Studies of the interaction of the cytoplasmic determinants involved in sensitivity to Helminthosporium maydis race T are hampered by the fact that only maternal cytoplasm is transmitted after sexual crosses. Hybrid cytoplasms can, however, be produced by asexual fusion of protoplasts. We have therefore studied the effect of the toxin produced by H. maydis race T (HmT toxin) on fused and unfused protoplasts from plants with different cytoplasms.

HmT toxin has a clearcut cytoplasm-specific effect on cultured protoplasts. Protoplasts from corn plants with T cytoplasm collapse after 1-3 days of exposure to low levels of toxin while protoplasts with N, C, or S cytoplasm are unaffected by long-term treatment with high toxin levels (Plant Physiol. 61:420-424). Survival of protoplasts from oat leaf mesophyll and soybean callus is also unaffected by HmT toxin.

Toxin-sensitive T cytoplasm protoplasts can be distinguished from toxin-resistant protoplasts by visual markers. For example, green T cytoplasm leaf protoplasts look very different from non-green protoplasts isolated from callus or albino leaves. When mixtures of such protoplasts are treated with HmT toxin, the T cytoplasm protoplasts collapse while the others survive. Thus HmT toxin can be used to distinguish and select the toxin-resistant protoplasts in a mixed protoplast population.

Fusion of green T cytoplasm protoplasts with non-green soybean callus protoplasts can be induced by brief treatment with 30% polyethylene glycol (PEG) 6000, followed by rinsing in a high calcium, high pH solution. Fused corn-soybean protoplasts are recognized by the simultaneous presence of large corn chloroplasts and the dense cytoplasm characteristic of the soybean protoplasts. Such corn-soybean fusion products, which have a combination of T and non-T cytoplasm, survive at least 1-2 weeks in toxin levels which rapidly collapse all unfused T protoplasts. Survival of the corn-soybean fusion products in medium ± toxin is comparable. Similar results are seen when green T cytoplasm corn protoplasts are fused with non-green N cytoplasm corn leaf protoplasts from albino plants with the Cl gene. Many of the corn-soybean fusion products change shape (as soybean protoplasts typically do), and several have divided.

HmT toxin causes rapid ultrastructural damage to isolated T (but not N) mitochondria and to mitochondria within T cytoplasm roots and protoplasts (Tiss. & Cell 9:167). We are therefore examining the mitochondrial populations within toxin-treated corn-soybean fusion products. We are also exposing T cytoplasm protoplasts to isolated N mitochondria under conditions that facilitate organelle uptake in hopes of altering the response of treated protoplasts to HmT toxin. These studies may help pinpoint the location of the cytoplasmic genes for sensitivity to HmT toxin.

E. D. Earle and V. E. Gracen

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