Accumulation of Viroid RNA in Tumor Cells After Double Infection by Agrobacterium tumefaciens and Citrus Exocortis Viroid

SEMANCIK, J. S., L. K. GRILL, and E. L. CIVEROLO. 1978. Accumulation of viroid RNA in tumor cells after double infection by Agrobacterium tumefaciens and citrus exocortis viroid. Phytopathology 68: 1288-1292. The presence of citrus exocortis viroid (CEV) in tumors RNase-resistant ' I-CEV following molecular hybridization induced by A. tumefaciens in CEV-infected tomato has been of nucleic acid preparations from CEV-infected tissue. detected by infectivity of pathogenic RNA and visualization Primary tumors contained higher levels of the viroid than did of viroid RNA after polyacrylamide gel electrophoresis. apex tissue or fruit. In neoplastic cell cultures CEV can be These data correlate directly with the increased level of detected through four subculture passages. The low-molecular-weight (10' daltons) RNAs classed needle puncture of the hypocotyl at a point above the site as "viroids" present a unique opportunity to study the of CEV inoculation about 3-7 days after inoculation with process of pathogenesis by a minimal infectious molecule the viroid. (7). The dramatic expression of biological activity can be Tumor culture.-Tumors induced by A. tumefaciens compared to a transformed condition with respect to the were excised from 1to 2-mo-old infections on tomato permanent alteration in normal cell development (12). hypocotyls. After surface sterilization with 10% The major obstacles to the study of viroid synthesis and hypochlorite and 1 drop of Tween-80 in 100 ml, the tumor pathogenesis center on the extremely low concentration tissue was cut into 2to 3-mm cubes and dipped into of the pathogenic RNA and the asynchrony of intact sodium hypochlorite solution and 70% ethanol for 1-3 plant systems (13). min each before being placed on minimal medium (6) The accumulation of the citrus exocortis viroid in supplemented with neomycin sulfate (50 mg/liter), neoplastic plant cell growth following double inoculation penicillin G (80 mg/liter), streptomycin sulfate (50 with CEV and Agrobacterium tumefaciens, introduces a mg/liter), and 50,000 units/liter polymyxin B sulfate. novel system for the investigation of interaction of the Neoplastic outgrowths were subcultured on nonsuppleviroid with the host cell as coordinated with cell division. mented minimal medium. All operations were carried out in a laminar flow hood (Microvoid IIC) and cultures were MATERIALS AND METHODS maintained in the dark at 28-30 C. Nucleic acid extraction and analysis.-Tomato plant Culture and bioassay of citrus exocortis viroid.-CEV apex tissue, fruit, and primary tumors as well as cultured was rub-inoculated to the first true leaves of tomato neoplastic tumors were phenol-extracted under (Lycopersicon esculentum Mill 'Rutgers') seedlings and conditions previously reported (10). Detection of viroid by stem puncture with a tRNA-like preparation (10) from RNA was performed either by 5% cylindrical or slab polyinfected tissue. Infectivity was determined by bioassay on acrylamide gel electrophoresis (5, 9). Molecular Gynura aurantiaca (13). hybridization with 'I-CEV (kindly provided by Culture and inoculation of Agrobacterium Elizabeth Dickson) was performed utilizing conditions of tumefaciens.Cultures of Agrobacterium tumefaciens 4X SSC (SSC: 0.15 M NaC1/0.015 M sodium citrate at (ATCC 15955) were maintained at 4 C on 2.3% nutrient pH7) and 50% formamide at 42 C as recently reported (3). agar (Difco) slants containing 0.3% beef extract and 0.5% peptone. Inoculum was prepared by incubating a seeded flask containing 50 ml of 0.8% nutrient broth, 0.5% RESULTS sucrose, 0.1% yeast extractand 0.002 M MgSO4 for 18-24 Effect of double infection on whole plant hr at 28-30 C (with rotary shaking). Cells were collected, washed with water, and adjusted to contain about 1-3 X system.-Under standard conditions of being inoculated 106 cells/ml. Tomato seedlings were inoculated by a single with the citrus exocortis viroid, tomato does not demonstrate the sensitivity reported for G. aurantiaca 00032-949X/78/000 232$03.00/0 (13). Inoculum which produces a 90-100% infection in G. Copyright © 1978 The American Phytopathological Society, 3340 aurantiaca inoculated by stem slashing, induces viroid Pilot Knob Road, St. Paul, MN 55121. All rights reserved, symptoms on only 30-50% of combined stem-punctured