Long-Term Potato Virus X (PVX)-Based Transient Expression of Recombinant GFP Protein in Nicotiana benthamiana Culture In Vitro

Abstract

Plant molecular farming has a great potential to produce valuable proteins. Transient expression technology provides high yields of recombinant proteins in greenhouse-grown plants, but every plant must be artificially agroinfiltrated, and open greenhouse systems are less controlled. Here, we propose to propagate agrobacteria-free plants with high-efficient long-term self-replicated transient gene expression in a well-controlled closed in vitro system. Nicotiana benthamiana plant tissue culture in vitro, with transient expression of recombinant GFP, was obtained through shoot induction from leaf explants infected by a PVX-based vector. The transient expression occurs in new tissues and regenerants due to the natural systemic distribution of viral RNA carrying the target gene. Gene silencing was delayed in plants grown in vitro, and GFP was detected in plants for five to six months. Agrobacteria-free, GFP-expressing plants can be micropropagated in vitro (avoiding an agroinfiltration step), “rejuvenated” through regeneration (maintaining culture for years), or transferred in soil. The mean GFP in the regenerants was 18% of the total soluble proteins (TSP) (0.52 mg/g of fresh leaf weight (FW). The highest value reached 47% TSP (2 mg/g FW). This study proposes a new method for recombinant protein production combining the advantages of transient expression technology and closed cultural systems.

Construction of viral-based expression vectors for high-level production of human interferon alpha 2b in plants

“…The difference between the hINF production using different expression systems was highly reliable ( P < 0.01). For an agroinfiltration procedure, we used the first tree fully expended leaves from the apex as these leaves were shown the best producers of recombinant proteins (Sindarovska and Kuchuk 2021 ). We estimated that the total yield of hINF that can be obtained from the three leaves of one plant is 0.671 ± 0.106 mg. We also confirmed by ELISA method that recombinant hINF accumulated in upper uninfected leaves due to systemic distribution of viral RNA carrying the inf α2b gene.…”

“…Preparation of Agrobacterium suspension and agroinfiltration procedure were performed as described earlier (Sindarovska and Kuchuk 2021 ). Suspension of agrobacterial cells harboring the pPVX-INF plasmid vector (long or short version) (Fig.…”

“…The fresh leaf tissues were weighed and ground into fine powder at + 4 °C in a pre-cooled mortar with a pestle and then two volumes of ice-cold extraction buffer (w/v) were added (Sindarovska and Kuchuk 2021 ). To purify the protein fraction, two rounds of centrifugation were performed at 16,000 g for 10–15 min at + 4 °C.…”

“…While simple expression systems where the target gene is controlled by 35S promoter of cauliflower mosaic virus (CaMV) are often used for transient gene expression in plants (Norkunas et al 2018 ; Pang et al 2019 ; Phan et al 2020 ), improved expression systems that include viral genome elements, such as genes coding viral polymerases, replication initiation protein, coat and movement proteins, and suppressors of gene silencing, can enhance the expression of the target genes, and increase the final yield of recombinant proteins (Jiang et al 2019 ; Malla et al 2021 ; Phakham et al 2021 ; Poborilova et al 2020 ; Sheludko et al 2007 ; Sindarovska and Kuchuk 2023 ; Yamada et al 2020 ; Yamamoto et al 2018 ). Tobacco mosaic virus and potato virus X genomes are often used to create improved expression cassettes and to achieve high levels of a target protein through transient gene expression (Baró et al 2022 ; Dickmeis et al 2014 ; Lindbo 2007a , 2007b ; Marillonnet et al 2004 ; Shi et al 2019 ; Sindarovska and Kuchuk 2021 , 2023 ; Torti et al 2021 ).…”

Nicotiana benthamiana as a model plant host for Xylella fastidiosa: Control of infections by transient expression and endotherapy with a bifunctional peptide

“…On the contrary, constitutive expression of BP134 in rice plants had strong negative impact on rice fitness ( Nadal et al., 2012 ). No yield quantification was performed in our study because when peptides are expressed using a virus-based vector system, transient production levels of heterologous peptides differ between plants, as it depends on physical factors influencing the viral vector infection, environmental conditions, and the physiological state of each plant ( Sindarovska and Kuchuk, 2021 ). In the present study, absence of self-cleavage of the FMDV 2A sequence to obtain the free AMPs was also observed ( Kovalskaya et al., 2011 ), which can be due to the amino acid sequence, as the ratio of fused CP and non-fused viral CP depends on it ( Dickmeis et al., 2015 ).…”