Engineering high-level transient expression of heterologous proteins in plant cell suspensions

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Low protein product titers have thus far limited the application of Agrobacterium tumefaciens for the transient expression of heterologous proteins in plant cell suspensions and hairy root cultures. The overall objectives of this work were to overcome this limitation by: (1) increasing protein product titers in cell suspensions and hairy root cultures by harnessing A. tumefaciens to efficiently deliver replicating RNA viral vectors, and (2) identifying the physical and physiological factors important for reproducible, high-level transient expression in cell suspensions

Replicating vectors derived from Potato virus X (PVX) and Tobacco rattle virus (TRV) were modified to contain the reporter gene beta-glucuronidase (GUS) with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX construct retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-genome PVX vector. Co-delivery of the minimal PVX vector with p19 of Tomato bushy stunt virus and HC-Pro of Tobacco etch virus increased transient GUS expression by 40-80%. In hairy roots, a vector derived from TRV that was capable of systemic movement increased GUS accumulation by 150- fold relative to the analogous PVX vector.

A modified TRV-based vector and a transgenic host with integrated suppression of posttranscriptional gene silencing were investigated as strategies to further increase transient protein expression in hairy roots. A TRV vector was modified to retain the viral helper protein 2b, which is required for nematode transmission. In N. benthamiana hairy roots, the modified TRV vector resulted in GUS expression levels comparable to the control vector without the 2b gene. In contrast, Nicotiana tabacum cv. Xanthi hairy roots transgenic for HC-Pro yielded 5-fold more GUS from the modified TRV vector than control roots.

Prior to the development of the TRV vectors, a study was conducted to determine if movement-deficient PVX vectors could be complemented in trans by the Tobacco mosaic virus 30K movement protein (MP). Unexpectedly, transient co-delivery of TMV MP with the minimal PVX vector resulted in reduced transient GUS expression in cell suspensions and hairy roots of N. benthamiana.

Improved productivity in plant cell suspensions was achieved by manipulating the host, co-culture environment, and non-replicating expression vectors to produce conditions more favorable for Agrobacterium-mediated transient GUS expression. Modification of the N. glutinosa cell suspension host by transformation with Agrobacterium rhizogenes did not have a significant effect on average transient GUS expression across multiple independent cell lines. In N. benthamiana, doubling the concentration of potassium phosphate resulted in a 5-fold increase in transient GUS expression. Removal of the plant selectable marker cassette from a nonreplicating vector with a constitutive promoter decreased the T-DNA size by one-third and increased transient GUS expression by almost 50%.

This work established the feasibility of utilizing Agrobacterium-mediated delivery of replicating RNA viral vectors for amplified heterologous protein expression in plant tissue cultures. A TRV vector capable of systemic movement represents the first and only vector to achieve transient GUS expression levels in hairy roots comparable to those observed in plant cell suspensions. Considering the inconsistent absolute transient expression levels intrinsic to plant cell suspensions, hairy root cultures may be a preferable host for reproducible transient protein expression. The advantages and limitations of this approach are discussed in the context of engineering a safe, cost-effective, and scalable protein production platform.