The promoter and 5'-untranslated region (5'UTR) play a key role in determining the efficiency of recombinant protein expression in plants. Comparative experiments are used to identify suitable elements but these are usually tested in transgenic plants or in transformed protoplasts/suspension cells, so their relevance in whole-plant transient expression systems is unclear given the greater heterogeneity in expression levels among different leaves. Furthermore, little is known about the impact of promoter/5'UTR interactions on protein accumulation. We therefore established a predictive model using a design of experiments (DoE) approach to compare the strong double-enhanced Cauliflower mosaic virus 35S promoter (CaMV 35SS) and the weaker Agrobacterium tumefaciens Ti-plasmid nos promoter in whole tobacco plants transiently expressing the fluorescent marker protein DsRed. The promoters were combined with one of three 5'UTRs (one of which was tested with and without an additional protein targeting motif) and the accumulation of DsRed was measured following different post-agroinfiltration incubation periods in all leaves and at different leaf positions. The model predictions were quantitative, allowing the rapid identification of promoter/5'UTR combinations stimulating the highest and quickest accumulation of the marker protein in all leaves. The model also suggested that increasing the incubation time from 5 to 8 days would reduce batch-to-batch variability in protein yields. We used the model to identify promoter/5'UTR pairs that resulted in the least spatiotemporal variation in expression levels. These ideal pairs are suitable for the simultaneous, balanced production of several proteins in whole plants by transient expression.

The production of recombinant proteins in plants is often achieved by transient expression, e.g. following the injection or vacuum infiltration of Agrobacterium tumefaciens into tobacco leaves. We investigated the associated plant defence responses, revealing that callose deposition is triggered by T-DNA transfer and that subsets of secondary metabolites accumulate in response to mechanical wounding or the presence of bacteria. We also tested the ability of five co-expressed type III effector proteins from Pseudomonas syringae to modulate these defence responses and increase the yield of two model proteins, the fluorescent marker DsRed and monoclonal antibody 2G12. HopF2 and AvrRpt2 induced necrotic lesions 5 days post-injection (dpi) even at low doses (OD600 nm  = 0.0078), and increased the concentration of certain secondary metabolites. HopAO1 significantly reduced the number of callose deposits at 2 dpi compared to cells expressing DsRed and 2G12 alone, whereas HopI1 reduced the concentration of several secondary metabolites at 5 dpi compared to cells expressing DsRed and 2G12 alone. Co-expression with HopAO1, AvrPtoB or HopI1 increased the concentrations of DsRed and 2G12 increased by ~6% but this was not a significant change. In contrast, HopF2 and AvrRpt2 significantly reduced the concentrations of DsRed and 2G12 by 34% and 22%, respectively. Our results show that type III effector proteins can modulate plant defence responses and secondary metabolite profiles but that transient co-expression is not sufficient to increase the yields of target recombinant proteins in tobacco.