Transient expression of an scFvG8 antibody in plants and characterization of its effects on the virulence factor pthA of Xanthomonas citri subsp. citri.

Citrus bacterial canker, caused by Xanthomonas citri subsp. citri (Xcc), is a major disease of citrus plants, causing a significant loss in the citrus industry. The pthA is a bacterial effector protein mediates protein-protein and protein-DNA interactions and modulates host transcription. Injection of pthA effector protein into the host cell induces the expression of the susceptibility gene CsLOB1 which is required for citrus canker disease development. In this study, we described in planta expression of a specific anti-pthA single-chain variable fragment (scFv) recombinant antibody, scFvG8, and assessed its function using molecular docking, immunoblotting, and indirect enzyme-linked immunosorbent assay (ELISA). Based on the results, homology-based molecular docking suggested that at least eight intermolecular hydrogen bonds are involved in pthA-scFvG8 interactions. Immunoblotting and indirect ELISA results reconfirmed specific binding of scFvG8 to pthA protein. Moreover, gene fragment encoding scFvG8 was cloned into plant expression vector and transiently expressed in leaves of Nicotiana tabacum cv. Samson by agroinfiltration method. Transient expression of scFvG8 (at the expected size of 35 kDa) in N. tabacum leaves was confirmed by western blotting. Also, immunoblotting and indirect ELISA showed that the plant-derived scFvG8 had similar activity to purified scFvG8 antibody in detecting pthA. Additionally, in scFvG8-expressing tobacco leaves challenged with Xcc, a reduction (for up to 70%) of hypersensitive response (HR) possibly via direct interaction with pthA, was observed in the necrotic leaf area compared to control plants infected with empty vector. The results obtained in this study confirm that scFvG8 can suppress the function of pthA effector protein within plant cells, thus the induction of stable expression of scFvG8 in lime trees can be considered as an appropriate approach to confer resistance to Xcc.