Amplicon-plus Targeting Technology (APTT) for rapid production of a highly unstable vaccine protein in tobacco plants

SummaryTransient transfection of plants by vacuum infiltration of Agrobacterium vectors represents the state of the art in plant-based protein manufacturing; however, the complexity and cost of this approach restrict it to pharmaceutical proteins. We demonstrated that simple spraying of Nicotiana plants with Agrobacterium vectors in the presence of a surfactant can substitute for vacuum inoculation. When the T-DNA of Agrobacterium encodes viral replicons capable of cell-to-cell movement, up to 90% of the leaf cells can be transfected and express a recombinant protein at levels up to 50% of total soluble protein. This simple, fast and indefinitely scalable process was successfully applied to produce cellulases, one of the most volume-and cost-sensitive biotechnology products. We demonstrate here for the first time that representatives of all hydrolase classes necessary for cellulosic biomass decomposition can be expressed at high levels, stored as silage without significant loss of activity and then used directly as enzyme additives. This process enables production of cellulases, and other potential high-volume products such as noncaloric sweetener thaumatin and antiviral protein griffithsin, at commodity agricultural prices and could find broad applicability in the large-scale production of many other cost-sensitive proteins.

Section: Discussionmentioning

confidence: 99%

A novel and fully scalable Agrobacterium spray‐based process for manufacturing cellulases and other cost‐sensitive proteins in plants

Hahn

Giritch

Bartels

et al. 2014

“…Hypervirulent strains of Agrobacterium, such as the succinamopine-type EHA105 and AGL1, improve transformation efficiencies (Hellens et al, 2000;Zhi et al, 2015), and might increase agro-inoculation success rates. The chrysopinetype strain Chry5 was originally isolated from chrysanthemum (Shao et al, 2018) and its derivative strain CryX was reported to have agro-inoculation efficiencies 100-1000 times higher than those of commonly used Agrobacterium strains ( (Azhakanandam et al, 2007;Giritch et al, 2013;Gleba et al, 2014;Grimsley et al, 1986;Hahn et al, 2015;Krenek et al, 2015). Viruses might be asymptomatic, vertically inherited in host plants and lack cell-to-cell movement (Roossinck, 2010); these aspects should be taken into account during experimental design and result evaluation.…”

Section: and Inoculation Efficiencymentioning

confidence: 99%

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

Pasin

Menzel

Daròs

2019

Summary Recent metagenomic studies have provided an unprecedented wealth of data, which are revolutionizing our understanding of virus diversity. A redrawn landscape highlights viruses as active players in the phytobiome, and surveys have uncovered their positive roles in environmental stress tolerance of plants. Viral infectious clones are key tools for functional characterization of known and newly identified viruses. Knowledge of viruses and their components has been instrumental for the development of modern plant molecular biology and biotechnology. In this review, we provide extensive guidelines built on current synthetic biology advances that streamline infectious clone assembly, thus lessening a major technical constraint of plant virology. The focus is on generation of infectious clones in binary T‐ DNA vectors, which are delivered efficiently to plants by Agrobacterium . We then summarize recent applications of plant viruses and explore emerging trends in microbiology, bacterial and human virology that, once translated to plant virology, could lead to the development of virus‐based gene therapies for ad hoc engineering of plant traits. The systematic characterization of plant virus roles in the phytobiome and next‐generation virus‐based tools will be indispensable landmarks in the synthetic biology roadmap to better crops.

“…Both transgenic expression and infection with recombinant viruses have been shown to be efficient systems for the production of foreign proteins in plants (Cañizares et al, 2005;Streatfield, 2007). The transformation of plants with a full-length viral cDNA, which is then transcribed into an RNA that can be amplified by the viral replication machinery ('amplicon'), integrates the stability of the transgenic system with the high copy numbers of viral expression vectors (Azhakanandam et al, 2007). However, constitutive viral replication can cause disease symptoms, impeding the appro- and Baulcombe, 1997).…”

Section: Discussionmentioning

confidence: 99%

“…They frequently yielded low viral accumulation levels, mainly because plants displaying disease symptoms could be counterselected during the transformation process and/or because the amplicon transgene induced an efficient antiviral RNA silencing response (Angell and Baulcombe, 1997; Liu et al ., 2004). Therefore, special strategies, such as the co‐expression of strong silencing suppressors (Mallory et al ., 2002; Azhakanandam et al ., 2007) or the use of chemically controlled promoters (Mori et al ., 2001), have been necessary in order to obtain amplicon lines with high expression levels.…”

Section: Introductionmentioning

confidence: 99%

A temperature‐controlled amplicon system derived from Plum pox potyvirus

Dujovny

Vallí

Calvo

et al. 2008

SummaryThe control of replication can facilitate a viral amplicon to reach high expression levels by enabling the virus to escape host defence mechanisms and reducing the deleterious effects of viral infection. We have developed a novel system to regulate amplicon expression by controlling the temperature of plant growth. Nicotiana benthamiana plants were transformed at two different temperatures with a cDNA copy of the Plum pox potyvirus genome harbouring the open reading frame 2 of Porcine circovirus 2 between the nuclear inclusion protein b and coat protein coding sequences. Although transformation at 27 ° C mainly yielded nonexpressing amplicons, lines with a tight control of amplicon expression were obtained. Viral replication was not detected in these plants when germinated at 28 ° C, but was observed when the plants were shifted to 20 ° C. In lines transformed at 24 ° C, although the amplicon was expressed at 28 ° C, viral accumulation was low and caused only minor growing defects. Viral replication was enhanced in these plants by shifting the temperature to 20 ° C; under such conditions, the amplicon reached higher and more persistent expression levels than in plants transformed at 27 ° C. These results demonstrate the utility of temperature regulation to control viral amplicon expression.