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Re-Annotated Nicotiana Benthamiana Gene Models for Enhanced bioRxiv preprint doi: https://doi.org/10.1101/373506; this version posted July 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 4.0 International license. 1 Re-annotated Nicotiana benthamiana gene models for 2 enhanced proteomics and reverse genetics 3 4 Jiorgos Kourelis1, Farnusch Kaschani2, Friederike M. Grosse-Holz1, Felix Homma1, 5 Markus Kaiser2, Renier A. L. van der Hoorn1 6 1Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, South Parks Road, 7 OX1 3RB Oxford, UK; 2Chemische Biologie, Zentrum fur Medizinische Biotechnologie, Fakultät für 8 Biologie, Universität Duisburg-Essen, Essen, Germany. 9 Nicotiana benthamiana is an important model organism and representative of the 10 Solanaceae (Nightshade) family. N. benthamiana has a complex ancient allopolyploid 11 genome with 19 chromosomes, and an estimated genome size of 3.1Gb. Several draft 12 assemblies of the N. benthamiana genome have been generated, however, many of the 13 gene-models in these draft assemblies appear incorrect. Here we present a nearly non- 14 redundant database of 42,855 improved N. benthamiana gene-models. With an 15 estimated 97.6% completeness, the new predicted proteome is more complete than the 16 previous proteomes. We show that the database is more sensitive and accurate in 17 proteomics applications, while maintaining a reasonable low gene number. As a proof- 18 of-concept we use this proteome to compare the leaf extracellular (apoplastic) 19 proteome to a total extract of leaves. Several gene families are more abundant in the 20 apoplast. For one of these apoplastic protein families, the subtilases, we present a 21 phylogenetic analysis illustrating the utility of this database. Besides proteome 22 annotation, this database will aid the research community with improved target gene 23 selection for genome editing and off-target prediction for gene silencing. 24 25 Keywords: Solanaceae // Genome annotation // Nicotiana benthamiana // Proteomics // Subtilases 26 27 Database availability. The database has been uploaded at Oxford Research Archives (ORA) and can be 28 downloaded from this link: https://deposit.ora.ox.ac.uk/datasets/uuid:f09e1d98-f0f1-4560-aed4-a5147bc7739d. 29 We hope that this database will be accessible via the SolGenomics database in the near future. 30 31 Introduction 56 includes important crops such as potato (Solanum 32 Nicotiana benthamiana has risen to prominence as 57 tuberosum), tomato (Solanum lycopersicum), 33 a model organism for several reasons. First, N. 58 eggplant (Solanum melongena), and pepper 34 benthamiana is highly susceptible to viruses, 59 (Capsicum ssp.), as well as tobacco (Nicotiana 35 resulting in highly efficient virus-induced gene- 60 tabacum) and petunia (Petunia ssp.). 36 silencing (VIGS) for rapid reverse genetic screens 61 N. benthamiana belongs to the 37 (Senthil-Kumar and Mysore, 2014). This 62 Suaveolentes section of the Nicotiana genus, and 38 hypersusceptibility to viruses is due to an ancient 63 has an ancient allopolyploid origin (>10Mya) 39 disruptive mutation in the RNA-dependent RNA 64 accompanied by chromosomal re-arrangements 40 polymerase 1 gene (Rdr1), present in the lineage 65 resulting in a complex genome with 19 41 of N. benthamiana which is used in laboratories 66 chromosomes in the haploid genome – reduced 42 around the world (Bally et al., 2015). Reverse 67 from the ancestral allotetraploid 24 chromosomes 43 genetics using N. benthamiana have confirmed 68 - and an estimated haploid genome size of ~3.1Gb 44 many genes important for disease resistance (Wu 69 (Leitch et al., 2008; Goodin et al., 2008; Wang 45 et al., 2017; Senthil‐ Kumar et al., 2018). 70 and Bennetzen, 2015). There are four independent 46 Additionally, N. benthamiana is highly amenable 71 draft assemblies of the N. benthamiana genome 47 to the generation of stable transgenic lines 72 (Bombarely et al., 2012; Naim et al., 2012), as 48 (Clemente, 2006; Sparkes et al., 2006) and to 73 well as a de-novo transcriptome generated from 49 transient expression of transgenes (Goodin et al., 74 short-read RNAseq (Nakasugi et al., 2014). These 50 2008). This easy manipulation has facilitated rapid 75 datasets have greatly facilitated research in N. 51 forward genetic screens and has established N. 76 benthamiana, allowing for efficient prediction of 52 benthamiana as the plant bioreactor of choice for 77 off-targets of VIGS (Fernandez-Pozo et al., 2015) 53 the production of biopharmaceuticals (Stoger et 78 and genome editing using CRISPR/Cas9 (Liu et 54 al., 2014). Finally, N. benthamiana is a member of 79 al., 2017), as well as RNAseq and proteomics 55 the Solanaceae (Nightshade) family which 80 studies (Grosse-Holz et al., 2018). These draft 1 bioRxiv preprint doi: https://doi.org/10.1101/373506; this version posted July 25, 2018. 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 4.0 International license. 81 assemblies are, however, several years old and 141 redundancy using CD-HIT-EST and combined the 82 gene annotations have not been updated since. 142 gene-models into a single database (NbA) 83 Furthermore, in the course of our research we 143 containing 41,651 gene-models (Figure 1, Step 84 realized that many of the gene models in these 144 3). We next compared the predicted proteome 85 draft assemblies are incorrect, and that putative 145 derived from our NbA database against the 86 pseudo-genes are often annotated as protein- 146 published predicted proteomes using a proteomics 87 encoding genes. This is exacerbated because these 147 dataset from a full-leaf extract or apoplastic fluid 88 draft assemblies are highly fragmented and that N. 148 (samples described further in the manuscript). 89 benthamiana has a complex origin. Furthermore, 149 Proteins for which peptides were identified in the 90 the de-novo transcriptome assembly has a high 150 published proteomes but not in our NbA database 91 proportion of chimeric transcripts. Because of 151 were extracted and re-annotated in the draft 92 incorrect annotations, extensive processing is 152 genomes as described above and added to our 93 required to select target genes for reverse genetic 153 NbA database resulting in the NbB database 94 approaches such as gene silencing and editing, or 154 containing 42,884 gene-models (Figure 1; Step 4, 95 for phylogenetic analysis of gene families. We 155 1233 additional entries). Finally, missing 96 realized that gene annotation in several other 156 BUSCOs (Benchmarking Universal Single-Copy 97 species in the Nicotiana genus were much better 157 Orthologues) (Simão et al., 2015; Waterhouse et 98 (Xu et al., 2017; Sierro et al., 2013; Sierro et al., 158 al., 2018) were re-annotated in our database as 99 2014), and decided to re-annotate the available N. 159 above, together with the manual curation of 100 benthamiana draft genomes using these gene 160 several gene-families in which several duplicated 101 models as a template. The gene models obtained 161 genes were removed (see Material & Methods) to 102 in this way were extracted into a single non- 162 obtain our final NbC database containing 42,853 103 redundant database with improved gene models. 163 entries (Figure 1; Step 5). 104 Here we show that this database is more accurate 164 105 and sensitive for proteomics, facilitates 165 The new proteome database is more complete, 106 phylogenetic analysis of gene families, and may 166 more sensitive and accurate, and relatively small 107 be useful for genome editing and VIGS on- and 167 We next compared the predicted proteome 108 off-target prediction. 168 database to the published predicted proteomes. 109 169 We also included our Nicotiana_db95 proteome 110 Results and Discussion 170 database in this comparison. The published 111 Re-annotation of gene-models in the N. 171 proteomes included the predicted proteomes from 112 benthamiana genome assemblies 172 the Niben0.4.4 and Niben1.0.1 draft genomes, a 113 For the annotation of gene-models in the different 173 previously described curated database in which 114 N. benthamiana draft genomes, we chose to use 174 gene-models from Niben1.0.1 were corrected 115 Scipio (Keller et al., 2008). Scipio refines the 175 using RNAseq reads (Grosse-Holz et al., 2018), 116 transcription start-site, exon-exon boundaries, and 176 and the predicted proteome derived from the de- 117 the stop-codon position of protein sequences 177 novo transcriptomes (Nakasugi et al., 2014). 118 aligned to the genome using BLAT (Keller et al., 178 We used BUSCO (Simão et al., 2015; 119 2008). Importantly, given that the input protein 179 Waterhouse et al., 2018) as a quality measure to 120 sequences are well-annotated, this method is more 180 estimate the completion of our database as 121 accurate and sensitive than other gene prediction 181 compared to the published predicted proteomes 122 methods (Keller et al., 2011). Because the 182 (Figure 2a). The BUSCO set used contains 1440 123 efficiency of this process correlates with 183 highly conserved plant genes which are expected 124 phylogenetic distance, we took the predicted 184 to be predominantly found in a single-copy 125 protein sequences from recently sequenced 185 (Simão et al., 2015). Nicotiana_db95 has one 126 Nicotiana species (Figure 1) (Sierro et al., 2013; 186 fragmented and nine missing BUSCOs, indicating 127 Xu et al., 2017). We then used CD-HIT at a 95% 187 that at best we should be able to identify 99.3% of 128 identity cut-off to reduce the redundancy in this 188 the N.
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