The transcriptome, extracellular proteome and active secretome of agroinfiltrated Nicotiana benthamiana uncover a large, diverse protease repertoire.

Infiltration of disarmed Agrobacterium tumefaciens into leaves of Nicotiana benthamiana (agroinfiltration) facilitates quick and safe production of antibodies, vaccines, enzymes and metabolites for industrial use (molecular farming). However, yield and purity of proteins produced by agroinfiltration are hampered by unintended proteolysis, restricting industrial viability of the agroinfiltration platform. Proteolysis may be linked to an immune response to agroinfiltration, but understanding of the response to agroinfiltration is limited. To identify the proteases, we studied the transcriptome, extracellular proteome and active secretome of agroinfiltrated leaves over a time course, with and without the P19 silencing inhibitor. Remarkably, the P19 expression had little effect on the leaf transcriptome and no effect on the extracellular proteome. 25% of the detected transcripts changed in abundance upon agroinfiltration, associated with a gradual up-regulation of immunity at the expense of photosynthesis. By contrast, 70% of the extracellular proteins increased in abundance, in many cases associated with increased efficiency of extracellular delivery. We detect a dynamic reprogramming of the proteolytic machinery upon agroinfiltration by detecting transcripts encoding for 975 different proteases and protease homologs. The extracellular proteome contains peptides derived from 196 proteases and protease homologs, and activity-based proteomics displayed 17 active extracellular Ser and Cys proteases in agroinfiltrated leaves. We discuss unique features of the N. benthamiana protease repertoire and highlight abundant extracellular proteases in agroinfiltrated leaves, being targets for reverse genetics. This data set increases our understanding of the plant response to agroinfiltration and indicates ways to improve a key expression platform for both plant science and molecular farming.