Plant Cell, Vol. 10, 209-218, Copyright © 1998, American Society of Plant Physiologists
The Self-Incompatibility Phenotype in Brassica Is Altered by the Transformation of a Mutant S Locus Receptor Kinase
Richard J. Stahla,
MaryAnne Arnoldob,
Tracy L. Glavin1,a,
Daphne R. Goringc, and
Steven J. Rothsteina
a Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario N1G 2W1, Canada
b Pioneer Hi-Bred Production Limited, Canola Research Station, 12111 Mississauga Road, Georgetown, Ontario L7G 4S7, Canada
c Biology Department, York University, 4700 Keele Avenue, North York, Ontario M3J 1P3, Canada
Correspondence to:
Steven J. Rothstein, srothste{at}uoguelph.ca (E-mail), 519-837-2075 (fax).
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ABSTRACT |
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The self-incompatible (SI) Brassica napus line W1, which carries the 910 S allele, was transformed with an inactive copy of the 910 S locus receptor kinase (SRK) gene. Two transformed lines were analyzed based on their heritable ability to set self-seed. The first line was virtually completely self-compatible (SC), and reciprocal pollinations with the original W1 line demonstrated that only the stigma side of the SI phenotype was altered. An analysis of the expression of endogenous SRK-910 demonstrated that the mechanism of transgene action is via gene suppression. Furthermore, the expression of the S locus glycoprotein gene present in the 910 allele (SLG-910), SLG-A10, which is derived from a nonfunctional S allele, and an S locus–related gene were also suppressed. When the transgene was crossed into another SI line carrying the A14 S allele, it was also capable of suppressing the expression of the endogenous genes and of making this line SC. The second transgenic line studied was only partly SC. In this case as well, only the stigma phenotype was affected, although no gene suppression was detected for endogenous SRK-910 or SLG-910. In this line, the expression of the transgene most likely was causing the change in phenotype, and no effect was observed when this transgene was crossed into the other SI line. Therefore, this work reinforces the hypothesis that the SRK gene is required, but only for the stigma side of the SI phenotype, and that a single transgene can alter the SI phenotype of more than one S allele.
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INTRODUCTION |
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Brassica napus is an amphidiploid plant that is normally self-compatible (SC), whereas its presumptive progenitors, B. oleracea and B. rapa, are diploid self-incompatible (SI) species (Downey and Rakow 1987 
). Self-incompatibility in Brassica spp is of the sporophytic type in which the pollen parent genotype determines the phenotype of the pollen. Many SI alleles have been identified genetically, and pollen derived from a plant that carries the same allele as that present in the stigma is rejected (Bateman 1955 ; Ockendon 1974 ). These alleles are generally codominant with each other, and self-incompatibility is dominant over self-compatibility (Ockendon 1974 , Ockendon 1982 ).
SI alleles can be transferred into B. napus from the progenitor species via interspecific crosses, with the resulting individuals being repeatedly backcrossed to the B. napus parent, thus generating SI lines (Goring et al. 1992a , Goring et al. 1992b ). The two SI lines used in this study are called W1 and T2. W1 carries the 910 S allele (Goring et al. 1992a ), and T2 carries the A14 S allele (Goring et al. 1992b ). Other than the region involved in self-incompatibility, these lines are virtually isogenic pairs, with the original SI lines used as the recurrent parent (for these lines, W1 is similar to Westar and T2 is similar to Topas).
The recognition of self-pollen and its subsequent rejection involve a very rapid process. For strong S alleles, like the 910 allele, very little of the pollen will even hydrate, thus preventing subsequent germination. The pollen that does germinate forms tubes that grow for a short distance and virtually never penetrate the stigma surface. Given the sporophytic nature of Brassica spp self-incompatibility, it has been postulated that some factor is produced in the tapetum or premeiotically in the diploid meiocytes of the parental plant (Pandey 1958 ) and that this factor is present on the outer surface of the pollen exine and is recognized by something on the stigma surface that prevents the normal pollen germination and fertilization processes from occurring (de Nettancourt 1977 ).
Two genes have been found to be associated with the SI phenotype. The first to be discovered codes for the S locus glycoprotein (SLG), which is expressed primarily in stigma papilla cells, although some transcript has been detected in anther tissue (Kandasamy et al. 1989 ). The sequences of the SLG gene from different S loci vary considerably (80 to 90% similarity), which is reasonable for a gene that encodes a product that might be involved in self-recognition. The second gene codes for a protein called the S locus receptor kinase (SRK) (Stein et al. 1991 ). This is a protein with a receptor-like domain very similar in sequence to the SLG, a transmembrane domain and a kinase domain that has been shown to have serine/threonine kinase activity (Goring and Rothstein 1992 ). The receptor portion has some similarity in sequence to immunoglobulin-like receptor domains in animal cells (Glavin et al. 1994 ) and shows a similar sequence heterogeneity between genes derived from different alleles as that seen for SLG (Glavin et al. 1994 ). This gene is also expressed in the stigma papilla cells (Stein et al. 1991 ; Goring and Rothstein 1992 ). There are a variety of SRK-like genes in Brassica spp (Kumar and Trick 1994 ) as well as genes similar to the SLG genes that have been shown to be unlinked to self-incompatibility (Lalonde et al. 1989 ). The functions of most of these genes are unknown, although the expression of one of these has been correlated with wound and pathogen response (Pastuglia et al. 1997 ).
The following hypothesis for self-recognition has been formulated based on these experimental observations and on what is known about receptor kinase interactions with their specific ligands in mammalian cells. The SRK protein would form a receptor complex in the stigma papilla cells that recognizes an allele-specific ligand present on the pollen surface. The SLG protein either would be involved in bringing the ligand into contact with the SRK protein or would serve as part of the receptor complex. The pollen ligand is thought to be a protein that is encoded by a separately linked gene and that is recognized only by the receptor complex encoded by the same allele. Alternatively, in theory, the ligand could be SLG itself activated in the pollen by the binding of another molecule (Stein et al. 1991 ; Goring and Rothstein 1992 , Goring and Rothstein 1996 ).
Although this hypothesis is reasonable, a number of aspects of this model have not been experimentally demonstrated. The involvement of the SRK and SLG in self-incompatibility has been supported by their linkage to the S locus and also by an analysis of plants selected for self-compatibility, with neither SLG (Nasrallah et al. 1992 ) nor SRK (Nasrallah et al. 1994 ) being expressed in these plants at normal levels. In addition, SC B. napus has an S allele with an SRK gene having a 1-bp deletion that would encode a truncated protein product (Goring et al. 1993 ), and transformation of a B. campestris SLG gene into B. oleracea was found to disrupt the SI phenotype in some lines (Toriyama et al. 1991 ). However, whereas the DNA sequence diversity of the SRK and SLG genes implies that these are most likely involved in self-recognition, this has not been demonstrated biochemically, nor has the pollen ligand been identified. Finally, the process by which recognition of pollen carrying the same allele leads to its rejection has not been delineated, although it has been demonstrated that SRK binds to thioredoxin-h, and this might be part of the signal transduction process (Bower et al. 1996 ).
We wished to develop a transgene that would prevent the functioning of SRK. In mammalian cells, considerable work has been done with tyrosine receptor kinases. In these cases, the binding of the specific ligand leads to the dimerization of the receptor kinase and thus stimulates kinase activity, which in turn leads to the change in phenotype through a variety of signal transduction processes (Ullrich and Schlessinger 1990 ). In a number of cases, the expression of a mutant receptor kinase turned out to prevent the functioning of the wild-type receptor kinase. For example, in mouse, several mutant forms of the c-Kit receptor tyrosine kinase exist. One particular mutation, W41, produces normal levels of the c-Kit protein, but with reduced autophosphorylation activity due to a point mutation in the kinase domain (Reith et al. 1990 ). Because W41/+ heterozygous mice show a stronger dominant phenotype compared with other loss-of-function c-Kit mutations, the W41 mutation was proposed to prevent signaling by the functional c-Kit protein because of the formation of defective heterodimers (Reith et al. 1990 ).
Previously, we had shown that a single amino acid substitution mutation in the kinase domain of the SRK prevented kinase activity in vitro (Goring and Rothstein 1992 ). Therefore, we made the same mutation in SRK-910 and transformed this mutant gene into the W1 line carrying this allele. Two SC transgenic lines were analyzed in detail and found to be affected only in the stigma side of the SI phenotype. In one case, the presence of the transgene led to the suppression of the expression of the SRK-910 and SLG-910 genes as well as the corresponding genes present in other alleles. In the other case, the expression of the endogenous genes was not significantly affected, and in this case, it is likely that the expression of the mutant SRK was having an effect. This work strongly supports the importance of SRK for the stigma side of the SI phenotype but at the same time does not exclude the involvement of the SLG in the SI pathway.
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RESULTS |
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Chimeric Genes and Plant Transformation
The kinase coding region of the SRK-910 gene was modified so that the codon at position 557 now codes for an alanine instead of a lysine. This substitution had earlier been shown to prevent kinase activity completely when it was expressed in Escherichia coli (Goring and Rothstein 1992 ). Mutated SRK-910 was then remade so that the only difference between the wild-type and mutant versions was this substitution. Our assumption was that this mutant version would be able to be assembled into a receptor complex, but the deficiency in kinase activity would prevent it from functioning. Two regulatory regions were used to express the mutant SRK-910, as shown in Figure 1. The first used the promoter from the SLG-A10 gene, which is expressed at very high levels in stigma tissue (Goring et al. 1992b ). The second was the promoter from the S locus–related SLR1 gene, which is expressed at even higher levels in stigmas (Franklin et al. 1996 ) and had been shown to work well in transgenic plants.
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Figure 1.
Schematic Representation of the Transgene Constructs.
Shaded areas designate the transmembrane domain. H, HindIII site.
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These chimeric genes were transformed into the SI cultivar W1, which carries the 910 allele, and the resulting transformants were analyzed for their ability to set self-seed. Of the 25 transformants that were transformed with the SLG-A10 mutant SRK construct, only one showed a heritable increase in the production of self-seed. Of the 11 SLR1 mutant SRK-transformed lines tested, two showed an increase in the amount of self-seed, although only one of these did so in a consistent fashion in the progeny. Therefore, one line transformed with each of the two constructs was studied further.
Phenotype of the Lines Transformed with the Mutant SRK Gene
The transformed lines were analyzed for their ability to set self-seed, the level of fertility when cross-pollinated with the W1 line, and the ability of the pollen to germinate and form pollen tubes. The A10 mutant SRK-transformed line had only a slightly lower level of seed set as the SC line Westar had when it was self-pollinated. Of the 17 individuals that carried the transgene tested over two generations, the average self-seed set was 19 per pod compared with ~29 per pod for Westar, as shown in Table 1. The W1 line, derived from an introgression of the S locus from B. rapa into Westar, in general shows a noticeably weaker vigor compared with Westar. This could account in part for the overall inability of these transgenic W1 plants to be fully restored to the fertility of Westar. There were 11 individual plants derived from the original transgenic line tested in which the transgene was no longer present because of segregation, and all of these were completely SI, demonstrating that the change in phenotype was caused by the transgene. When reciprocal crosses were done with the W1 line, pollen from W1 was able to fertilize the transgenic line, yielding an average of 22 seeds per pod. However, pollen from the transgenic line was rejected by the W1 line with virtually no seed set (Table 1).
An analysis of pollen germination and tube growth corresponded exactly with the seed set results, with self-pollen and pollen from W1 both being able to germinate on the transgenic line and their pollen tubes having normal growth. Using fluorescence microscopy of aniline blue–stained tissues, Figure 2A and Figure 2C show that this transgenic line, when crossed with W1 pollen, closely resembles the typical SC response seen in Westar (Figure 2E). In contrast, pollen from the transgenic line would not germinate on W1 stigmas (Figure 2B and Figure 2D), and these pollinations led to the typical production of excess callose on the stigma surface, which one normally sees in an incompatible response as in W1 selfed (Figure 2F). Therefore, only the stigma side of the SI phenotype was affected by the transgene, with no noticeable change in the pollen phenotype.
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Figure 2.
Analysis of Pollinated B. napus Stigma Tissue by Using Fluorescence Microscopy.
(A) Stigma of an A10 mutant SRK-transformed plant pollinated by W1.
(B) Stigma of a W1 plant pollinated by the A10 mutant SRK transgenic W1 line.
(C) Enlarged view of stigma papilla cells shown in (A).
(D) Enlarged view of stigma papilla cells shown in (B).
(E) Stigma of a Westar plant pollinated by a transgenic line demonstrating an SC response.
(F) Stigma of a W1 self-pollinated plant demonstrating an SI response.
(A) to (F) represent 16-hr postpollinated tissue stained with aniline blue and visualized under UV light to detect callose accumulation. Bars = 50 µm.
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The second transgenic line with the SLR1 mutant SRK gene did not show a complete restoration of self-fertility. The average seed set, although much higher than for the SI W1 line, was ~25% of that seen for the SC Westar line (Ta-ble 1). Therefore, in this case, there was only a partial breakdown in the SI phenotype. Crosses between this line and W1 again demonstrated that only the stigma phenotype was affected, with seed being formed only when the W1 pollen was used to fertilize the transgenic line and the reciprocal cross giving minimal seed set (Table 1). In this case, when either self-pollen or W1 pollen was applied to stigmas of the transgenic line, a strong callose reaction occurred, and only a small percentage of pollen would germinate and form pollen tubes (data not shown). This supports the notion that this line is intermediate in phenotype between SC and SI lines in the stigma, with the pollen phenotype not being affected.
The phenotypes of the seed pods produced after self-pollination for the SC line Westar, for the original SI line W1, for the W1 line transformed with the A10 mutant SRK transgene, and for the W1 line transformed with the SLR1 mutant SRK transgene are shown in Figure 3A to D. As can be seen, the fully developed pods for the W1 line transformed with the A10 mutant SRK transgene (Figure 3C) are very similar to those seen in the SC line Westar (Figure 3A). The pods produced by the SLR1 mutant SRK transgene are consistently reduced in size and seed content (Figure 3D).
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Figure 3.
Comparison of Mature Self-Seeded B. napus Pods of Two Transgenic Lines and Two Control Plants.
(A) B. napus cv Westar (SC).
(B) B. napus cv W1 (SI).
(C) W1 carrying the A10 mutant SRK transgene.
(D) W1 carrying the SLR1 mutant SRK transgene.
Bar = 1 cm.
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Detection of the Mutant SRK Transgenes by DNA Gel Blot Analysis
To determine the presence and approximate copy number of the A10 mutant SRK and SLR1 mutant SRK transgenes, DNA blots of HindIII-digested genomic DNA from progeny derived from each of the two transgenic lines were probed with the SLG-910 cDNA, which cross-hybridizes with SRK-910 and the transgenes (Figure 4). Because these two lines were no longer segregating, they were presumably homozygous for the transgene. All of the progeny derived from the A10 mutant SRK transgenic line produced the expected 4.5-kb band, whereas the progeny from the SLR1 mutant SRK line yielded a fragment of 5.4 kb. By comparing the relative intensity of these bands to the 3.6- and 6.4-kb bands representing the single-copy haploid genome of the SRK-910 and SLG-910 genes in WI, we estimate one copy per haploid genome for the A10 mutant SRK transgene and approximately five copies per haploid genome for the SLR1 mutant SRK transgene. The low-stringency wash conditions also enabled the detection of the A10 S locus genes present in Westar (SRK-A10 and SLG-A10 are on 9.6- and 8.4-kb fragments, respectively) due to cross-hybridization.
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Figure 4.
Genomic DNA Gel Blot Analysis of the Transgenic Lines of B. napus cv W1.
DNA was restricted by HindIII to excise the transgene. Lanes 1 to 4 represent plants containing the A10 mutant SRK transgene (the 4.5-kb fragment is indicated by the letter a), and lanes 5 to 7 contain the SLR1 mutant SRK transgene (the 5.4-kb fragment is indicated by the letter b). The blot was probed with the SLG-910 cDNA. The numbers at left indicate the positions of 1-kb DNA ladder bands (Bethesda Research Laboratories).
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Expression of the SLG and SRK Genes in the A10 Mutant SRK Line
To determine the mechanism by which the A10 mutant SRK transgene was affecting the phenotype, the levels of the SRK-910 and SLG-910 transcripts present in floral buds were analyzed by using reverse transcription–polymerase chain reaction (RT-PCR) and RNA gel blot analyses, respectively (Figure 5 and Figure 6). It is clear from these results that the mutant SRK transgene is working via cosuppression, with both the SLG and SRK transcripts being virtually completely absent. The receptor portion of the SRK-910 gene is 90% similar in sequence to the SLG-910 coding region, and this is clearly sufficient to induce the cosuppression effect. As predicted, individual plants having lost the transgene through segregation were observed to express close to wild-type levels of each affected transcript (data not shown).
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Figure 5.
RT-PCR Analysis of Transcripts of the A10 Mutant SRK Transgenic W1 Line.
RNA samples were reverse transcribed and then amplified for 25 cycles with gene-specific primers. The three individual W1 plants carrying the A10 mutant SRK transgene are labeled 1, 2, and 3. The designations at right indicate the copy of the SRK gene amplified by PCR. Blots were probed with the SRK-910 gene. The numbers at left indicate the positions of the 1-kb DNA ladder (Bethesda Research Laboratories). W1* denotes a self-seeded progeny from a transgenic plant in which the transgene was lost through segregation. wt, wild type.
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Figure 6.
RNA Gel Blot Analysis of the A10 Mutant SRK Transgenic W1 Line.
The three individual W1 plants carrying the A10 mutant SRK transgene are labeled 1, 2, and 3. The B. napus cDNA probes are indicated at right. The positions of the 18S (1.8-kb) and 25S (3.4-kb) rRNA bands are marked at left.
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In B. napus, there is also the nonfunctional A10 allele present in the a genome (Goring et al. 1993 ) (the 910 allele was originally derived from B. rapa and is thus in the c genome). The level of the SLG-A10 transcript was also analyzed using RNA gel blot hybridization (Figure 6). It is clear that this transcript was also present at very low levels. SRK-910 is 85% similar in DNA sequence to the SLG-A10 coding region. Therefore, the transgene suppresses the expression of SLG-910 and SRK-910 as well as SLG-A10, which is present at the S locus in the other genome of B. napus.
Brassica spp also contain SLR genes that have been shown to be unlinked to the S locus, and it has been demonstrated that an antisense gene that prevents SLR1 expression level has no effect on the SI phenotype (Franklin et al. 1996 ). One such example is the highly conserved SLR1 gene, which has only 66% sequence homology with the receptor domain of the SRK-910 transgene. RNA gel blot hybridization was performed using the SLR1-A29 cDNA probe (Goring et al. 1992b ). The abundant SLR1 transcript was affected, but the level of suppression was not as absolute as with the SLG-910 and SLG-A10 transcripts (Figure 6).
Effect of the SLR1 Mutant SRK Transgene on Expression of the SLG and SRK Genes
The expression of SLG-910 and SRK-910 was analyzed in the line carrying the SLR1 mutant SRK transgene. In this case, there was no significant decrease in the expression of these genes, as seen in Figure 7A and Figure 7B. The mutant SRK transgene is expressed (see Figure 7C). However, even though the SLR1 promoter used is expressed at high levels (Franklin et al. 1996 ), transgene expression could not be detected on an RNA gel blot (data not shown) and is likely in a range similar to that of wild-type SRK-910 (Figure 7B). Therefore, in this case, cosuppression is not likely to be the mechanism involved in the partial breakdown of the SI phenotype. Instead, the expression of the transgene and presumably the production of the mutant SRK-910 protein must have an effect, although the change in phenotype is not complete.
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Figure 7.
RNA and RT-PCR Analyses of the SLR1 Mutant SRK Transgenic W1 Line.
The two individual W1 plants carrying the SLR1 mutant SRK transgene are labeled 1 and 2.
(A) and (D) RNA gel blots probed with the B. napus gene indicated at right.
(B) and (C) Blots containing cDNA PCR amplified with primers specific for the gene indicated at right and probed with the SRK-910 gene.
In (B), the 0.3-kb band represents the amplified fragment of the mature RNA transcript, whereas the larger fragments are due to incomplete splicing of one or both of two small introns. The positions of the 18S (1.8-kb) and 25S (3.4-kb) rRNA bands are marked on the RNA blots, whereas the 1-kb DNA ladder (Bethesda Research Laboratories) bands are marked on the cDNA gel blots. wt, wild type.
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Transfer of the Mutant SRK Transgenes into a Line with a Different Functional S Allele
B. napus line T2 carries the A14 S allele through introgression into the line Topas (Goring et al. 1992b ). Progeny from the original transgenic line carrying the A10 mutant SRK chimeric gene were used as a pollen donor in a cross with T2. Progeny from this cross would be expected to have the transgene, the 910 S allele present in the W1 line, and the A14 S allele found in T2 all present in a heterozygous state. This indeed turned out to be the case when these were analyzed for their presence by PCR and by their segregation into the subsequent generation (data not shown). As shown in Table 2, these plants set self-seed at a high rate only slightly lower than the SC line Topas and much higher than is seen for the T2 x W1 hybrid line. This line, which is heterozygous for the A14 and 910 S alleles, produces somewhat greater seed set than either the T2 or W1 parental lines. The first T2-backcrossed generation (BC-1) of these hybrids was also analyzed for self-seed set as well as for seeding rates when reciprocally crossed with T2 (see Table 3). The BC-1 generation plants containing the transgene exhibited a strong SC stigma phenotype indicated by the high seed set values, whereas the plants without the transgene were clearly SI. The pollen of the BC-1 plants was ineffective in fertilizing the T2 line.
Expression of SLG-A14 was analyzed, and its transcript was present at very low levels, as was the SLG-910 in the original hybrid plants (Figure 8). As expected, transcript levels of both of these genes were restored in hybrid plants that had lost the kinase mutant transgene (Figure 8). SLG-A14 is 83% similar in its coding sequence to SRK-910. Therefore, not only can the A10 mutant SRK transgene prevent the functioning of the 910 S allele, but it can also prevent the phenotypic expression of a different functional allele.
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Figure 8.
RNA Gel Blot Analysis of the T2 x W1 Hybrid Carrying the A10 Mutant SRK Transgene.
The three individual hybrid plants are labeled 1, 2, and 3; (+) and (-) indicate the presence or absence of the transgene. The B. napus cDNA probes are indicated at right. The positions of the 18S (1.8-kb) and 25S (3.4-kb) rRNA bands are marked at left.
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To determine whether the SLR1 mutant SRK transgene was operating in an allele-specific manner, this transgene also was crossed into the SI line T2. In contrast to the A10 mutant SRK transgene, the seed set phenotype of this hybrid line showed no increase in self-seed values when compared with an equivalent T2 x W1 hybrid line lacking this transgene (Table 2). Analysis of the expression levels of SRK-A14 and SLG-A14 (Figure 9A and Figure 9B) revealed no significant effect on the A14 allele by the presence of this SLR1 mutant SRK transgene. Similarly, the expression of SRK-910 and SLG-910 was not affected in these plants (data not shown). Finally, using RT-PCR, the expression of this transgenic copy of SRK-910 was detected at roughly equal abundance in these hybrids as in the original transgenic line (data not shown).
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Figure 9.
RNA and RT-PCR Analyses of the T2 x W1 Hybrid Carrying the SLR1 Mutant SRK Transgene.
The three individual hybrid plants carrying the SLR1 mutant SRK transgene are labeled 1, 2, and 3.
(A) Gel blot containing the cDNA PCR product amplified with primers specific for the SRK-A14 gene and probed with the same gene.
(B) and (C) RNA gel blots probed with the genes indicated at right. The positions of the 18S (1.8-kb) rRNA bands are marked on the RNA gel blots; the 1-kb DNA ladder (Bethesda Research Laboratories) bands are marked on the cDNA blot. W1+ denotes a W1 plant carrying the SLR1 mutant SRK transgene.
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DISCUSSION |
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The SI phenotype requires the specific recognition and subsequent rejection of pollen derived from a plant carrying the same S allele as that present in the female plant. The role of the SRK gene as the receptor that recognizes a pollen factor has been implied by the structure of the SRK protein (Stein et al. 1991 ; Glavin et al. 1994 ), by analysis of several SC lines in which this gene is downregulated (Nasrallah et al. 1994 ) or altered in the coding region (Goring et al. 1993 ), and by its linkage to the S locus (Stein et al. 1991 ; Goring and Rothstein 1992 ). Furthermore, the variability in the coding sequence in this gene from different alleles is consistent with its role in allele-specific recognition. Recently, it has been shown that transformation of SRK gene constructs into B. oleracea led to the suppression of SRK and SLG expression and the loss of the self-incompatibility phenotype (Conner et al. 1997 ). The change in the SI phenotype caused by the transformation of the mutant SRK gene into B. napus described here strongly supports this hypothesis.
It is clear that the effect of the transgene is only on the stigma side of the SI phenotype. This is true in the A10 mutant SRK-transformed line, in which the expression of both the SLG and SRK genes is reduced to very low levels, as well as in the SLR1 mutant SRK plants, in which the transgene does not effect endogenous gene expression of either gene. Therefore, it seems likely that both SRK and the SLG only function in the stigma and that there must be some other gene involved in the pollen phenotype. This is supported further by some preliminary results in which an SRK antisense gene was tested by using a promoter expressed only in tapetal tissue. Surprisingly, one line was SC, but only in the stigma part of the phenotype (R. Stahl and S. Rothstein, unpublished results). This is presumably due to the site of insertion of the transgene and supports the notion that SRK is only involved in the stigma part of the phenotype.
There are a wide array of transgenes that have been found to suppress the expression of endogenous genes (reviewed in Flavell 1994 ). The mechanism of this suppression has been found to be post-transcriptional in most studies, although in some cases it may be due to modifications in the gene itself, such as methylation (Jorgensen 1990 ). In the case of the A10 mutant SRK-transformed line, the transgene suppresses the expression of the endogenous SRK-910 gene but also suppresses expression of the SLG-910 gene and genes from other alleles. These range from 83 to 100% in similarity to the transgene. A wide array of SLG- and SRK-like genes have been found in Brassica spp (Lalonde et al. 1989 ; Kumar and Trick 1994 ) as well as in other species. For example, the SLR1 gene is related in sequence to SLG, but it has a different chromosomal location. It has been shown that a marked decrease in its expression due to the transformation of an antisense gene does not lead to a noticeable change in phenotype (Franklin et al. 1996 ). On the other hand, the SRK-like genes that are present in other places in the genome have been postulated to have a function in development (Kumar and Trick 1994 ) or in wound response and pathogen defense (Pastuglia et al. 1997 ). It has been hypothesized that for multigene families, there must be a mechanism in place that protects the entire set of genes from suppression during genetic crosses (Flavell 1994 ). However, this is clearly not the case here, where a single transgene can decrease the expression of an entire set of genes having considerable sequence heterogeneity. Even the SLR1 gene, which is only 66% similar in sequence, showed considerable decrease in expression, although not as complete as that seen for the more similar SLG and SRK genes.
A dominant-negative mutation is one in which the expression of the mutant gene affects the functioning of the wild-type gene product. Generally, this occurs when the wild-type protein functions as a multimer and in which the assembly of the mutant monomer with the wild-type prevents function. The simplest explanation of the breakdown of self-incompatibility in the SLR1 mutant SRK line is that the mutant SRK gene product is functioning in this fashion. This is supported by the observation that this transgene failed to have an effect on plants carrying a different S allele. One would not expect multimers to form between proteins derived from different alleles. Whether this also implies that SRK works as a dimer, as do tyrosine receptor kinases in animal cells, requires additional biochemical analysis of this protein.
In conclusion, the mutant SRK transgene can prevent the self-incompatibility phenotype. The mechanism of its action in one of the transgenic lines is clearly via cosuppression; in the other, the most likely explanation is that self-compatibility is caused by the expression of the mutant protein. In either case, only the female side of the self-incompatibility phenotype is affected, even when the expression of both SLG and SRK is reduced to very low levels. This strongly supports the hypothesis that another gene besides these must be responsible for the pollen phenotype. It is interesting that a single transgene can suppress the expression of a wide array of related genes and prevent the functioning of multiple S alleles. Given the large number of SLG- and SRK-related genes, the possibility is raised that this type of suppression could have arisen naturally, thus creating cryptic S alleles.
During the evolution of the Brassica spp, this ability to suppress the self-incompatibility phenotype would have been very advantageous on occasion. For example, the generation of the allotetraploid species B. napus involved an interspecific cross between a B. oleracea and a B. rapa individual, both of which are SI species. The ability of the newly formed allotetraploid species to efficiently set self-seed would have been crucial in this initial speciation stage, and suppression of SLG and SRK gene expression is a simple mechanism by which this could have occurred. Later, gene mutation such as that seen in extant lines (Goring et al. 1993 ) could have occurred to ensure the maintenance of the SC phenotype. Finally, it is likely that the A10 mutant SRK transgene can suppress the SI phenotype for virtually any Brassica spp S allele, and this should prove useful for the development of commercially valuable lines in several Brassica spp.
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METHODS |
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Plant Transformation and the Analysis of Progeny
The self-incompatible (SI) Brassica napus cv W1 carrying the 910 S locus (Goring et al. 1992a ) was used as the recipient of the two transgene constructs. The 2.8-kb W1 cDNA containing the coding region and polyadenylation sequence of the S locus receptor kinase SRK-910 gene had been previously modified by polymerase chain reaction (PCR) mutagenesis to introduce a lysine-to-alanine substitution at codon 557 (Goring and Rothstein 1992 ). This fragment was ligated to the 1.7-kb regulatory region of the S locus glycoprotein SLG-A10 gene from B. napus subsp oleifera (Goring et al. 1993 ) and the 2.6-kb promoter region on the SLR1 gene from B. napus (Franklin et al. 1996 ). The assembled chimeric genes were inserted as HindIII fragments into the plant transformation vector pALL TKREP (Pioneer Hi-Bred, Johnston, IA). Plant transformations were performed using Agrobacterium tumefaciens–mediated methods (Moloney et al. 1989 ).
Selected individual plants exhibiting a self-compatible (SC) phenotype were studied in detail, and self-progeny from up to three successive generations were studied. Both transgenes were introduced into a second SI B. napus cultivar, T2, carrying the A14 allele (Goring et al. 1992b ) through an interspecific cross with the transgenic W1 line (as male). Backcrossed generations were made to the T2 parent (as female) to study the segregation of SI phenotype versus genotype in the progeny. The transgenes and the S locus alleles were screened by PCR, using gene-specific primers in addition to DNA gel blot analysis. The SI B. napus progenitor cultivars Westar (Agriculture Canada, Saskatoon, Canada) and Topas (Svalof AB, Savlov, Sweden) were used as controls. All plants were grown in growth cabinets under controlled conditions providing 16 hr of light at 18°C and 8 hr of dark at 15°C. Pollination bags were applied 2 days before anthesis for both self- and cross-pollination. When required, forced pollinations were made on emasculated premature flowers to circumvent the expression of the SI phenotype; otherwise, cross-pollinations were performed 2 days after emasculation onto fully open flowers. Self-incompatibility was tested by measuring seed set and by fluorescence microscopy of 16-hr postpollinated stigmas stained with aniline blue (Kho and Baer 1968 ). Pollen tube growth was also observed in detail by laser scanning confocal microscopy under autofluorescing conditions.
Genomic DNA Analysis
Genomic DNA was extracted from floral bud or young leaf tissue by using a rapid alkali boiling method (Klimyuk et al. 1993 ) for screening by PCR or by the method of McGarvey and Kaper 1991 , except that the cetyltrimethylammonium bromide concentration was increased to 3% for DNA gel blot analysis. Approximately 6 to 8 µg of DNA was digested with HindIII, size fractioned through a 0.7% agarose gel, and transferred to a Biodyne B membrane (Pall, East Hills, NY) in 10 x SSC (1 x SSC is 150 mM NaCl and 15 mM sodium citrate). DNA was cross-linked to the membrane by using the UV Stratalinker (Stratagene, La Jolla, CA). Prehybridization and hybridization steps were performed as described previously (Goring et al. 1992b ). Membranes were washed for 30 min at a final stringency of 0.2 x SSC and 0.1% SDS at 60°C. Full-length purified cDNA fragments labeled by random priming (Feinberg and Vogelstein 1983 ) were used as probes.
RNA Analysis
Total RNA was purified from 5- to 6-mm-long floral buds, using the guanidinium isothiocyanate phenol chloroform extraction method of Ausubel et al. 1987 . Yields of RNA were quantified by UV spectrophotometry. Ten micrograms of each RNA sample was fractioned through a 1.2% agarose–formaldehyde gel and transferred to a Biodyne B membrane, as was done previously. Cross-linking and hybridization of RNA filters were the same as was done for the genomic blots. Final washing conditions consisted of 30 min in 0.2 x SSC and 0.1% SDS at 65°C. Equal loading of RNA samples was demonstrated by rehybridization of the membranes, using either the 18S rRNA or the ß-ATPase genes isolated from B. napus as probes.
The analysis of the low-abundance SRK-910 and SRK-A14 genes, including the transgenes, required reverse transcription (RT) followed by PCR amplification before blotting and probing, as was done for the genomic gel blots. One microgram of DNase I–treated total RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories), according to the supplier's instructions. For the 910 allele, the two different first-strand primers were designed to terminate at the introduced mutation site, thereby creating the specificity between the transgenic and endogenous copies of the SRK-910 gene. The sequence of the wild- type SRK-910–specific primer consisted of 5'-CCGACGT T T T TGATA-GCCT T T T-3', whereas the mutant SRK-910 primer read 5'-CCGACG-T T T T TGATAGCCT TGC-3'. The first-strand primer for the SRK-A14 gene was composed of 5'-TATACAACCAGTCCCACC AT-3'. Half of each cDNA sample was amplified for 25 cycles with the addition of a gene-specific upstream primer: 5'-TCTGCT TCT TATGATCATGT TC-3' for SRK-910 or 5'-AAAGGGATCT T TCCAGCGT-3' for SRK-A14. Each cycle typically consisted of 20 sec at 94°C, 15 sec at 57°C, and 25 sec at 72°C, and 5 or 10% of the PCR product was subjected to gel electrophoresis and DNA gel blot analysis. After hybridization, membranes were washed in 0.1 x SSC and 0.1% SDS at 65°C. Control lanes included an RNA sample without reverse transcriptase and a genomic DNA positive to demonstrate primer specificity.
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FOOTNOTES |
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1 Current address: Department of Biology, McMaster University, 1830 Main Street West, Hamilton, Ontario L8S 1H5, Canada. 
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ACKNOWLEDGMENTS |
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We thank Nic Bate for useful discussions. This work was funded by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada and Pioneer Hi-Bred to S.J.R. T.L.G. was supported by graduate scholarships from the NSERC.
Received December 1, 1997; accepted December 17, 1997.
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ABSTRACT
INTRODUCTION
