Molecular responses of agroinfiltrated Nicotiana benthamiana leaves expressing suppressor of silencing P19 and influenza virus-like particles.

The production of influenza vaccines in plants is achieved through transient expression of viral hemagglutinins (HAs), a process mediated by the bacterial vector Agrobacterium tumefaciens. HA proteins are then produced and matured through the secretory pathway of plant cells, before being trafficked to the plasma membrane where they induce formation of virus-like particles (VLPs). Production of VLPs unavoidably impacts plant cells, as do viral suppressors of RNA silencing (VSRs) that are co-expressed to increase recombinant protein yields. However, little information is available on host molecular responses to foreign protein expression. This work provides a comprehensive overview of molecular changes occurring in Nicotiana benthamiana leaf cells transiently expressing the VSR P19, or co-expressing P19 and an influenza HA. Our data identifies general responses to Agrobacterium-mediated expression of foreign proteins, including shutdown of chloroplast gene expression, activation of oxidative stress responses and reinforcement of the plant cell wall through lignification. Our results also indicate that P19 expression promotes salicylic acid (SA) signalling, a process dampened by co-expression of the HA protein. While reducing P19 level, HA expression also induces specific signatures, with effects on lipid metabolism, lipid distribution within membranes and oxylipin-related signalling. When producing VLPs, dampening of P19 responses thus likely results from lower expression of the VSR, crosstalk between SA and oxylipin pathways, or a combination of both outcomes. Consistent with the upregulation of oxidative stress responses, we finally show that reduction of oxidative stress damage through exogenous application of ascorbic acid improves plant biomass quality during production of VLPs.

A drug-resistant mutation in plant target of rapamycin validates the specificity of ATP-competitive TOR inhibitors in vivo.

Kinases are major components of cellular signaling pathways, regulating key cellular activities through phosphorylation. Kinase inhibitors are efficient tools for studying kinase targets and functions, however assessing their kinase specificity in vivo is essential. The identification of resistant kinase mutants has been proposed to be the most convincing approach to achieve this goal. Here, we address this issue in plants via a pharmacogenetic screen for mutants resistant to the ATP-competitive TOR inhibitor AZD-8055. The eukaryotic TOR (Target of Rapamycin) kinase is emerging as a major hub controlling growth responses in plants largely thanks to the use of ATP-competitive inhibitors. We identified a dominant mutation in the DFG motif of the Arabidopsis TOR kinase domain that leads to very strong resistance to AZD-8055. This resistance was characterized by measuring root growth, photosystem II (PSII) activity in leaves and phosphorylation of YAK1 (Yet Another Kinase 1) and RPS6 (Ribosomal protein S6), a direct and an indirect target of TOR respectively. Using other ATP-competitive TOR inhibitors, we also show that the dominant mutation is particularly efficient for resistance to drugs structurally related to AZD-8055. Altogether, this proof-of-concept study demonstrates that a pharmacogenetic screen in Arabidopsis can be used to successfully identify the target of a kinase inhibitor in vivo and therefore to demonstrate inhibitor specificity. Thanks to the conservation of kinase families in eukaryotes, and the possibility of creating amino acid substitutions by genome editing, this work has great potential for extending studies on the evolution of signaling pathways in eukaryotes.