Protein processing in Nicotiana benthamiana by Vacuolar Processing Enzymes and other proteases

Thesis

Protein processing by proteases is an irreversible post-translational modification that shapes the proteomes of all organisms. The outcomes of protein processing by proteases have diverse effects on protein function. This thesis describes the investigation of the landscape of protein processing in the model plant Nicotiana benthamiana.

Firstly, this thesis presents a snapshot of processed proteoforms in leaves captured by two degradomic techniques: PROtein TOpography Migration Analysis Platform (PROTOMAP) and Terminal Amine Isotopic Labelling of Substrates (TAILS). In combination these techniques revealed putative processing of 58% of the 4769 identified protein groups. Several case studies of putative processing events are discussed, which illustrate the value of this dataset as a resource to support future studies of plant proteins.

Secondly, this thesis explores the roles of two major vacuolar processing enzymes (VPEs) in leaves, NbVPE1a and NbVPE1b, are explored. VPEs are described as positive regulators of programmed cell death (PCD). Using a CRISPR-Cas9 generated vpe1a/b double mutant in N. benthamiana we demonstrate that NbVPE1a and NbVPE1b are responsible for the major VPE activities in leaves. Despite this, vpe1a/b mutants were not compromised in hypersensitive response PCD, nor in responses to salt, nitrogen, heat or ER stresses.

Finally, to elucidate the contribution of NbVPEs to protein processing, High-Efficiency Undecanal-based N-Termini EnRichment (HUNTER) was used to identify substrates of NbVPE1a/b. The N-terminome captured by HUNTER revealed significant changes in protease activity between the vpe1a/b and WT leaves and a clear preference of overexpressed NbVPE1b for asparaginase activity. Several candidate NbVPE substrates were identified, including the receptor-like protein NbFERONIA and papain-like cysteine proteases NbRD21 and NbALP. These were subject to further investigation.

Together this thesis presents an insight into general protein processing in leaves and the specific contribution of NbVPEs to the protease activities and stress responses of N. benthamiana.

Authors: Thomas, EL

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English