THE REQUIREMENT OF PHYSICAL CONTACT FOR MOSS GAMETOPHORE INDUCTION BY AGROBACTERIUM TUMEFACIENS

Gametophore induction in the moss Pylaisiella selwynii by Agrobacterium tumefaciens strain B6 does not occur in parabiotic chambers when the moss and bacterium are separated by filters of 0.22 um pore diameter. The combination of moss plus bacteria which gives increased gametophore formation also had no effect on sterile moss plants separated from the mixture by these filters. Heat-killed B6 cells inhibit gametophore induction by viable B6 cells when added prior to or with the viable bacteria but not when added 6 or more hr later. The concentration dependence of both gametophore induction by viable B6 cells and inhibition of this process by heat-killed B6 cells appears linear, suggesting that each response may require only one particle. Gametophore induction by Agrobacterium, therefore, requires physical contact comparable to the bacteria-site attachment step demonstrated essential for tumor induction in higher plants and shows similar concentration dependence. The size of the moss and the absznce of a wound requirement for this response makes the Agrobacterium-moss system particularly advantageous for further resolution of the site attachment step in Agrobacterium tumorigenesis. IN HIGHER PLANTS tumor induction by Agrobacterium tumefaciens proceeds only after bacterial attachment to a specific host site exposed by wounding (Lippincott and Lippincott, 1969). Spiess, Lippinco'tt, and Lippincott (1971) found that A. tumefaciens strain B6, which is highly tumorigenic, promotes gametophore formation in the moss Pylaisiella selwynii, and that the number of gametophores increases with bacterial concentration. Because this response does not require any apparent wounding and because several low molecular weight compounds can induce gametophore formation, it was assumed that similar substances produced by the bacteria might be responsible for this effect (Spiess, Lippincott, and Lippincott, 1972, 1973). This paper describes experiments to determine whether a diffusible Agrobacterium product or direct bacterium-moss contact is required for strain B6-induced gametophore formation. The data indicate that a process similar to the site attachment step in tumor induction in higher plants is essential for this response. ' Received for publication 18 February 1975. These investigations were supported in part by a Brown-Hazen grant from the Research Corporation and by PHS grants CA05387 from the National Cancer Institute, AI12149 from the National Institute of Allergy and Infectious Diseases and Biomedical Sciences Support Grant FR7028. Name and address for correspondence: L. D. Spiess, Department of Biology, Lake Forest College, Lake Forest, Illinois 60045. MATERIALS AND METHODS-Pylaisiella selwynii was cultured as described previously (Spiess et al., 1973) and treated with bacteria 17 days after sowing the spores. Stationary phase (48 hr) cultures of Agrobacterium tumefaciens (Smith and Town.) Conn, strain B6, were grown as described by Lippincott and Lippincott (1966). Bacterial cultures were washed prior to use by two centrifugations for 30 min at 10,000 x g, with resuspension in sterile moss culture medium after each centrifugation. Sterile technique was used throughout. The concentration of viable bacteria in these washed suspensions was estimated by serial dilution and plating. For inhibition experiments portions of washed bacteria cultures were heat-killed by a 35-min treatment in an 80 C water bath and then quickly cooled in ice water (Lippincott and Lippincott, 1969). Viable and heated bacteria were combined and 0.5 ml added to the moss plants in 5cm petri dishes containing 4.5 ml of moss medium. In experiments with delayed addition of bacteria, 0.25 ml of viable and/or heated bacteria were added to each dish at the times indicated. In all cases concentrations of the two types of bacteria were adjusted prior to addition so that the final concentration of each in the moss culture medium was as indicated. Flanges of parabiotic chambers (Bellco Glass Co. # 1945) were thickly coated with silicone stop-cock lubricant and two 0.22-uxm Millipiore filters were placed between the two chambers.