CRISPR/Cas9-mediated knockout of the RDR6 gene in Nicotiana benthamiana for efficient transient expression of recombinant proteins

Avian influenza A/H7N9 is a subtype of influenza viruses that has been detected in birds in the past. This subtype has not been previously seen in either animals or human until it was found in March 2013 in China. However, since then, infections of modified H7N9 in both human and birds have been observed such as H7N2, H7N3 and H7N5 subtypes. Hemagglutinin (HA) is the most important antigen for induction of immunity, and is a target to develop influenza vaccines. In this study, the gene encoding HA protein was sucessfully inserted into pRTRA vectors to generate 35S-HA-histag-cmyc-ELP-KDEL and 35S-HApII-histag-cmyc-ELP-KDEL cassettes in the absence and presence of trimer motif GCN4, respectively in order to enhance accumulation of recombinant proteins in plants. These cassettes were inserted into pCB301 binary vectors and transformed into Agrobacterium tumefaciens in order to carry out the transient expression in Nicotiana benthamiana leaves. Five days after infiltration, the total soluble protein was extracted and analysed in SDS-PAGE. The HA-ELP and (HApII-ELP)3 recombinant proteins were detected by Western blot using anti-cmyc monoclonal antibody. These results showed that HA protein fused ELP was observed in monomeric and trimeric forms. The plant extract containing hemagglutinin protein in monomeric and trimeric forms were characterized bio-function using hemagglutination assay. The plant extract containing hemagglutinin protein in trimeric form agglutinated chicken red blood cells at concentration of 94 µg protein/ml whereas the plant extract containing hemagglutinin protein in monomeric form did not agglutinate chicken red blood cells at the same of protein concentration.

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Repression of the DCL2 and DCL4 genes in Nicotiana benthamiana plants for the transient expression of recombinant proteins

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2017.02.019 ◽

2017 ◽

Vol 124 (2) ◽

pp. 215-220 ◽

Cited By ~ 4

Author(s):

Kouki Matsuo ◽

Takeshi Matsumura

Keyword(s):

Transient Expression ◽

Recombinant Proteins ◽

Nicotiana Benthamiana

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Covalent protein display on Hepatitis B core-like particles in plants through the in vivo use of the SpyTag/SpyCatcher system

Scientific Reports ◽

10.1038/s41598-020-74105-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Hadrien Peyret ◽

Daniel Ponndorf ◽

Yulia Meshcheriakova ◽

Jake Richardson ◽

George P. Lomonossoff

Keyword(s):

Hepatitis B ◽

Cost Saving ◽

Capsid Protein ◽

Recombinant Proteins ◽

Nicotiana Benthamiana ◽

Vaccine Development ◽

Binding Partner ◽

Gfp Fluorescence ◽

Display Systems

Abstract Virus-like particles (VLPs) can be used as nano-carriers and antigen-display systems in vaccine development and therapeutic applications. Conjugation of peptides or whole proteins to VLPs can be achieved using different methods such as the SpyTag/SpyCatcher system. Here we investigate the conjugation of tandem Hepatitis B core (tHBcAg) VLPs and the model antigen GFP in vivo in Nicotiana benthamiana. We show that tHBcAg VLPs could be successfully conjugated with GFP in the cytosol and ER without altering VLP formation or GFP fluorescence. Conjugation in the cytosol was more efficient when SpyCatcher was displayed on tHBcAg VLPs instead of being fused to GFP. This effect was even more obvious in the ER, showing that it is optimal to display SpyCatcher on the tHBcAg VLPs and SpyTag on the binding partner. To test transferability of the GFP results to other antigens, we successfully conjugated tHBcAg VLPs to the HIV capsid protein P24 in the cytosol. This work presents an efficient strategy which can lead to time and cost saving post-translational, covalent conjugation of recombinant proteins in plants.

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Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽

10.3390/plants10030524 ◽

2021 ◽

Vol 10 (3) ◽

pp. 524

Author(s):

Bingqi Wu ◽

Zhiting Chen ◽

Xiaohui Xu ◽

Ronghua Chen ◽

Siwei Wang ◽

...

Keyword(s):

Gene Expression ◽

Transient Expression ◽

Nicotiana Benthamiana ◽

Heterologous Gene Expression ◽

Expression System ◽

Functional Characterization ◽

Transient Gene Expression ◽

High Mobility ◽

Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

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