Molecular Characterization of Osmotin and Four Plasmodesmata Callose Binding Protein Homologs from Arabidopsis in Turnip Mosaic Virus Infection
Abstract
Plant viruses enter neighboring cells through plasmodesmata (PD), plasma membrane-lined microchannels that traverse the cell wall, to establish systemic infection. This intercellular movement process relies on the coordinated action of virus-encoded proteins and host proteins, especially PD-localized ones. To better understand the involvement of PD in viral infection, our lab previously conducted a quantitative, comparative proteomic study on the PD-enriched fraction from Nicotiana benthamiana leaves infected by turnip mosaic virus (TuMV). Osmotin (OSM, a PR-5 related protein) and plasmodesmata callose-binding protein 3 (PDCB3) are among the significantly differentially accumulated proteins in response to TuMV infection. In this thesis, I employed the TuMV-Arabidopsis thaliana pathosystem and characterized OSM (AtOSM34) and four PDCB homologs (AtPDCB1, AtPDCB2, AtPDCB3 and AtPDCB5) from Arabidopsis in TuMV infection.
I found that in Arabidopsis, AtOSM34 expression is upregulated, while AtPDCBs are downregulated after TuMV infection, consistent with previous proteomic data derived from N. benthamiana. Deficiency of osmotin in A. thaliana and N. benthamiana inhibits TuMV infection, whereas overexpression of AtOSM34 promotes viral replication and intercellular movement. Knockout of AtPDCB5 promotes TuMV infection but knockout of any of the other three AtPDCBsdoes not affect TuMV infection. As PDCB1, PDCB2 and PDCB3 may have functional redundancy, I generated double and triple knockout mutants. Only the triple knockout mutant shows enhanced TuMV infection, suggesting theses three AtPDCBs highly likely do have functional redundancy. Moreover, overexpression of AtPDCBs inhibits TuMV infection and reduces PD permeability by stimulating callose deposition. Overexpression of AtPDCB5 inhibits TuMV intercellular movement. In this thesis I also found that AtOSM34 and AtPDCBs are localized at PD and redistributed to virus replication complexes (VRCs) in TuMV-infected cells. Recruitment of AtOSM34 and AtPDCBs to VRCs is likely through the interaction with 6K2, the virus-encoded integral membrane protein that induces the formation of VRC-associated membrane structures. Overexpression of AtOSM34 increases PD permeability, reduces PD callose deposition and promotes TuMV intercellular movement. Overexpression of AtOSM34 also compromises antiviral resistance mediated by reactive oxygen species (ROS) burst. Taken together, these data suggest that OSM functions as a proviral host factor and PDCBs play antiviral roles in TuMV infection.