Plasmid-dependent temperature-sensitive phase in crown gall tumorigenesis

Two methods have been used to show that the temperature-sensitive (ts) genes for plasmid maintenance located on pTiC58 are responsible for the presence of a specific 37 degrees C ts period during crown gall tumorigenesis. Tumors induced by Agrobacterium tumefaciens strain C58 become resistant to 32 degrees C by 96 hr after infection, indicating that the "inception phase" of crown gall is complete at that time. However, C58 tumors remain sensitive to 37 degrees C until 168 hr after infection. When pTiC58 is replaced in strain C58 by a plasmid that is temperature resistant for maintenance within the bacterium at 37 degrees C (B(6)806) the tumors induced by the new strain (A277) become resistant to 37 degrees C by 96 hr after infection. Furthermore, when pTiC58 is selected for temperature resistance in strain C58 at 37 degrees C, the tumors induced by the new strain, C58(TR), become resistant to 37 degrees C approximately 40 hr earlier than do tumors induced by the parent strain C58. These results prove that the 37 degrees C ts period (96-168 hr after infection) is coded for ts plasmid maintenance genes located on pTiC58. Incorporation of [(3)H]-thymidine into pTiC58 in strain C58 is specifically inhibited at 37 degrees C compared to chromosomal DNA and is not inhibited at 32 degrees C. The data presented support a mechanism in which the 37 degrees C, ts period between 96 and 168 hr after infection is due to the requirement for the plasmid-coded maintenance genes involved in plasmid replication within incipient tumor cells. The fact that the 37 degrees C ts period ends after 168 hr indicates a transient requirement for the ts gene product, perhaps due to the stable integration of plasmid DNA into plant genetic elements by that time.