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.
Department of Biological Sciences, University of Maryland Baltimore County, Catonsville, Maryland 21228.
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Rogler