Nonstructural proteins of Tobacco rattle virus which have a role in nematode-transmission:: expression pattern and interaction with viral coat protein

RNA 2 of Tobacco rattle virus isolate PpK20 encodes the viral coat protein (CP) and two nonstructural proteins of 40 kDa ('40K protein') and 32.8 kDa ('32.8K'). The 40K protein is required for transmission of the virus by the vector nematode Paratrichodorus pachydermus whereas the 32.8K protein may be involved in transmission by other vector nematode species. An antiserum was raised against the 40K protein expressed in E. coli and used to study the expression and subcellular localization of this protein in infected Nicotiana benthamiana plants. The timecourse of the expression of the 40K protein in leaves and roots was similar to that of CP and both proteins were similarly distributed over the 1000 g pellet, 30 000 g pellet and 30 000 g supernatant fractions of leaf and root homogenates. Using the yeast two-hybrid system, a strong interaction between CP subunits was observed and weaker interactions between CP and the 32.8K protein and between CP and the 40K protein were detected. A deletion of the C-terminal 19 amino acids of CP interfered with the CP-40K interaction but not with CP-32.8K or CP-CP interactions, whereas a C-terminal deletion of 79 amino acids interfered with CP-40K and CP-32.8K interactions but not with the CP-CP interaction. As the C terminus of CP is known to be involved in nematode-transmission of tobraviruses, the data support the hypothesis that interactions between CP and RNA 2-encoded nonstructural proteins play a role in the transmission process.