isolated from moss panels and green roof mosses and their virulence toward the model moss, P. patens .......................................................................................................................................20 4.1.1 Fungal genera in moss panels and on green, mossy roofs .......................................................20 4.1.2 Effects of isolated fungi on the model moss, P. patens .............................................................21 4.2 Fungi that infect cultivated moss can also cause disease in crop plants ........................................25 4.2.1 Two fungal isolates pathogenic to mosses cause disease in crop plants ..................................25 4.2.2 Fusarium oxysporum and Alternaria alternata do not cause damage to vascular plants ........26 4.3 Screening of part of the Physcomitrella mutant collection reveals a role for an NAD(P)-binding Rossman fold protein in chitosan-induced signaling............................................................................27 4.3.1 The Physcomitrella mutant collection as a source for candidate genes in chitosan-induced signaling pathways ............................................................................................................................27 4.3.2 One mutation is located within an NAD(P)-binding Rossmann fold protein..........................28 4.3.3 An NAD(P)-binding Rossmann fold protein is involved in chitosan-induced peroxidase activity and lipoxygenase expression ................................................................................................29