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

Abstract

SummaryThe 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.

Heterologous expression of influenza haemagglutinin leads to early and transient activation of the unfolded protein response in Nicotiana benthamiana

“…To better define molecular responses in N. benthamiana leaves expressing influenza VLPs, we previously conducted a proteomics survey using isobaric tags for relative and absolute quantitation (iTRAQ) labelling (Hamel et al ., 2023 ). At 6 DPI, this revealed enhanced accumulation of UPR proteins, including PDIs, BiPs, and CRTs (Table 1 ).…”

“… Using iTRAQ labelling, changes in protein abundance were evaluated in mock‐infiltrated leaves, leaves expressing P19 only, or leaves co‐expressing P19 and the HA protein from pandemic influenza virus strain H5 Indonesia (H5/A/Indonesia/05/2005; Hamel et al ., 2023 ). Using mock samples as a control, pairwise comparisons were performed for P19 and H5 samples.…”

“…Our previous transcriptomics and proteomics investigation at 6 DPI had also revealed up‐regulation of cytosolic heat shock proteins (HSPs), a response seen following expression of P19 only, but not following co‐expression of P19 and H5 (Hamel et al ., 2023 ). One noticeable exception to this was the gene model Niben101Scf04331g09018, which encodes a molecular chaperone of the HSP90 family.…”

“…Using influenza HA protein H5, we recently reported that expression of VLPs results in a unique molecular signature that affects metabolism and fitness of plant cells at 6 days post‐infiltration (DPI; Hamel et al ., 2023 ). In addition to the shutdown of chloroplast gene expression and to the activation of immune pathways, proteomics revealed that H5 expression results in the accumulation of UPR proteins, including PDIs, BiPs, and CRTs.…”

“…Enforced expression of a complex secreted protein such as H5 was expected to trigger the UPR so that plant cells can manage stress associated to the transient expression system, including increased needs for both recombinant and endogenous defence protein secretion. Interestingly, thorough monitoring of the transcriptome at 6 DPI did not reveal induction of UPR genes, suggesting that increased accumulation of UPR proteins was either independent of transcriptional regulation, or that UPR gene up‐regulation occurred earlier, before returning to levels that prevented their detection at 6 DPI (Hamel et al ., 2023 ).…”