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

Friederike Grosse‐Holz,Renier A.L Van Der Hoorn,Steven Kelly,Farnusch Kaschani,Markus Kaiser,Svenja Blaskowski

doi:10.1111/pbi.12852

Friederike Grosse‐Holz, Renier A.L Van Der Hoorn + Show 4 more

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https://doi.org/10.1111/pbi.12852

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Abstract

SummaryInfiltration 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.

Highlights

Agroinfiltration of Nicotiana benthamiana is widely applied to transiently express proteins, either as biopharmaceutcials, for other industrial use or to study their functions
Searching the extracellular proteome mass spectrometry (MS) spectra with our curated proteome, we identified peptides corresponding to 30 proteins more than with the best published database, showing that the curation improved interpretation of experimental data (Appendix S1)
25% of the full leaf mRNA transcriptome changes in abundance, associated with an immune response mounted at the expense of photosynthesis. 70% of all extracellular proteins increase in abundance and their predicted functions confirm that an extracellular immune response occurs

Summary

Introduction

Agroinfiltration of Nicotiana benthamiana (a relative of tobacco) is widely applied to transiently express proteins, either as biopharmaceutcials, for other industrial use or to study their functions. Agrobacterium delivers the T-DNA to the nucleus of its host plant, where genes are expressed within a few days upon agroinfiltration. Co-expression of several transgenes is achieved by mixing Agrobacterium cultures delivering these different transgenes before agroinfiltration. Production of biopharmaceuticals (molecular farming) (Stoger et al, 2014) in agroinfiltrated N. benthamiana offers speed, scalability and low risk of contamination with human pathogens when compared to classical insect or mammalian cell culture systems. Speed and simplicity of agroinfiltration are leveraged for high-throughput screening of fluorescently tagged proteins to study their subcellular localization (Martin et al, 2009)

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