DEVELOPMENTAL BIOLOGY 143,173-184 (1991) A Brassica S-Locus Gene Promoter Targets Toxic Gene Expression and Cell Death to the Pistil and Pollen of Transgenic Nicotiana MARYK.THORSNESS,MUTHUGAPATTI K. KANDASAMY,MIKHAIL E. NASRALLAH,ANDJUNE B. NASRALLAH Section of Plant Biology, Division of Biological Sciences, Cornell University, Ithaca, New York l&53 Accepted September 2.5, 1990 The S-locus glycoprotein gene of Brassica is derived from the genetic locus that controls the self-incompatibility response and the specific recognition between pollen and stigma. The promoter of this gene was used to direct expression of the diphtheria toxin A chain gene and the Escherichia coli P-glucuronidase gene in transgenic Nicotiana tabacum. Expression of the promoter in cells of the pistil and in pollen suggests that a single gene may direct the self-incompati- bility response in the two interacting cell types. Additionally, the fusion genes were expressed gametophytically in the heterologous host species, Nicotiana, rather than sporophytically as expected for Brassica. Thus, although the genes involved in self-incompatibility in Brassica and Nicotiana are not homologous in their coding regions, signals for expression of these genes are apparently conserved between the two genera. Our analysis of toxic gene fusion transfor- mants shows that genetic ablation is useful for probing developmental processes and for studying temporal and spatial patterns of gene expression in plants. 0 1991 Academic Press, Inc. INTRODUCTION Genetic models of the pollen-stigma interaction of self-incompatibility predict expression of the control- ling locus, the S-locus, in two cell types of the flower. In members of the family Brassicaceae, the interacting cells are the pollen and the receptive papillar cells of the stigma surface. When the same S-allele is active in the two cell types, pollen germination at the stigma surface and the invasive growth of pollen tubes into the papillar cell wall are inhibited, and self-pollination is blocked. This is in contrast to the self-incompatibility response in Solanaceous species, in which pollen tube growth is inhibited after invasion and growth into the style (re- viewed in Ebert et al, 1989; Nasrallah and Nasrallah, 1989). Molecular analyses of self-incompatibility in species belonging to the Brassicaceae and the Solanaceae have defined the expression of S-locus derived genes in the pistil. In Bras&a oleracea, the S-locus glycoprotein (SLG)’ gene is derived from the S-locus. It is expressed specifically in the papillar cells of the stigma (Nasrallah et al, 1988), and its glycoprotein products accumulate in the papillar cell walls at the site of pollen contact (Kan- dasamy et al., 1989). When this gene is introduced into Nicotiana tabacum, a self-compatible member of the family Solanaceae, it is expressed in the secretory zone ’ Abbreviations used: SLG, S-locus glycoprotein; DT-A, diphtheria toxin A chain; GUS, P-glucuronidase; X-Glut, 5-bromo-4-chloro-3-in- doyl O-D-glucuronide; DAPI, 4’-6-diamidino-2-phenylindole; km’, kana- mycin resistant; km”, kanamycin sensitive. of the stigma, the transmitting tissue of the style, and the placental epidermis of the ovary (Moore and Nasral- lah, 1990; Kandasamy et al, 1990). This pattern of ex- pression is consistent with inhibition of incompatible pollen at the level of pollen tube growth within the style, as occurs in the Solanaceae, and is similar to the pattern of expression reported for the S-associated gene of the self-incompatible species N. alata (Cornish et al., 198’7; Anderson et al., 1989). The timing and site of S-gene expression during pol- len development is expected to differ between the Bras- sicaceae and the Solanaceae. In the Brassicaceae, ex- pression is expected to occur in a sporophytic tissue of the anther, since pollen incompatibility phenotype is determined sporophytically by the diploid genome of the parent plant. This prediction was borne out in the self-compatible crucifer Arabidopsis, which expressed a Brassica SLG promoter:reporter gene fusion sporo- phytically in the anther tapetum (Toriyama et al, 1991). In the Solanaceae the S-gene is expected to be expressed in the pollen grain, since pollen incompatibility pheno- type is determined gametophytically by the haploid ge- nome of the pollen grain (reviewed in Heslop-Harrison, 1975). In this study, we applied the method of genetic abla- tion to delete the specific cell types that express the SLG gene in transgenic N. tabacum. Our goals were to test the genetic model that predicts SLG gene expression during pollen development and to probe the evolution- ary relationship between sporophytic and gametophytic self-incompatibility. Another objective was to test the feasibility of using genetic ablation as an experimental 173 0012-1606/91 $3.00 Copyright CC 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.