APPLICATION OF RECOMBINANT PROTEIN TOOLS TO ASSAY POLEROVIRUS P0 PROTEIN INTERACTIONS WITH THE AUTOPHAGY PATHWAY

The Poleroviruses are plant viruses that have a broad range of hosts that include important crop species. Significant losses in crop yields occur annually due to polerovirus infection. Successful infection is due to the virus protein P0 which was shown to be a viral suppressor of RNA silencing (VSR) that suppresses the RNA-silencing pathway by disruption of the RNA-induced silencing complex (RISC) formation. Prior research showed that P0 acts as an F-box protein within the Skp1-Cullin-1-F-box (SCF) E3 ubiquitin ligase complex that marks proteins with polyubiquitin for degradation.P0-mediated degradation of Argonaut 1 (AGO1) was shown to require tagging with the K63-linked polyubiquitin via the autophagy pathway . In this research, two novel tools (NbATG4BDN:Flag and NbATG8c:EGFP:HA) were created to study P0-mediated degradation in Nicotiana benthamiana. The dominant-negative mutant NbATG4BDN was expected to be a biological autophagy inhibitor when it infiltrated into the plants based on the corresponding mutation in the human ATG4 protein. NbATG8c:EGFP:HA was used as a readout for ATG8 cleavage by ATG4 protease activity in the autophagy pathway. No significant inhibition of autophagy was observed with NbATG4BDN infiltration in the presence of P0, expressed alone or in the context of TuYV infection, and P0 VSR activity appeared unaffected. However, P0 co-expression with NbATG8c:EGFP:HA showed a decrease in ATG8 cleavage by ATG4, which might indicate direct binding of P0 to ATG8 that is preventing its processing. This observation may indicate a new role for P0 through a physical interaction with ATG8 that could direct target proteins like AGO1 to the autophagosome for degradation.