Elevated temperature differentially affects virulence, VirB protein accumulation, and T-pilus formation in different Agrobactetium tumefaciens and Agrobactetium vitis strains

That gene transfer to plant cells is a temperature-sensitive process has be en known for more than 50 years. Previous work indicated that this sensitiv ity results from the inability to assemble a functional T pilus required fo r T-DNA and protein transfer to recipient cells. The studies reported here extend these observations and more clearly define the molecular basis of th is assembly and transfer defect. T-pilus assembly and virulence protein acc umulation were monitored in Agrobacterium tumefaciens strain C58 at differe nt temperatures ranging from 20 degreesC to growth-inhibitory 37 degreesC. Incubation at 28 degreesC but not at 26 degreesC strongly inhibited extrace llular assembly of the major T-pilus component VirB2 as well as of pilus-as sociated protein VirB5, and the highest amounts of T pili were detected at 20 degreesC. Analysis of temperature effects on the cell-bound virulence ma chinery revealed three classes of virulence proteins. Whereas class I prote ins (VirB2, VirB7, VirB9, and VirB10) were readily detected at 28 degreesC, class II proteins (VirB1, VirB4, VirB5, VirB6, VirB8, VirB11, VirD2, and V irE2) were only detected after cell growth below 26 degreesC. Significant l evels of class III proteins (VirB3 and VirD4) were only detected at 20 degr eesC and not at higher temperatures. Shift of virulence-induced agrobacteri a from 20 to 28 or 37 degreesC had no immediate effect on cell-bound T pili or on stability of most virulence proteins. However, the temperature shift caused a rapid decrease in the amount of cell-bound VirB3 and VirD4, and V irB4 and VirB11 levels decreased next. To assess whether destabilization of virulence proteins constitutes a general phenomenon, levels of virulence p roteins and of extracellular T pili were monitored in different A. tumefaci ens and Agrobacterium vitis strains grown at 20 and 28 degreesC. Levels of many virulence proteins were strongly reduced at 28 degreesC compared to 20 degreesC, and T-pilus assembly did not occur in all strains except "temper ature-resistant" Ach5 and Chry5. Virulence protein levels correlated well w ith bacterial virulence at elevated temperature, suggesting that degradatio n of a limited set of virulence proteins accounts for the temperature sensi tivity of gene transfer to plants.