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The Proteolytic Machinery of the Plant Apoplast:
from Basic Understanding to Improved Recombinant Protein Production

Final Report Summary - GREENPROTEASES (The Proteolytic Machinery of the Plant Apoplast:from Basic Understanding to Improved Recombinant Protein Production)

The model plant Nicotiana benthamiana is increasingly used in industry to produce pharmaceutical proteins such as vaccines, therapeutic antibodies and peptide hormones. Proteins can be quickly produced in this system upon transient expression, which is achieved by infiltrating Agrobacterium tumefaciens (agroinfiltration), carrying genes of interest. Glyco engineering of this expression platform has resulted in the production of recombinant proteins with humanized glycans, that are delivered into the extracellular space (apoplast) of agroinfiltrated leaves. However, although several proteins are efficiently produced, many other proteins initially accumulate and then disappear via cleavage products. The aim of this project was to deplete the responsible proteases to improve this expression platform, and to study the role of these secreted proteases in plant immunity.
We first characterized the transcriptome and secreted proteome of agroinfiltrated plants and identified 196 putative proteases in the apoplast, much more than anticipated (published in PBJ, 2017). Activity-based proteomics using chemical probes targeting the active site of proteases confirmed that many of these proteases are active enzymes. Many of the detected apoplastic proteases were depleted by virus-induced gene silencing, but this strategy did not prevent the degradation of recombinant glycoproteins. Co-expression with protease inhibitors, however, did increase recombinant protein levels. Upon testing >25 candidate protease inhibitors we identified three unrelated protease inhibitors that boost recombinant protein levels (published in PBJ, 2018).
We found that protease inhibitor SlCYS8 targets nine cysteine proteases (published in PBJ, 2019). These proteases were removed from N. benthamiana genome by CRSPR/Cas9 genome editing. These lines have no developmental phenotypes even though they lack multiple proteases and have an altered proteolytic profile, but these lines remain to be characterized for improved recombinant protein expression.
Our studies of secreted papain-like proteases revealed that they are activated by secreted subtilase-like proteases, which remove the autoinhibitory prodomain of papain-like proteases by cleavage after aspartic acid residues (published in PNAS, 2020). Activation of these proteases was blocked by silencing subtilase SBT5.2 in N. benthamiana, or with subtilase inhibitor EPI1, which is produced by the notorious potato blight pathogen Phytophthora infestans, presumably to prevent activation of immune proteases in the host. Similarly, these proteases are activated by multiple subtilases in tomato, that belong to a different subfamily of SBT5.2 illustrating the complexity of the extracellular proteolytic network, probably providing robust immunity.
We also investigated which apoplastic proteases release peptide elicitors from pathogen proteins that are recognized by plants to trigger immune responses. Pathogenic bacteria are recognized by plants through the detection of peptides released from flagellin. We discovered that the proteolytic release of elicitor peptides is preceeded by the deglycosylation of the flagellin polymer, mediated by an apoplastic glycosidase (published in Science, 2019). Plant pathogens avoid recognition by producing inhibitors targeting this glycosidase and by producing glycans that are insensitive to the glycosidase.
We also improved the genome annotation of N. benthamiana (published in BMC-PB, 2019) and established methods to detect glycoprotein degradation and active metalloproteases, and supported visiting scientists with their studies on plant proteases. Overall, this project generated genetic and proteomic tools to study proteases and uncovered the complexity of the extracellular proteolytic network in N. benthamiana and related crop plants. This protease network contributes to robust immunity but also causes major challenges in improving protein expression.