A homology-guided, genome-based proteome for improved proteomics in the alloploid Nicotiana benthamiana

Abstract

BackgroundNicotiana benthamiana is an important model organism of the Solanaceae (Nightshade) family. Several draft assemblies of the N. benthamiana genome have been generated, but many of the gene-models in these draft assemblies appear incorrect.ResultsHere we present an improved proteome based on the Niben1.0.1 draft genome assembly guided by gene models from other Nicotiana species. Due to the fragmented nature of the Niben1.0.1 draft genome, many protein-encoding genes are missing or partial. We complement these missing proteins by similarly annotating other draft genome assemblies. This approach overcomes problems caused by mis-annotated exon-intron boundaries and mis-assigned short read transcripts to homeologs in polyploid genomes. With an estimated 98.1% completeness; only 53,411 protein-encoding genes; and improved protein lengths and functional annotations, this new predicted proteome is better in assigning spectra than the preceding proteome annotations. This dataset is more sensitive and accurate in proteomics applications, clarifying the detection by activity-based proteomics of proteins that were previously predicted to be inactive. Phylogenetic analysis of the subtilase family of hydrolases reveal inactivation of likely homeologs, associated with a contraction of the functional genome in this alloploid plant species. Finally, we use this new proteome annotation to characterize the extracellular proteome as compared to a total leaf proteome, which highlights the enrichment of hydrolases in the apoplast.ConclusionsThis proteome annotation provides the community working with Nicotiana benthamiana with an important new resource for functional proteomics.Electronic supplementary materialThe online version of this article (10.1186/s12864-019-6058-6) contains supplementary material, which is available to authorized users.

Identification of two subtilisin‐like serine proteases engaged in the degradation of recombinant proteins in Nicotiana benthamiana

“…these SBTs were cloned from reverse-transcribed N. benthamiana RNA and found to closely match those predicted from the most current N. benthamiana gene assemblies [27,28].…”

Abscisic Acid Connects Phytohormone Signaling with RNA Metabolic Pathways and Promotes an Antiviral Response that Is Evaded by a Self-Controlled RNA Virus

“…We used a similar bioinformatics workflow to test whether P1Pro infection affects AS and host transcriptomic diversity. An augmented assembly of the N. benthamiana transcriptome was generated by merging reported transcriptome datasets ( Nakasugi et al, 2014 ; Kourelis et al, 2019 ) and transcripts assembled from our RNA-seq data. This augmented transcriptome was used to examine transcript abundance and the diversity of P1Pro or PPV samples from inoculated N. benthamiana plants ( Supplemental Figure 6 ).…”

Efficient Confirmation of Plant Viral Proteins and Identification of Specific Viral Strains by nanoLC-ESI-Q-TOF Using Single-Leaf-Tissue Samples

“…For each tested plant host, either reference proteome with all isoforms was downloaded from the UniProt protein knowledgebase [ 49 ] (accessed 3 January 2020) or, if not present, all available protein sequences for the given taxonomic plant genus from UniProt were used instead. For Nicotiana benthamiana , a homology-guided proteome [ 39 ] derived from Nicotiana tabacum was also included (see UniProt identifiers in Table 1 ). For each tested plant host, a host range property was searched in Plant Viruses Online [ 34 ] and Descriptions of Plant Viruses [ 35 , 36 ] online databases, and a list of potential virus pathogens for the tested plant genus was assembled.…”

“… * total count of identified MaxQuant proteinGroups containing only host proteins, without contaminants, proteins from reverse database, and without proteins only identified by site (by their peptide mass only, without supporting MS/MS spectrum); ** see also Supplementary File S2 ; *** see also Supplementary File S3 **** see Supplementary File S4 for method details; * 1 used extraction protocol without RuBisCO depletion step; * 2 tested against UP000084051 reference proteome of Nicotiana tabacum (73,605 proteins) together with assembled proteome for Nicotiana benthamiana (ref. [ 39 ], NbD_AA—53,411 proteins, NbE_AA—74,802 proteins); * 3 no reference proteome is available in UniProt database, and thus, tested against all available protein sequences of Prunus genus; * 4 no reference proteome is available in UniProt database, and thus, tested against all available protein sequences of Chenopodium genus. …”