SUMMARYGenome-scale metabolic models (GEMs) provide a functional view of the complex network of biochemical reactions in the living cell. Initially mainly applied to reconstruct the metabolism of model organisms, the availability of increasingly sophisticated reconstruction methods and more extensive biochemical databases now make it possible to reconstruct GEMs for less wellcharacterized organisms, and have the potential to unravel the metabolism in pathogen-host systems. Here, we present a GEM for the oomycete plant pathogen Phytophthora infestans as a first step towards an integrative model with its host. We predict the biochemical reactions in different cellular compartments and investigate the gene-protein-reaction associations in this model to obtain an impression of the biochemical capabilities of P. infestans. Furthermore, we generate life stage-specific models to place the transcriptomic changes of the genes encoding metabolic enzymes into a functional context. In sporangia and zoospores, there is an overall down-regulation, most strikingly reflected in the fatty acid biosynthesis pathway. To investigate the robustness of the GEM, we simulate gene deletions to predict which enzymes are essential for in vitro growth. This model is an essential first step towards an understanding of P. infestans and its interactions with plants as a system, which will help to formulate new hypotheses on infection mechanisms and disease prevention.

The oomycete pathogen Phytophthora infestans causes potato and tomato late blight, a disease that is a serious threat to agriculture. P. infestans is a hemibiotrophic pathogen, and during infection, it scavenges nutrients from living host cells for its own proliferation. To date, the nutrient flux from host to pathogen during infection has hardly been studied, and the interlinked metabolisms of the pathogen and host remain poorly understood. Here, we reconstructed an integrated metabolic model of P. infestans and tomato (Solanum lycopersicum) by integrating two previously published models for both species. We used this integrated model to simulate metabolic fluxes from host to pathogen and explored the topology of the model to study the dependencies of the metabolism of P. infestans on that of tomato. This showed, for example, that P. infestans, a thiamine auxotroph, depends on certain metabolic reactions of the tomato thiamine biosynthesis. We also exploited dual-transcriptome data of a time course of a full late blight infection cycle on tomato leaves and integrated the expression of metabolic enzymes in the model. This revealed profound changes in pathogen-host metabolism during infection. As infection progresses, P. infestans performs less de novo synthesis of metabolites and scavenges more metabolites from tomato. This integrated metabolic model for the P. infestans-tomato interaction provides a framework to integrate data and generate hypotheses about in planta nutrition of P. infestans throughout its infection cycle. IMPORTANCE Late blight disease caused by the oomycete pathogen Phytophthora infestans leads to extensive yield losses in tomato and potato cultivation worldwide. To effectively control this pathogen, a thorough understanding of the mechanisms shaping the interaction with its hosts is paramount. While considerable work has focused on exploring host defense mechanisms and identifying P. infestans proteins contributing to virulence and pathogenicity, the nutritional strategies of the pathogen are mostly unresolved. Genome-scale metabolic models (GEMs) can be used to simulate metabolic fluxes and help in unravelling the complex nature of metabolism. We integrated a GEM of tomato with a GEM of P. infestans to simulate the metabolic fluxes that occur during infection. This yields insights into the nutrients that P. infestans obtains during different phases of the infection cycle and helps in generating hypotheses about nutrition in planta.

Botrytis cinerea is a plant-pathogenic fungus producing apothecia as sexual fruiting bodies. To study the function of mating type (MAT) genes, single-gene deletion mutants were generated in both genes of the MAT1-1 locus and both genes of the MAT1-2 locus. Deletion mutants in two MAT genes were entirely sterile, while mutants in the other two MAT genes were able to develop stipes but never formed an apothecial disk. Little was known about the reprogramming of gene expression during apothecium development. We analyzed transcriptomes of sclerotia, three stages of apothecium development (primordia, stipes, and apothecial disks), and ascospores by RNA sequencing. Ten secondary metabolite gene clusters were upregulated at the onset of sexual development and downregulated in ascospores released from apothecia. Notably, more than 3,900 genes were differentially expressed in ascospores compared to mature apothecial disks. Among the genes that were upregulated in ascospores were numerous genes encoding virulence factors, which reveals that ascospores are transcriptionally primed for infection prior to their arrival on a host plant. Strikingly, the massive transcriptional changes at the initiation and completion of the sexual cycle often affected clusters of genes, rather than randomly dispersed genes. Thirty-five clusters of genes were jointly upregulated during the onset of sexual reproduction, while 99 clusters of genes (comprising >900 genes) were jointly downregulated in ascospores. These transcriptional changes coincided with changes in expression of genes encoding enzymes participating in chromatin organization, hinting at the occurrence of massive epigenetic regulation of gene expression during sexual reproduction.

Along with Plasmopara destructor, Peronosopora belbahrii has arguably been the economically most important newly emerging downy mildew pathogen of the past two decades. Originating from Africa, it has started devastating basil production throughout the world, most likely due to the distribution of infested seed material. Here, we present the genome of this pathogen and results from comparisons of its genomic features to other oomycetes. The assembly of the nuclear genome was around 35.4 Mbp in length, with an N50 scaffold length of around 248 kbp and an L50 scaffold count of 46. The circular mitochondrial genome consisted of around 40.1 kbp. From the repeat-masked genome, 9,049 protein-coding genes were predicted, out of which 335 were predicted to have extracellular functions, representing the smallest secretome so far found in peronosporalean oomycetes. About 16% of the genome consists of repetitive sequences, and, based on simple sequence repeat regions, we provide a set of microsatellites that could be used for population genetic studies of P. belbahrii. P. belbahrii has undergone a high degree of convergent evolution with other obligate parasitic pathogen groups, reflecting its obligate biotrophic lifestyle. Features of its secretome, signaling networks, and promoters are presented, and some patterns are hypothesized to reflect the high degree of host specificity in Peronospora species. In addition, we suggest the presence of additional virulence factors apart from classical effector classes that are promising candidates for future functional studies.

15 16 2 ORIGINALITY & SIGNFICANCE STATEMENT 17The intimate interaction between pathogens and their hosts exerts strong selection 18 pressure leading to rapid adaptation. How this shapes the metabolism of pathogens is 19 largely unknown. Here, we used comparative genomics to systematically characterize 20 the metabolisms of animal and plant pathogenic oomycetes, a group of eukaryotes 21 comprising many important animal and plant pathogens with significant economic and 22 ecological impact. Core-and pan-genome as well as metabolic network analyses of 23 distantly related oomycetes and their non-pathogenic relatives revealed considerable 24 lifestyle-and lineage-specific adaptations. Extreme lifestyle adaptation could be 25 observed in the metabolism of obligate biotrophic oomycetes -a group of pathogens 26 that require a living host for proliferation. The metabolic networks of obligate biotrophic 27 oomycetes reflect profound patterns of reductive evolution, converging to a loss the 28 same metabolic enzymes during acquisition of an obligate parasitic lifestyle. These 29 findings contribute to a be better understanding of oomycete evolution and the 30 relationship between metabolism and lifestyle adaptation. 31 32 SUMMARY 33 Pathogen-host symbiosis drives metabolic adaptations. Animal and plant pathogenic 34 oomycetes are thought to adapt their metabolism to facilitate interactions with their 35 hosts. Here, we performed a large-scale comparison of oomycete metabolism and 36 uncovered considerable variation in oomycete metabolism that could be linked to 37 3 differences in lifestyle. Pathway comparisons revealed that plant pathogenic oomycetes 38 can be divided in two parts; a conserved part and an accessory part. The accessory 39 part could be associated with the degradation of plant compounds produced during 40 defence responses. Obligate biotrophic oomycetes have smaller metabolic networks, 41 and this group displays converged evolution by repeated gene losses affecting the 42 same metabolic pathways. A comparison of the metabolic networks of obligate 43 biotrophic oomycetes with those of plant pathogenic oomycetes as a whole revealed 44 that the losses of metabolic enzymes in biotrophs are not random and that the network 45 of biotrophs contracts from the periphery inwards. Our analyses represent the first 46 metabolism-focused comparison of oomycetes at this scale and will contribute to a 47 better understanding of the evolution and relationship between metabolism and lifestyle 48 adaptation. 49 4 INTRODUCTION 50