In planta proximity-dependent biotin identification (BioID) identifies a TMV replication co-chaperone NbSGT1 in the vicinity of 126 kDa replicase.

Tobacco mosaic virus (TMV) is a positive, single-stranded RNA virus. It encodes two replicases (126 kDa and 183 kDa), a movement protein and a coat protein. These proteins interact with host proteins for successful infection. Some host proteins such as eEF1α, Tm-1, TOM1, 14-3-3 proteins directly interact with Tobamovirus replication proteins. There are host proteins in the virus replication complex which do not interact with viral replicases directly, such as pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase. We have used Proximity-dependent biotin identification (BioID) technique to screen for transient or weak protein interactions of host proteins and viral replicase in vivo. We transiently expressed BirA* tagged TMV 126 kDa replicase in TMV infected Nicotiana benthamiana plants. Among 18 host proteins, we identified NbSGT1 as a potential target for further characterization. Silencing of NbSGT1 in N. benthamiana plants increased its susceptibility to TMV infection, and overexpression of NbSGT1 increased resistance to TMV infection. There were weak interactions between NbSGT1 and TMV replicases but no interaction between them was found in Y2H assay. It suggests that the interaction might be transient or indirect. Therefore, the BioID technique is a valuable method to identify weak or transient/indirect interaction(s) between pathogen proteins and host proteins in plants. BIOLOGICAL SIGNIFICANCE: TMV is a well characterized positive-strand RNA virus model for study of virus-plant host interactions. It infects >350 plant species and is one of the significant pathogens of crop loss globally. Many host proteins are involved in TMV replication complex formation. To date there are few techniques available for identifying interacting host proteins to viral proteins. There is limited knowledge on transient or non-interacting host proteins during virus infection/replication. In this study, we used agroinfiltration-mediated in planta BioID technique to identify transiently or non-interacting host proteins to viral proteins in TMV-infected N. benthamiana plants. This technique allowed us to identify potential candidate proteins in the vicinity of TMV 126 kDa replicase. We have selected NbSGT1 and its overexpression suppresses TMV replication and increase plant resistance. NbSGT1 is believed to interact transiently or indirectly with TMV replicases in the presence of Hsp90/Hsp70. BioID is a novel and powerful technique to identify transiently or indirectly interacting proteins in the study of pathogen-host interactions.