bioRxiv preprint doi: https://doi.org/10.1101/582205; this version posted March 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 The Zymoseptoria tritici ORFeome: a functional genomics 2 community resource 3 4 Yogesh Chaudhari*,a, Timothy C. Cairns*,b, Yaadwinder Sidhua, Victoria Attaha, 5 Graham Thomasa, Michael Csukaic, Nicholas J. Talbotd, David J. Studholmea,e, Ken 6 Haynesa,f. 7 *Authors contributed equally to this study 8 aBiosciences, University of Exeter, Exeter EX4 4QD, United Kingdom 9 bCurrent address; Tianjin Institute of Industrial Biotechnology, Chinese Academy of 10 Sciences, Tianjin, 300308, China 11 cSyngenta, Jealott’s Hill International Research Centre, Bracknell, RG42 6EY 12 dCurrent address; The Sainsbury Laboratory, University of East Anglia, Norwich 13 Research Park, NR47UH, United Kingdom 14 eCorresponding author ([email protected]) 15 fAuthor deceased 16 Emails: 17 [email protected] 18 [email protected] 19 [email protected] 20 [email protected] 21 [email protected] 22 [email protected] 23 [email protected] 24 [email protected] 25 1 bioRxiv preprint doi: https://doi.org/10.1101/582205; this version posted March 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 26 Abstract: 27 Libraries of protein-encoding sequences can be generated by identification of open 28 reading frames (ORFs) from a genome of choice that are then assembled into 29 collections of plasmids termed ORFeome libraries. These represent powerful 30 resources to facilitate functional genomic characterization of genes and their encoded 31 products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, 32 which causes the most serious disease of wheat in temperate regions of the world. 33 We screened the genome of strain IP0323 for high confidence gene models, 34 identifying 4075 candidates from 10,933 predicted genes. These were amplified from 35 genomic DNA, cloned into the Gateway® Entry Vector pDONR207, and sequenced, 36 providing a total of 3022 quality-controlled plasmids. The ORFeome includes genes 37 predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins 38 (n = 171), in addition to genes residing at dispensable chromosomes (n= 122), or those 39 that are preferentially expressed during plant infection (n = 527). The ORFeome 40 plasmid library is compatible with our previously developed suite of Gateway® 41 Destination vectors, which have various combinations of promoters, selection 42 markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for 43 functional genomics, and offers unparalleled opportunities to understand the biology 44 of Z. tritici. 45 46 Keywords: ORFeome, Zymoseptoria tritici, Mycosphaerella graminicola, functional 47 genomics. 48 49 Dedication: This paper is dedicated to the memory of Ken Haynes who led the study 50 and was an outstanding fungal biologist, as well as an inspirational colleague, friend 51 and mentor to his fellow co-authors. 52 53 54 2 bioRxiv preprint doi: https://doi.org/10.1101/582205; this version posted March 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 55 Introduction 56 Fungal pathogens kill more people per year than malaria, and result in crop destruction 57 or post-harvest spoilage that destroys enough food to feed approximately 10% of the 58 population (1, 2). Technological advances in fungal genomics, transcriptomics, 59 proteomics, metabolomics, bioinformatics, and network analyses, however, now 60 enable pathogenic fungi to be studied as integrated systems, providing unparalleled 61 opportunities to understand their biology (3, 4). Functional genomic approaches, which 62 define the function and interactions of genes and their encoded products at a genome 63 or near-genome level, are increasingly used to dissect host pathogen interactions, 64 virulence factors, drug resistance, and infectious growth during fungal disease (5–9). 65 However, a significant constraint to conducting functional genomic experiments are 66 high reagent and labour costs, due to the necessity to study thousands, or tens of 67 thousands of genes from a given fungal pathogen. 68 In order to obviate this challenge, community accessible libraries have been 69 developed, which consist of hundreds or thousands of either individual genes, null 70 mutant or over-expression strains, which ultimately enable facile and high throughput 71 experimentation by the end user at a minimal expense (10–17). ORFeomes are 72 collections of open reading frames (ORFs) that are encoded in a library of plasmid 73 vectors. These resources have been generated for several model organisms, including 74 humans, Escherichia coli, Caenorhabditis elegans, and Arabidopsis thaliana (18–23). 75 ORFeomes have also been developed for the fungal kingdom, including fission yeast 76 (18), budding yeast (24) and, most recently, the human pathogenic yeast Candida 77 albicans (8). Usually, ORFeomes are compatible with the Gateway® cloning 78 technology (Invitrogen), which enables rapid and high throughput recombinase-based 79 transfer of an ORF coding sequence to generate expression vectors (25, 26). 80 Community access to hundreds or thousands of such plasmids in a single library 81 enables highly flexible generation of expression vectors for high-throughput functional 82 genomic experiments. 83 The filamentous ascomycete fungus Zymoseptoria tritici (previously Mycosphaerella 84 graminicola) (27) causes Septoria tritici blotch, an important foliar disease of wheat 85 (28). Z. tritici is a significant threat to international food security, and even with access 86 to some resistant wheat cultivars and frequent fungicide applications, the estimated 3 bioRxiv preprint doi: https://doi.org/10.1101/582205; this version posted March 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 87 average yield losses due to this pathogen are still 10% (28). Even more strikingly, 88 approximately 70% of agricultural fungicides in Europe are deployed to control just this 89 single disease (29), which likely drives triazole resistance in fungal pathogens of 90 humans as well as plants (30). These challenges are compounded by very high levels 91 of genome plasticity and gene flow between populations of Z. tritici (31, 32). While 92 recent efforts have characterized the underlying cellular biology and infectious growth 93 (33, 34), transcriptionally deployed secondary metabolite loci (35, 36), and 94 components of the secreted effector arsenal [37-40], the vast majority of genes and 95 encoded proteins remain uncharacterized in the laboratory (27). 96 Recently, there has been a community-wide effort to develop numerous tools, 97 techniques, and resources for Z. tritici. This research toolkit includes mutants in the 98 non-homologous end joining pathway for highly efficient gene targeting (41), 99 optimization of conditional expression systems (42), a range of fluorescent 100 translational gene fusion for sub-cellular localization studies (43, 44), optimized 101 virulence assays (45), and a suite of Gateway® Destination vectors (46, 47). These 102 Gateway® destination vectors have been validated using a pilot Gateway® Entry 103 library to generate 32 over-expression mutants, demonstrating the role of a fungal 104 specific transcription factor for in vitro hyphal growth (48). 105 In this study, we report the generation of an improved functional genomics community 106 resource to supplement these tools, by generating a Gateway® compatible Z. tritici 107 ORFeome, which to our knowledge is the first such library for a plant infecting fungus. 108 This library is compatible with numerous Gateway® destination vectors that have 109 multiple functionality in Z. tritici, including numerous selection markers, epitope tags, 110 and promoters (26, 47). For ORFeome construction, we firstly screened the IP0323 111 reference genome for high confidence gene models, yielding 4075 candidate ORFs 112 from a possible 10,933 predicted genes. These were PCR amplified from genomic 113 DNA and cloned into the Gateway® Entry vector pDONR207. Quality of ORF 114 sequences was verified by a combination of Sanger and Illumina sequencing, yielding 115 3022 plasmids that passed quality control checks with 100% sequence verification. 116 The Z. tritici ORFeome described in this study is freely available to the research 117 community. This resource can be rapidly utilized to interrogate the broadest aspects 118 of Z. tritici biology, including identification of novel drug targets, mechanisms of drug 4 bioRxiv preprint doi: https://doi.org/10.1101/582205; this version posted March 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 119 detoxification and resistance, and pathogen virulence factors, which may ultimately 120 enable development of new disease control strategies. 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 5 bioRxiv preprint doi: https://doi.org/10.1101/582205; this version posted March 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.