Biophysical characterisation of a designed TMV coat protein mutant, R46G, that elicits a moderate hypersensitivity response in Nicotiana sylvestris

The hypersensitivity resistance response directed by the N' gene in Nicotia na sylvestris is elicited by the tobacco mosaic virus (TMV) coat protein R4 6G, but not by the U1 wild-type TMV coat protein. In this study, the struct ural and hydrodynamic properties of R46G and wild-type coat proteins were c ompared for Variations that may explain N' gene elicitation. Circular dichr oism spectroscopy reveals no significant secondary or tertiary structural d ifferences between the elicitor and nonelicitor coat proteins. Analytical u ltracentrifugation studies, however, do show different concentration depend encies of the weight average sedimentation coefficients at 4 degrees C. Vir al reconstitution kinetics at 20 degrees C were used to determine viral ass embly rates and as an initial assay of the rate of 20S formation, the oblig ate species for viral reconstitution. These kinetic results reveal a decrea sed lag time for reconstitution performed with R46G that initially lack the 20S aggregate. However, experiments performed with 20S initially present r eveal no detectable differences indicating that the mechanism of viral asse mbly is similar for the two coat protein species. Therefore, an increased r ate of 20S formation from R46G subunits may explain the differences in the viral reconstitution lag times. The inferred increase in the rate of 20S fo rmation is verified by direct measurement of the 20S boundary as a function of time at 20 degrees C using velocity sedimentation analysis. These resul ts are consistent with the interpretation that there may be an altered size distribution and/or lifetime of the small coat protein aggregates in elici tors that allows N. sylvestris to recognize the invading virus.