Transient Gene Expression for the Characteristic Signal Sequences and the  Estimation of the Localization of Target Protein in Plant Cell

Berestovoy, M. A.; Tyurin, A. A.; Кабардаева, К. В.; Sidorchuk, Yuriy V.; Фоменков, А. А.; Носов, А. В.; Goldenkova-Pavlova, I. V.

doi:10.21769/bioprotoc.2738

Cited by 6 publications

(7 citation statements)

References 1 publication

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“…The full length cDNAs of rice HDAC genes were subcloned into the pCambia 1301 vector to create the GFP fusions. Protoplast isolation and transient expression were performed by following the AgI-PrI method ( 33 ). Briefly, the A. tumefaciens cells were infiltrated into abaxial epidermis of 6-week-old tobacco ( N. benthamiana ) leaves using a syringe without a needle.…”

Section: Methodsmentioning

confidence: 99%

Histone deacetylases control lysine acetylation of ribosomal proteins in rice

Liu

Chen

et al. 2021

Nucleic Acids Research

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Lysine acetylation (Kac) is well known to occur in histones for chromatin function and epigenetic regulation. In addition to histones, Kac is also detected in a large number of proteins with diverse biological functions. However, Kac function and regulatory mechanism for most proteins are unclear. In this work, we studied mutation effects of rice genes encoding cytoplasm-localized histone deacetylases (HDAC) on protein acetylome and found that the HDAC protein HDA714 was a major deacetylase of the rice non-histone proteins including many ribosomal proteins (r-proteins) and translation factors that were extensively acetylated. HDA714 loss-of-function mutations increased Kac levels but reduced abundance of r-proteins. In vitro and in vivo experiments showed that HDA714 interacted with r-proteins and reduced their Kac. Substitutions of lysine by arginine (depleting Kac) in several r-proteins enhance, while mutations of lysine to glutamine (mimicking Kac) decrease their stability in transient expression system. Ribo-seq analysis revealed that the hda714 mutations resulted in increased ribosome stalling frequency. Collectively, the results uncover Kac as a functional posttranslational modification of r-proteins which is controlled by histone deacetylases, extending the role of Kac in gene expression to protein translational regulation.

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Section: Methodsmentioning

confidence: 99%

Histone deacetylases control lysine acetylation of ribosomal proteins in rice

Liu

Chen

et al. 2021

Nucleic Acids Research

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“…The transformed bacteria were selected on a medium containing kanamycin and designated according to the expression vector used for the transformation. The transformed agrobacteria were used for agroinfiltration into the abaxial epidermis of the leaves of 6-week-old N. benthamiana with a syringe without a needle as earlier described [20,21]. After the agroinfiltration, the plants were cultivated under the same conditions for 7 days.…”

Section: Engineering Of Plant Expression Vectorsmentioning

confidence: 99%

A High Throughput Assay of Lichenase Activity with Congo Red Dye in Plants

Tyurin

Suhorukova

Deineko

et al. 2021

Preprint

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Background : To elucidate the functional role of regulatory sequences and contexts identified and predicted during omics studies in the complex mechanisms of gene expression regulation, experimental verification methods in a plant cell are required. For this, the method of transient expression of reporter genes fused with the tested sequences is widely used. Several reporter systems are available that have shown good performance in the studies including the thermostable lichenase Clostridium thermocellum . However, each reporter system has its own limitations with respect to the quantification of the protein product of a reporter gene and, in particular, for the use of high-throughput approaches. Results: In this study, we report the design a high throughput fluorometric method for quantification of thermostable lichenase C. thermocellum using Congo red and further experimental verification of its relevance and efficiency in assessment of the functional role of regulatory sequences in the plant cell. Conclusion: The specific interaction between the dye Congo red and β -D-glucans formed the background for designing a high-throughput fluorometric assay for quantification of C. thermocellum thermostable lichenase as a reporter protein for plants. This assay (i) makes it possible to precisely measure the amount of reporter protein in a plant sample; (ii) has shown a high sensitivity for quantification of thermostable lichenase; (iii) is more time- and cost-efficient as compared with the Somogyi–Nelson assay; and (iv) is to the least degree dependent on the presence of the tested buffer components as compared with the Somogyi–Nelson assay.

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“…Knop's solution was used as a nutrient medium. The pVIG-E, pVIG-D9, pVIG-D9E, pVIG-Lch-D9, pVIG-Lch-D9E, pVIG-D9-ER, and pVIG-D9E-ER vectors were used to transform the Agrobacterium tumefaciens GV3101 strain as described earlier (Berestovoy et al 2018). Transformed bacteria were selected on a medium containing kanamycin and designated according to the expression vector used for the transformation.…”

Section: Plantmentioning

confidence: 99%

“…Transformed bacteria were selected on a medium containing kanamycin and designated according to the expression vector used for the transformation. The transformed agrobacteria were used for agroinfiltration into the abaxial epidermis of the leaves of six-week-old N. benthamiana and N. excelsior plants by means of a syringe without a needle as described earlier (Berestovoy et al 2018). After the agroinfiltration, the plants were cultured under the same conditions for seven days.…”

Section: Plantmentioning

confidence: 99%

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Altered fatty acid composition of Nicotiana benthamiana and Nicotiana excelsior leaves under transient overexpression of the cyanobacterial desC gene

Berestovoy¹,

Pavlenko²,

Tyurin³

et al. 2020

Biologia plant.

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Transient heterologous gene expression in two model plant species, Nicotiana benthamiana and N. excelsior, has been used to study the localization of the heterologous Δ9 acyl-lipid desaturase (Δ9 desaturase) of Synechococcus vulcanus in different cell compartments and its functional activity in the cases of the cytosol, chloroplast, and endoplasmic reticulum (ER) localization. The functional activity and substrate specificity of the heterologous desaturase under the conditions of transient expression have been confirmed by comparison of fatty acid (FA) profiles. The Δ9 desaturase, responsible for the synthesis of oleic and palmitoleic acids, has also been shown to strongly promote the accumulation of polyunsaturated FAs. The results convincingly demonstrate that the Δ9 desaturase of the thermophilic cyanobacterium transiently expressed in two Nicotiana species considerably alters lipid metabolism in their leaves towards a higher FA unsaturation. The functional activity of Δ9 desaturase depends on both the model plant species, N. benthamiana or N. excelsior, and the cellular localization of the enzyme. The method of transient expression of heterologous genes in plants is highly effective, inexpensive, and not time-consuming, which makes it attractive for estimating the functional activity and/or substrate specificity of heterologous desaturases.

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Transient Gene Expression for the Characteristic Signal Sequences and the Estimation of the Localization of Target Protein in Plant Cell

Cited by 6 publications

References 1 publication

Histone deacetylases control lysine acetylation of ribosomal proteins in rice

Histone deacetylases control lysine acetylation of ribosomal proteins in rice

A High Throughput Assay of Lichenase Activity with Congo Red Dye in Plants

Altered fatty acid composition of Nicotiana benthamiana and Nicotiana excelsior leaves under transient overexpression of the cyanobacterial desC gene

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