pH Gradient Mitigation in the Leaf Cell Secretory Pathway Attenuates the Defense Response of Nicotiana benthamiana to Agroinfiltration

Partial neutralization of the Golgi lumen pH by the ectopic expression of influenza virus M2 proton channel is useful to stabilize acid-labile recombinant proteins in plant cells, but the impact of pH gradient mitigation on host cellular functions has not been investigated. Here, we assessed the unintended effects of M2 expression on the leaf proteome of Nicotiana benthamiana infiltrated with the bacterial gene vector Agrobacterium tumefaciens. An isobaric tags for relative and absolute quantification quantitative proteomics procedure was followed to compare the leaf proteomes of plants agroinfiltrated with either an "empty" vector or an M2-encoding vector. Leaves infiltrated with the empty vector had a low soluble protein content compared to noninfiltrated control leaves, associated with increased levels of stress-related proteins but decreased levels of photosynthesis associated proteins. M2 expression partly compromised these effects of agroinfiltration to restore soluble protein content in the leaf tissue, associated with restored levels of photosynthesis-associated proteins and reduced levels of stress-related proteins in the apoplast. These data illustrate the cell-wide influence of the Golgi lumen pH homeostasis on the leaf proteome of N. benthamiana responding to microbial challenge. They also underline the relevance of assessing the eventual unintended effects of accessory proteins used to modulate specific cellular or metabolic functions in plant protein biofactories.