SummaryGametophytic self-incompatibility (GSI) systems involving the expression of stylar ribonucleases have been described and extensively studied in many plant families including the Solanaceae, Rosaceae and Scrophulariaceae. Pollen recognition and rejection is governed in the style by specific ribonucleases called SRNases, but in many self-incompatibility (SI) systems, modifier loci that can modulate the SI response have been described at the genetic level. Here, we present at the molecular level, the isolation and characterization of two Solanum chacoense homologues of the Nicotiana HT modifier that had been previously shown to be necessary for the SI reaction to occur in N. alata (McClure et al., 1999). HT homologues from other solanaceous species have also been isolated and a phylogenetic analysis reveals that the HT genes fall into two groups. In S. chacoense, these small proteins named ScHT-A and ScHT-B are expressed in the style and are developmentally regulated during anthesis identically to the S-RNases as well as following compatible and incompatible pollination. To elucidate the precise role of each HT isoform, antisense ScHT-A and RNAi ScHT-B lines were generated. Conversion from SI to self-compatibility (SC) was only observed in RNAi ScHT-B lines with reduced levels of ScHT-B mRNA. These results confirm the role of the HT modifier in solanaceous SI and indicate that only the HT-B isoform is directly involved in SI.

Self-incompatibility (SI) in angiosperms is a genetic mechanism that promotes outcrossing through rejection of self-pollen. In the Solanaceae, SI is determined by a multiallelic S locus whose only known product is an S RNase. S RNases show a characteristic pattern of five conserved and two hypervariable regions. These are thought to be involved in the catalytic function and in allelic specificity, respectively. When the Solanum chacoense S12S14 genotype is transformed with an S11 RNase, the styles of plants expressing significant levels of the transgene reject S11 pollen. A previously characterized S RNase, S13, differs from the S11 RNase by only 10 amino acids, four of which are located in the hypervariable regions. When S12S14 plants were transformed with a chimeric S11 gene in which these four residues were substituted with those present in the S13 RNase, the transgenic plants acquired the S13 phenotype. This result demonstrates that the S RNase hypervariable regions control allelic specificity.

A novel WRKY-like transcription factor was isolated from a screen for weakly expressed mRNAs in ovules in the self-incompatible wild potato species Solanum chacoense Bitt. This protein, named ScWRKY1, consisted of 525 amino acids and can be classified as a WRKY group-I member, having two WRKY domains. It is expressed at low levels in stems, roots, and petals, and expressed at much higher levels in leaves. Interestingly, although barely detectable in developing seeds, it is strongly and transiently expressed in fertilized ovules bearing late torpedo-staged embryos, suggesting a specific role during embryogenesis.

SummaryCultivated tomato (Lycopersicon esculentum), a self-compatible species, evolved from self-incompatible (SI) species in the genus Lycopersicon following a breakdown of the self-incompatibility system. In order to elucidate the molecular basis of this breakdown in L. esculentum, we ®rst analysed the stylar proteins with an in-gel assay for ribonuclease activity and 2D-PAGE. No S-RNase protein or its activity was detected in the style of L. esculentum. We then introduced the S6-RNase gene from an SI relative, L. peruvianum, into L. esculentum. However, the styles of transgenic plants expressing S 6 -RNase at levels comparable to those found in the L. peruvianum style were unable to reject self-pollen and L. peruvianum pollen in an allele-speci®c manner. This indicated that defect in the S-RNase expression was not the sole reason for the loss of self-incompatibility in tomato. The asparagine-rich HT protein, originally identi®ed from the style of Nicotiana alata, is the other stylar factor involved in selfincompatibility reaction. We cloned and sequenced two distinct genes encoding HT-A and HT-B proteins from L. peruvianum (LpHT-A and LpHT-B) and L. esculentum (LeHT-A and LeHT-B). A frame shift mutation in the coding sequence of LeHT-A and a stop codon in the ORF of LeHT-B were found, and no LeHT-B transcript was detected in the style of L. esculentum. The results suggest that the breakdown of self-incompatibility in cultivated tomato is associated with loss-of-function mutations in both S-RNase and HT genes.

In some families of flowering plants, a single self-incompatibility (S) locus prevents the fertilization of flowers by pollen from the same plant. Selfincompatibility of this type involves the interaction of molecules produced by the S locus in pollen with those present in the female tissues (pistil). Until recently, the pistil products of the S locus were known in only two families, the Brassicaceae (which indudes the cabbages and mustards) and Solanaceae (potatoes and tomatoes). A paper in this issue of the Proceedings describes the molecules acted with self-incompatibility in a third family, the Papaveraceae (poppies Self-incompatibility is a relatively simple and genetically defined example of cellcell recognition in plants. Self-incompatible plants are able to distinguish between self pollen and nonself pollen within the female reproductive tissue (the pistil) and arrest the further growth of self pollen (see ref. 5 and the references therein). By recognizing and rejecting self pollen before fertilization, self-incompatible plants promote outbreeding and maintain genetic variability, a factor considered important in the evolutionary success of flowering plants (6). The molecular genetics of two types of self-incompatibility, gametophytic and sporophytic selfincompatibility, have been studied intensively (5, 7).Gametophytic self-incompatibility is well-characterized in plants from the family Solanaceae-such as the ornamental tobacco (Nicotiana alata), petunia (Petunia inflata and Petunia hybrida), potato (Solanum tuberosum and Solanum chacoense), and wild tomato (Lycopersicon peruvianum). Some molecular information is also available for gametophytically selfincompatible species from the Papaveraceae, Rosaceae, and Scrophulariaceae (Fig. 1, see below). In each case, selfincompatibility is controlled by a single genetic locus (S locus) with many alleles. Rejection of pollen occurs when the single S allele present in the haploid pollen grain matches either of the S alleles present in the diploid tissues of the pistil. Anderson and others (10) showed that the S loci ofN. 1992 alata and L. peruvianum encode extracellular glycoproteins that are abundant within the pistil, and the discovery that these glycoproteins are ribonucleases related to extracellular ribonucleases of some fungi was a major surprise (11). Until recently, evidence for the involvement of these glycoproteins (now called S-RNases) in self-incompatibility was indirect and relied on a number of correlations: for example, the genes that encode S-RNases cosegregate with alleles ofthe S locus, and the timing of expression of S-RNases is coincident with the onset of self-incompatibility in the pistil (5). There is now direct evidence that S-RNases determine the selfincompatibility phenotype of the pistil and that the ribonuclease activity of these glycoproteins is required for rejection of incompatible pollen (refs. 12 and 13; J. Royo, Y. Kowyama, and A.E.C., unpublished work). These findings strengthen the view that if S-RNases enter incompat...

'Self-incompatibility (SI) in angiosperms is a genetic mechanism that promotes outcrossing through rejection of selfpollen. In the Solanaceae, SI is determined by a multiallelic S locus whose only known product is an S RNase. S RNases show a characteristic pattern of five conserved and two hypervariable regions. These are thought to be involved in the catalytic function and in allelic specificity, respectively. When the Solanum chacoense Si&, genotype is transformed with an Sll RNase, the styles of plants expressing significant levels of the transgene reject Sll pollen. A previously characterized S RNase, S13, differs from the Sl1 RNase by only 10 amino acids, four of which are located in the hypervariable regions. When S1Si4 plants were transformed with a chimeric Sii gene in which these four residues were substituted with those present in the SI, RNase, the transgenic plants acquired the SI3 phenotype. This result demonstrates that the S RNase hypervariable regions control allelic specificity.

The pathogenesis-related gene PR-7Oa (formerly STH-2) is induced in various organs of potato after wounding, elicitor treatment, or infection by Phytophthora infestam. Deletion analysis of the promoter of the PR-lOa gene enabled us to identify a 50-bp region, located between positions -155 and -105, necessary for the elicitor responsiveness of the P-glucuronidase reporter gene in transgenic potato plants. Within this region, a 30-bp sequence, located between positions -135 and -105, was necessary for the activation of the promoter by the elicitor. However, strong promoter activity after elicitor treatment required the presence of a 20-bp sequence located between positions -155 and -135. The region between -135 and -105 was specifically recognized by two nuclear factors, PBF-1 (PR-lOa Binding Eactor 1) and PEF-2, and binding of PBF-1 ,was coordinated with the accumulation of the PR-lOa mRNA. Gel shift assays using nuclear extracts pretreated with sodium deoxycholate or alkaline phosphatase suggested that P B M is a multimeric factor in which at least one of the constituent proteins can be phosphorylated. Treatment with alkaline phosphatase also indicated that binding of PBF-1 is positively regulated by phosphorylation and that it is phosphorylated only in tissues in which PR-7Oa is expressed. The use of protein phosphatase and kinase inhibitors in vivo provided additional evidence that wounding and elicitor treatment induce the phosphorylation of PBF-1 and that this phosphorylation is associated with gene activation.

Gametophytic self-incompatibility in plants involves rejection of pollen when pistil and pollen share the same allele at the S locus. This locus is highly multiallelic, but the mechanism by which new functional S alleles are generated in nature has not been determined and remains one of the most intriguing conceptual barriers to a full understanding of self-incompatibility. The S(11) and S(13) RNases of Solanum chacoense differ by only 10 amino acids, but they are phenotypically distinct (i.e., they reject either S(11) or S(13) pollen, respectively). These RNases are thus ideally suited for a dissection of the elements involved in recognition specificity. We have previously found that the modification of four amino acid residues in the S(11) RNase to match those in the S(13) RNase was sufficient to completely replace the S(11) phenotype with the S(13) phenotype. We now show that an S(11) RNase in which only three amino acid residues were modified to match those in the S(13) RNase displays the unprecedented property of dual specificity (i.e., the simultaneous rejection of both S(11) and S(13) pollen). Thus, S(12)S(14) plants expressing this hybrid S RNase rejected S(11), S(12), S(13), and S(14) pollen yet allowed S(15) pollen to pass freely. Surprisingly, only a single base pair differs between the dual-specific S allele and a monospecific S(13) allele. Dual-specific S RNases represent a previously unsuspected category of S alleles. We propose that dual-specific alleles play a critical role in establishing novel S alleles, because the plants harboring them could maintain their old recognition phenotype while acquiring a new one.