The metagenomes of root nodules in actinorhizal plants A bioinformatic study of endophytic bacterial communities Ellen Fasth Bachelor thesis, 15 ECTS Degree project in Bachelor’s program of Biology, 180 ECTS Spring 2021 Abstract Actinorhizal plants are in symbiosis with the nitrogen-fixating soil bacterium Frankia, which forms nodules in the plant root. However, several studies also report other endophytic bacteria appearing in the nodules, but their function and interaction with the host plant or Frankia is not yet understood. This thesis used a bioinformatic approach to investigate the metagenomes of eighteen actinorhizal nodule samples to find out which bacteria are present, how the microbiomes differed from each other, and if the genomes of non-Frankia inhabitants could give indications of any functions. The results showed that the bacterial composition, richness, and diversity differed among the samples, especially between the samples sequenced from the field versus those primarily cultivated in a greenhouse. All samples had a substantial number of sequencing reads belonging to non-Frankia endophytes, such as strains of Enterobacteria, Pseudomonas, Streptomyces, Micromonospora, Mycobacteria and Pseudonocardia. There seemed to be a common microbial community shared among the plants on a family level, since no significant difference was found in the core microbiomes between the field and greenhouse groups. Some sequences found in the metagenomes were annotated as potential functions of the fellow travellers, such as antibiotic synthesis, proteins involved in regulating abiotic stresses, but also probable plant damaging compounds rather associated with pathogens than symbionts. Key words: Actinorhizal plants, Frankia, endosymbiont, root nodules, metagenomes, bioinformatics Preface I would like to thank Katharina Pawlowski and Fede Berckx from the Department of Ecology, Environment and Plant Sciences at Stockholm University, for letting me in on this project and assisting me through it. Table of Contents Abstract .......................................................................................................................... 2 Preface ............................................................................................................................ 2 1 Introduction and background ...................................................................................... 4 1.1 THE NEED FOR NITROGEN ...................................................................................................................... 5 1.2 ACTINORHIZAL PLANTS ......................................................................................................................... 5 1.3 THE FRANKIA GENUS ........................................................................................................................... 6 1.4 UNDETERMINED SYMBIOSES – THE FELLOW TRAVELLERS ....................................................................... 7 1.5 THE METAGENOMIC WORKFLOW ........................................................................................................... 8 1.6 CHALLENGES WITH METAGENOMIC DATA .............................................................................................. 9 1.7 PURPOSE AND THESIS QUESTIONS ........................................................................................................ 10 2 Material and method .................................................................................................. 10 2.1 FIELD SAMPLING, PROPAGATION, AND SEQUENCING ............................................................................. 10 2.2 MGX TAXONOMIC ANALYSIS ............................................................................................................... 11 2.3 DATA NORMALIZATION ....................................................................................................................... 12 2.4 FUNCTIONAL GENOMIC ANALYSIS ........................................................................................................ 12 2.5 STATISTICAL ANALYSIS ........................................................................................................................ 12 3 Results ....................................................................................................................... 13 3.1 RAREFACTION RESULT ........................................................................................................................ 13 3.2 BACTERIA COMPOSITION AND DIVERSITY ............................................................................................. 13 3.3 FUNCTIONAL ANNOTATION ................................................................................................................. 18 4 Discussion .................................................................................................................. 19 4.1 POTENTIAL FUNCTIONS OF THE FELLOW TRAVELLERS .......................................................................... 23 4.2 CONCLUSION ..................................................................................................................................... 26 5 References .................................................................................................................. 27 6 Appendix .................................................................................................................... 31 APPENDIX 1. BIODIVERSITY INDICES ......................................................................................................... 31 APPENDIX 2. TOP TEN FAMILIES PER SAMPLE ............................................................................................ 31 APPENDIX 3. CORE MICROBIOME READS ................................................................................................... 32 3 1 Introduction and background All plant species are coupled with a microbial community, and the symbiotic interactions between them have during the last decades received more attention (Abdelfattah et al. 2021). The microbiome is known to have advantageous effects on the health of its host plant contributing to for example increased growth or better stress tolerance, while receiving a carbohydrate-rich refuge in return (Franco et al. 2007; Trivedi et al. 2020). The improved understanding of microbes is much due to the modern DNA sequencing techniques that has enabled the identification of non-cultivatable bacteria, accounting for around 99% of the total, by taking whole metagenomes directly from their natural environment (Solden, Lloyd, and Wrighton 2016). This has allowed formerly hidden micro-world of many different niches, such as the concealed symbionts occupying plants, to be unravelled and plant-microbe interactions to be seen in a new light. The microbiome’s impact on its organism is of growing interest and understanding its influence on plants can have many vital applications in for example agriculture. The current conventional agricultural system is fully dependent on industrial fertilizers to provide the crops with sufficient nutrients, such as phosphorus and nitrogen, to the extent that about half of the human population today could not exist without it (Hoffman et al. 2014). Indeed, nitrogen is the main compound that limits plant growth. Despite being readily available in the atmosphere, it exists in an inaccessible form for plants, for which in some plant species is solved by having nitrogen-fixing prokaryotes to provide it for them. Nitrogen-fixing symbiosis with endophytic bacteria such as the one between legumes (Fabaceae) and rhizobia is well known, but less is known about the one occurring between actinorhizal plants and the soil bacterium Frankia. Just as in the legume-rhizobia interaction, the host plant forms root nodules to encompass Frankia intracellularly, where nitrogen-fixation takes place to convert the inert N2 to the much-needed NH3. However, more evidence points towards the fact that Frankia is not alone. Metagenomic and isolation studies continuously detect the presence of other endophytic bacteria inhabiting the nodule, such as other actinobacteria like Streptomyces, Micromonospora and Nocardia, but whose function is not entirely understood (Qin et al. 2009; Trujillo et al. 2015). When several of these bacteria lack the mechanisms needed for nitrogen-fixation, the question arises whether these fellow travellers may have any other beneficial functions for the plant hosting them. It is by some argued that the plant and its microbiome should be seen as one entity – a holobiont – concerning the large impact different microorganisms have on plants’ growth and fitness (Guerrero, Margulis, and Berlanga 2013). It could therefore prove fruitful to investigate not only a single species, but the whole ecosystem found in the nodule environment. An increased understanding of new important plant symbionts has many applications in both biotechnology and agriculture such as for improving nutrient acquisition, where for example the intense use of fertilizers comes with serious ecological consequences like eutrophication (Canfield, Glazer, and Falkowski 2010). Facing an increasing world population in combination with climate change, alternative, sustainable solutions need to be examined in order to guarantee the global food security. Further explorations of whole metagenomes derived from plants can give new insights into endophytic bacteria’s relations with each other and their genes or metabolites that influence plant fitness. 4