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Trans-inactivation of gene expression in plants
Trans-inactivation of gene expression in plants Jan M. Kooter and Joseph N. M. Mol Vrije University, A m s t e r d a m , The Netherlands Gene silencing induced by antisense RNA has been extremely helpful in characterizing the cellular functions of a number of plant gene products. The introduction of extra sense gene copies may lead not only to protein overproduction but also, surprisingly, to gene silencing. Elucidation of the mechanisms responsible for both types of gene silencing may reveal novel mechanisms by which gene expression is controlled. Current Opinion in Biotechnology 1993, 4:1 66-171
Introduction The selective suppression of gene activity by antisense and sense transgenes in plants is an effective method to generate mutant p h e n o t y p e s artificially. This approach is beneficial not only to studies of experimental systems but also to the genetic modification of crop plants. Despite the growing n u m b e r of successful applications, the mechanisms b y which the genes are suppressed are poorly understood as yet. Gene repression induced by antisense RNA seems to be very straightforward; however, observations thus far indicate that several u n k n o w n factors determine the efficiency. The finding that transgenes can influence each other's expression and the expression of resident genes in transgenic plants (a p h e n o m e n o n alternatively called co-suppression, trans-inactivation or sense suppression) indicates that there is still a lot to learn about nuclear processes involved in gene regulation and in g e n o m e maintenance. Here, w e will focus on recent applications of antisense and sense genes in transgenic plants and we will review the progress made in understanding the mechanisms underlying the trans-interactions. Several other detailed reviews on antisense RNA inhibition and trans-inactivation have b e e n published recently [1.-4.].
Gene suppression mediated by antisense RNA Unraveling gene function and plant physiology It is relatively easy to obtain a collection of cDNAs b y heterotogous hybridizations, differential screening procedures and polymerase chain reaction (PCR)-based strategies, but their functional characterization is more difficult. In the last five years, the function of several genes has b e e n determined b y manipulating their expression using antisense RNA. Antisense RNA is
thought to induce a dramatic decrease in the level of its complementary mRNA by the formation of a double-stranded RNA intermediate. Antisense RNA induced gene suppression has b e e n extremely helpful in the characterization of tomato fruit specific cDNAs [1"]. For example, antisense genes helped to identify a cDNA called pTOM13 that encodes the ethylene-forming enzyme, 1-amino cyclopropane 1-carboxylic acid (ACC) oxidase [5], and the cDNA pTOM5 which was found to encode the enzyme prephytoene pyrophosphate synthase [6], which is involved in the synthesis of the carotenoid lycopene responsible for red tomato coloration. The primary role of ethylene in fruit ripening was demonstrated b y the expression of ACC synthase antisense cDNA [7"] and ACC oxidase antisense cDNA [5]. The inhibition of ethylene synthesis b y these antisense genes delayed the ripening process. The expression of an u n k n o w n cDNA (E8) in the antisense orientation in tomato led to an increase in ethylene levels [8] and shows that the control of ethylene synthesis can b e very refined. To increase the shelf-life of tomatoes and to increase their resistance to bruising, antisense polygalacturonase genes [1"] and antisense pectin methylesterase genes [9] have b e e n used to inhibit steps in the depolymerization of cell wall pectins. Moreover, these studies will provide a better understanding of the physiology of fruit softening in general. The antisense RNA approach has proved to b e an important tool in other aspects of plant physiology as it allows the selective manipulation of the level of a single protein. In this way, interesting results have b e e n achieved that would otherwise have b e e n difficult to obtain [10-15]. The use of viral antisense genes to generate virus resistance in plants has b e e n successful [16]. However, RNA and DNA viruses behave differently with regard
Abbreviations ACC--l-amino cyclopropane 1-carboxytic acid; CaMV--cauliflower mosaic virus; DFR--dihydroflavonol 4-reductase; GUS~-glucuronidase; NOS---nopaline synthase; PAL--phenylalanine ammonia lyase; T-DNA transfer DNA.
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TranMnadivation of gene expressionin plants Kooter and Mol to sensitivity. The DNA viruses, which unfortunately make u p only a small fraction of all plant viruses, seem to be more susceptible to antisense RNA suppression than RNA viruses (for a review, see [17"]).
Manipulating flower pigmentation:antisenseRNA is not the only player The inhibition of pigment synthesis in flowers by antisense RNA can be monitored visually. The suppression of the flavonoid genes chalcone synthase (chs) [18] and dihydroflavonol 4-reductase (dfr) (R van Blokland, JM Kooter, JNM Mol and M Stam, unpublished data) [4"] by antisense genes in petunia can lead to both pure white flowers and surprisingly to partially white flowers [18]. In the latter case, the antisense RNAs w o r k in some flower sectors but not in others, even though the antisense genes are transcribed in both parts at the same rate (R van Blokland, JM Kooter, JNM Mol and M Stam, unpublished data) [4"]. This m a y indicate that the presence of antisense RNA is not sufficient for gene suppression and that additional factors are involved. The area in which the genes are suppressed is influenced by gibberellic acid, B9, which is an inhibitor of gibberellic acid synthesis, and b y light, only w h e n applied to very young flower buds in which the flavonoid genes are not yet expressed [19]. We believe, therefore, that the patterns are established very early in corolla development. Whether or not such local antisense RNA effects occur in other systems remains to be determined. The observation that the levels of antisense RNA do not always correlate with the level of suppression indicates that the presence of antisense RNA m a y not be sufficient in other systems either.
Gene suppression by homologous transgenes In the past few years, several studies have b e e n reported regarding gene silencing in plants b y homologous sequences. However, the molecular mechanisms involved are not understood, although several possibilities have b e e n put forward [3",4",20]. Part of the problem might be that, even though the end result is the same, it is not clear to what extent the results obtained with different systems can be c o m p a r e d because the mechanisms could be different. A key factor in all these systems seems to be the sequence h o m o l o g y between the suppressed and the suppressing genes, as if some kind of homology-sensing machinery is operating [3"]. For the sake of clarity, several types of gene suppression are recognized: mutual transgene suppression, unidirectional transgene suppression, and the suppression of resident genes by homologous transgenes.
Transgene inactivation and methylation Mutual suppression of transgenes occurs in plants that contain multiple copies of the transgene. This explains the reverse correlation that is often seen b e t w e e n gene copy number and expression level. Low expression is very often correlated with increased methylation of the transfer DNAs (T-DNAs) [21,22,23"] and it is well established that methylated DNA is not transcribed or poorly so. L i n n e t al. [22] observed this p h e n o m e n o n with a maize dihydroflavonol 4-reductase (DFR) gene in Petunia, and H o b b s et al. [21] saw it with a ~-glucuronidase (GUS) transgene in tobacco. In the latter case, plants with either a low or a high GUS expression level were obtained. The low expressors contained at least two T-DNAs per genome, which were more methylated than the single copies found in the high expressors. The expression of h o m o z y g o u s low expressors was even more reduced than that of the hemizygotes suggesting that an allelic interaction of s o m e kind leads to even stronger suppression. However, GUS expression in homozygous high expressors was almost twice as high as that of hemizygotes, which would b e expected if the two alleles are independent entities. The observed differences indicate, however, that the correlation between the transgene c o p y n u m b e r per nucleus and the total expression level is not tight. The structure of the integrated T-DNAs (single copy, or in direct or inverted tandem repeats) and the genomic environment of the T-DNA m a y establish the methylation state and expression properties of the transgenes. Mittelsten Scheid et al. [24.] observed that in Arabidops~s 50°/0 of the homozygous plants transgenic for a hygromycin gene failed to transmit the resistance phenotype to the progeny, even though the transgenes are inherited in a Mendelian fashion. This h a p p e n e d only with plants that contained multicopy (5-10) T-DNA integrations. W h e n crossed with a wild type or another sensitive transgenic plant, 6% of the progeny became hygromycin resistant showing reactivation. Because of the low percentage, this might be due to the reactivation of a single hygromycin resistance gene instead of a reactivation of all the genes. In contrast to other cases, the inactive states could not be correlated with increased T-DNA methylation. However, the methylation of important promoter sequences m a y have gone unnoticed as transgene inactivation by methylation has b e e n observed in Arabidopsis [25].
Double transformations and gene silencing Matzke et al. [26,27] found that the introduction of a second T-DNA (T-DNA ID construct into tobacco already containing a T-DNA (T-DNA I) could lead to the suppression of genes located on T-DNA I. The suppressed T-DNA I genes are methylated only w h e n T-DNA II is present. If T-DNA II is crossed out, the T-
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Plantbiotechnology DNA I genes b e c o m e demethylated and reactivated. This reaction is rarely completed in the first generation of the offspring. The dynamics and metastable nature of this type of transgene inactivation are reminiscent of paramutations [3",23"]. The study of transgenes m a y therefore contribute to a better understanding of paramutation p h e n o m e n a in plants. H o w can a second T-DNA located at another genomic position affect the methylation of the first T-DNA? The only sequences that both T-DNAs had in c o m m o n were the nopaline synthase (NOS) promoter and some T-DNA sequences. This suggests that the homology b e t w e e n the DNA sequences of the two T-DNAs, even though they are not transcribed, triggers the silencing, in this case in only one direction. Goring et al. [28,] retransformed a plant that already contained a nos gene, with a truncated nos gene and found that nos mRNA production was suppressed in all transformants. Whether or not expression of the partial nos gene was also suppressed is not known. The hygromycin resistance gene next to the partial nos gene was expressed. The suppression of nos could not b e correlated with methylation of the nos promoters. It will be necessary to analyze nascent RNA to find out whether, in this case, suppression results from promoter inactivation or from a post-transcriptional event.
Post-transcriptional gene suppression Suppression of 13-1,3-glucanase [29"q and chitinase transgene expression [301 is only observed in plants that are homozygous for the transgene. The 13-1,3glucanase suppression has b e e n examined in detail, showing that the transgenes are transcribed in a normal fashion. This means that, in this case, the suppression is a post-transcriptional process. An allelic interaction of s o m e kind is probably not involved because 13-1,3glucanase expression was also suppressed in haploid plants. Silencing therefore seems to b e correlated with the transgene dose per genome. Alternatively, a natural mechanism of g e n e regulation might be induced b y the extremely high 13-1,3-glucanase activity. The unperturbed expression of resident tobacco 13-1,3-glucanases seems to argue against such a mechanism. Elkind et al. [311 generated tobacco plants that were transgenic for a phenylalanine a m m o n i a lyase (PAL) gene from bean (Pbaseolus vulgaris). The plants which had an abnormal p h e n o t y p e [311 expressed the b e a n PAL genes at high levels but failed to accumulate the tobacco PAL transcripts even though the tobacco genes were transcribed (NJ Bate, JA Pallas, Y E1Mnd, CJ Lamb, abstract 9, Tenth J o h n Innes Symposium, 'The Chromosome', Norwich, September 1992). In this case, gene silencing is also unidirectional, but it is opposite to that of the 13-1,3-glucanase: the resident tobacco PAL genes are suppressed but not the PAL transgenes. To what extent the difference in sequence h o m o l o g y between the PAL genes played a role is not known, but this could
be an important factor in the suppression process in general. The introduction of polygalacturonase transgenes into tomato [32], and chalcone synthase [33,34] and dihydroflavonol 4-reductase [34] transgenes into P e t u n i a leads to suppression of the resident genes and the transgenes. In the case of flavonoid gene suppression, the p h e n o t y p e s were similar to those obtained with antisense gene constructs. The flowers w e r e pure white or contained pigmented sectors. Although the corresponding mRNAs were strongly reduced in the white sectors, the resident flavonoid genes w e r e transcribed at normal rates (R van Blokland, JM Kooter, JNM Mol and M Stam, unpublished data) [4"]. The transgenes were also transcribed irrespective of whether or not suppression took place, so trans-inactivation of these genes also occurs post-transcriptionally. It is not yet k n o w n if the polygalacturonase genes in tomato are suppressed in a similar manner. Co-suppression of the flavonoid genes correlated with neither the transcription level of the transgenes nor the c o p y n u m b e r of the transgenes. This indicates that the transcript levels are not important for co-suppression and that it could be sufficient that the transgenes are transcribed. Silencing of flavonoid [4"] and of polygalacturonase [32] genes is obtained using truncated transgenes, indicating that the suppression is not the result of some negative feedback control b y gene products or b y altered metabolite levels. The effect of transgenes on gene suppression resembles the proposed allelic interactions of the semi-dominant n i vea alleles of A n t i r r h i n u m [35"]. These alleles suppress the expression of the wild-type gene in trans. Interestingly, the semi-dominant alleles were created b y DNA rearrangements resulting from transp o s o n activity which m a y have induced a 'disturbed' chromatin structure, perhaps similar to the effect of transgene insertions. Another example of 'natural' suppression is found in the P e t u n i a h yb ri d a variety 'Red Star' w h o s e flowers have alternating white and red sectors. The white sectors do not accumulate chs mRNA; however, the e n d o g e n o u s chs genes are transcribed at the normal rates (R v a n Blokland, JM Kooter, JNM Mol and M Stam, unpublished data) [4"]. This indicates that the localized failure to accumulate chs mRNA is also caused by a post-transcriptional process, like that in chs or d f r co-suppressed flower tissue. The nature of the mutation of 'Red Star' is not known.
Is there a role for antisense RNA in sense suppression? In cases where methylation is responsible for gene inactivation, it is not very likely that antisense RNA plays a role. However, it is conceivable that it is involved in the post-transcriptional suppression of gene activity. We noticed that the sense suppression of flavonoid
Trans.inactivation of gene expression in plants Kooter and Mol genes in petunia has several features in c o m m o n with antisense suppression [4"]. It has been suggested that antisense RNAs transcribed accidentally from the transgenes could be responsible for sense suppression [36"]. In petunia flowers, antisense chs transcripts can indeed be detected [4",37"], but their presence can not be correlated with a particular phenotype. The amounts of antisense RNAs detected were very low and it therefore seems unlikely that each chs mRNA or precursor can be tagged by an antisense RNA for degradation. On the other hand, one may speculate that a low level of antisense RNA synthesized at one locus may facilitate the association of other genomic loci where complementary RNAs are produced. Subsequent events may lead to a collapse of mRNA production. However, it remains to be determined whether or not antisense RNA is capable of performing such a function. Whatever the effect of antisense RNA, if any, there are cases where antisense RNA can not readily explain gene suppression, even though the suppression occurs post-transcriptionally [29"']. It is conceivable that chromatin structure and the nuclear and genomic position of the genes involved are important parameters, especially if we assume that homologous or complementary sequences have to detect each other and interact. A compatible interaction leading to suppression may therefore be difficult to obtain, which could explain w h y in many cases complete sense and antisense suppression occurs rather infrequently.
petunia. H o w the sensing of homologous sequences eventually leads to inactivation, either by methylation or by inducing other processes, remains to be determined but the search for answers may reveal a number of unexpected and novel ways by which gene expression is controlled in plants.
Acknowledgements We thank the Netherlands Organization of Scientific Research (NWO) for their support. JMK is supported by a research fellowship from the Royal Netherlands Academy of Sciences.
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Conclusions In trans, suppression of plant genes is a very effective tool for manipulating gene expression. Instead of using the constitutive CaMV 35S promoter, one can consider using an RNA polymerase III promoter to enhance suppression induced by antisense RNA [38"]. The expanding collection of tissue and cell type specific promoters opens up ways to repress genes very selectively [38",39]. The examples described in this review show that silencing of genes by expressing homologous sense sequences seems as good an approach to manipulating gene expression as the use of antisense gene constructs. It will probably b e c o m e the method of choice in experimental systems because transfection of a complete sense gene construct will yield a collection of plants displaying all possible expression levels. However, w e are faced with a number of burning questions regarding the mechanism of both antisense and sense suppression. For example, it remains to be determined whether or not all antisense genes act by antisense RNA. It is conceivable that in some cases they work like sense genes because they share sequence homology with endogenous genes and that suppression is independent of the direction of transcription. This may explain the similarities between antisense and sense gene induced suppression of flavonoid genes in
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L t ~ F, HEIDMANN I, SAEDLER H, MEYER P: E p i g e n e t i c C h a n g e s i n t h e E x p r e s s i o n o f t h e Maize A1 G e n e in Petunia hybrida: Role o f N n r n h e r s o f I n t e g r a t e d G e n e Copies a n d State o f M e t h y l a t i o n . Mol Gen Genet 1990, 222:329-336. MEYER P, LINN F, HEIDMANN I, MEYER H, NIEDENHOF I, SAEDLER H: E n d o g e n o u s a n d E n v i r o n t n e n t a l F a c t o r s I n f l u e n c e 35S p r o m o t e r M e t h y l a t i o n o f a Maize A1
JORGENSEN R: I n t r o d u c t i o n o f Synthase Gene into Petunia Co-supression of Homologous Cell 1990, 2:279-289.
Trans-inactivation o f gene e x p r e s s i o n in plants K o o t e r a n d Mol This paper describes the behavior of semi-dominant alleles of the cbs gene in Antirrbinum majus that inhibit cbs expression 25-50-fold in a heterozygous plant. The alleles act in transwhich resembles transinactivation by transgenes. The semi-dominant alleles are the result of extensive DNA rearrangements, possibly caused by transposon activity. 36.
GRIERSON D, FRAY RG, HAMILTON AJ, SMITH CJS, WATSON CF: Does Co-Suppression o f Sense Genes in Transgenic P l a n t s I n v o l v e A n t i s e n s e RNA. Trends Biotechnol 1991, 9:122-123. This paper speculates on the possibility that co-suppression is caused by antisense RNA. Models are presented that m a y explain antisense RNA production from the transgenes. MOL J, vAN BLOKLANDR, KOOTERJ: More A b o u t Co-Supp r e s s i o n . Trends Biotechnol 1991, 9:182-183. Discusses the presence of antisense transcripts in transgenic plants containing sense chs transgenes.
BOURQUEJE, FOLK 3grR: Suppression o f Gene Express i o n i n P l a n t C e l l s utili~|ng Antisense Sequences Transcribed b y RNA P o l y m e r a s e HI. Plant Mol Bto11992, 19:641-647. An RNA polymerase III promoter w a s u s e d to express a chloramphenicol acetyl transferase antisense gene to suppress chloramphenical acetyl transferase expression in carrot protoplasts. The antisense inhibition with the RNA pol III promoter gene w a s in s o m e cases five-fold greater than with an antisense construct expressed by RNA pol II from the CaMV 35S promoter. 38.
,
39.
VAN DER MEER IM, STAM M, VAN TUNEN AJ, MOL JNM, STUITJE AR: Antisense Inhibition o f Flavonoid Biosynthesis i n P e t u n i a Anthers Results i n Male Sterility. Plant Cell 1992, 4:253-262.
37.
JM Kooter and JNM Mol, Department of Genetics, Vrije Universiteit Amsterdam, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands.
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Introduction The selective suppression of gene activity by antisense and sense transgenes in plants is an effective method to generate mutant p h e n o t y p e s artificially. This approach is beneficial not only to studies of experimental systems but also to the genetic modification of crop plants. Despite the growing n u m b e r of successful applications, the mechanisms b y which the genes are suppressed are poorly understood as yet. Gene repression induced by antisense RNA seems to be very straightforward; however, observations thus far indicate that several u n k n o w n factors determine the efficiency. The finding that transgenes can influence each other's expression and the expression of resident genes in transgenic plants (a p h e n o m e n o n alternatively called co-suppression, trans-inactivation or sense suppression) indicates that there is still a lot to learn about nuclear processes involved in gene regulation and in g e n o m e maintenance. Here, w e will focus on recent applications of antisense and sense genes in transgenic plants and we will review the progress made in understanding the mechanisms underlying the trans-interactions. Several other detailed reviews on antisense RNA inhibition and trans-inactivation have b e e n published recently [1.-4.].
Gene suppression mediated by antisense RNA Unraveling gene function and plant physiology It is relatively easy to obtain a collection of cDNAs b y heterotogous hybridizations, differential screening procedures and polymerase chain reaction (PCR)-based strategies, but their functional characterization is more difficult. In the last five years, the function of several genes has b e e n determined b y manipulating their expression using antisense RNA. Antisense RNA is
thought to induce a dramatic decrease in the level of its complementary mRNA by the formation of a double-stranded RNA intermediate. Antisense RNA induced gene suppression has b e e n extremely helpful in the characterization of tomato fruit specific cDNAs [1"]. For example, antisense genes helped to identify a cDNA called pTOM13 that encodes the ethylene-forming enzyme, 1-amino cyclopropane 1-carboxylic acid (ACC) oxidase [5], and the cDNA pTOM5 which was found to encode the enzyme prephytoene pyrophosphate synthase [6], which is involved in the synthesis of the carotenoid lycopene responsible for red tomato coloration. The primary role of ethylene in fruit ripening was demonstrated b y the expression of ACC synthase antisense cDNA [7"] and ACC oxidase antisense cDNA [5]. The inhibition of ethylene synthesis b y these antisense genes delayed the ripening process. The expression of an u n k n o w n cDNA (E8) in the antisense orientation in tomato led to an increase in ethylene levels [8] and shows that the control of ethylene synthesis can b e very refined. To increase the shelf-life of tomatoes and to increase their resistance to bruising, antisense polygalacturonase genes [1"] and antisense pectin methylesterase genes [9] have b e e n used to inhibit steps in the depolymerization of cell wall pectins. Moreover, these studies will provide a better understanding of the physiology of fruit softening in general. The antisense RNA approach has proved to b e an important tool in other aspects of plant physiology as it allows the selective manipulation of the level of a single protein. In this way, interesting results have b e e n achieved that would otherwise have b e e n difficult to obtain [10-15]. The use of viral antisense genes to generate virus resistance in plants has b e e n successful [16]. However, RNA and DNA viruses behave differently with regard
Abbreviations ACC--l-amino cyclopropane 1-carboxytic acid; CaMV--cauliflower mosaic virus; DFR--dihydroflavonol 4-reductase; GUS~-glucuronidase; NOS---nopaline synthase; PAL--phenylalanine ammonia lyase; T-DNA transfer DNA.
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TranMnadivation of gene expressionin plants Kooter and Mol to sensitivity. The DNA viruses, which unfortunately make u p only a small fraction of all plant viruses, seem to be more susceptible to antisense RNA suppression than RNA viruses (for a review, see [17"]).
Manipulating flower pigmentation:antisenseRNA is not the only player The inhibition of pigment synthesis in flowers by antisense RNA can be monitored visually. The suppression of the flavonoid genes chalcone synthase (chs) [18] and dihydroflavonol 4-reductase (dfr) (R van Blokland, JM Kooter, JNM Mol and M Stam, unpublished data) [4"] by antisense genes in petunia can lead to both pure white flowers and surprisingly to partially white flowers [18]. In the latter case, the antisense RNAs w o r k in some flower sectors but not in others, even though the antisense genes are transcribed in both parts at the same rate (R van Blokland, JM Kooter, JNM Mol and M Stam, unpublished data) [4"]. This m a y indicate that the presence of antisense RNA is not sufficient for gene suppression and that additional factors are involved. The area in which the genes are suppressed is influenced by gibberellic acid, B9, which is an inhibitor of gibberellic acid synthesis, and b y light, only w h e n applied to very young flower buds in which the flavonoid genes are not yet expressed [19]. We believe, therefore, that the patterns are established very early in corolla development. Whether or not such local antisense RNA effects occur in other systems remains to be determined. The observation that the levels of antisense RNA do not always correlate with the level of suppression indicates that the presence of antisense RNA m a y not be sufficient in other systems either.
Gene suppression by homologous transgenes In the past few years, several studies have b e e n reported regarding gene silencing in plants b y homologous sequences. However, the molecular mechanisms involved are not understood, although several possibilities have b e e n put forward [3",4",20]. Part of the problem might be that, even though the end result is the same, it is not clear to what extent the results obtained with different systems can be c o m p a r e d because the mechanisms could be different. A key factor in all these systems seems to be the sequence h o m o l o g y between the suppressed and the suppressing genes, as if some kind of homology-sensing machinery is operating [3"]. For the sake of clarity, several types of gene suppression are recognized: mutual transgene suppression, unidirectional transgene suppression, and the suppression of resident genes by homologous transgenes.
Transgene inactivation and methylation Mutual suppression of transgenes occurs in plants that contain multiple copies of the transgene. This explains the reverse correlation that is often seen b e t w e e n gene copy number and expression level. Low expression is very often correlated with increased methylation of the transfer DNAs (T-DNAs) [21,22,23"] and it is well established that methylated DNA is not transcribed or poorly so. L i n n e t al. [22] observed this p h e n o m e n o n with a maize dihydroflavonol 4-reductase (DFR) gene in Petunia, and H o b b s et al. [21] saw it with a ~-glucuronidase (GUS) transgene in tobacco. In the latter case, plants with either a low or a high GUS expression level were obtained. The low expressors contained at least two T-DNAs per genome, which were more methylated than the single copies found in the high expressors. The expression of h o m o z y g o u s low expressors was even more reduced than that of the hemizygotes suggesting that an allelic interaction of s o m e kind leads to even stronger suppression. However, GUS expression in homozygous high expressors was almost twice as high as that of hemizygotes, which would b e expected if the two alleles are independent entities. The observed differences indicate, however, that the correlation between the transgene c o p y n u m b e r per nucleus and the total expression level is not tight. The structure of the integrated T-DNAs (single copy, or in direct or inverted tandem repeats) and the genomic environment of the T-DNA m a y establish the methylation state and expression properties of the transgenes. Mittelsten Scheid et al. [24.] observed that in Arabidops~s 50°/0 of the homozygous plants transgenic for a hygromycin gene failed to transmit the resistance phenotype to the progeny, even though the transgenes are inherited in a Mendelian fashion. This h a p p e n e d only with plants that contained multicopy (5-10) T-DNA integrations. W h e n crossed with a wild type or another sensitive transgenic plant, 6% of the progeny became hygromycin resistant showing reactivation. Because of the low percentage, this might be due to the reactivation of a single hygromycin resistance gene instead of a reactivation of all the genes. In contrast to other cases, the inactive states could not be correlated with increased T-DNA methylation. However, the methylation of important promoter sequences m a y have gone unnoticed as transgene inactivation by methylation has b e e n observed in Arabidopsis [25].
Double transformations and gene silencing Matzke et al. [26,27] found that the introduction of a second T-DNA (T-DNA ID construct into tobacco already containing a T-DNA (T-DNA I) could lead to the suppression of genes located on T-DNA I. The suppressed T-DNA I genes are methylated only w h e n T-DNA II is present. If T-DNA II is crossed out, the T-
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Plantbiotechnology DNA I genes b e c o m e demethylated and reactivated. This reaction is rarely completed in the first generation of the offspring. The dynamics and metastable nature of this type of transgene inactivation are reminiscent of paramutations [3",23"]. The study of transgenes m a y therefore contribute to a better understanding of paramutation p h e n o m e n a in plants. H o w can a second T-DNA located at another genomic position affect the methylation of the first T-DNA? The only sequences that both T-DNAs had in c o m m o n were the nopaline synthase (NOS) promoter and some T-DNA sequences. This suggests that the homology b e t w e e n the DNA sequences of the two T-DNAs, even though they are not transcribed, triggers the silencing, in this case in only one direction. Goring et al. [28,] retransformed a plant that already contained a nos gene, with a truncated nos gene and found that nos mRNA production was suppressed in all transformants. Whether or not expression of the partial nos gene was also suppressed is not known. The hygromycin resistance gene next to the partial nos gene was expressed. The suppression of nos could not b e correlated with methylation of the nos promoters. It will be necessary to analyze nascent RNA to find out whether, in this case, suppression results from promoter inactivation or from a post-transcriptional event.
Post-transcriptional gene suppression Suppression of 13-1,3-glucanase [29"q and chitinase transgene expression [301 is only observed in plants that are homozygous for the transgene. The 13-1,3glucanase suppression has b e e n examined in detail, showing that the transgenes are transcribed in a normal fashion. This means that, in this case, the suppression is a post-transcriptional process. An allelic interaction of s o m e kind is probably not involved because 13-1,3glucanase expression was also suppressed in haploid plants. Silencing therefore seems to b e correlated with the transgene dose per genome. Alternatively, a natural mechanism of g e n e regulation might be induced b y the extremely high 13-1,3-glucanase activity. The unperturbed expression of resident tobacco 13-1,3-glucanases seems to argue against such a mechanism. Elkind et al. [311 generated tobacco plants that were transgenic for a phenylalanine a m m o n i a lyase (PAL) gene from bean (Pbaseolus vulgaris). The plants which had an abnormal p h e n o t y p e [311 expressed the b e a n PAL genes at high levels but failed to accumulate the tobacco PAL transcripts even though the tobacco genes were transcribed (NJ Bate, JA Pallas, Y E1Mnd, CJ Lamb, abstract 9, Tenth J o h n Innes Symposium, 'The Chromosome', Norwich, September 1992). In this case, gene silencing is also unidirectional, but it is opposite to that of the 13-1,3-glucanase: the resident tobacco PAL genes are suppressed but not the PAL transgenes. To what extent the difference in sequence h o m o l o g y between the PAL genes played a role is not known, but this could
be an important factor in the suppression process in general. The introduction of polygalacturonase transgenes into tomato [32], and chalcone synthase [33,34] and dihydroflavonol 4-reductase [34] transgenes into P e t u n i a leads to suppression of the resident genes and the transgenes. In the case of flavonoid gene suppression, the p h e n o t y p e s were similar to those obtained with antisense gene constructs. The flowers w e r e pure white or contained pigmented sectors. Although the corresponding mRNAs were strongly reduced in the white sectors, the resident flavonoid genes w e r e transcribed at normal rates (R van Blokland, JM Kooter, JNM Mol and M Stam, unpublished data) [4"]. The transgenes were also transcribed irrespective of whether or not suppression took place, so trans-inactivation of these genes also occurs post-transcriptionally. It is not yet k n o w n if the polygalacturonase genes in tomato are suppressed in a similar manner. Co-suppression of the flavonoid genes correlated with neither the transcription level of the transgenes nor the c o p y n u m b e r of the transgenes. This indicates that the transcript levels are not important for co-suppression and that it could be sufficient that the transgenes are transcribed. Silencing of flavonoid [4"] and of polygalacturonase [32] genes is obtained using truncated transgenes, indicating that the suppression is not the result of some negative feedback control b y gene products or b y altered metabolite levels. The effect of transgenes on gene suppression resembles the proposed allelic interactions of the semi-dominant n i vea alleles of A n t i r r h i n u m [35"]. These alleles suppress the expression of the wild-type gene in trans. Interestingly, the semi-dominant alleles were created b y DNA rearrangements resulting from transp o s o n activity which m a y have induced a 'disturbed' chromatin structure, perhaps similar to the effect of transgene insertions. Another example of 'natural' suppression is found in the P e t u n i a h yb ri d a variety 'Red Star' w h o s e flowers have alternating white and red sectors. The white sectors do not accumulate chs mRNA; however, the e n d o g e n o u s chs genes are transcribed at the normal rates (R v a n Blokland, JM Kooter, JNM Mol and M Stam, unpublished data) [4"]. This indicates that the localized failure to accumulate chs mRNA is also caused by a post-transcriptional process, like that in chs or d f r co-suppressed flower tissue. The nature of the mutation of 'Red Star' is not known.
Is there a role for antisense RNA in sense suppression? In cases where methylation is responsible for gene inactivation, it is not very likely that antisense RNA plays a role. However, it is conceivable that it is involved in the post-transcriptional suppression of gene activity. We noticed that the sense suppression of flavonoid
Trans.inactivation of gene expression in plants Kooter and Mol genes in petunia has several features in c o m m o n with antisense suppression [4"]. It has been suggested that antisense RNAs transcribed accidentally from the transgenes could be responsible for sense suppression [36"]. In petunia flowers, antisense chs transcripts can indeed be detected [4",37"], but their presence can not be correlated with a particular phenotype. The amounts of antisense RNAs detected were very low and it therefore seems unlikely that each chs mRNA or precursor can be tagged by an antisense RNA for degradation. On the other hand, one may speculate that a low level of antisense RNA synthesized at one locus may facilitate the association of other genomic loci where complementary RNAs are produced. Subsequent events may lead to a collapse of mRNA production. However, it remains to be determined whether or not antisense RNA is capable of performing such a function. Whatever the effect of antisense RNA, if any, there are cases where antisense RNA can not readily explain gene suppression, even though the suppression occurs post-transcriptionally [29"']. It is conceivable that chromatin structure and the nuclear and genomic position of the genes involved are important parameters, especially if we assume that homologous or complementary sequences have to detect each other and interact. A compatible interaction leading to suppression may therefore be difficult to obtain, which could explain w h y in many cases complete sense and antisense suppression occurs rather infrequently.
petunia. H o w the sensing of homologous sequences eventually leads to inactivation, either by methylation or by inducing other processes, remains to be determined but the search for answers may reveal a number of unexpected and novel ways by which gene expression is controlled in plants.
Acknowledgements We thank the Netherlands Organization of Scientific Research (NWO) for their support. JMK is supported by a research fellowship from the Royal Netherlands Academy of Sciences.
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Conclusions In trans, suppression of plant genes is a very effective tool for manipulating gene expression. Instead of using the constitutive CaMV 35S promoter, one can consider using an RNA polymerase III promoter to enhance suppression induced by antisense RNA [38"]. The expanding collection of tissue and cell type specific promoters opens up ways to repress genes very selectively [38",39]. The examples described in this review show that silencing of genes by expressing homologous sense sequences seems as good an approach to manipulating gene expression as the use of antisense gene constructs. It will probably b e c o m e the method of choice in experimental systems because transfection of a complete sense gene construct will yield a collection of plants displaying all possible expression levels. However, w e are faced with a number of burning questions regarding the mechanism of both antisense and sense suppression. For example, it remains to be determined whether or not all antisense genes act by antisense RNA. It is conceivable that in some cases they work like sense genes because they share sequence homology with endogenous genes and that suppression is independent of the direction of transcription. This may explain the similarities between antisense and sense gene induced suppression of flavonoid genes in
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GORINGDR, THOMSON L, ROTHSTEIN SJ: T r a n s f o r m a t i o n o f a P a r t i a l N o p a l i n e S y n t h a s e G e n e i n t o T o b a c c o Suppresses the Expression of a Resident Wild-Type Gene. Proc Natl Acad Sct USA 1991, 88:1770-1774. This p a p e r describes the suppression of a nos gene in tobacco by the introduction of a second T-DNA containing a partial nos gene. Suppression was observed in all transformants b u t n o n e of t h e m carried a methylated nos promoter. 29. •,
DECARVALHOF, GHEYSEN G, KUSHNIRS, VANMONTAGUM, INZE D, CASTRESANAC: Suppression o f ~ - l , 3 - G l u c a n a s e T r a n s g e n e E x p r e s s i o n i n H o m o z y g o u s P l a n t s . EMBO J 1992, 11:2595-2602. This paper gives a detailed analysis of [~-l,3-glucanase t m n s g e n e suppression. The suppression is the result of a post-transcriptional process a n d is observed only in h o m o z y g o u s a n d in haploid plants. The latter is a novel finding which seems to exclude a m e c h a n i s m that involves an allelic interaction of s o m e kind. 30.
NEUHAUSJ-M, AHL-GOY P, HINZ U, FLORES S, MEINS FJ: H i g h Level E x p r e s s i o n o f a T o b a c c o C h i t i n a s e G e n e i n Nicotiana sylvestris. S u s c e p t i b i l i t y o f T r a n s g e n i c P l a n t s to C e r c o s p o r a nicotianae I n f e c t i o n . Plant Mol Btol 1991~ 16:141-151.
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ELKINDY, EDWARDSR, MAVANDADM, HEDRICK SA, RIBAK O, DIXON RA, LAMB CJ: A b n o r m a l P l a n t D e v e l o p m e n t a n d Down-Regulation of Phenylpropanoid Biosynthesis in Transgenic Tobacco Cont~inlttg a Heterologous Phenyi a l a t l i n e Ammoxxla-Lyase Gene. Proc Natl Acad Sct USA 1990, 87:9057-9061.
32.
SMITHCJS, WATSONCF, BIRD CR, RAY J, SCHUCHW, GRIERSON D: E x p r e s s i o n o f a T r u n c a t e d T o m a t o P o l y g a i a c t u r onase Gene Inhibits Expression of the Endogenous G e n e i n P l a n t s . Mol Gen Genet 1990, 224:477-481.
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NAPOLI C, LEMIEUX C, a Chimeric Chalcone Results in Reversible G e n e s in Trans. Plant
34.
VAN DER KROL AR, Mum LA, BELD M, MOL JNM, STUITJE AR: F l a v o n o i d G e n e s i n P e t u n i a : A d d i t i o n o f a L i m i t e d Nnmher of Gene Copies May Lead to a Suppression o f G e n e E x p r e s s i o n . Plant Cell 1990, 2:291-299.
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BOLI~A~ J, CARPENTER R, COEN ES: Allelic I n t e r a c t i o n s at t h e N i v e a Locus o f A n t i r r b i n u m . Plant Cell 1991, 3:1327-1336.
DE LANGE P, GERATSAGM, MOL Chalcone Synthase Genes in Variable Transgene Expres220:204-212.
L t ~ F, HEIDMANN I, SAEDLER H, MEYER P: E p i g e n e t i c C h a n g e s i n t h e E x p r e s s i o n o f t h e Maize A1 G e n e in Petunia hybrida: Role o f N n r n h e r s o f I n t e g r a t e d G e n e Copies a n d State o f M e t h y l a t i o n . Mol Gen Genet 1990, 222:329-336. MEYER P, LINN F, HEIDMANN I, MEYER H, NIEDENHOF I, SAEDLER H: E n d o g e n o u s a n d E n v i r o n t n e n t a l F a c t o r s I n f l u e n c e 35S p r o m o t e r M e t h y l a t i o n o f a Maize A1
JORGENSEN R: I n t r o d u c t i o n o f Synthase Gene into Petunia Co-supression of Homologous Cell 1990, 2:279-289.
Trans-inactivation o f gene e x p r e s s i o n in plants K o o t e r a n d Mol This paper describes the behavior of semi-dominant alleles of the cbs gene in Antirrbinum majus that inhibit cbs expression 25-50-fold in a heterozygous plant. The alleles act in transwhich resembles transinactivation by transgenes. The semi-dominant alleles are the result of extensive DNA rearrangements, possibly caused by transposon activity. 36.
GRIERSON D, FRAY RG, HAMILTON AJ, SMITH CJS, WATSON CF: Does Co-Suppression o f Sense Genes in Transgenic P l a n t s I n v o l v e A n t i s e n s e RNA. Trends Biotechnol 1991, 9:122-123. This paper speculates on the possibility that co-suppression is caused by antisense RNA. Models are presented that m a y explain antisense RNA production from the transgenes. MOL J, vAN BLOKLANDR, KOOTERJ: More A b o u t Co-Supp r e s s i o n . Trends Biotechnol 1991, 9:182-183. Discusses the presence of antisense transcripts in transgenic plants containing sense chs transgenes.
BOURQUEJE, FOLK 3grR: Suppression o f Gene Express i o n i n P l a n t C e l l s utili~|ng Antisense Sequences Transcribed b y RNA P o l y m e r a s e HI. Plant Mol Bto11992, 19:641-647. An RNA polymerase III promoter w a s u s e d to express a chloramphenicol acetyl transferase antisense gene to suppress chloramphenical acetyl transferase expression in carrot protoplasts. The antisense inhibition with the RNA pol III promoter gene w a s in s o m e cases five-fold greater than with an antisense construct expressed by RNA pol II from the CaMV 35S promoter. 38.
,
39.
VAN DER MEER IM, STAM M, VAN TUNEN AJ, MOL JNM, STUITJE AR: Antisense Inhibition o f Flavonoid Biosynthesis i n P e t u n i a Anthers Results i n Male Sterility. Plant Cell 1992, 4:253-262.
37.
JM Kooter and JNM Mol, Department of Genetics, Vrije Universiteit Amsterdam, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands.
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