Expressing self-incompatibility RNases (S-RNases) in transgenic plants.
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Animals↗
Biomedical subjects
B A McClure
Publications and source records attributed to B A McClure.
RNaseI from Escherichia coli cannot substitute for S-RNase in rejection of Nicotiana plumbaginifolia pollen.
Unilateral incompatibility often occurs between self-incompatible (SI) species and their self-compatible (SC) relatives. For example, SI Nicotiana alata rejects pollen from SC N. plumbaginifolia, but the reciprocal pollination is compatible. This interspecific pollen rejection system closely resembles intraspecific S-allele-specific pollen rejection. However, the two systems differ in degree of specificity. In SI, rejection is S-allele-specific, meaning that only a single S-RNase causes rejection of pollen with a specific S genotype. Rejection of N. plumbaginifolia pollen is less specific, occurring in response to almost any S-RNase. Here, we have tested whether a non-S-RNase can cause rejection of N. plumbaginifolia pollen. The Escherichia coli rna gene encoding RNAseI was engineered for expression in transgenic (N. plumbaginifolia x SC N. alata) hybrids. Expression levels and pollination behavior of hybrids expressing E. coli RNaseI were compared to controls expressing SA2-RNase from N. alata. Immunoblot analysis and RNase activity assays showed that RNaseI and SA2-RNase were expressed at comparable levels. However, expression of SA2-RNase caused rejection of N. plumbaginifolia pollen, whereas expression of RNaseI did not. Thus, in this system, RNase activity alone is not sufficient for rejection of N. plumbaginifolia pollen. The results suggest that S-RNases may be specially adapted to function in pollen rejection.
Amino Acid Sequence↗
Expressing foreign genes in the pistil: a comparison of S-RNase constructs in different Nicotiana backgrounds.
Transgenic plant experiments have great potential for extending our understanding of the role of specific genes in controlling pollination. Often, the intent of such experiments is to over-express a gene and test for effects on pollination. We have examined the efficiency of six different S-RNase constructs in Nicotiana species and hybrids. Each construct contained the coding region, intron, and downstream sequences from the Nicotiana alata S(A2)-RNase gene. Among the six expression constructs, two utilized the cauliflower mosaic virus (CaMV) 35S promoter with duplicated enhancer, and four utilized promoters from genes expressed primarily in pistils. The latter included promoters from the tomato Chi2;1 and 9612 genes, a promoter from the N. alata S(A2)-RNase gene, and a promoter from the Brassica SLG-13 gene. Some or all of the constructs were tested in N. tabacum, N. plumbaginifolia, N. plumbaginifolia x SI N. alata S(C10)S(c10) hybrids, N. langsdorffii, and N. langsdorffii x SC N. alata hybrids. Stylar specific RNase activities and S(A2)-RNase transcript levels were determined in transformed plants. Constructs including the tomato Chi2;1 gene promoter or the Brassica SLG-13 promoter provided the highest levels of S(A2)-RNase expression. Transgene expression patterns were tightly regulated, the highest level of expression was observed in post-anthesis styles. Expression levels of the S(A2)-RNase transgenes was dependent on the genetic background of the host. Higher levels of S(A2)-RNase expression were observed in N. plumbaginifolia x SC N. alata hybrids than in N. plumbaginifolia.
Cloning, Molecular↗
Antisense suppression of S-RNase expression in Nicotiana using RNA polymerase II- and III-transcribed gene constructs.
In the Solanaceae, self-incompatibility is controlled by a single, multi-allelic ('S') locus. One product of this locus is a ribonuclease, the S-RNase, which is expressed predominantly in mature pistils and has recently been shown to cause allele-specific pollen rejection in transgenic plants. Hybrid Nicotiana plumbaginifolia x N. alata plants were used to test the effects of antisense suppression of the SA2-RNase from N. alata using three different gene constructs: two driven by RNA polymerase II-transcribed promoters, and the third, containing a truncated soybean tRNA (met-i) gene, transcribed by RNA polymerase III. All three constructs caused suppression of S-RNase activity in the transgenic plants. Unexpectedly, the CaMV 35S promoter was more effective for antisense suppression than the tissue specific tomato ChiP promoter. Antisense suppression of S-RNase correlated with low sense SA2 transcript levels and high antisense SA2 transcript levels. Untransformed hybrids that contained the N. alata SA2 allele were incompatible with N. alata SA2 pollen, while transgenic plants with suppressed SA2 gene expression accepted the pollen. The utility of this hybrid plant system for studying some aspects of antisense gene suppression is discussed.
Chimera↗
S-RNase expressed in transgenic Nicotiana causes S-allele-specific pollen rejection.
Many angiosperms employ self-incompatibility systems to prevent inbreeding. The simple genetics of such systems have made them attractive models of plant cellular communication. Implicit in the single locus genetics is that only one or a few gene products are necessary for recognition and rejection of incompatible pollen. Results in the sporophytic system of the Brassicaceae suggest that different S-locus products are responsible for the pollen and pistil parts of the recognition and rejection response. In solanaeceous plants, which have a gametophytic self-incompatibility system, the S locus product responsible for the pollen portion of the interaction has not been identified, but ribonucleases encoded by the S-locus (S-RNases) are strongly implicated in the style part of the recognition and rejection reaction. In Nicotiana alata, pollen recognition and rejection occur if its S-allele matches either S-allele in the style. The putative stylar S-RNase is abundant in the transmitting tract, and pollen rejection may be related to action of S-RNase on pollen RNAs. Efforts to understand the molecular basis for pollen recognition and rejection have been limited by the lack of a system for manipulating and expressing S-RNases. Here we use the promoter of a style-expressed gene from tomato to obtain high levels of S-RNase expression in transgenic Nicotiana. Recognition and rejection of N. alata pollen S-alleles occur faithfully in the transgenic plants. Our results show that S-RNases alone are sufficient for pollen rejection in this system.
Alleles↗
S-locus products in Nicotiana alata pistils are subject to organ-specific post-transcriptional processing but not post-translational processing.
The expression of genes encoding self-incompatibility ribonucleases (S-RNases) in Nicotiana alata were examined at both protein and RNA level for organ specificity. S-RNases recovered from stigmas and styles were indistinguishable by SDS-PAGE, chromatographic behaviour and RNase specific activity. The pistil S transcripts are heterogeneous in size, the stigma transcript being shorter and more heterogeneous than the transcripts in the style and ovary. RNase H analysis shows that this organ-specific difference is mainly in the length of the polyadenylate tail. By sequence analysis of cloned cDNAs we show that the transcript present in the stigma is derived from the same gene as the transcript in the style.
Gene Expression Regulation, Enzymologic↗
Tissue-print hybridization for detecting RNA directly.
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Antisense Elements (Genetics)↗
Molecular and evolutionary aspects of self-incompatibility in flowering plants.
Self-incompatibility (SI) is widely distributed in flowering plants. In this review, early work on the biology, genetics and distribution of SI is summarized. Approaches to understanding the molecular genetics of SI have been made in two systems-Solanaceous species, for example Nicotiana alata, which have gametophytic systems of SI, and Brassica spp, which have sporophytic systems of SI. The information in both systems is derived from cDNAs that encode pistil glycoproteins (S-glycoproteins) that segregate with S-genotype. Comparison of the sequence data indicates that the gametophytic and sporophytic systems of SI probably arose independently during the evolution of angiosperms. The S-glycoproteins of a solanaceous plant Nicotiana alata, are ribonucleases (RNases). Whether the RNase activity is directly involved in the characteristic arrest of pollen tube growth during self-(incompatible) pollination, is not known. An alternative possibility is that the RNase was 'recruited' during evolution for a function in SI, without involvement of its catalytic function. The nature of the S-gene in pollen is not yet known for either the gametophytic or sporophytic SI systems. This is a key piece of information that will be required to progress our understanding of how the growth of a pollen tube bearing a particular S-allele is arrested within the style bearing an identical S-allele, but is not arrested within the style bearing other S-alleles.
Amino Acid Sequence↗
Self-incompatibility: a self-recognition system in plants.
Self-incompatibility (SI), a genetically controlled mechanism to prevent inbreeding in plants, offers a relatively simple model system for studying the interactions between plant cells or between a plant cell and the secreted product or products of another cell. Examples of two major types of SI, gametophytic and sporophytic, have been studied by cloning cDNAs corresponding to glycoproteins of the female tissues that segregate with particular variants encoded by the putative S locus. These secreted glycoproteins are envisaged to interact with the currently undescribed pollen component to cause arrest of pollen tube growth.
Amino Acid Sequence↗
Rapid redistribution of auxin-regulated RNAs during gravitropism.
Gravitropism, the bending of plants in response to gravity, is caused by differential growth rates on two sides of a responding organ. The general belief, although somewhat controversial, is that auxins play a major role in gravitropism by controlling the rate of cell extension. The tissue print technique was used to ascertain the distribution of auxin-regulated RNAs during the gravitropic response of soybean hypocotyls. In vertically oriented seedlings, auxin-regulated RNAs are symmetrically distributed in the elongating region of the hypocotyl. In horizontally orientated seedlings the distribution becomes asymmetrical within 20 minutes and the greatest asymmetry coincides with the onset of rapid bending. The results provide a clear correlation between the dynamic expression of genes under auxin control and a morphogenetic phenomenon traditionally known as an auxin response.
Gene Expression Regulation, Plant↗
Transcription, organization, and sequence of an auxin-regulated gene cluster in soybean.
We have characterized a soybean gene cluster that encodes a group of auxin-regulated RNAs (small auxin up RNAs). DNA sequencing of a portion of the locus reveals five homologous genes, spaced at intervals of about 1.25 kilobases and transcribed in alternate directions. At least three of the genes are transcriptionally regulated by auxin. An increase in the rate of transcription is detected 10 min after application of auxin to soybean elongating hypocotyl sections. Each of the genes contains an open reading frame that could encode a protein of 9 kilodaltons to 10.5 kilodaltons. Sequence comparisons among the five genes reveal several areas of high homology. Two regions of high homology begin about 250 base pairs upstream of the open reading frames and two regions of homology have been identified in sequences downstream of the open reading frames. One of the latter sequences occurs in the 3'-untranslated region of the RNAs. The other occurs far downstream, 618 base pairs to 741 base pairs from the stop codon. Conservation of these sequences among the five different genes suggests that they may be important for the regulation of expression of the genes.
Amino Acid Sequence↗
A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA.
A simple new procedure was described for producing a sequential series of overlapping clones for use in DNA sequencing. The technique used single-stranded M13 DNA and complementary DNA oligomers to form specific cleavage and ligation substrates. It was, therefore, independent of the sequence of the DNA cloned into the vector. Deletions of varying sizes were generated from one end of the insert through the 3' to 5' exonuclease activity of T4 DNA polymerase. The approximate size of the deletion and therefore the starting point for DNA sequencing could be estimated by electrophoresis of the subcloned phage DNA on a agarose gel. This greatly reduced the number of templates that must be sequenced to obtain a complete sequence. The entire procedure could be carried out in one tube in less than a day. The procedure was used to subclone and sequence the maize mitochondrial 18 S rDNA and 5' flanking region (2622 bases) in less than a week. Other applications of oligomers and single-stranded DNA in the construction of insertions, deletions, and cDNAs are discussed.
Base Sequence↗
Style self-incompatibility gene products of Nicotiana alata are ribonucleases.
Self-incompatibility in flowering plants is often controlled by a single nuclear gene (the S-gene) having several alleles. This gene prevents fertilization by self-pollen or by pollen bearing either of the two S-alleles expressed in the style. Sequence analysis shows that three alleles of the S gene of Nicotiana alata encode style glycoproteins with regions of defined homology. Two of the homologous regions also show precise homology with ribonucleases T2 (ref. 4) and Rh (ref. 5). We report here that glycoproteins corresponding to the S1, S2, S3, S6 and S7 alleles isolated from style extracts of N. alata are ribonucleases. These style S-gene-encoded glycoproteins account for most of the ribonuclease activity recovered from style extracts. The ribonuclease specific activity of style extracts of the self-incompatible species N. alata is 100-1,000-fold higher than that of the related self-compatible species N. tabacum. These observations implicate ribonuclease activity in the mechanism of gametophytic self-incompatibility.
Alleles↗