Manipulation of the Nicotiana benthamiana immune response to Agrobacterium tumefaciens to improve plat molecular farming
Keywords: Agrobacterium tumefaciens
molecular farming
Nicotiana benthamiana
plant immunity

Nicotiana benthamiana is used as an expression platform for recombinant protein production using Agrobacterium tumefaciens-mediated transient expression. This method has successfully produced multiple recombinant proteins. However, the use of A. tumefaciens for transient expression poses a problem because it elicits an immune response from N. benthamiana which hampers molecular farming yields. In this research we aimed to improve understanding of the interaction between N. benthamiana and A. tumefaciens and how this interaction can be manipulated to improve molecular farming. This was achieved firstly by studying how apoplastic alkalinization is detrimental to A. tumefaciens. Apoplastic alkalinization was achieved by manipulating the activity of the H+-ATPase named Autoinhibited H+-ATPase isoform 2 (AHA2). AHA2 activity was manipulated by over-expression of the RAPID ALKALINIZATION FACTOR-1 peptide (RALF1) and expression of the AHA2-S899A mutant. Both strategies resulted in alkalinization of the apoplast. We found that apoplastic alkalinization triggered upregulation of pathogenesis-related proteins, mediated by salicylic acid signalling. We demonstrated that these alkalinization-induced plant defence responses were detrimental to A. tumefaciens and reduced accumulation of recombinant proteins. Next, we developed a high-throughput ‘Traffic Light Screen’ to evaluate new strategies to improve molecular farming and uncover how plant immunity affects A. tumefaciens. Using this screen, we investigated the impact of co-expressing 130 RxLR effectors on transient expression and A. tumefaciens. We identified six effectors which, when co-expressed, increased fluorescence of secreted RFP. Two of these six effectors also increased accumulation of a recombinantly expressed IgG antibody, VRC01. However, when co-expressed in an industrial scale agroinfiltration, the most promising effector did not increase final yields of VRC01 produced. We then used the ‘Traffic Light Screen’ to assess the impacts of inhibiting plant defence responses through depletion of genes involved in plant immunity. This identified that silencing of genes ICS (encoding isochorismate synthase), NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 and CHITIN ELICITOR RECEPTOR KINASE 1 improved transient expression. These three genes are promising candidates to pursue further. We also revealed that the receptor NbCORE is essential for perception of the MAMP derived from bacterial cold shock protein (CSP), called csp22, in older N. benthamiana plants and that depletion of NbCORE enables the use of older plants with greater biomass for transient expression. This research demonstrates that manipulating the interaction between N. benthamiana and A. tumefaciens is an effective strategy to improve molecular farming. We also demonstrate the need to study this interaction further to identify plant defence responses that hamper molecular farming in order to find new strategies to increase recombinant protein yields.