The Plant Cell, Vol. 9, 1495-1 504, August 1997 O 1997 American Society of Plant Physiologists A Transcriptionally Active State 1s Required for Post-Transcriptional Silencing (Cosuppression) of Nitrate Reductase Host Genes and Transgenes Hervé Vaucheret,all Laurent Nussaume,a~2 Jean-Christophe Palauqui,a lsabelle Quilléré,b and Taline Elmayana13 a Laboratoire de Biologie Cellulaire, Centre INRA de Versailles, F-78026 Versailles Cedex, France Laboratoire du Métabolisme, Centre INRA de Versailles, F-78026 Versailles Cedex, France Using tobacco nitrate reductase cosuppression as a model system of post-transcriptional gene silencing, we analyzed the influence of DNA and RNA dosages both together and independently. For this purpose, zero, one, two, or four active or transcriptionally silenced copies of a cauliflower mosaic virus 35SNia2 transgene were combined by transformation and subsequent crosses with zero, one, two, three, or four active, disrupted, or transcriptionally repressed copies of the wild-type host Nia genes. The analysis of the corresponding transgenic lines revealed that (1) the percentage of isogenic plants that are affected by cosuppression depends directly upon the relative dosage of both host gene and transgene; (2) transcriptional silencing of the 35SNia transgene impedes cosuppression; and (3) the absence of host gene transcription reduces the frequency of cosuppression or delays its triggering. Taken together, these results indi- cate that transgene DNA per se is not sufficient to trigger post-transcriptional cosuppression of nitrate reductase host genes and transgenes. The requirement for a transcriptionally active state is discussed with respect to both the RNA dosage and the DNA-DNA pairing hypotheses. INTRODUCTION Cosuppression, that is, the coordinated inactivation of (par- tially) homologous transgenes and host genes in transgenic plants, is one of the most fascinating problems discovered by plant molecular biologists in the past few years (reviewed in Flavell, 1994; Matzke and Matzke, 1995; Meyer, 1995; Baulcombe and English, 1996). This phenomenon was origi- nally described as the consequence of the introductionof ad- ditional copies of a gene (encoding chalcone synthase [CHS]) or of a transgene consisting of the corresponding cDNA under the control of the cauliflower mosaic virus 35s pro- moter (Napoli et al., 1990; van der Krol et al., 1990). Further analysis revealed that this phenomenon occurs at the post- transcriptional level, that is, that host genes and transgenes are correctly transcribed in the nucleus, whereas the corre- sponding mRNAs do not accumulate in the cytosol (de Carvalho et al., 1992; Van Blokland et al., 1994).Cosuppres- sion has now been described for severa1 plant genes in sev- era1 species by using either transcribed transgenes (van Blokland et al., 1994; de Carvalho Niebel et al., 1995; Kunz ’ To whom correspondenceshould be addressed. E-mail vauchere@ versailles.inra.fr; fax 33-1 -30-83-30-99. ‘Current address: CEA, Cadarache, 13108 St. Paul lez Durance Cedex, France. Current address: UA Phytopharmacie, INRA, BV 1540, 21034 Dijon Cedex, France. et al., 1996) or, more surprisingly, promoterless transgenes (van Blokland et al., 1994). This latter report suggests that post-transcriptionalcosuppression may be initiated by DNA- DNA pairing between host genes and transgenes, leading to the production of aberrant RNAs that activate the specific degradation of all homologous RNAs (Baulcombe and En- glish, 1996). Post-transcriptionalsilencing of foreign reporter genes has also been reported (Dehio and Schell, 1994; lngelbrechtet al., 1994; Elmayan and Vaucheret, 1996). Elmayan and Vaucheret (1996) reported that silencing affected all of the transgenic lines carrying a foreign uidA transgene under the control of a 35s promoter with a double enhancer, irrespective of their T-DNA copy number. In addition, silencing was observed in haploid plants carrying a single copy of the 35s X 2-uidA transgene, thus indicating that neither host genehransgene nor transgenehransgene DNA-DNA pairing is required for the triggering of silencing in this case. Therefore, silencing may re- sult from the accumulation of RNAs produced by the trans- gene above a threshold level that triggers an increase in the turnover and/or a specific degradation of all homologous RNAs (Meins, 1989; Dehio and Schell, 1994; Dougherty and Parks, 1995; Elmayan and Vaucheret, 1996). Transgenic plants exhibiting resistance to RNA viruses were also obtained when a transgene consisting of a cDNA
Transcript
The Plant Cell, Vol. 9, 1495-1 504, August 1997 O 1997 American Society of Plant Physiologists
A Transcriptionally Active State 1s Required for Post-Transcriptional Silencing (Cosuppression) of Nitrate Reductase Host Genes and Transgenes
a Laboratoire de Biologie Cellulaire, Centre INRA de Versailles, F-78026 Versailles Cedex, France Laboratoire du Métabolisme, Centre INRA de Versailles, F-78026 Versailles Cedex, France
Using tobacco nitrate reductase cosuppression as a model system of post-transcriptional gene silencing, we analyzed the influence of DNA and RNA dosages both together and independently. For this purpose, zero, one, two, or four active or transcriptionally silenced copies of a cauliflower mosaic virus 35SNia2 transgene were combined by transformation and subsequent crosses with zero, one, two, three, or four active, disrupted, or transcriptionally repressed copies of the wild-type host Nia genes. The analysis of the corresponding transgenic lines revealed that (1) the percentage of isogenic plants that are affected by cosuppression depends directly upon the relative dosage of both host gene and transgene; (2) transcriptional silencing of the 35SNia transgene impedes cosuppression; and (3) the absence of host gene transcription reduces the frequency of cosuppression or delays its triggering. Taken together, these results indi- cate that transgene DNA per se is not sufficient to trigger post-transcriptional cosuppression of nitrate reductase host genes and transgenes. The requirement for a transcriptionally active state is discussed with respect to both the RNA dosage and the DNA-DNA pairing hypotheses.
INTRODUCTION
Cosuppression, that is, the coordinated inactivation of (par- tially) homologous transgenes and host genes in transgenic plants, is one of the most fascinating problems discovered by plant molecular biologists in the past few years (reviewed in Flavell, 1994; Matzke and Matzke, 1995; Meyer, 1995; Baulcombe and English, 1996). This phenomenon was origi- nally described as the consequence of the introduction of ad- ditional copies of a gene (encoding chalcone synthase [CHS]) or of a transgene consisting of the corresponding cDNA under the control of the cauliflower mosaic virus 35s pro- moter (Napoli et al., 1990; van der Krol et al., 1990). Further analysis revealed that this phenomenon occurs at the post- transcriptional level, that is, that host genes and transgenes are correctly transcribed in the nucleus, whereas the corre- sponding mRNAs do not accumulate in the cytosol (de Carvalho et al., 1992; Van Blokland et al., 1994). Cosuppres- sion has now been described for severa1 plant genes in sev- era1 species by using either transcribed transgenes (van Blokland et al., 1994; de Carvalho Niebel et al., 1995; Kunz
’ To whom correspondence should be addressed. E-mail vauchere@ versailles.inra.fr; fax 33-1 -30-83-30-99. ‘Current address: CEA, Cadarache, 13108 St. Paul lez Durance Cedex, France. Current address: UA Phytopharmacie, INRA, BV 1540, 21 034 Dijon
Cedex, France.
et al., 1996) or, more surprisingly, promoterless transgenes (van Blokland et al., 1994). This latter report suggests that post-transcriptional cosuppression may be initiated by DNA- DNA pairing between host genes and transgenes, leading to the production of aberrant RNAs that activate the specific degradation of all homologous RNAs (Baulcombe and En- glish, 1996).
Post-transcriptional silencing of foreign reporter genes has also been reported (Dehio and Schell, 1994; lngelbrecht et al., 1994; Elmayan and Vaucheret, 1996). Elmayan and Vaucheret (1996) reported that silencing affected all of the transgenic lines carrying a foreign uidA transgene under the control of a 35s promoter with a double enhancer, irrespective of their T-DNA copy number. In addition, silencing was observed in haploid plants carrying a single copy of the 35s X 2-uidA transgene, thus indicating that neither host genehransgene nor transgenehransgene DNA-DNA pairing is required for the triggering of silencing in this case. Therefore, silencing may re- sult from the accumulation of RNAs produced by the trans- gene above a threshold level that triggers an increase in the turnover and/or a specific degradation of all homologous RNAs (Meins, 1989; Dehio and Schell, 1994; Dougherty and Parks, 1995; Elmayan and Vaucheret, 1996).
Transgenic plants exhibiting resistance to RNA viruses were also obtained when a transgene consisting of a cDNA
1496 The Plant Cell
fragment of the viral genome was introduced under thecontrol of the 35S promoter (Lindbo et al., 1993; Smith et al.,1994; Mueller et al., 1995). High levels of resistance to homo-logous viruses were observed in plants in which the tran-scription of the transgene was detected in the nucleus,although the corresponding mRNAs did not accumulate inthe cytosol (English et al., 1996; Goodwin et al., 1996; Sijenet al., 1996). Because replication of the viral RNA takes placein the cytosol, it has been suggested that post-transcrip-tional silencing phenomena occur in the cytosol.
Several studies have suggested that cosuppression andrelated phenomena are dosage dependent. The frequencyand/or the intensity of cosuppression is increased in plantshomozygous for the transgene locus, as compared with hemi-zygous plants (de Carvalho et al., 1992; Hart et al., 1992;Angenent et al., 1993; Dorlhac de Borne et al., 1994), and inplants carrying a combination of unlinked transgene loci, ascompared with parental lines (de Carvalho Niebel et al., 1995;Palauqui and Vaucheret, 1995; Jorgensen et al., 1996). In ad-dition, silencing occurs similarly in haploid and homozygousplants, thus indicating such a dosage effect (de Carvalho etal., 1992; Elmayan and Vaucheret, 1996). Finally, some stud-ies have revealed that the expression of host genes is re-quired for the triggering of cosuppression (Smith et al.,1990; Dorlhac de Borne et al., 1994). In this study, we testedDNA and RNA dosage effects both together and indepen-dently. We show that transgene DNA per se is not sufficientto trigger nitrate reductase (NR) cosuppression and that atranscriptionally active state is required.
RESULTS
Silencing of a 35S-/V/a2 Transgene Occurs in anNR-Deficient Nicotians plumbaginifolia Mutant
The NR-deficient mutant E23 of N. plumbaginifolia is im-paired in the production of a full-length Nia mRNA (Pouteauet al., 1989) because of the insertion of the retrotransposonTnp2 at amino acid 154 of the coding sequence (Vaucheretet al., 1992a). The Nia transcript is processed in the 5'-longterminal repeat of the retrotransposon, thus leading to atruncated Nia-Tnp2 transcript that can be detected on RNAgel blots by using a probe situated upstream of the insertionsite (Vaucheret et al., 1992a). Mutant E23 NR activity was re-stored by introducing either a genomic clone of the tobaccoNia2 gene (Vaucheret et al., 1990) or the tobacco Nia2 cDNAunder the control of the 35S promoter (Vincentz andCaboche, 1991). Among the various transgenic plants carry-ing the latter construct inserted at a single locus, one, 30C,carrying a single copy of the T-DNA (Figure 1), showed signsof silencing and was retained for further analysis. A homozy-gous descendant, 30C.1, was identified in its self-progeny.Plants derived by subsequent self-fertilizations were grownunder different physiological conditions. When grown in the
35S probe Npt probe 1 k b
:rr 3' Npt II 5'
NS S
• 1.3kb
35S probe Npl probe
Figure 1. T-DNA Configuration in Silenced and Nonsilenced De-scendants of Plant 30C.DNA was extracted from a green nonsilenced (NS) or a chlorotic si-lenced (S) descendant of plant 30C, cut with EcoRV, and hybridizedwith either a 35S probe or an Npt probe. The restriction map of theT-DNA and the position of the probes are indicated above. LB andRB, left and right borders, respectively; V, EcoRV.
greenhouse during the winter under limited light conditions,plants showed stable and uniform NR activity (Quillere et al.,1994), whereas plants grown in the greenhouse during thesummer belong to two classes. A major class (68%) consistsof green plants with high NR activity. A minor class (32%) con-sists of chlorotic plants with no NR activity (data not shown),suggesting some impairment in the function of the 35S-Matransgene.
Silencing of the 35S-/V/a2 Transgene Occursat Each Generation
To check whether the T-DNA was absent, rearranged, or si-lenced in chlorotic plants, DNA was extracted from onegreen and one chlorotic plant. DNA gel blot analysis indi-cated no detectable rearrangements (Figure 1). In addition,seeds were harvested from 10 green and 10 chlorotic plantsand sown in vitro on medium supplemented with either chlo-rate (the 35S-N/a2 transgene confers sensitivity to chlorate)
Nitrate Reductase Cosuppression 1497
or kanamycin (the nopaline synthase Nos-neomycin phospho-transferase Npt transgene carried on the same T-DNA con-fers resistance to kanamycin). Both types of plants werehomozygous for the transgene locus, as shown by uniformresistance of their progenies to kanamycin and uniform sen-sitivity to chlorate (data not shown). Seeds of both types ofplants were sown in the greenhouse the next summer. Asegregation of ~70% green plants and 30% chloroticNR-deficient plants was observed in the progeny of bothtypes and similarly in subsequent progenies (data notshown). These results demonstrate that silencing of the35S-A//a transgene in N. plumbaginifolia is reversible at mei-osis and occurs at the same frequency in each generation.These results confirm previous genetic and molecular re-sults obtained with transgenic tobacco plants. These stud-ies suggest that silencing is not due to DMA rearrangementsor ploidy variations (Dorlhac de Borne et al., 1994; Palauquiand Vaucheret, 1995).
NR Silencing Is Post-Transcriptional
RNA was extracted from leaves of the NR-deficient mutantE23, wild-type plants, and green and chlorotic descendantsof plant 30C.1 and hybridized with an Nia probe situated up-stream (Figure 2) or downstream (Figure 3) of the insertionsite of the retrotransposon. Full-length Nia transcripts accu-mulated in green descendants and in wild-type plants butnot in the mutant E23 and chlorotic descendants. Nuclearrunoff experiments were also performed with the same tis-sues to determine whether silencing occurs at the transcrip-tional or post-transcriptional level (Figure 4). The absence ofthe full-length Nia transcript in mutant E23 allowed us to an-alyze only the transcription of the transgene in line 30C.1 or
PBHDI E2330C.I
NS S
30C. I xPBHDINS S
Nia
Figure 3. Steady State Levels of Host and Transgenic Nia Tran-scripts in Silenced and Nonsilenced Descendants of Plant 30C.
RNA was extracted from leaves of a wild-type plant (PBHD1), mutantE23, and green nonsilenced (NS) or chlorotic silenced (S) descendantsof plant 30C.1 by selfing or crossing with PBHD1. Five microgramsof total RNA was hybridized with an Ma probe situated downstreamof the insertion site of retrotransposon Tnp2 in mutant E23.
only the transcription of the host gene in the wild type by us-ing a probe situated downstream of the insertion site of theretrotransposon. In addition, the presence of a single insertedcopy of the 35S-Ma2 transgene (Figure 1) allowed us to com-pare the rate of transcription of the host Nia gene with that ofthe 35S-/V/'a2 transgene. Runoff results indicated that the35S-Ma2 transgene is transcribed at a 3.5-fold higher rate inplant 30C.1 than is the host Ma gene in a wild-type plant. Re-sults also indicated that the 35S-/V/a2 transgene is tran-scribed similarly in the nucleus of green descendants thataccumulate Nia mRNA and in chlorotic descendants that donot accumulate the corresponding transcript. Therefore, si-lencing of the 35S-/V/a2 transgene is post-transcriptional.
Accumulation of the Truncated Nia-Tnp2 TranscriptIs Not Affected by Silencing, Whereas theAccumulation of the Wild-Type Nia TranscriptIs Affected
30C.INS
30C.I 30C.IS S
• full-length Nia
- truncated Nia-Tnp2
Figure 2. Steady State Levels of Host Nia-Tnp2 and Transgenic NiaTranscripts in Silenced and Nonsilenced Descendants of Plant 30C.
RNA was extracted from leaves of a green nonsilenced (NS) descen-dant of plant 30C.1, a wild-type plant (PBHD1), mutant E23, and twochlorotic silenced (S) descendants of plant 30C.1. Five microgramsof total RNA was hybridized with an Nia probe situated upstream ofthe insertion site of retrotransposon Tnp2 in mutant E23.
We reported previously that only a truncated Nia-Tnp2 tran-script accumulates in the NR-deficient mutant E23. Theabundance of this transcript is low. In addition, it is consid-erably reduced when a functional Nia host gene is introducedby crossing, because transcription of the Nia promoter issubjected to feedback regulation by end products of the ni-trate assimilation pathway (Vaucheret et al., 1992a). As ex-pected, the accumulation of the truncated Nia-Tnp2 transcriptwas very low (below detectable levels) in the green descen-dant of line 30C.1, whereas it could be detected in mutantE23, in which end products of the nitrate assimilation path-way failed to accumulate. Unexpectedly, it accumulated to asimilar level in mutant E23 and in chlorotic descendants ofline 30C.1 (Figure 2), thereby suggesting that the truncatedNia-Tnp2 transcript is not recognized as a target by the cel-lular machinery involved in silencing and RNA degradation.
To test whether the accumulation of the truncated Nia-Tnp2 transcript in silenced plants is due to truncation of thetranscript or whether it is a particularity of the N. plumbagini-folia Nia transcript, plant 30C.1 was crossed with a wild-type
1498 The Plant Cell
PBHDI E23 30C.I S 30C.1NS
B100% nd 337% 370%
pBS
Nia
rDNA
Nia/rDNA
Figure 4. Nuclear Transcriptional Levels of the Nia Host Gene andTransgene in Silenced and Nonsilenced Transgenic N. plumbagini-folia Plants.
Runoff experiments were performed with the leaves of a wild-typeplant (PBHD1), mutant E23, and green nonsilenced (NS) or chloroticsilenced (S) 30C.1 transgenic plants. Labeled RNA was hybridizedwith 2 n.g of double-stranded vector DNA containing the followinggene-specific sequences: rDNA; Nia, NR probe situated downstreamof the insertion site of retrotransposon Tnp2 in mutant E23; and pBS,empty pBluescript SK+ (Stratagene) vector. Each DNA was slottedtwice. Signals were quantified using lower exposure autoradiography.Percentages at the bottom indicate wild-type plants, nd, not detectable.
plant. Hybrid plants were grown in the greenhouse during thesummer. Among 200 plants, two were chlorotic. RNA was ex-tracted from green and chlorotic plants and hybridized withan Nia probe situated downstream of the insertion site ofTnp2. The Ma probe provides better signals than does theprobe situated upstream of the insertion site. No full-lengthtranscripts accumulated in chlorotic hybrids (Figure 3), thusindicating that the wild-type N. plumbaginifolia Nia transcript issubjected to cosuppression in transgenic N. plumbaginifoliaplants, as was found previously with the wild-type N. tabacumNia transcript in transgenic N. tabacum plants (Dorlhac deBorne et al., 1994; Palauqui and Vaucheret, 1995).
The Frequency of Silencing Depends on Both 35S-/V/a2Transgene and Wild-Type Nia Host Gene Copy Numbers
The frequency of NR silencing, that is, the percentage ofchlorotic plants, was measured in isogenic sibling progeniescarrying either zero, one, or two copies of the 35S-Ma2transgene in combination with zero, one, or two copies ofthe wild-type Ma gene. For this purpose, one line homozy-gous for both the transgene locus and the wild-type Magene was generated (see Methods) and named 30C.14.Isogenic sibling progenies were obtained by selfing orcrossing one with a single plant of each genotype as follows:30C.14.1 (35S-Ma2/35S-Ma2, Ma/Ma), 30C.1.1 (35S-Ma2/35S-Ma2, nia-Tnp2/nia-Tnp2), PBHD1 (-/-, Ma/Ma), andE23 (-/-, nia-Tnp2/nia-Tnp2).
Plants were grown in the greenhouse during the summer.The number of plants becoming chlorotic was determinedthroughout the life of the plants. As observed previously withtransgenic tobacco plants, chlorosis appeared between ger-mination and flowering but not later (Dorlhac de Borne et al.,1994; Palauqui and Vaucheret, 1995). In contrast to tobaccoplants that can become partially chlorotic at low frequency(Palauqui et al., 1996), transgenic N. plumbaginifolia plantsalways became completely chlorotic. The frequencies of si-lencing are reported in Table 1. This comparison of lines car-rying the same number of copies of the wild-type host geneshowed that the frequency of silencing increases when thenumber of copies of the 35S-Ma2 transgene increases(35S-Ma2/35S-Ma2, Ma/Ma at 100% versus 35S-Ma2/-,Ma/Ma at 2%; 35S-Ma2/35S-Ma2, nia-Tnp2/nia-Tnp2 at29% versus 35S-Ma2/-, nia-Tnp2/nia-Tnp2 at 1%). Simi-larly, the comparison of lines carrying the same number ofcopies of the 35S-Ma2 transgene showed that the fre-quency of silencing increases when the number of copies ofthe wild-type host gene increases (35S-Ma2/35S-Ma2, Ma/Ma at 100% versus 35S-Ma2/35S-Ma2, Nia/nia-Tnp2 at 50%versus 35S-Ma2/35S-Ma2, nia-Tnp2/nia-Tnp2 at 29%).
These results confirm that host genes and transgenes cancooperate in the triggering of cosuppression. Nevertheless,in our study, the influence of the wild-type host gene seemsto be weaker than that of the 35S-Ma2 transgene. Becausethe transcription rate of the 35S-M'a2 transgene is 3.5-foldhigher than that of the wild-type host Ma gene (see the re-sults of the runoff experiments in Figure 4), this result agreeswith a threshold hypothesis involving the dosage of a geneproduct (probably RNA).
Table 1. Determination of NR Cosuppression Frequencies inIsogenic Lines of N. plumbaginifolia Carrying Zero, One, or TwoCopies of the Wild-Type Nia Gene in Combination with Zero, One, orTwo Copies of a 35S-/V/a2 Transgene
SiblingProgenies3
30C.14.1 selfed30C.14.1 x 30C.1.130C.1.1 selfed30C.14.1 x PBHD130C.1.1 x PBHD130C.1.1 x E23PBHD1 selfedPBHD1 x E23E23 selfed
Wild-Type NiaCopyNumber
210210210
35S-N/a2CopyNumber
222111000
Number ofSilencedPlants
100/10050/10029/100
2/1001/1000/1000/1000/100
-a Isogenic sibling progenies were obtained by selfing or crossing onewith a single plant of each genotype as follows: 30C14.1 (35S-A//a2/35S-W/a2, Nia/Nia), 30C.1.1 (35S-N/a2/35S-/V/a2, nia-Tnp2/nia-Tnp2), E23 (-/-, nia-Tnp2/nia-Tnp2), and PBHD (-/-, Nia/Nia).One hundred plants of each combination were grown in the green-house at the same time.
Nitrate Reductase Cosuppression 1499
Table 2. Determination of NR Cosuppression Frequencies in lntraspecific or lnterspecific Hybrids Carrying One or Two Copies of a 35SNia2 Locus in a Triploid or Tetraploid Background
Sibling Progeniesa Copy Number Copy Number Genomes Genome Plants Host Loci Transgene Loci Number of Haploid Loci per Haploid Number of Silenced
27-44.7.8 selfed 4 2 4 1.5 24/1 O0 27-44.7.8 X PBDG 4 1 4 1.2 0/1 O0 27-44.7.8 X N. glauca 3 1 3 1.33 10/1 O0 27-44.7.8 x N. sylvestris 3 1 3 1.33 9/1 O0
a lsogenic sibling progenies were obtained by selfing or crossing one with a single plant of each genotype as follows: 27-44.7.8 (35S-Nia2/35S- Nia2, NiallNial, NiaZ/NiaZ), PBDG (-/-, Nial/Nial, NiaZlNiaP), N. glauca (-/-, Nia/Nia), and N. sylvestris (-/-, Nia/Nia). One hundred plants of each combination were grown in the greenhouse at the same time.
Frequency of Silencing Depends on the Cellular Ploidy Leve1
The results given above indicate that when the cellular ploidy level remains constant, the frequency of cosuppres- sion depends on both host gene and transgene copy num- bers. To test the influence of the cellular ploidy level, we performed interspecific crosses between wild-type Nicofiana diploid species and a transgenic tobacco (N. tabacum) line carrying the 35s-Nia2 transgene. Tobacco is an allotetraploid (or amphidiploid) species containing 48 chromosomes, which is a result of the fusion of the genomes of its two ancestors, N. sylvestris (24 chromosomes) and N. tomentosiformis (24 chromosomes). Thus, tobacco carries four allelic copies of the Nia gene, with two alleles coming from each ancestor.
The transgenic tobacco line 27-44.7 is homozygous for a single transgene locus carrying two or three copies of the 35s-Nia2 transgene (Palauqui and Vaucheret, 1995). A sin- gle plant derived from this line (27-44.7.8; 48 chromosomes) was self-fertilized, backcrossed with a wild-type tobacco plant (PBD6; 48 chromosomes), or crossed with two diploid Nicofiana species, N. sylvestris (24 chromosomes) and N. glauca (24 chromosomes). The number of chlorotic plants was determined after 3 months of growth in the greenhouse (Table 2). None of the plants carrying one copy of the 35S- Nia2 transgene locus and four copies of the host Nia genes in a tobacco background (48 chromosomes) showed cosup- pression, whereas 24 of 100 plants carrying two copies of the 35s-Nia2 transgene locus and four copies of the host Nia genes in a tobacco background (48 chromosomes) showed cosuppression, thus confirming a dose effect. Inter- mediate frequencies of cosuppression, that is, nine or 10 plants of 100, were obtained using hybrids carrying one copy of the 35s-Nia transgene locus and three copies of the host Nia genes in an N. sylvestris and N. tabacum or N. glauca and N. tabacum hybrid background (36 chromo- somes). Although it is possible that outcrossing with differ- ent species indirectly affects cosuppression, this result suggests that the frequency of cosuppression depends on the ratio between host gene and transgene copy numbers and the number of haploid genomes. This is consistent with the hypothesis of gene dosage.
Transcriptional Silencing of the 35s-Nia2 Transgene Impedes Cosuppression of Nia Host Genes and Transgenes
To test whether a modification of transgene expression without changes in copy number, genomic position, or cellu- lar ploidy level can also influence the frequency of cosup- pression, we crossed a transgenic tobacco line exhibiting cosuppression of host Nia genes and 35s-Nia2 transgenes with a transgenic tobacco line carrying a 35s-specific si- lencing locus. Tobacco line 271.5.8 is homozygous for a transgene locus (locus 271) that carries a 35s-RiN trans- gene consisting of the nitrite reductase cDNA cloned in an antisense orientation under the control of the 35s promoter. This transgene is transcriptionally silent and impedes in frans the expression of the host nitrite reductase genes at the post-transcriptional level and the expression of any transgene driven by the 35s promoter, irrespective of its po- sition and of the nature of the coding sequence, at the tran- scriptional level (Vaucheret, 1993; Elmayan and Vaucheret, 1996; Park et al., 1996). Plant 271 5.8 was retransformed by a construct carrying the bean nitrite reductase gene that is insensitive to silencing by locus 271 and that restores nitrite reductase activity (lhierry and Vaucheret, 1996). Transgenic line 271-22-2.1 1 is homozygous for locus 271 and homozy- gous for the bean nitrite reductase gene.
Transgenic line 30-1 8.9 is homozygous for two transgene loci, each carrying two copies of the 35SNia transgene (Palauqui and Vaucheret, 1995). This line shows cosuppres- sion of Nia host genes and transgenes with a frequency of 100%. A single plant derived from this line (30-18.9.6) was crossed with either a wild-type plant (PBD6) or plant 271 -22- 2.1 1. The silencing effect of locus 271 on the different copies of the 35s-Nia2 transgene carried by plant 30-18.9 was ana- lyzed by sowing hybrid seeds on medium supplemented with chlorate (the 35s-Nia2 transgene confers sensitivity to chlor- ate). None of the hybrid seedlings carrying locus 271 died, whereas all hybrid seedlings lacking locus 271 died on this medium, indicating that locus 271 totally inhibits the expres- sion of all copies of the 35s-Nia2 transgene carried by plant 30-18.9. Plants were grown in the greenhouse, and the per- centage of chlorotic plants was determined (Table 3). As
1500 The Plant Cell
Table 3. Determination of NR Cosuppression Frequencies in Isogenic Lines of N. tabacum Carrying One or Two Copies of a 35S-W/a2 Locusand Zero or One Copy of a 35S-Specific Transcriptional Silencing Locus (271)
Sibling Progenies3
30-1 8.9.6 selfed30-18.9.6 x PBD630-18.9.6 x 271-22.2.11
Host Loci CopyNumber
444
Transgene Loci CopyNumber
422
Transcription of theTransgene
YesYesNo
Loci per HaploidGenome21.51.5
Number of SilencedPlants
100/10015/1000/100
a Isogenic sibling progenis were obtained by selfing or crossing one with a single plant of each genotype as follows: 30-18.9.6 (35S-N/'a2/35S-W/a2, -/-, Nia1/Nia1, Nia2/Nia2), 271-22-2.11 (-/-, 271/271, Nia1/Nia1, Nia2/Nia2), and PBD6 (-/-, -/-, Nia1/Nia1, Nia2/Nia2). One hundredplants of each combination were grown in the greenhouse at the same time.
found previously, transgenic plant 30-18.9.6 when selfedshowed 100% silencing, whereas plants resulting from thebackcross with wild-type plant PBD6 showed Cosuppressionwith a frequency of 15% (Palauqui and Vaucheret, 1995).Conversely, none of the plants resulting from the cross withplant 271-22-2.11 showed Cosuppression, suggesting thattranscriptional silencing of the 35S-Ma2 transgene by the271 locus impedes the triggering of Cosuppression of Niahost genes and transgenes.
RNA was extracted from leaves of wild-type plants, greenand chlorotic plants resulting from the cross between line30-18.9.6 and PBD6, and plants resulting from the cross be-tween line 30-18.9.6 and line 271-22-2.11 and hybridized withan Nia probe (Figure 5). No Ma mRNA accumulated in chlo-rotic 30-18.9.6 x PBD6 plants, whereas Ma mRNA accumu-lated to a much higher level in green 30-18.9.6 x PBD6plants than it did in wild-type plants. Conversely, Ma mRNAaccumulated to the same level in 30-18.9.9 x 271-22-2.11plants as it did in wild-type PBD6 plants. This result con-firms that the expression of the 35S-Ma2 transgene is abol-ished by the 35S-specific silencing locus of plant 271, asshown above by the results of the chlorate tests and previ-ously by a runoff analysis with 35S-uidA and 35S-/ipf trans-genes silenced by this locus (Elmayan and Vaucheret, 1996;Park et al., 1996). This result demonstrates that the pres-ence of the 35S-Ma2 transgene per se is not sufficient totrigger Cosuppression and suggests that the transgene mustbe in a transcriptionally active state.
duction of the coding sequence of a host gene under thecontrol of the strong 35S promoter, this phenomenon hasbeen primarily considered as a response of the plant to theoverexpression of the corresponding gene.
RNA is supposed to be the target of Cosuppression be-cause nuclear runoff experiments have shown that cosup-pression does not affect the transcription of host genes andtransgenes but occurs at a post-transcriptional level (VanBlokland et al., 1994; de Carvalho Niebel et al., 1995; Kunzet al., 1996). To explain the absence of RNA accumulation inthe cytoplasm of silenced plants, several groups have devel-oped the working hypothesis of a "biochemical switch" or"threshold hypothesis" (Meins, 1989; Dehio and Schell,1994; Dougherty and Parks, 1995). According to this hy-pothesis, cells must be able to count the products of theirgenes and to trigger the specific degradation of RNAs whenone gene product accumulates above a certain thresholdlevel. However, English et al. (1996) disagreed with this hy-pothesis and reported no difference in the level of transgenetranscription between silenced and nonsilenced transgeniclines. In addition, the post-transcriptional silencing of hostCHS genes by a weakly transcribed or (untranscribed) pro-moterless CHS transgene has been reported (Van Bloklandet al., 1994). Alternative hypotheses were therefore proposedthat involved DNA-DNA interactions leading to the productionof aberrant RNAs by transgenes and/or host genes, and thesubsequent targeting and degradation of these aberrant
DISCUSSION
Cosuppression is a potential problem that every plant biolo-gist must keep in mind when supernumerary/multiple (re-combinant) copies of an endogenous gene are introducedinto the corresponding host by transformation or crossing.Approximately 15 examples of Cosuppression of homolo-gous host genes and transgenes have been reported to datein the literature, and this must constitute a minor representa-tion of problems encountered by breeding companies(Rnnegan and McElroy, 1994). Because most of the exam-ples of Cosuppression have been reported after the intro-
PBD6
30-18.9.6\PBD6 30-18.9.6 xS NS 2 7 l - 2 2 - 2 . l l
Nia
Figure 5. Effect of a 35S-Specific Transcriptional Silencing Locuson the Nia Steady State Levels in Transgenic N. tabacum Plants.RNA was extracted from leaves of a wild-type plant (PBD6) andgreen nonsilenced (NS) or chlorotic silenced (S) progenies of thecrosses 30-18.9.6 x PBD6 and 30-18.9.6 x 271-22-2.11 (271-22-2.11is homozygous for a 35S-specific transcriptional silencing locus).Five micrograms of total RNA was hybridized with an M'a probe.
Nitrate Reductase Cosuppression 1501
RNAs and of every homologous mRNA (Flavell, 1994; Van Blokland et al., 1994; Baulcombe and English, 1996).
In an attempt to elucidate whether an increase in DNA copies or an overproduction of the corresponding RNA is re- sponsible for the triggering of cosuppression, we designed experiments to modify DNA and RNA dosages, both to- gether and independently. For this purpose, we used the model system of NR cosuppression. This system allows a rapid study of cosuppression based on phenotypic analysis because a deficiency in NR is cell autonomous and leads to an easily detectable chlorosis of the leaves (Gabard et al., 1987). lntroduction of a 35s-Nia2 transgene into the diploid species N. plumbaginifolia or the allotetraploid species N. tabacum leads to efficient cosuppression of host Nia genes and 35s-Nia2 transgenes (Dorlhac de Borne et al., 1994; Palauqui and Vaucheret, 1995; Palauqui et al., 1996; this work). We attempted to modify host gene and transgene DNA dosage by crossing transgenic lines carrying different numbers of active transgene loci with wild-type plants of various ploidy levels. In addition, we attempted to modify host gene or transgene RNA dosage (without modifying DNA dosage) by using transgenic line 271 carrying a 35S- specific transcriptional silencing locus, by using an NR-defi- cient mutant line carrying a host Nia gene disrupted by the insertion of a retrotransposon, or by growing plants under physiological conditions that induce or repress transcription of wild-type host Nia genes.
A dosage effect was clearly demonstrated using various combinations of diploid, triploid, and tetraploid plants. N. plumbaginifolia is a true diploid species carrying a single Nia locus per haploid genome, whereas N. tabacum, com- monly named tobacco, is an allotetraploid (or amphidiploid) species carrying two Nia loci per haploid genome (Vaucheret et al., 1989). Transgenic tobacco lines homozygous for a 35SNia2 transgene inserted at a single Iocus, that is, carry- ing a total of 1.5 Nia loci per haploid genome, showed co- suppression with an efficiency varying between O and 57%: cosuppression affected a limited fraction of an isogenic population of plants in each generation, but none of the lines reached 100% (Palauqui and Vaucheret, 1995). Only the transgenic lines homozygous for two transgene loci (carry- ing a total of two Nia loci per haploid genome) showed 100% cosuppression (Palauqui and Vaucheret, 1995). In ad- dition, transgenic lines of N. plumbaginifolia homozygous for a 35s-Nia2 transgene inserted at a single locus (carrying a total of two Nia loci per haploid genome) showed cosup- pression with 100% efficiency. Triploid hybrids hemizygous for one transgene locus (carrying a total of 1.33 Nia loci per haploid genome) showed frequencies of cosuppression be- tween those of tetraploid lines hemizygous for one transgene locus (carrying a total of 1.2 Nia loci per haploid genome) and tetraploid lines homozygous for one transgene locus (carry- ing a total of 1.5 Nia loci per haploid genome). Taken together, these results indicate that the relative dosage of host genes and transgenes per cell plays an important role in the effi- ciency of triggering of cosuppression. However, because both
the host genes and transgenes in these plants are transcrip- tionally active, DNA and RNA dosages vary concomitantly, and it is not possible to determine whether cosuppression results from a DNA dosage effect or from an RNA dosage effect.
To test the RNA dosage hypothesis, we used transcrip- tionally inactive forms of the host gene or transgenes to modify RNA dosage without modifying DNA dosage. First, introgression of a 35s-specific silencing locus into a trans- genic tobacco line that usually shows cosuppression of the host Nia genes and the 35s-Nia2 transgene led to silencing of the 35s-Nia2 transgene and completely prevented the triggering of host gene cosuppression, thus suggesting that transgene transcription is required for NR cosuppression. Second, the replacement of the wild-type Nia allele of N. plumbaginifolia by a mutant allele carrying the retrotrans- poson Tnp2 inserted at amino acid 154 (leading to the for- mation of a truncated Nia-Tnp2 transcript, which is not subject to cosuppression) significantly decreased the fre- quency of NR silencing, thus suggesting that transcription of a full-length transcript by the host gene also contributes to the efficiency of triggering. Finally, we previously reported that growing plants on medium deprived of nitrate (which is required to induce the transcription of Nia host genes) sig- nificantly delays the triggering of cosuppression (Dorlhac de Borne et al., 1994), thus confirming the contribution of host gene transcription. These results suggest that the dosage effect reported here may be related to RNA and not to DNA.
However, these inactive forms of the 35s-Nia2 transgene and of the host Nia genes may not be able to undergo the DNA-DNA pairing event that initiates the production of aber- rant RNAs by one or the other partner (according to the DNA-DNA pairing hypothesis of Baulcombe and English, 1996), thus impeding the triggering of cosuppression. The chromatin structure of the host Nia gene disrupted by the Tnp2 retrotransposon may be different from that of the wild- type allele, thus affecting pairing with the 35s-Nia2 trans- gene. Similarly, the chromatin structure of the wild-type host Nia gene may also be different when the gene is transcribed (with nitrate) or is not transcribed (without nitrate), thus af- fecting pairing with the 35SNia2 transgene. In addition, the chromatin structure of the 35S-Nia2 transgene may be differ- ent because of transcriptional silencing by the tobacco locus 271. Indeed, we previously reported that the 271 locus trig- gers de novo methylation in the 35s promoter of target trans- genes (Vaucheret, 1993; Park et al., 1996). It is conceivable that such a modification affects the pairing of the 35SNia2 transgene with the host Nia genes. Therefore, the results de- scribed here may be interpreted according to the DNA-DNA pairing hypothesis as well as the RNA dosage hypothesis.
To summarize, we observed that the frequency of NR co- suppression (1) decreases when full-length transcription of a host gene is impeded (while the host gene is still present at the DNA level but in a different state, i.e., disrupted or inactive), (2) is abolished when transgene transcription is impeded (while the transgene is still present at the DNA level, but in a
1502 The Plant Cell
different state, i.e., silenced in trans), (3) increases when the copy number of functional host gene loci increases, (4) in- creases when the copy number of functional transgene loci increases, and (5) increases when the ratio of (trans)gene copy number per haploid genome increases. These results strongly suggest that a transcriptionally active state of the 35s-Nia2 transgene, and to a lesser extent of the host Nia genes, is necessaty to trigger cosuppression of NR. Whether this reflects an RNA dosage mechanism or a DNA-DNA pairing mechanism remains to be determined.
Because post-transcriptional silencing of endogenous genes has been reported not only in the case of transcribed transgenes (Van Blokland et al., 1994; de Carvalho Niebel et al., 1995; Kunz et al., 1996) but also in the case of weakly transcribed or (untranscribed) promoterless transgenes (van Blokland et al., 1994), DNA-DNA pairing seems more attrac- tive. However, efficient post-transcriptional gene silencing has also been reported in hemizygous or haploid plants car- rying a single copy of the 35s X 2-uidA transgene, in which DNA-DNA pairing cannot occur (Elmayan and Vaucheret, 1996). These two contradictoty results suggest that there may be two alternative ways to trigger post-transcriptional silenc- ing. One way involves RNA overproduction by the transgene. If we consider that a small fraction of the transcribed RNA consists of naturally aberrant RNAs (because of the infidelity of the RNA polymerase), an increase in transcription using a very strong promoter will increase both the fraction of normal RNA and aberrant RNAs. Cosuppression could then be trig- gered when the amount of aberrant RNAs reaches a particular threshold level that activates the cellular machinery involved in RNA degradation. Alternatively, sometimes transcribed or untranscribed transgenes might be able to interact directly with endogenous genes by DNA-DNA pairing. This interac- tion might lead to a change in the ratio between normal and aberrant RNAs produced by host genes and/or the trans- genes, thereby allowing aberrant RNAs to reach the threshold level that triggers post-transcriptional silencing without re- quiring strong transgene transcription.
METHODS
Plant Material and Genetic Analysis
Transgenic plant 30C was obtained by Agrobacterium tumefaciens- mediated transformation of the Nicotiana plumbaginifolia E23 mutant (Gabard et al., 1987), using a cauliflower mosaic virus 35s-Ma2 transgene linked to a pNos-Npt selectable marker (Vincentz and Caboche, 1991). Genetic analyses were performed by sowing sur- face-sterilized seeds in vitro on medium supplemented with either 50 mg/L kanamycin or 6 mM chlorate to indicate that the transgene had been inserted ata single locus. Homozygous line 30C.1 was selected by scoring F, progeny for 100% resistance to kanamycin and 100% sensitivity to chlorate.
Plant 30C.14 was obtained as follows. Plant 30C.1 was crossed with a wild-type plant (N. plumbaginifolia cv PBHDI). The hybrid
plant was selfed. Twenty-four plants from the progeny were grown in the greenhouse and selfed. Plants whose progeny showed in vitro a 3:l resistant-to-sensitive segregation ratio on medium supple- mented with either kanamycin or chlorate and a 100% ability to grow on medium containing nitrate as sole nitrogen source were desig- nated as being hemizygous for the transgene locus and homozygous for the host wild-type Nia gene. One of these plants was selfed. Twelve progeny of this self-cross were grown in the greenhouse and selfed. Plants from populations showing 100% resistance to kana- mycin and 100% sensitivity to chlorate in vitro were retained as being homozygous for both the host wild-type Nia gene and the 30C trans- gene locus. One of these plants was retained and named 30C.14.
The transgenic plant 30-1 8 was obtained by Agrobacterium-medi- ated transformation of ‘N. tabacum cv PBD6, using the 35S-Nia2 transgene linked to a pNos-Npt selectable marker (Dorlhac de Borne et al., 1994). Transgenic line 30-18.9 was derived by selfing and was previously shown to be homozygous for two 35s-Nia2 loci by ge- netic and molecular analysis (Palauqui and Vaucheret, 1995).
Transgenic plant 271 -22-2 was obtained by Agrobacterium-medi- ated transformation of transgenic lhe 271 5.8 (Vaucheret et al., 1992b; Vaucheret, 1993), using the bean nitrite reductase gene linked to a pNos-Npt selectable marker (Thierry and Vaucheret, 1996). Line 271 5.8 is homozygous for a transgene locus that inactivates host ni- trite reductase genes at the post-transcriptional level and any 35S- driven transgenes at the transcriptional level (Elmayan and Vaucheret, 1996; Park et al., 1996). Transgenic line 271 -22-2.1 1 was derived by selfing and shown to be homozygous for the bean gene that restores nitrite reductase activity.
Growth Conditions and Determination of the Frequency of Cosuppression
The different combinations of host and transgene loci were obtained by self-fertilization or crossing using a single plant derived from each of the following homozygous lines: 30C.1 . I , 30C.14.1, E23, PBHDI, 27-44.7.8, 30-18.9.6, 271-22-2.1 1, and PBD6. For cosuppression analysis, 100 plants were sown in vitro and transferred to the green- house after 1 month of growth. Plants were grown in the greenhouse during the summer under natural conditions or during the winter at 23°C with a 16-hr-light and 8-hr-dark photoperiod and 120 pE sec-I lighting. Plants were watered with a nutrient solution as de- scribed by Cok and Lesaint (1971). The number of chlorotic (si- lenced) plants was determined every 2 weeks. To produce sufficient material for molecular analysis, silenced plants were grafted onto wild-type tobacco stocks.
RNA Gel Blot Analysis and Nuclear Runoff Transcription Assays
DNA and RNA were extracted from leaves at the beginning of the day period, as described previously (Vaucheret et al., 1992b). DNA gel blot analysis was performed using 35s or Npt probes (Palauqui and Vaucheret, 1995). RNA gel blot analysis was performed using a 1.6-kb EcoRl interna1 fragment or a 0.2-kb Sacl-Sacl fragment lo- cated at the 5’ end of the Nia2 cDNA (Vincentz and Caboche, 1991).
Nuclei were isolated from leaf tissue essentially as described by Luthe and Quatrano (1980), with three exceptions: (1) leaves were homogenized in Honda buffer containing 2.5% Triton X-100, and the nuclear pellet was dissolved in Honda buffer without Triton X-100; (2) most nuclei were found in the pellet after centrifugation in a discon- tinuous Percoll gradient; and (3) purified nuclei were washed and re-
Nitrate Reductase Cosuppression 1503
suspended in 20 mM Hepes, pH 7.2,5 mM MgC12, 2 mM DTT, and 30% glycerol. Nuclei from 10 g of N. plumbaginifolia leaf tissue were resuspended in 4 mL of that mix and stored in 0.25-mL aliquots at
Nuclear runoff and nascent RNA hybridizations were performed acccording to Hagen and Guilfoyle (1985). One hundred milliliters of 3 x transcription buffer (plus heparin) were added to 200 mL of nu- clear suspension (107 nuclei) with 250 mCi of 32P-UTP (3000 Ci mmol-I; New England Nuclear, Beverly, MA). Labeled RNA was re- suspended in I .5 mL of hybridization buffer.
Samples of 2 mg of double-stranded DNAs corresponding to a 1.6-kb EcoRl interna1 fragment of the Nia2 cDNA and to rDNA were applied to a Hybond N (Amersham) filter by dot blotting (model SRC 96 minifold I dot blotter; Schleicher & Schuell). Prehybridization, hy- bridization, and washing were performed as described previously (Vaucheret et al., 1992). Analysis was performed by scanning three different exposures from runoff autoradiography with an EASY. RH3 camera, and signals were quantified by using the EASY. program (Herlab GmbH, Wiesloch, Germany).
-80°C.
ACKNOWLEDGMENTS
We thank Jean-Marie Pollien for taking care of the plants in the greenhouse and lan Small for critical reading of the manuscript.
Received February 24, 1997; accepted May 7, 1997.
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DOI 10.1105/tpc.9.8.1495 1997;9;1495-1504Plant Cell
H. Vaucheret, L. Nussaume, J. C. Palauqui, I. Quillere and T. Elmayanof Nitrate Reductase Host Genes and Transgenes.
A Transcriptionally Active State Is Required for Post-Transcriptional Silencing (Cosuppression)
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