Intercellular spread of Cowpea mosaic virus (CPMV) occurs via movement tubules inserted into the cell wall spanning plasmodesmata (PD) channels. These tubules are composed solely of viral movement proteins (MPs) and carry virus particles to neighbouring cells. The insertion of movement tubules into PD requires severe structural modification of these channels. These modifications are thought to rely on the concerted action of host and viral proteins. The aim of this thesis research has been to identify those host factors involved in the intercellular transport of CPMV.
Movement tubules collected from protoplasts, isolated plants cells without cell walls, were analysed by tandem mass spectrometry. This analysis revealed that seven host proteins associated specifically with these movement structures. Among these tubule-associated host proteins were CDC48-type AAA-proteins, heat shock protein (HSP) 60 and HSP70 isoforms. These three proteins were selected for further analysis in order to test their potential role in CPMV infection and spread. Targeted gene silencing of either CDC48 or HSP70 proteins caused a reduction in viral titres and also reduced the spread of CPMV in infected leaves. These results show that altered expression of these two tubule-associated host proteins influences the viral infection cycle.
Furthermore, interactions between the MP of CPMV and both plasma membrane intrinsic proteins (PIPs) and plasmodesmata-located proteins (PDLPs) were investigated using fluorescently labelled fusion proteins. An interaction between the MP and PIP-isoforms could not be established, however, MPs were found to co-localise with PDLP1 in PD. Using fluorescence lifetime imaging (FRET-FLIM) the molecular interaction between PDLPs and viral MPs was established in PD. This interaction was, however, only found in plant tissues and was not observed in protoplasts. This suggests that the interaction is restricted to structural context of the PD. Further experiments in protoplasts showed that co-localisation of MPs with PDLP1 was not required for the formation of movement tubules in these cells, which supports the hypothesis that the role of PDLP in plant virus intercellular transport is restricted to the PD.