Dataset Information

Genome and transcriptome characterization of the glycoengineered Nicotiana benthamiana line ?XT/FT.

ABSTRACT:

Background

The allotetraploid tobacco species Nicotiana benthamiana native to Australia has become a popular host for recombinant protein production. Although its usage grows every year, little is known on this plant's genomic and transcriptomic features. Most N. benthamiana accessions currently used in research lack proper documentation of their breeding history and provenance. One of these, the glycoengineered N. benthamiana line ?XT/FT is increasingly used for the production of biopharmaceutical proteins.

Results

Based on an existing draft assembly of the N. benthamiana genome we predict 50,516 protein -encoding genes (62,216 transcripts) supported by expression data derived from 2.35 billion mRNA-seq reads. Using single-copy core genes we show high completeness of the predicted gene set. We functionally annotate more than two thirds of the gene set through sequence homology to genes from other Nicotiana species. We demonstrate that the expression profiles from leaf tissue of ?XT/FT and its wild type progenitor only show minimal differences. We identify the transgene insertion sites in ?XT/FT and show that one of the transgenes was inserted inside another predicted gene that most likely lost its function upon insertion. Based on publicly available mRNA-seq data, we confirm that the N. benthamiana accessions used by different research institutions most likely derive from a single source.

Conclusions

This work provides gene annotation of the N. benthamiana genome, a genomic and transcriptomic characterization of a transgenic N. benthamiana line in comparison to its wild-type progenitor, and sheds light onto the relatedness of N. benthamiana accessions that are used in laboratories around the world.

SUBMITTER: Schiavinato M

PROVIDER: S-EPMC6642603 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Genome and transcriptome characterization of the glycoengineered Nicotiana benthamiana line ΔXT/FT.

Schiavinato Matteo M Strasser Richard R Mach Lukas L Dohm Juliane C JC Himmelbauer Heinz H

BMC genomics 20190719 1

<h4>Background</h4>The allotetraploid tobacco species Nicotiana benthamiana native to Australia has become a popular host for recombinant protein production. Although its usage grows every year, little is known on this plant's genomic and transcriptomic features. Most N. benthamiana accessions currently used in research lack proper documentation of their breeding history and provenance. One of these, the glycoengineered N. benthamiana line ΔXT/FT is increasingly used for the production of biopha ...[more]

PMID: 31324144

Similar Datasets

Project description:BackgroundTobacco curly shoot virus (TbCSV) is a monopartite begomovirus associated with betasatellite (Tobacco curly shoot betasatellite, TbCSB), which causes serious leaf curl disease on tomato and tobacco in China. It is interesting that TbCSV induced severe upward leaf curling in Nicotiana benthamiana, but in the presence of TbCSB, symptoms changed to be downward leaf curling. However, the mechanism of interactions between viral pathogenicity, host defense, viral-betasatellite interactions and virus-host interactions remains unclear.MethodsIn this study, RNA-seq was used to analyze differentially expressed genes (DEGs) in N. benthamiana plants infected by TbCSV (Y35A) and TbCSV together with TbCSB (Y35AB) respectively.ResultsThrough mapping to N. benthamiana reference genome, 59,814 unigenes were identified. Transcriptome analysis revealed that a total of 4081 and 3196 DEGs were identified in Y35AB vs CK (control check) and Y35A vs CK, respectively. Both GO and KEGG analyses were conducted to classify the DEGs. Ten of the top 15 GO terms were enriched in both DEGs of Y35AB vs CK and Y35A vs CK, and these enriched GO terms mainly classified into three categories including biological process, cellular component and molecular function. KEGG pathway analysis indicated that 118 and 111 pathways were identified in Y35AB vs CK and Y35A vs CK, respectively, of which nine and six pathways were significantly enriched. Three major pathways in Y35AB vs CK involved in metabolic pathways, carbon metabolism and photosynthesis, while those in Y35A vs CK were related to Ribosome, Glyoxylate and dicarboxylate metabolism and DNA replication. We observed that 8 PR genes were significantly up-regulated and 44 LRR-RLK genes were significantly differentially expressed in Y35A treatment or in Y35AB treatment. In addition, 7 and 13 genes were identified to be significantly changed in biosynthesis and signal transduction pathway of brassinosteroid (BR) and jasmonic acid (JA) respectively.ConclusionsThese results presented here would be particularly useful to further elucidate the response of the host plant against virus infection.

Project description:BACKGROUND:Nicotiana benthamiana has been widely used in laboratories around the world for studying plant-pathogen interactions and posttranscriptional gene expression silencing. Yet the exploration of its transcriptome has lagged behind due to the lack of both adequate sequence information and genome-wide analysis tools, such as DNA microarrays. Despite the increasing use of high-throughput sequencing technologies, the DNA microarrays still remain a popular gene expression tool, because they are cheaper and less demanding regarding bioinformatics skills and computational effort. RESULTS:We designed a gene expression microarray with 103,747 60-mer probes, based on two recently published versions of N. benthamiana transcriptome (v.3 and v.5). Both versions were reconstructed from RNA-Seq data of non-strand-specific pooled-tissue libraries, so we defined the sense strand of the contigs prior to designing the probe. To accomplish this, we combined a homology search against Arabidopsis thaliana proteins and hybridization to a test 244k microarray containing pairs of probes, which represented individual contigs. We identified the sense strand in 106,684 transcriptome contigs and used this information to design an Nb-105k microarray on an Agilent eArray platform. Following hybridization of RNA samples from N. benthamiana roots and leaves we demonstrated that the new microarray had high specificity and sensitivity for detection of differentially expressed transcripts. We also showed that the data generated with the Nb-105k microarray may be used to identify incorrectly assembled contigs in the v.5 transcriptome, by detecting inconsistency in the gene expression profiles, which is indicated using multiple microarray probes that match the same v.5 primary transcripts. CONCLUSIONS:We provided a complete design of an oligonucleotide microarray that may be applied to the research of N. benthamiana transcriptome. This, in turn, will allow the N. benthamiana research community to take full advantage of microarray capabilities for studying gene expression in this plant. Additionally, by defining the sense orientation of over 106,000 contigs, we substantially improved the functional information on the N. benthamiana transcriptome. The simple hybridization-based approach for detecting the sense orientation of computationally assembled sequences can be used for updating the transcriptomes of other non-model organisms, including cases where no significant homology to known proteins exists.

Project description:BackgroundNicotiana benthamiana has been widely used for transient gene expression assays and as a model plant in the study of plant-microbe interactions, lipid engineering and RNA silencing pathways. Assembling the sequence of its transcriptome provides information that, in conjunction with the genome sequence, will facilitate gaining insight into the plant's capacity for high-level transient transgene expression, generation of mobile gene silencing signals, and hyper-susceptibility to viral infection.Methodology/resultsRNA-seq libraries from 9 different tissues were deep sequenced and assembled, de novo, into a representation of the transcriptome. The assembly, of 16GB of sequence, yielded 237,340 contigs, clustering into 119,014 transcripts (unigenes). Between 80 and 85% of reads from all tissues could be mapped back to the full transcriptome. Approximately 63% of the unigenes exhibited a match to the Solgenomics tomato predicted proteins database. Approximately 94% of the Solgenomics N. benthamiana unigene set (16,024 sequences) matched our unigene set (119,014 sequences). Using homology searches we identified 31 homologues that are involved in RNAi-associated pathways in Arabidopsis thaliana, and show that they possess the domains characteristic of these proteins. Of these genes, the RNA dependent RNA polymerase gene, Rdr1, is transcribed but has a 72 nt insertion in exon1 that would cause premature termination of translation. Dicer-like 3 (DCL3) appears to lack both the DEAD helicase motif and second dsRNA binding motif, and DCL2 and AGO4b have unexpectedly high levels of transcription.ConclusionsThe assembled and annotated representation of the transcriptome and list of RNAi-associated sequences are accessible at www.benthgenome.com alongside a draft genome assembly. These genomic resources will be very useful for further study of the developmental, metabolic and defense pathways of N. benthamiana and in understanding the mechanisms behind the features which have made it such a well-used model plant.

Project description:BackgroundNicotiana benthamiana is an allo-tetraploid plant, which can be challenging for de novo transcriptome assemblies due to homeologous and duplicated gene copies. Transcripts generated from such genes can be distinct yet highly similar in sequence, with markedly differing expression levels. This can lead to unassembled, partially assembled or mis-assembled contigs. Due to the different properties of de novo assemblers, no one assembler with any one given parameter space can re-assemble all possible transcripts from a transcriptome.ResultsIn an effort to maximise the diversity and completeness of de novo assembled transcripts, we utilised four de novo transcriptome assemblers, TransAbyss, Trinity, SOAPdenovo-Trans, and Oases, using a range of k-mer sizes and different input RNA-seq read counts. We complemented the parameter space biologically by using RNA from 10 plant tissues. We then combined the output of all assemblies into a large super-set of sequences. Using a method from the EvidentialGene pipeline, the combined assembly was reduced from 9.9 million de novo assembled transcripts to about 235,000 of which about 50,000 were classified as primary. Metrics such as average bit-scores, feature response curves and the ability to distinguish paralogous or homeologous transcripts, indicated that the EvidentialGene processed assembly was of high quality. Of 35 RNA silencing gene transcripts, 34 were identified as assembled to full length, whereas in a previous assembly using only one assembler, 9 of these were partially assembled.ConclusionsTo achieve a high quality transcriptome, it is advantageous to implement and combine the output from as many different de novo assemblers as possible. We have in essence taking the 'best' output from each assembler while minimising sequence redundancy. We have also shown that simultaneous assessment of a variety of metrics, not just focused on contig length, is necessary to gauge the quality of assemblies.