Virus‐induced gene silencing database for phenomics and functional genomics in <i>Nicotiana benthamiana</i>

Senthil‐Kumar, Muthappa; Wang, Mingyi; Chang, Junil; Ramegowda, Venkategowda; Pozo, Olga del; Liu, Yule; Doraiswamy, Vanthana; Lee, Hee-Kyung; Ryu, Choong-Min; Wang, Keri; Xu, Ping; Eck, Joyce Van; Chakravarthy, Suma; Dinesh‐Kumar, Savithramma P.; Martin, Gregory B.; Mysore, Kirankumar S.

doi:10.1002/pld3.55

Cited by 20 publications

(14 citation statements)

References 35 publications

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“…This hypersusceptibility to viruses is due to an ancient disruptive mutation in the RNA-dependent RNA polymerase 1 gene ( Rdr1 ), present in the lineage of N. benthamiana which is used in laboratories around the world [1]. Reverse genetics using N. benthamiana have confirmed many genes important for disease resistance [43, 58]. Second, N. benthamiana is highly amenable to the generation of stable transgenic lines [6, 48] and to transient expression of transgenes [17].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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“…This hypersusceptibility to viruses is due to an ancient disruptive mutation in the RNA-dependent RNA polymerase 1 gene (Rdr1), present in the lineage of N. benthamiana which is used in laboratories around the world (Bally et al, 2015). Reverse genetics using N. benthamiana have confirmed many genes important for disease resistance (Wu et al, 2017;Senthil-Kumar et al, 2018). Second, N. benthamiana is highly amenable to the generation of stable transgenic lines (Clemente, 2006;Sparkes et al, 2006) and to transient expression of transgenes (Goodin et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Homology-guided re-annotation improves the gene models of the alloploidNicotiana benthamiana

Kourelis

Kaschani

et al. 2018

Preprint

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Nicotiana 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. Here we present an improved re-annotation of the Niben1.0.1 draft genome assembly guided by gene models from other Nicotiana species. 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 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 mis-annotated to be inactive. Phylogenetic analysis of the subtilase family of hydrolases reveal a pseudogenisation of likely homeologs, associated with a contraction of the functional genome in this alloploid plant species. We use this gene annotation to assign extracellular proteins in comparison to a total leaf proteome, to display the enrichment of hydrolases in the apoplast.

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“…In addition, TRV:hsc70 plants also showed slight necrosis. Senthil-Kumar et al (2018) reported that the VIGS phenotypes could be varied and visible, such as leaf chlorosis, spotted cell death, stunted growth, leaf curling, leaf crinkling, and leaf mottling. They have developed a VIGS phenomics and functional genomics database.…”

Section: Trv-derived Vigs Vector Construct Containing Target Fragmentmentioning

confidence: 99%

“…They have developed a VIGS phenomics and functional genomics database. The database contains DNA sequence information derived from over 4,000 N. benthamiana VIGS clones and the associated silencing phenotype for approximately 1,300 genes (Senthil-Kumar et al 2018). Such a database could be used as a reference and overcomes the disadvantage of VIGS assay since it very often does not produce a uniform silencing throughout the plant.…”

Section: Trv-derived Vigs Vector Construct Containing Target Fragmentmentioning

confidence: 99%

Validating Plant Genes Involved in Pepper Yellow Leaf Curl Indonesia Virus Infection Using VIGS in Model Plant Nicotiana benthamiana

2020

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<p>Pepper yellow leaf curl disease caused by Pepper yellow leaf curl Indonesia virus (PepYLCIV) has become a challenge to chili pepper cultivation. Development of resistant variety by utilizing recessive resistance gene is expected to control the disease in the field. This study aimed to validate three plant genes, namely deltaCOP, hsc70, and BAM1, in PepYLCIV infection by applying Virus-induced Gene Silencing (VIGS) in a model plant, wild type Nicotiana benthamiana. PepYLCIV and construct of Tobacco rattle virus (TRV) which induced silencing of each gene were co-inoculated into N. benthamiana plants through agroinfiltration. Gene expression and the relative amount of viral DNA were determined by quantitative reverse transcription PCR (qRT-PCR) and quantitative PCR (qPCR), respectively, at 15 days post inoculation. The results showed a decreased level of deltaCOP, hsc70, and BAM1 expressions to 66.4%, 53.0%, and 47.0%, respectively, compared to that in the control (100%). Silencing of the three genes decreased the accumulation of PepYLCIV to 0.1%, 18.4%, and 63.0%, respectively, compared to that in the control. deltaCOP and hsc70 genes were indicated to be involved in the viral infection and could be good candidate genes for obtaining chili pepper varieties resistant to PepYLCIV. This result affirmed that the reverse genetics technique could be an alternative approach for identifying plant genes involved in viral infection, including PepYLCIV. The use of an infectious clone in this study allows the virus inoculations could be carried out without rearing and maintaining its natural vector, hence reduces the risk of virus transmission to healthy plants.</p>

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Virus‐induced gene silencing database for phenomics and functional genomics in Nicotiana benthamiana

Cited by 20 publications

References 35 publications

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

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

Homology-guided re-annotation improves the gene models of the alloploidNicotiana benthamiana

Validating Plant Genes Involved in Pepper Yellow Leaf Curl Indonesia Virus Infection Using VIGS in Model Plant Nicotiana benthamiana

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