Technique for Using Isolated Corn Root Cap Cells in a Simple , Quantitative Assay for the Pathotoxin Produced by Helminthosporium maydis Race

Hawes, M. C. 1983. Technique for using isolated corn root cap cells in a simple, quantitative assay for the pathotoxin produced by Helminthosporium maydis race T. Phytopathology 73:1184-1187. Root cap cells from corn with Texas male sterile (Tms) cytoplasm were kill Tms cells within 6-8 hr and 50 ng/ ml or more caused nearly 100% cell much more sensitive to HMT-toxin, the pathotoxin produced by death within 10-12 hr. At least 50 /g/ml was required to kill N-cytoplasm Helminthosporium maydis race T, than cells from corn with normal (N) corn root cap cells. The rate of cell death was influenced by cell cytoplasm. Root cap cells were isolated by gentle agitation of the root in concentration, but not by plasmolysis. Isolated root cap cells provided a water, and the isolated cells were used to assay HMT-toxin. Each root cap simple, sensitive, quantitative assay for HMT-toxin. The assay was used to yielded 3-4 X 103 cells, which werejudged to be 98-100% viable by staining compare the lethal effects of HMT-toxin with cell death of oats caused by with fluorescein diacetate. Toxin-induced cell death was highly victorin, the pathotoxin produced by H. victoriae. temperature-dependent. At 35 C, 5 ng HMT-toxin per milliliter began to Additional key words: maize, temperature, Zea mays. Certain plant pathogens produce pathotoxins, compounds that have been evaluated several times (18,21,23,24); none combines play important causal roles in plant diseases. Pathotoxins are simplicity with sensitivity, quantitativeness, and speed. Corn root valuable research tools, since they can be used in lieu of pathogens cap cells from lines with the Texas male sterility (Tms) factor are to eliminate many of the complications of working with two much more sensitive to HMT-toxin than are those of lines with actively metabolizing organisms. Selective pathotoxins (20) are normal (N) cytoplasm (1). The purpose of this study was to adapt especially useful, since they can be used as components in model the root cap cell assay for use with H MT-toxin and corn and to use systems for studying pathogen specificity and the molecular basis the assay to determine the influence of temperature, plasmolysis, for disease resistance. and cell concentration on the lethal effects of HMT-toxin. The Recently, increased attention to toxin chemistry has led to the results were compared with data from a similar study of the effect of availability of several purified selective pathotoxins (3,14,15). victorin on oat root cap cells. However, lack of standard quantitative assays has hampered efforts to interpret results with toxins from different laboratories, MATERIALS AND METHODS and has limited progress in elucidating pathotoxin modes of action HMT-toxin. Purified HMT-toxin was a gift from J. M. Daly (23). Most assays have employed intact plants or tissues, with (Laboratory of Agricultural Biochemistry, University of Nebraska, consequent problems of uneven penetration and variation in Lincoln). Stock solutions of 10 mg/ml or 10 /Ag/ml in sensitivity of different cell types within a given tissue. Protoplasts dimethylsulfoxide (DMSO) were stored at room temperature. can be exposed uniformly, and they respond to toxins (5,8). Toxin was diluted from DMSO into water, and an equal amount of However, results with toxin-treated protoplasts have been too DMSO was added to control preparations. variable and inconsistent to provide quantitative bioassays (9,23). Corn seedlings. All studies were conducted using single lots of Knudson (13) reported that detached root cap cells can survive W64A Tms and W64A N-cytoplasm seeds, which were a gift from for weeks in a simple nutrient medium. An assay for victorin, the Vernon Gracen (Department of Plant Breeding and Biochemistry, pathotoxin produced by H. victoriae, was developed using isolated Cornell University, Ithaca, NY). To minimize microbial oat root cap cells maintained in water (9,10). The assay, based on contamination, aseptic protocol was used at all stages of victorin-induced cell death, was rapid, quantitative, and very germination. Seeds were soaked for 10 min in 0.05% sodium simple. It was used to demonstrate that victorin-induced cell death hypochlorite, rinsed thoroughly, and soaked for 1 hr in water. They is temperature-dependent and is inhibited by plasmolysis (10). were then placed embryo downward between sheets of moistened Root cap cells were compared with root cap protoplasts to show germination paper in 90-mm-diameter petri dishes, and were that the presence of the cell wall does not influence the lethal effects incubated at 25-27 C for 36-40 hr, or until the roots were 8-10 mm of victorin. In addition, root cap cells were compared with other cell in length. types to show that cells from different tissues of susceptible plants Isolation of cells. Root cap cells were harvested by a can vary significantly in sensitivity to victorin (9). modification of the procedure used to isolate oat root cap cells (10). HMT-toxin, the pathotoxin produced by H. maydis, race T, has Eight to 10 holes (5 mm in diameter) were punched in a vinyl mesh been studied more extensively than any other pathotoxin except screen. The screen was placed over a petri dish and secured with a victorin. The activity of this compound has been measured by at rubber band. Seedlings were placed, roots down, through the holes least 40 different methods (7). The four most commonly used assays and enough water was added to the dish so that 2-3 mm of the root tips was submerged. After 5-10 min, the dish was agitated manually The publication costs of this article were defrayed in part by page charge payment. This in a gentle circular motion for approximately 1 min. The cells article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. § released by this process were transferred to a beaker, and the cells 1734 solely to indicate this fact. were agitated vigorously for 5-10 min using a stream of air ©1983 The American Phytopathological Society introduced through a hypodermic needle. The cells were then